WO2023187725A1

WO2023187725A1 - Targeted delivery of transgenes in plants

Info

Publication number: WO2023187725A1
Application number: PCT/IB2023/053217
Authority: WO
Inventors: Foley L.S. MARQUESS; Grant Nathan WORONUK
Original assignee: Relica Genomics Inc
Current assignee: Relica Genomics Inc
Priority date: 2022-03-30
Filing date: 2023-03-30
Publication date: 2023-10-05
Anticipated expiration: 2024-09-30

Abstract

The present invention is directed to a transformed plant and a method for producing and using the same, or components thereof.

Description

TARGETED DELIVERY OF TRANSGENES IN PLANTS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63/325,347, filed

March 30, 2022, the contents of which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] Chloroplast transformation is an attractive approach for developing transgenic plants where high ectopic expression levels of exogenous materials is desired. Each plant cell has 10,000 copies of chloroplast genome from which to express constructs (Shahid and Daniell, 2016), and transplastomic expression studies have shown to produce as much as >70% of total soluble protein (Ruhlman et al., 2010, see also McBride et al., 1995). Other advantages include maternal inheritance that decreases transgene dispersal (Heifetz, 2000), polycistronic expression per transformation event (Hanson et al., 2013), and reduced gene silencing resulting from homologous recombination (see Adem et al., 2017 and references therein). However, methods for generating transgenic plants via chloroplast transformation to date has been demonstrated only in a limited number of plant species, e.g., the Nicotiana genus (Rigano et al., 2012).

SUMMARY OF THE INVENTION

[0003] The present disclosure provides, among other things, methods of transforming a plant and compositions including transformed plants or portions thereof (e.g., an exogenous protein or fragment thereof produced by a plant encompassed in the present disclosure).

[0004] In one aspect of the disclosure, methods of transforming a plant include providing a nucleic acid material and transforming a chloroplast in a plant cell with the nucleic acid material. In some embodiments, a nucleic acid material includes an expression cassette comprising, e.g., in 5’ to 3’ orientation a first (5’) targeting sequence, a promoter sequence, an exogenous nucleic acid sequence, and a second (3’) targeting sequence. In some embodiments, an expression cassette also includes one or more additional components such as a selection sequence and/or an enhancer sequence. In some embodiments, a nucleic acid material is complexed with a carrier for enhanced delivery to the chloroplast genome. In some embodiments, the first (5’) targeting sequence and/or the second (3’) targeting sequence comprises 16S ribosomal gene DNA sequence of the plant. In some embodiments, the first (5’) targeting sequence and/or the second (3’) targeting sequence comprising 16S ribosomal gene DNA sequence of the plant contains at least one mutation relative to the native plant 16S ribosomal gene DNA sequence.

[0005] In some embodiments, the first (5’) targeting sequence comprises 16S ribosomal gene DNA sequence of the plant containing at least one mutation relative to the native plant 16S ribosomal gene DNA sequence. In some embodiments, the second (3’) targeting sequence comprises 23S ribosomal gene DNA sequence of the plant. In some embodiments, the second (3’) targeting sequence comprises 23 S ribosomal gene DNA sequence of the plant comprises at least one mutation relative to the native plant 23 S ribosomal gene DNA sequence.

[0006] In some embodiments, the method further comprises expressing the exogenous nucleic acid sequence, wherein the expression occurs, at least in part, in a chloroplast. In some embodiments, transformation occurs, at least in part, through homologous recombination.

[0007] In some embodiments, transforming the chloroplast comprises contacting the plant cell with the nucleic acid material. In some embodiments, contacting the plant cell comprises use of biolistics or gene gun, use of chloroplast targeting sequences/peptides, cell penetrating peptides, use of a carrier such as a functionalized nanoparticle, electroporation, chemical-mediated transfection (e.g. using polyethylene gylcol), or any combination thereof. In some embodiments, contacting comprises culturing the nucleic acid material in a solution comprising the plant cell for at least 1 hour. In some embodiments, contacting comprises introducing the nucleic acid material into the plant via syringe injection. In some embodiments, syringe injection comprises surface leaf infusion through a needleless syringe. In some embodiments, syringe injection comprises stem injection through a needled syringe. In some embodiments, contacting comprises applying a vacuum and/or compression to the plant cell.

[0008] In some embodiments, the at least one mutation comprises a mutation that confers antibiotic resistance in the plant. In some embodiments, antibiotic resistance comprises resistance to one or more of lincomycin, spectinomycin, streptomycin, kanamycin, gentamycin, neomycin, Beta lactam resistance, and any combination thereof. In some embodiments, the antibiotic resistance comprises resistance to lincomycin, spectinomycin, and streptomycin [0009] In some embodiments, the plant is millet, sorghum, wheat, maize, barley, triticale, or alfalfa.

[0010] In some embodiments, the 16S ribosomal gene DNA sequence of the first (5’) targeting sequence comprises a core sequence that has 90% sequence identity to the sequence corresponding to positions 95,395-95,672 of the millet plastid genome sequence (GenBank Accession No. KU343177.1), or the sequence represented SEQ ID NO: 21 or 135. In some embodiments, the 16S ribosomal gene DNA sequence of the first (5’) targeting sequence comprises a core sequence corresponding to positions 95, 395-95, 672of the millet plastid genome sequence (GenBank Accession No. KU343177.1 or the sequence represented by SEQ ID NO: 21 or 135). In some embodiments, the at least one mutation comprises a C to A nucleotide substitution at position 95,395 and/or an A to a C nucleotide substitution at position 95,672 of the millet plastid genome sequence (GenBank Accession No. KU343177.1).

[0011] In some embodiments, the first and second targeting sequences comprise SEQ ID NO: 13, 133, or 85 and SEQ ID NO: 14 or 87, respectively.

[0012] In some embodiments, the 23 S ribosomal gene DNA sequence of the second (3’) targeting sequence comprises a core sequence that has at least 90% identity to the sequence corresponding to positions 100,566-100,594 (GenBank Accession No. KU343177.1) or SEQ ID NO: 89. In some embodiments, the 23 S ribosomal gene DNA sequence of the second (3’) targeting sequence comprises a core sequence corresponding to positions 100,566-100,594 (GenBank Accession No. KU343177.1) or SEQ ID NO: 89. In some embodiments, the at least one mutation relative to the native plant 23S ribosomal gene DNA sequence comprises one or more of: (i) a Gto A nucleotide substitution at position 100,566; (ii) an A to a G nucleotide substitution at position 100,593; and (iii) an A to G nucleotide substitution at position 100,594 of the millet plastid genome sequence (GenBank Accession No. KU343177.1).

[0013] In some embodiments, the 16S ribosomal gene DNA sequence of the first (5’) targeting sequence comprises a core sequence that has at least 90% identity to the sequence corresponding to positions 96,895-97,172 of the sorghum chloroplast genome sequence (GenBank Accession No. NC 008602.1) or SEQ ID NO: 20. In some embodiments, the 16S ribosomal gene DNA sequence of the first (5’) targeting sequence comprises a core sequence corresponding to positions 96,895-97,172 of the sorghum chloroplast genome sequence (GenBank Accession No. NC 008602.1) or SEQ ID NO: 20. In some embodiments, the at least one mutation comprises a C to A nucleotide substitution at position 96,895 and/or an A to a C nucleotide substitution at position 97,172 of the sorghum chloroplast genome sequence (GenBank Accession No. NC_008602.1).

[0014] In some embodiments, the 23 S ribosomal gene DNA sequence of the second (3’) targeting sequence comprises a core sequence that has at least 90% identity to the sequence corresponding to positions 102,072-102,100 of the sorghum chloroplast genome sequence (GenBank Accession No. NC_008602.1) or SEQ ID NO: 94. In some embodiments, the 23S ribosomal gene DNA sequence of the second (3’) targeting sequence comprises a core sequence corresponding to positions 102,072-102,100 of the sorghum chloroplast genome sequence (GenBank Accession No. NC 008602.1) or SEQ ID NO: 94. In some embodiments, the at least one mutations relative to the native plant 23S ribosomal gene DNA sequence comprises one or more of: (i) a Gto A nucleotide substitution at position 102,072; (ii) an A to a G nucleotide substitution at position 102,098; and (iii) an A to G nucleotide substitution at position 102,099 of the sorghum chloroplast genome sequence (GenBank Accession No. NC_008602.1).

[0015] In some embodiments, wherein the first and second targeting sequences comprise SEQ ID NO: 11, 132, or 90 and SEQ ID NO: 12 or 92, respectively.

[0016] In some embodiments, the 16S ribosomal gene DNA sequence of the first (5’) targeting sequence comprises a core sequence that has at least 90% sequence identity to the sequence corresponding to positions 99,019 -99,297 of the alfalfa plastid genome sequence (GenBank Accession No. NC_042841.1), positions 38,069-38,097 of the alfalfa plastid genome sequence GenBank KU321681.1, or SEQ ID NO: 22. In some embodiments, the 16S ribosomal gene DNA sequence of the first (5’) targeting sequence comprises a core sequence corresponding to positions 99,019 -99,297 of the alfalfa plastid genome sequence (GenBank Accession No. NC_042841.1), positions 38,069-38,097 of the alfalfa plastid genome sequence GenBank KU321681.1, or SEQ ID NO: 22. In some embodiments, the at least one mutation comprises a C to A nucleotide substitution at position 99,019 and/or an A to a C nucleotide substitution at position 99,297 of the alfalfa plastid genome sequence (GenBank Accession No. NC_042841.1).

[0017] In some embodiments, the 23 S ribosomal gene DNA sequence of the second (3’) targeting sequence comprises a core sequence that has at least 90% identity to the sequence corresponding to positions 38,069-38,097 of the alfalfa plastid genome sequence GenBank Accession No. KU321683.1 or SEQ ID NO: 99. In some embodiments, the 23S ribosomal gene DNA sequence of the second (3’) targeting sequence comprises a core sequence corresponding to positions 38,069-38,097 of the alfalfa plastid genome sequence GenBank Accession No. KU321683.1 or SEQ ID NO: 99. In some embodiments, the at least one mutation relative to the native plant 23 S ribosomal gene DNA sequence comprises one or more of: (i) a Gto A nucleotide substitution at position 38,069; (ii) an A to a G nucleotide substitution at position 38,096; and (iii) an A to G nucleotide substitution at position 38,097 of the alfalfa plastid genome sequence GenBank Accession No. KU321683.1.

[0018] In some embodiments, the first and second targeting sequences comprise SEQ ID NO: 17, 136, or 95 and SEQ ID NO: 18 or 97, respectively.

[0019] In some embodiments, a promoter sequence is selected from PpsbA, Prrn, Prna, psaA, PrbcL, CaMV35S, rbcS, and any combination thereof. In some embodiments, the promoter sequence is a Prrn promoter sequence [comprising SEQ ID NO: 1 or GenBank: MF580999.1],

[0020] In some embodiments, the nucleic acid material further comprises at least one enhancer sequence. In some embodiments, the at least one enhancer sequence is selected from a sequence encoding: ggagg, rrn 5’UTR, T7genel0 5’ UTR (GenBank: EU520588.1), LrbcL 5’UTR, LatpB 5’UTR, Tobacco mosaic virus omega prime 5’UTR (GenBank: KM507060.1), Lcry9Aa2 5’UTR, atpl 5’UTR, psbA 5’UTR, cry2a, rrnB, rpsl6, petD, psbA, pabA, and any combination thereof. In some embodiments, the at least one enhancer sequences comprises a sequence selected from SEQ ID NOs: 2, 4, and 6.

[0021] In some embodiments, the nucleic acid material further comprises a selection sequence. In some embodiments, a selection sequence is or comprises a nucleic acid sequence encoding: a His tag, GUS uidA lacz, green fluorescent protein, yellow fluorescent protein, red fluorescent protein, cyan fluorescent protein, and any combination thereof. In some embodiments, a selection sequence is or comprises a yellow fluorescent protein (YFP, GenBank: GQ221700.1), enhanced green fluorescent protein eGFP (e.g., GenBank: U55761.1: 97-816), red fluorescent protein (DsRED, GenBank: KY426960.1), or cyan fluorescent protein (CFP, GenBank: HQ993060.1). In some embodiments, the His tag comprises the sequence CATCACCATCACCATCAC-TAA (SEQ ID NO: 100), CATCATCATCATCATCAT (SEQ ID NO: 101), CATCACCATCACCATCAC (SEQ ID NO: 8), or a fragment or variant thereof. [0022] In some embodiments, the exogenous nucleic acid material is or comprises a RNA oligonucleotide, a DNA oligonucleotide, a plasmid, and any combination thereof. In some embodiments, the nucleic acid material comprises two or more exogenous nucleic acid sequences. In some embodiments, the exogenous nucleic acid sequence encodes a peptide comprising a sequence that is at least 90% identical to a leukotoxin A (ItkA) protein) according to Genbank: DQ672338.1, or a fragment or variant thereof. In some embodiments, the exogenous nucleic acid sequence comprises a sequence encoding at least one region of ItkA selected from the group consisting of PL1, PL4, or a fragment or variant thereof. In some embodiments, the exogenous nucleic acid sequence comprises a PL1 sequence comprising SEQ ID NO: 51 encoding an amino acid sequence SEQ ID NO: 52 and/or a nucleic acid sequence comprising SEQ ID NO: 53 encoding an amino acid sequence SEQ ID NO: 54, or a fragment or variant thereof.

[0023] In some embodiments, the exogenous nucleic acid sequence further comprises a termination sequence. In some embodiments, the termination sequence comprises a sequence encoding rpsl6 (GenBank: MF580999.1) or a portion or fragment thereof.

[0024] Another aspect of the disclosure provides, a plant comprising a nucleic acid material comprising an expression cassette comprising in 5’ to 3’ orientation a first (5’) sequence; a promoter sequence; an exogenous nucleic acid sequence; and a second (3’) sequence, wherein at least one exogenous nucleic acid sequence is expressed, at least in part, in the chloroplast of the plant. In some embodiments, the first (5’) sequence and/or the second (3’) sequence comprises 16S ribosomal gene DNA sequence of the plant. In some embodiments, the first (5’) sequence and/or the second (3’) sequence comprising 16S ribosomal gene DNA sequence of the plant contains at least one mutation relative to the native plant 16S ribosomal gene DNA sequence.

[0025] In some embodiments, the first (5’) sequence and/or the second (3’) sequence comprises 23S ribosomal gene DNA sequence of the plant. In some embodiments, the first (5’) sequence and/or the second (3’) sequence comprising the 23 S ribosomal gene DNA sequence of the plant contains at least one mutation relative to the native plant 23 S ribosomal gene DNA sequence. In some embodiments, the first (5’) sequence comprises 16S ribosomal gene DNA sequence of the plant containing at least one mutation relative to the native plant 16S ribosomal gene DNA sequence. In some embodiments, the second (3’) sequence comprises 23 S ribosomal gene DNA sequence of the plant. In some embodiments, the second (3’) sequence comprises 23 S ribosomal gene DNA sequence of the plant comprises at least one mutation relative to the native plant 23 S ribosomal gene DNA sequence.

[0026] In some embodiments, the at least one mutation comprises a mutation that confers antibiotic resistance in the plant. In some embodiments, the antibiotic resistance comprises resistance to one or more of lincomycin, spectinomycin, streptomycin, kanamycin, gentamycin, neomycin, Beta lactam resistance, and any combination thereof. In some embodiments, the antibiotic resistance comprises resistance to lincomycin, spectinomycin, and streptomycin.

[0027] In some embodiments, the plant is millet, sorghum, wheat, maize, barley, triticale, or alfalfa.

[0028] In some embodiments, the 16S ribosomal gene DNA sequence of the first (5’) sequence comprises a core sequence that has 90% sequence identity to the sequence corresponding to positions 95,395-95,672 of the millet plastid genome sequence (GenBank Accession No. KU343177.1) or the sequence represented SEQ ID NO: 21 or 135. In some embodiments, the 16S ribosomal gene DNA sequence of the first (5’) sequence comprises a core sequence corresponding to positions 95,395-95,672 of the millet plastid genome sequence (GenBank Accession No. KU343177.1) or the sequence represented SEQ ID NO: 21 or 135. In some embodiments, the at least one mutation comprises a C to A nucleotide substitution at position 95,395 and/or an A to a C nucleotide substitution at position 95,672 of the millet plastid genome sequence (GenBank Accession No. KU343177.1).

[0029] In some embodiments, the 23 S ribosomal gene DNA sequence of the second (3’) sequence comprises a core sequence that has at least 90% identity to the sequence corresponding to positions 100,566-100,594 of the millet plastid genome sequence GenBank Accession No. KU343177.1 or SEQ ID NO: 89. In some embodiments, the 23S ribosomal gene DNA sequence of the second (3’) sequence comprises a core sequence corresponding to positions 100,566-100,594 of the millet plastid genome sequence GenBank Accession No. KU343177.1) or SEQ ID NO: 89. In some embodiments, the at least one mutation relative to the native plant 23 S ribosomal gene DNA sequence comprises one or more of: (i) a G to A nucleotide substitution at position 100,566; (ii) an A to a G nucleotide substitution at position 100,593; and (iii) an A to G nucleotide substitution at position 100,594 of the millet plastid genome sequence (GenBank Accession No. KU343177.1). [0030] In some embodiments, the first and second sequences comprise SEQ ID NO: 13, 133, or 85 and SEQ ID NO: 14 or 87, respectively.

[0031] In some embodiments, the 16S ribosomal gene DNA sequence of the first (5’) sequence comprises a core sequence that has at least 90% identity to the sequence corresponding to positions 96,895 -97,172 of the sorghum chloroplast genome sequence (GenBank Accession No. NC_008602.1 or SEQ ID NO: 20). In some embodiments, the 16S ribosomal gene DNA sequence of the first (5’) sequence comprises a core sequence corresponding to positions 96,895-97,172 of the sorghum chloroplast genome sequence (GenBank Accession No. NC 008602.1 or SEQ ID NO: 20). In some embodiments, the at least one mutation comprises a C to A nucleotide substitution at position 96,895 and/or an A to a C nucleotide substitution at position 97,172 of the sorghum chloroplast genome sequence (GenBank Accession No. NC_008602.1 or SEQ ID NO: 20).

[0032] In some embodiments, the 23 S ribosomal gene DNA sequence of the second (3’) sequence comprises a core sequence that has at least 90% identity to the sequence corresponding to positions 102,072-102,100 of the sorghum chloroplast genome sequence (GenBank Accession No. NC 008602.1) or SEQ ID NO: 94. In some embodiments, the 23S ribosomal gene DNA sequence of the second (3’) sequence comprises a core sequence corresponding to positions 102,072-102,100 of the sorghum chloroplast genome sequence (GenBank Accession No. NC 008602.1) or SEQ ID NO: 94. In some embodiments, the at least one mutations relative to the native plant 23 S ribosomal gene DNA sequence comprises one or more of: (i) a Gto A nucleotide substitution at position 102,072; (ii) an A to a G nucleotide substitution at position 102,098; and (iii) an A to G nucleotide substitution at position 102,099 of the sorghum chloroplast genome sequence (GenBank Accession No. NC 008602.1).

[0033] In some embodiments, the first and second sequences comprise SEQ ID NO: 11, 132, or 90 and SEQ ID NO: 12 or 92, respectively.

[0034] In some embodiments, the 16S ribosomal gene DNA sequence of the first (5’) sequence comprises a core sequence that has at least 90% sequence identity to the sequence corresponding to positions 99,019-99,297 of the alfalfa plastid genome sequence (GenBank Accession No.

NC 042841.1, positions 38,069-38,097 of the alfalfa plastid genome sequence GenBank KU321681.1, or SEQ ID NO: 22). In some embodiments, the 16S ribosomal gene DNA sequence of the first (5’) sequence comprises a core sequence corresponding to positions 99,019 -99,297 of the alfalfa plastid genome sequence (GenBank Accession No. NC 042841.1, positions 38,069-38,097 of the alfalfa plastid genome sequence GenBank KU321681.1, or SEQ ID NO: 22). In some embodiments, the at least one mutation comprises a C to A nucleotide substitution at position 99,019 and/or an A to a C nucleotide substitution at position 99,297 of the alfalfa plastid genome sequence (GenBank Accession No. NC_042841.1), positions 38,069-38,097 of the alfalfa plastid genome sequence GenBank KU321681.1, or SEQ ID NO: 22).

[0035] In some embodiments, the 23S ribosomal gene DNA sequence of the second (3’) sequence comprises a core sequence that has at least 90% identity to the sequence corresponding to positions 38,069-38,097 of the alfalfa plastid genome sequence GenBank Accession No. KU321683.1 or SEQ ID NO: 99. In some embodiments, the 23S ribosomal gene DNA sequence of the second (3’) sequence comprises a core sequence corresponding to positions 38,069-38,097 of the alfalfa plastid genome sequence GenBank Accession No. KU321683.1 or SEQ ID NO: 99. In some embodiments, the at least one mutation relative to the native plant 23 S ribosomal gene DNA sequence comprises one or more of: (i) a G to A nucleotide substitution at position 38,069; (ii) an A to a G nucleotide substitution at position 38,096; and (iii) an A to G nucleotide substitution at position 38,097 of the alfalfa plastid genome sequence GenBank Accession No. KU321683.1.

[0036] In some embodiments, the first and second sequences comprise SEQ ID NO: 17, 136, or 95 and SEQ ID NO: 18 or 97, respectively.

[0037] In some embodiments, a promoter sequence is selected from PpsbA, Prrn, Prna, psaA, PrbcL, CaMV35S, rbcS, and any combination thereof. In some embodiments, the promoter sequence is a Prrn promoter sequence (e.g., comprising SEQ ID NO: 1 or MF580999.1 :73-201).

[0038] In some embodiments, the nucleic acid material further comprises at least one enhancer sequence. In some embodiments, the at least one enhancer sequence is selected from a sequence encoding: ggagg, rrn 5’UTR, T7genel0 5’ UTR (GenBank: EU520588.1:5627-5689), LrbcL 5’UTR(Genbank EU224430.1: 1456-1512), LatpB 5’UTR(Genbank: EU224425.1: 2006-2095), Tobacco mosaic virus omega prime 5’UTR (GenBank: KM507060.1), Lcry9Aa2 5’UTR, atpl 5’UTR, psbA 5’UTR, cry2a, rrnB, rpsl6, petD, psbA, pabA, and any combination thereof. In some embodiments, the at least one enhancer sequences comprises a sequence selected from SEQ ID NOs: 2, 4, and 6. [0039] In some embodiments, the nucleic acid material further comprises a selection sequence. In some embodiments, a selection sequence is or comprises a nucleic acid sequence encoding: a His tag, GUS uidA lacz, green fluorescent protein, yellow fluorescent protein, red fluorescent protein, cyan fluorescent protein, and any combination thereof. In some embodiments, a selection sequence is or comprises a yellow fluorescent protein (YFP, GenBank: GQ221700.1), green fluorescent protein (eGFP, e.g., GenBank: U55761.1: 97-816), red fluorescent protein (DsRED, GenBank: KY426960.1), or cyan fluorescent protein (CFP, GenBank: HQ993060.1). In some embodiments, a His tag comprises the sequence CATC ACC ATCACCATCAC-TAA (SEQ ID NO: 100), CATCATCATCATCATCAT (SEQ ID NO: 101), CATCACCATCACCATCAC (SEQ ID NO: 8), or a fragment or variant thereof.

[0040] In some embodiments, the exogenous nucleic acid material, when integrated into the chloroplast genome of the plant, is or comprises cpDNA (chloroplast DNA), or RNA when transcribed within the chloroplast. In some embodiments, the nucleic acid material comprises two or more exogenous nucleic acid sequences. In some embodiments, the exogenous nucleic acid sequence encodes a peptide comprising a sequence that is at least 90% identical to a leukotoxin A (ItkA) protein) according to GenBank: DQ672338.1: 1-498 (PL1) and/or GenBank: DQ672338.1 :5638-6606 (PL4)), or a fragment or variant thereof. In some embodiments, the exogenous nucleic acid sequence comprises a sequence encoding at least one region of ItkA selected from the group consisting of PL1, PL4, or a fragment or variant thereof. In some embodiments, the exogenous nucleic acid sequence comprises a PL1 sequence comprising SEQ ID NO: 51 encoding an amino acid sequence SEQ ID NO: 52 and/or a nucleic acid sequence comprising SEQ ID NO: 53 encoding an amino acid sequence SEQ ID NO: 54, or a fragment or variant thereof.

[0041] In some embodiments, the exogenous nucleic acid sequence further comprises a termination sequence. In some embodiments, the termination sequence comprises a sequence encoding rpsl6 (GenBank: MF580999.1: 1769-1918 or SEQ ID NO: 9) or a portion or fragment thereof.

[0042] Another aspect of the disclosure provides a method of transforming a plant comprising providing a nucleic acid material conjugated (e.g., fused) to a carrier to form a complex; and transforming a chloroplast in a plant cell with the nucleic acid material. In some embodiments, the nucleic acid material comprises an expression cassette comprising in 5’ to 3’ orientation a first (5’) targeting sequence that corresponds to a region in the plant chloroplast genome, a promoter sequence, an exogenous nucleic acid sequence, and a second (3’) targeting sequence that corresponds to a region in the plant chloroplast genome that is 3’ of the sequence targeted by the first (5’) targeting sequence.

In some embodiments, the carrier comprises a chloroplast-targeting peptide (CTP). In some embodiments, the first (5’) targeting sequence comprises 16S or 23S ribosomal gene DNA sequence of the plant containing at least one mutation relative to the native plant 16S or 23 S ribosomal gene DNA sequence. In some embodiments, the first (5’) targeting sequence comprises 16S ribosomal gene DNA sequence of the plant containing at least one mutation relative to the native plant 16S ribosomal gene DNA sequence. In some embodiments, the second (3’) targeting sequence comprises 23S ribosomal gene DNA sequence of the plant containing at least one mutation relative to the native plant 23 S ribosomal gene DNA sequence.

[0043] In some embodiments, the at least one mutation comprises a mutation that confers antibiotic resistance in the plant. In some embodiments, antibiotic resistance comprises resistance to one or more of lincomycin, spectinomycin, streptomycin, kanamycin, gentamycin, neomycin, Beta lactam resistance, and any combination thereof. In some embodiments, the antibiotic resistance comprises resistance to lincomycin, spectinomycin, and streptomycin.

[0044] In some embodiments, the plant is millet, sorghum, wheat, maize, barley, triticale, or alfalfa.

[0045] In some embodiments, the CTP comprises a protein derived from one or more of: an Arabidopsis thaliana an outer envelope membrane protein; molecular weight of 34 d (OEP34) Genbank accession no. NP 850768.1), Pisium sativum functional homologue translocase of chloroplasts 34 (TOC34; Genbank accession no. Q41009.1), TOC34 proteins of sorghum (Genbank accession no. XP 021306533.1), millet (Genbank accession no. RLN39229.1), and Medicago truncatula (Genbank accession no. XP 003624825.1), or a fragment or variant thereof. In some embodiments, the CTP comprises the hydrophobic core region of the OEP34 protein. In some embodiments, a CTP comprises an OEP34 protein encoded by the amino acid sequence SEQ ID NO: 23 [ILAVEYFLW], SEQ ID NO: 24 [IFALQYLFLA], or SEQ ID NO: 25 [LFALEFLLIM], or a fragment or variant thereof.

[0046] In some embodiments, the carrier further comprises one or more cell-penetrating peptides (CPP)

[0047] In some embodiments, a CTP is conjugated to the positively charged amino acids at the N-terminus of the CPP to forma a CTP-CPP complex. In some embodiments, the CPP comprises a KH9 sequence (SEQ ID NO: 28 [KHKHKHKHKHKHKHKHKH]), BP100 (sequence: SEQ ID NO: 29 [KKLFKKILKYL] -amide) or K9 (SEQ ID NO: 30 [KKKKKKKKK]), or HPV33L2-DD447 (SEQ ID NO: 102 [SYDDLRRRRKRFPYFFTDVRVAA]). In some embodiments, the CTP-CPP complex comprises SEQ ID NO: 83 (Sorghum/millet KH9-OEP34 KHKHKHKHKHKHKHKHKHILAVEYFLVV) OR SEQ ID NO: 84 (Medicago KH9-OEP34 KHKHKHKHKHKHKHKHKHLFALEFLLIM). In some embodiments, the C-terminus of the CPP is conjugated to the nucleic acid material.

[0048] In some embodiments, transforming the chloroplast comprises contacting the plant cell with the complex comprises the carrier conjugated to the nucleic acid material. In some embodiments, contacting comprises applying a vacuum and/or compression. In some embodiments, contacting comprises introducing the nucleic acid material and carrier complex via syringe injection. In some embodiments, contacting comprises culturing the nucleic acid material and carrier complex in a solution comprising the plant (e.g., for at least 1 minute).

[0049] In some embodiments, the transformed exogenous nucleic acid sequence is expressed in the chloroplast of the plant cell. In some embodiments, the transformed exogenous nucleic acid sequence is integrated in the chloroplast genome of the plant cell. In some embodiments, the transformed exogenous nucleic acid sequence is stably integrated in the chloroplast genome of the plant cell. In some embodiments, the exogenous nucleic acid sequence encodes an exogenous protein, and wherein the transformed plant expresses the exogenous protein.

[0050] Another aspect of the disclosure provides a method of transforming a plant comprising providing a nucleic acid material complexed with a carrier and transforming a chloroplast in a plant cell with the nucleic acid material, where the nucleic acid material comprises an expression cassette comprising in 5’ to 3’ orientation a first (5’) targeting sequence that corresponds to a region in the plant chloroplast genome, a promoter sequence, an exogenous nucleic acid sequence, and a second (3’) targeting sequence that corresponds to a region in the plant chloroplast genome that is 3 ’ of the sequence targeted by the first (5’) targeting sequence. In some embodiments, the carrier comprises a nanotube that is positively charged (i.e., has a zeta potential) and is sized and dimensioned such that it is able to pass through the chloroplast envelope of the plant.

[0051] In some embodiments, a nanotube comprises a single-walled nanotube. In some embodiments, a nanotube is a single-walled carbon nanotube (SWCNT). In some embodiments, a nanotube is complexed with chitosan (CS-SWCNT). In some embodiments, the CS-SWCNT is PEGylated (CS^PEG-SWCNT).

[0052] In some embodiments, the nanotube comprises a zeta potential that is at least 10 mV. In some embodiments, the nanotube comprises a zeta potential that less than 20 mV. In some embodiments, the dimension of the nanotube comprises a length of between about 1.0 and 10.0 μm. In some embodiments, the dimension of the nanotube comprises a diameter of between about 1.0 to 2.0 nm.

[0053] In some embodiments, transforming the chloroplast comprises contacting the plant cell with the complex comprising the nanotube conjugated to the nucleic acid material. In some embodiments, contacting comprises introducing the nucleic acid material and carrier complex into the plant via syringe injection. In some embodiments, syringe injection comprises surface leaf infusion through a needleless syringe. In some embodiments, syringe injection comprises stem injection through a needled syringe. In some embodiments, contacting comprises applying a vacuum and/or compression to the plant cell.

[0054] In some embodiments, once the exogenous nucleic acid material enters the chloroplast of the plant, it separates from the nanotube. In some embodiments, the transformed exogenous nucleic acid sequence is expressed in the chloroplast of the plant cell. In some embodiments, the transformed exogenous nucleic acid sequence is integrated in the chloroplast genome of the plant cell. In some embodiments, the transformed exogenous nucleic acid sequence is stably integrated in the chloroplast genome of the plant cell. In some embodiments, the exogenous nucleic acid sequence encodes an exogenous protein, and wherein the transformed plant expresses the exogenous protein.

[0055] In some embodiments, the plant is millet, sorghum, wheat, maize, barley, triticale, or alfalfa.

[0056] In some embodiments, the transformed exogenous nucleic acid sequence is stably integrated within a region of the 16S or 23 S ribosomal gene DNA region.

Another aspect of the disclosure provides a method of transforming a plant comprising providing a nucleic acid material and transforming a chloroplast in a plant cell with the nucleic acid material, wherein the nucleic acid material comprises an expression cassette comprising, in 5’ to 3’ orientation a first 5’ targeting sequence, a promoter sequence, an exogenous nucleic acid sequence; and a second 3’ targeting sequence; and wherein the first (5’) targeting sequence and/or the second (3’) targeting sequence comprises 23 S ribosomal gene DNA sequence of the plant containing at least one mutation relative to the native plant 23S ribosomal gene DNA sequence. In some embodiments, the second (3’) targeting sequence comprises 23 S ribosomal gene DNA sequence of the plant. In some embodiments, the second (3’) targeting sequence comprises 23 S ribosomal gene DNA sequence of the plant comprising at least one mutation relative to the native plant 23 S ribosomal gene DNA sequence. In some embodiments, the first (5’) targeting sequence comprises 16S ribosomal gene DNA sequence of the plant containing at least one mutation relative to the native plant 16S ribosomal gene DNA sequence.

[0057] In some embodiments, the method comprises expressing the exogenous nucleic acid sequence, wherein the expression occurs, at least in part, in a chloroplast.

[0058] In some embodiments, transforming the chloroplast comprises contacting the plant cell with the nucleic acid material. In some embodiments, contacting the plant cell comprises use of biolistics or gene gun, use of chloroplast targeting sequences/peptides, cell penetrating peptides, use of a carrier such as a functionalized nanoparticle, electroporation, chemical-mediated transfection (e.g. using polyethylene gylcol), or any combination thereof.

[0059] In some embodiments, the at least one mutation comprises a mutation that confers antibiotic resistance in the plant. In some embodiments, antibiotic resistance comprises resistance to one or more of lincomycin, spectinomycin, streptomycin, kanamycin, gentamycin, neomycin, Beta lactam resistance, and any combination thereof. In some embodiments, the antibiotic resistance comprises resistance to lincomycin, spectinomycin, and streptomycin.

[0060] In some embodiments, the plant is millet, sorghum, wheat, maize, barley, triticale, or alfalfa.

[0061] In some embodiments, the 23S ribosomal gene DNA sequence of the second (3’) targeting sequence comprises a core sequence that has 90% sequence identity to the sequence corresponding to positions 100,566-100,594 of the 23S ribosomal gene of the millet plastid genome sequence (GenBank Accession No. KU343177.1 or SEQ ID NO: 89). In some embodiments, the 23 S ribosomal gene DNA sequence of the second (3’) targeting sequence comprises a core sequence corresponding to positions 100,566-100,594 of the 23S ribosomal gene of the millet plastid genome sequence (GenBank Accession No. KU343177.1 or SEQ ID NO: 89). In some embodiments, the at least one mutation comprises one or more of: (i) a G to A nucleotide substitution at position 100,566; (ii) an A to a G nucleotide substitution at position 100,593; and (iii) an A to G nucleotide substitution at position 100,594 of the millet plastid genome sequence (GenBank Accession No. KU343177.1 or SEQ ID NO: 89).

[0062] In some embodiments, the 16S ribosomal gene DNA sequence of the first (5’) targeting sequence comprises a core sequence that has 90% sequence identity to the sequence corresponding to positions 95,395-95,672 of the millet plastid genome sequence (GenBank Accession No. KU343177.1) or the sequence represented SEQ ID NO: 21 or 135. In some embodiments, the 16S ribosomal gene DNA sequence of the first (5’) targeting sequence comprises a core sequence corresponding to positions 95,395-95,672 of the millet plastid genome sequence (GenBank Accession No. KU343177.1), or the sequence represented by SEQ ID NO: 21 or 135. In some embodiments, the at least one mutation comprises a C to A nucleotide substitution at position 95,395 and/or an A to a C nucleotide substitution at position 95,672 of the millet plastid genome sequence (GenBank Accession No.

KU343177.1).

[0063] In some embodiments, the first and/or second targeting sequences comprises all or a portion of SEQ ID NO: 79.

[0064] In some embodiments, the first (5’) and second (3) targeting sequences comprise SEQ ID NO: 13, 133, or 85 and SEQ ID NO: 14 or 87, respectively.

[0065] In some embodiments, the 23S ribosomal gene DNA sequence of the second (3’) targeting sequence comprises a core sequence that has at least 90% identity to the sequence corresponding to positions 102,072-102,100 of the sorghum chloroplast genome sequence (GenBank Accession No. NC_008602.1 or SEQ ID NO: 94). In some embodiments, the 23S ribosomal gene DNA sequence of the second (3’) targeting sequence comprises a core sequence corresponding to positions 102,072-102,100 of the sorghum chloroplast genome sequence (GenBank Accession No. NC 008602.1 or SEQ ID NO: 94). In some embodiments, the at least one mutation comprises one or more of: (i) a G to A nucleotide substitution at position 102,072; (ii) an A to a G nucleotide substitution at position 102,098; and (iii) an A to G nucleotide substitution at position 102,099 of the sorghum chloroplast genome sequence (GenBank Accession No. NC 008602.1 or SEQ ID NO: 94). [0066] In some embodiments, the 16S ribosomal gene DNA sequence of the first (5’) targeting sequence comprises a core sequence that has at least 90% identity to the sequence corresponding to positions 96,895-97,172 of the sorghum chloroplast genome sequence (GenBank Accession No. NC 008602.1) or SEQ ID NO: 20. In some embodiments, the 16S ribosomal gene DNA sequence of the first (5’) targeting sequence comprises a core sequence corresponding to positions 96,895-97,172 of the sorghum chloroplast genome sequence (GenBank Accession No. NC 008602.1) or SEQ ID NO: 20. In some embodiments, the at least one mutation comprises a C to A nucleotide substitution at position 96,895 and/or an A to a C nucleotide substitution at position 97,172 of the sorghum chloroplast genome sequence (GenBank Accession No. NC_008602.1).

[0067] In some embodiments, the first and/or second targeting sequences comprises all or a portion of SEQ ID NO: 81. In some embodiments, the first and second targeting sequences comprise SEQ ID NO: 11, 132, or 90 and SEQ ID NO: 12 or 92, respectively.

[0068] In some embodiments, the 23 S ribosomal gene DNA sequence of the second (3’) targeting sequence comprises a core sequence that has at least 90% identity to the sequence corresponding to positions 38,069-38,097 of the alfalfa plastid genome sequence GenBank Accession No. KU321683.1 or SEQ ID NO: 99. In some embodiments, the 23S ribosomal gene DNA sequence of the second (3’) targeting sequence comprises a core sequence corresponding to positions 38,069-38,097 of the alfalfa plastid genome sequence GenBank Accession No. KU321683.1 or SEQ ID NO: 99. In some embodiments, the at least one mutation relative to the native plant 23 S ribosomal gene DNA sequence comprises one or more of: (i) a Gto A nucleotide substitution at position 38,069; (ii) an A to a G nucleotide substitution at position 38,096; and (iii) an A to G nucleotide substitution at position 38,097 of the alfalfa plastid genome sequence GenBank Accession No. KU321683.1.

[0069] In some embodiments, the 16S ribosomal gene DNA sequence of the first (5’) targeting sequence comprises a core sequence that has at least 90% sequence identity to the sequence corresponding to positions 99,019-99,297 of the alfalfa plastid genome sequence (GenBank Accession No. NC_042841.1), positions 38,069-38,097 of the alfalfa plastid genome sequence GenBank KU321681.1, or SEQ ID NO: 22. In some embodiments, the 16S ribosomal gene DNA sequence of the first (5’) targeting sequence comprises a core sequence corresponding to positions 99,019 -99,297 of the alfalfa plastid genome sequence (GenBank Accession No. NC_042841.1) or positions 38,069- 38,097 of the alfalfa plastid genome sequence GenBank KU321681.1 or SEQ ID NO: 22. In some embodiments, the at least one mutation comprises a C to A nucleotide substitution at position 99,019 and/or an A to a C nucleotide substitution at position 99,297 of the alfalfa plastid genome sequence (GenBank Accession No. NC_042841.1).

[0070] In some embodiments, the first and second targeting sequences comprise SEQ ID NO: 17, 136, or 95 and SEQ ID NO: 18 or 97, respectively.

[0071] In some embodiments, a promoter sequence is selected from PpsbA, Prrn, Prna, psaA, PrbcL, CaMV35S, rbcS, and any combination thereof. In some embodiments, the promoter sequence comprises Prrn (GenBank: MF580999.1 :73-201).

[0072] In some embodiments, the nucleic acid material further comprises at least one enhancer sequence. In some embodiments, the enhancer sequence comprises one or more of T7 phage gene 10 leader sequence (GenBank: EU520588.1:5627-5689), LrbcL (Genbank EU224430.1 : 1456-1512), and LatpB (Genbank: EU224425.1 : 2006-2095).

[0073] In some embodiments, the nucleic acid material further comprises a selection sequence. In some embodiments, a selection sequence is or comprises a yellow fluorescent protein (YFP, GenBank: GQ221700.1), enhanced green fluorescent protein (eGFP, GenBank: U55761.1: 97-816), red fluorescent protein (DsRED, GenBank: KY426960.1), or cyan fluorescent protein (CFP, GenBank: HQ993060.1). In some embodiments, the selection sequence comprises a His tag. In some embodiments, the His tag comprises the sequence CATCACCATCACCATCAC-TAA (SEQ ID NO: 100), SEQ ID NO: CATCATCATCATCATCAT (SEQ ID NO: 101), CATCACCATCACCATCAC (SEQ ID NO: 8) or a fragment or variant thereof.

[0074] In some embodiments, the exogenous nucleic acid material is or comprises a RNA oligonucleotide, a DNA oligonucleotide, a plasmid, and any combination thereof. In some embodiments, the exogenous nucleic acid sequence encodes a peptide comprising a sequence that is at least 90% identical to a leukotoxin A (ItkA) protein) according to GenBank: DQ672338.1: 1-498 and/or GenBank: DQ672338.1 : 5638-6606, or a fragment or variant thereof. In some embodiments, the exogenous nucleic acid sequence comprises a sequence encoding at least one region of ItkA selected from the group consisting of PL1, PL4, or a fragment or variant thereof. In some embodiments, the exogenous nucleic acid sequence comprises a PL1 sequence comprising SEQ ID NO: 51 encoding an amino acid sequence SEQ ID NO: 52 and/or a nucleic acid sequence comprising SEQ ID NO: 53 encoding an amino acid sequence SEQ ID NO: 54, or a fragment or variant thereof.

[0075] Any citations to publications, patents, or patent applications herein are incorporated by reference in their entirety. Any numerals used in this application with or without about/approximately are meant to cover any normal fluctuations appreciated by one of ordinary skill in the relevant art.

[0076] Other features, objects, and advantages of the present invention are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments of the present invention, is given by way of illustration only, not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the detailed description.

BRIEF DESCRIPTION OF THE DRAWING

[0077] The Figures described below, which together make up the Drawing, are for illustration purposes only, not for limitation.

[0078] FIG. 1 shows an example DNA construct for transformation into a sorghum chloroplast genome.

[0079] FIG. 2 shows an example DNA construct for transformation into a millet chloroplast genome.

[0080] FIG. 3 shows an example DNA construct for transformation into an alfalfa chloroplast genome.

[0081] FIG. 4 shows an exemplary targeting strategy for integrating an exogenous nucleic acid material into a host plant cell chloroplast genome using a first (5’) targeting sequence that includes 16S ribosomal gene sequence DNA.

[0082] FIG. 5 shows an exemplary targeting strategy for integrating an exogenous nucleic acid material into a host plant cell chloroplast genome using a first (5’) targeting sequence that includes 16S ribosomal gene sequence DNA and a second (3’) targeting sequence that includes 23S ribosomal gene sequence DNA. [0083] FIG. 6 shows size of exemplary individual carbon nanoparticles to have an average diameter between 1.0 to 2.0 um confirmed using by TEM.

[0084] FIG. 7 shows infusion of a mature sorghum plant via evaporation of nucleic acid material on the leaf adaxial surface (top). The bottom shows observation of fluorescence (labelled in cyan in silico) within surface of leaf exposed to single-walled carbon nanotubes with nucleic acid material. No observation of fluorescence was found on areas with carbon nanotubes without nucleic acid material or on areas treated with water (controls).

[0085] FIG. 8 shows PCR confirmation of the integration of nucleic acid material into the plastid genome in millet. DNA was extracted from treated millet microcalli, and long PCR was performed using integrated forward/reverse primer pairs - one inside the native plastid genome, and one within various parts of the exogenous nucleic acid sequence (strategy shown in Panel B). Panel (A) shows that all bands were observed (left), and matched the expected band sizes when properly integrated (right).

[0086] FIG. 9 shows PCR confirmation of the integration of nucleic acid material into the plastid genome in sudangrass (sorghum). DNA was extracted from treated sudangrass calli, and long PCR was performed using integrated forward/reverse primer pairs - one inside the native plastid genome, and one within various parts of the exogenous nucleic acid sequence (strategy shown in Panel B). Panel (A) shows that all bands observed (left), matched the expected band sizes if integrated (right).

DEFINITIONS

[0087] In this application, unless otherwise clear from context, (i) the term “a” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and/or”; (iii) the terms “comprising” and “including” may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; and (iv) the terms “about” and “approximately” may be understood to permit standard variation as would be understood by those of ordinary skill in the art; and (v) where ranges are provided, endpoints are included. [0088] About: The term “about” or “approximately”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0089] Administration: As used herein, the term “administration” typically refers to the administration of a composition to a subject or system (e.g., a non-human animal or plant). Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human or a non-human. If, for example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some particular embodiments, administration may comprises feeding a composition to a non-human animal. In some particular embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e. g. intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreal, etc. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time). In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time. In some particular embodiments, an animal may be fed a composition in a dosing regimen that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing. In some particular embodiments, an animal may be fed a composition continually over a period of time.

[0090] Agent: In general, the term “agent”, as used herein, may be used to refer to a compound or entity of any chemical class including, for example, a polypeptide, nucleic acid, saccharide, lipid, small molecule, metal, or combination or complex thereof. In appropriate circumstances, as will be clear from context to those skilled in the art, the term may be utilized to refer to an entity that is or comprises a cell or organism, or a fraction, extract, or component thereof. In some instances, as will be clear from context, the term may be used to refer to one or more entities that is man-made in that it is designed, engineered, and/or produced through action of the hand of man and/or is not found in nature. In some embodiments, an agent may be utilized in isolated or pure form; in some embodiments, an agent may be utilized in crude form. In some embodiments, potential agents may be provided as collections or libraries, for example that may be screened to identify or characterize active agents within them. In some cases, the term “agent” may refer to a compound or entity that is or comprises a polymer; in some cases, the term may refer to a compound or entity that comprises one or more polymeric moieties. In some embodiments, the term may refer to a compound or entity that lacks or is substantially free of any polymeric moiety.

[0091] Animal: As used herein refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans, of either sex and at any stage of develoμment. In some embodiments, "animal" refers to non-human animals, at any stage of develoμment. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, chicken, goat, a primate, and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms. In some embodiments, an animal may be a transgenic animal, genetically engineered animal, and/or a clone.

[0092] Antigen. The term “antigen”, as used herein, refers to an agent that elicits an immune response; and/or (ii) an agent that binds to a T cell receptor (e.g., when presented by an MHC molecule) or to an antibody. In some embodiments, an antigen elicits a humoral response (e.g., including production of antigen-specific antibodies); in some embodiments, an antigen elicits a cellular response (e.g., involving T-cells whose receptors specifically interact with the antigen). In some embodiments, an antigen binds to an antibody and may or may not induce a particular physiological response in an organism. In general, an antigen may be or include any chemical entity such as, for example, a small molecule, a nucleic acid, a polypeptide, a carbohydrate, a lipid, a polymer (in some embodiments other than a biologic polymer [e.g., other than a nucleic acid or amino acid polymer) etc. In some embodiments, an antigen is or comprises a polypeptide. In some embodiments, an antigen is or comprises a glycan. Those of ordinary skill in the art will appreciate that, in general, an antigen may be provided in isolated or pure form, or alternatively may be provided in crude form (e.g., together with other materials, for example in an extract such as a cellular extract or other relatively crude preparation of an antigen-containing source). In some embodiments, antigens utilized in accordance with the present invention are provided in a crude form. In some embodiments, an antigen is a recombinant antigen.

[0093] Associated: Two events or entities are “associated” with one another, as that term is used herein, if the presence, level, degree, type and/or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, microbe, etc.) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and/or form correlates with incidence of and/or susceptibility to the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and/or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.

[0094] Breed. As used herein, the term “breed” refers to a group of animals (e.g., cattle) having common ancestors and/or sharing certain distinguishable traits that are not shared animals of other breeds. Those skilled in the art are familiar with breed standards and/or characteristics. In many embodiments, a particular breed is produced and/or maintained by mating particular identified parent or parents with one another.

[0095] Carrier: As used herein, “carrier” or in some cases a “nanoparticle carrier” refers to a diluent, adjuvant, excipient, or vehicle with which a composition is administered. In some exemplary embodiments, carriers can include sterile liquids, such as, for example, water and oils, including oils of petroleum, animal, vegetable or synthetic origin, such as, for example, peanut oil, soybean oil, mineral oil, sesame oil and the like. In some embodiments, carriers are or include one or more solid components. In some embodiments, a carrier can include a nanoparticle. In some particular embodiments, a carrier can include a nanotube, such as a carbon nanotube, a single-walled nanotube, a chitosan wrapped nanotube, or any combination thereof.

[0096] Chloroplast: A type of plastid that contains chlorophyll and can carry out photosynthesis. A chloroplast contains multiple copies of a plant cell plastome. [0097] Chromosome. As used herein, the term “chromosome” refers to a DNA molecule, optionally together with associated proteins and/or other entities, for example as found in the nucleus of eukaryotic cells. Typically, a chromosome carries genes and functions (e.g., origin of replication, etc.) that permit it to transmit hereditary information.

[0098] Comparable: As used herein, the term “comparable” refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison there between so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

[0099] Composition: Those skilled in the art will appreciate that the term “composition” may be used to refer to a discrete physical entity that comprises one or more specified components. In general, unless otherwise specified, a composition may be of any form - e.g., gas, gel, liquid, solid, etc. In some embodiments, a composition may be used to refer to a plant that has been transformed to express an exogenous protein. In some embodiments, a composition may include a nucleic acid material. In some particular embodiments, a composition may include a nucleic acid conjugated to a carrier.

[0100] Comprising: A composition or method described herein as "comprising" one or more named elements or steps is open-ended, meaning that the named elements or steps are essential, but other elements or steps may be added within the scope of the composition or method. To avoid prolixity, it is also understood that any composition or method described as "comprising" (or which "comprises") one or more named elements or steps also describes the corresponding, more limited composition or method "consisting essentially of' (or which "consists essentially of') the same named elements or steps, meaning that the composition or method includes the named essential elements or steps and may also include additional elements or steps that do not materially affect the basic and novel characteristic(s) of the composition or method. It is also understood that any composition or method described herein as "comprising" or "consisting essentially of' one or more named elements or steps also describes the corresponding, more limited, and closed-ended composition or method "consisting of' (or "consists of') the named elements or steps to the exclusion of any other unnamed element or step. In any composition or method disclosed herein, known or disclosed equivalents of any named essential element or step may be substituted for that element or step.

[0101] Corresponding to. As used herein, the term “corresponding to” may be used to designate the position/identity of a structural element in a compound or composition through comparison with an appropriate reference compound or composition. For example, in some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as “corresponding to” a residue in an appropriate reference polymer. For example, those of ordinary skill will appreciate that, for purposes of simplicity, residues in a polypeptide are often designated using a canonical numbering system based on a reference related polypeptide, so that an amino acid "corresponding to" a residue at position 190, for example, need not actually be the 190^th amino acid in a particular amino acid chain but rather corresponds to the residue found at 190 in the reference polypeptide; those of ordinary skill in the art readily appreciate how to identify "corresponding" amino acids. For example, those skilled in the art will be aware of various sequence alignment strategies, including software programs such as, for example, BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH/GLSEARCH, Genoogle, HMMER, HHpred/HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE that can be utilized, for example, to identify “corresponding” residues in polypeptides and/or nucleic acids in accordance with the present disclosure.

[0102] Dosing regimen: Those skilled in the art will appreciate that the term “dosing regimen” may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which is separated in time from other doses. In some embodiments, individual doses are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).

[0103] Engineered: In general, the term “engineered” refers to the aspect of having been manipulated by the hand of man. For example, a polynucleotide is considered to be “engineered” when two or more sequences that are not linked together in that order in nature are manipulated by the hand of man to be directly linked to one another in the engineered polynucleotide. For example, in some embodiments of the present invention, an engineered polynucleotide comprises a regulatory sequence that is found in nature in operative association with a first coding sequence but not in operative association with a second coding sequence, is linked by the hand of man so that it is operatively associated with the second coding sequence. Comparably, a cell or organism is considered to be “engineered” if it has been manipulated so that its genetic information is altered (e.g., new genetic material not previously present has been introduced, for example by transformation, mating, somatic hybridization, transfection, transduction, or other mechanism, or previously present genetic material is altered or removed, for example by substitution or deletion mutation, or by mating protocols). As is common practice and is understood by those in the art, progeny of an engineered polynucleotide or cell are typically still referred to as “engineered” even though the actual manipulation was performed on a prior entity.

[0104] Excipient: as used herein, refers to a non-therapeutic agent that may be included in a pharmaceutical composition, for example to provide or contribute to a desired consistency or stabilizing effect. In some embodiments, suitable pharmaceutical excipients may include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

[0105] Expression: As used herein, “expression” of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and/or 3’ end formation); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein.

[0106] Functional: As used herein, a “functional” biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.

[0107] Fragment: A “fragment” of a material or entity as described herein has a structure that includes a discrete portion of the whole, but lacks one or more moieties found in the whole. In some embodiments, a fragment consists of such a discrete portion. In some embodiments, a fragment consists of or comprises a characteristic structural element or moiety found in the whole. In some embodiments, a polymer fragment comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomeric units (e.g., residues) as found in the whole polymer. In some embodiments, a polymer fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the monomeric units (e.g., residues) found in the whole polymer. The whole material or entity may, in some embodiments, be referred to as the “parent” of the fragment.

[0108] Gene. As used herein, the term “gene” refers to a DNA sequence in a chromosome that codes for a product (e.g., an RNA product and/or a polypeptide product). In some embodiments, a gene includes coding sequence (i.e., sequence that encodes a particular product); in some embodiments, a gene includes non-coding sequence. In some particular embodiments, a gene may include both coding (e.g., exonic) and non-coding (e.g., intronic) sequences. In some embodiments, a gene may include one or more regulatory elements that, for example, may control or impact one or more aspects of gene expression (e.g., cell-type-specific expression, inducible expression, etc.). [0109] Genome. As used herein, the term “genome” refers to the total genetic information carried by an individual organism or cell, represented by the complete DNA sequences of its chromosomes.

[0110] Heterologous: As used herein, “heterologous” with respect to sequence means a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.

[0111] Homology. As used herein, the term “homology” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar.

[0112] Host: The term “host” is used herein to refer to a system (e.g., a cell, organism, etc.) in which a polypeptide of interest is present. In some embodiments, a host is a system that is susceptible to infection with a particular infectious agent. In some embodiments, a host is a system that expresses a particular polypeptide of interest. In some embodiments, a host system is a plant.

[0113] Host cell, as used herein, refers to a cell into which exogenous nucleic acids, for example DNA or RNA (recombinant or otherwise) has been introduced. Persons of skill will understand, upon reading this disclosure, that such terms refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. In some embodiments, host cells include prokaryotic and eukaryotic cells selected from any of the Kingdoms of life that are suitable for expressing an exogenous nucleic acid (e.g., a recombinant nucleic acid sequence). In some embodiments, a host cell is a plant cell.

[0114] Identity. As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “substantially identical” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of a reference sequence. The nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons made with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.

[0115] “Improve," “increase'' , “inhibit” or “reduce”: As used herein, the terms “improve”,

“increase”, “inhibit’, “reduce”, or grammatical equivalents thereof, indicate values that are relative to a baseline or other reference measurement. In some embodiments, an appropriate reference measurement may be or comprise a measurement in a particular system (e.g., in a single individual) under otherwise comparable conditions absent presence of (e.g., prior to and/or after) a particular agent or treatment, or in presence of an appropriate comparable reference agent. In some embodiments, an appropriate reference measurement may be or comprise a measurement in comparable system known or expected to respond in a particular way, in presence of the relevant agent or treatment. [0116] Introduced: “Introduced” in the context of inserting a nucleic acid fragment (e.g., a recombinant DNA construct) into a cell, means "transfection" or "transformation" or "transduction" and includes reference to the incorporation of a nucleic acid fragment into a eukaryotic or prokaryotic cell where the nucleic acid fragment may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

[0117] In vitro'. The term “in vitro” as used herein refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multicellular organism.

[0118] In vivo: as used herein refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).

[0119] Nanoparticle: As used herein, the term “nanoparticle” refers to a particle having a diameter of less than 1000 nanometers (nm). In some embodiments, a nanoparticle has a diameter of less than 300 nm, as defined by the National Science Foundation. In some embodiments, a nanoparticle has a diameter of less than 100 nm as defined by the National Institutes of Health. In some embodiments, nanoparticles are micelles in that they comprise an enclosed compartment, separated from the bulk solution by a micellar membrane, typically comprised of amphiphilic entities which surround and enclose a space or compartment (e.g, to define a lumen). In some embodiments, a micellar membrane is comprised of at least one polymer, such as for example a biocompatible and/or biodegradable polymer. In some embodiments, a nanoparticle can be a nanotube.

[0120] Nanoparticle composition: Ns, used herein, the term “nanoparticle composition” refers to a composition that contains at least one nanoparticle and at least one additional agent or ingredient. In some embodiments, a nanoparticle composition contains a substantially uniform collection of nanoparticles as described herein. In some embodiments, a nanoparticle composition contains a nanoparticle conjugated to another agent (e.g a drug, agent, nucleic acid material).

[0121] Nucleic acid. As used herein, in its broadest sense, refers to any compound and/or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and/or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage. As will be clear from context, in some embodiments, "nucleic acid" refers to an individual nucleic acid residue (e.g., a nucleotide and/or nucleoside); in some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, a "nucleic acid" is or comprises RNA; in some embodiments, a "nucleic acid" is or comprises DNA. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodi ester backbone. For example, in some embodiments, a nucleic acid is, comprises, or consists of one or more "peptide nucleic acids", which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention. Alternatively or additionally, in some embodiments, a nucleic acid has one or more phosphorothioate and/or 5'-N-phosphoramidite linkages rather than phosphodiester bonds. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxy cytidine). In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5 -methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5- bromouridine, C 5 -fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 -propynyl-cytidine, C5- methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8- oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a nucleic acid comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein. In some embodiments, a nucleic acid includes one or more introns. In some embodiments, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long. In some embodiments, a nucleic acid is partly or wholly single stranded; in some embodiments, a nucleic acid is partly or wholly double stranded. In some embodiments a nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide. In some embodiments, a nucleic acid has enzymatic activity.

[0122] Nucleic Acid Material. As used herein, “a nucleic acid material” in its broadest sense, refers to any composition comprising a one or more nucleic acid substance, alone or in combination with another component or agent. In some embodiments, a nucleic acid material can include one or more exogenous nucleic acid sequences alone or in combination with one or more endogenous nucleic acid sequences. In some embodiments, a nucleic acid material can be a DNA construct.

[0123] Oral: The phrases “oral administration” and “administered orally” as used herein have their art-understood meaning referring to administration by mouth of a compound or composition. In some embodiments, oral administration may refer to feeding a non-human subject.

[0124] Operably linked, as used herein, refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control element "operably linked" to a functional element is associated in such a way that expression and/or activity of a functional element is achieved under conditions compatible with the control element. In some embodiments, "operably linked" control elements are contiguous (e.g, covalently linked) with the coding elements of interest; in some embodiments, control elements act in trans to or otherwise at a from the functional element of interest. In the context of two or more nucleic acid fragments, “operably linked” may refer, for example, to the association of two or more DNA fragments in a DNA construct so that the function of one, e.g. protein-encoding DNA, is controlled by the other, e.g. a promoter.

[0125] Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, an active agent is present in a unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a subject. In some embodiments, an active agent can be a transformed plant (e.g., a transgenic plant). In some embodiments, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.

[0126] Pharmaceutically acceptable: As used herein, the phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0127] Pharmaceutically acceptable carrier: As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.

[0128] Phenotype. As used herein, the term “phenotype” refers to a trait, or to a class or set of traits displayed by a cell or organism. In some embodiments, a particular phenotype may correlate with a particular allele or genotype. In some embodiments, a phenotype may be discrete; in some embodiments, a phenotype may be continuous. [0129] Plant: includes reference to whole plants, plant organs, plant tissues, seeds and plant cells and progeny of same. Plant cells include, without limitation, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. Plants of the present disclosure may include, without limitation, food crops, economic crops, vegetable crops, fruits, flowers, grasses, trees, industrial raw material crops, feed crops or medicine crops. In some embodiments, a plant may include a member of the Leguminosae plant family, the Poaceae plant family, or a combination thereof. Examples of plants in the Leguminosae plant family include, but are not limited to, alfalfa, peas, beans, and lentils. Examples of plants in the Poaceae plant family include, but are not limited to, corn, wheat, rice, sorghum, and millet.

[0130]

[0131] Plastid: A type of membrane-bound organelle found in cells of plants, algae, and other eukaryotic cells that commonly carry one or more of chlorophyll or other pigment(s), fats, proteins, starches, or other compounds.

[0132] Plastome: As used herein, a “plastome” refers to the genome of a plastid. Each chloroplast contains multiple copies of the plastome.

[0133] Progeny: comprises any subsequent generation of a plant or other living organism.

[0134] Polypeptide: As used herein refers to any polymeric chain of amino acids. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and/or produced through action of the hand of man. In some embodiments, a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may comprise or consist of only natural amino acids or only non-natural amino acids. In some embodiments, a polypeptide may comprise D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide’s N-terminus, at the polypeptide’s C-terminus, or any combination thereof. In some embodiments, such pendant groups or modifications may be selected from the group consisting of acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In some embodiments, a polypeptide may be cyclic, and/or may comprise a cyclic portion. In some embodiments, a polypeptide is not cyclic and/or does not comprise any cyclic portion. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or comprise a stapled polypeptide. In some embodiments, the term “polypeptide” may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides. For each such class, the present specification provides and/or those skilled in the art will be aware of exemplary polypeptides within the class whose amino acid sequences and/or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family. In some embodiments, a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and/or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; in some embodiments with all polypeptides within the class). For example, in some embodiments, a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and/or includes at least one region (e.g., a conserved region that may in some embodiments be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region usually encompasses at least 3-4 and often up to 20 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids. In some embodiments, a relevant polypeptide may comprise or consist of a fragment of a parent polypeptide. In some embodiments, a useful polypeptide as may comprise or consist of a plurality of fragments, each of which is found in the same parent polypeptide in a different spatial arrangement relative to one another than is found in the polypeptide of interest (e.g., fragments that are directly linked in the parent may be spatially separated in the polypeptide of interest or vice versa, and/or fragments may be present in a different order in the polypeptide of interest than in the parent), so that the polypeptide of interest is a derivative of its parent polypeptide. [0135] Promoter: As used herein, “promoter” refers to a DNA regulatory element for initializing transcription. A plant promoter is a promoter capable of initiating transcription in plant cells whether or not its origin is a plant cell, e.g. it is well known that Agrobacterium promoters are functional in plant cells. Thus, plant promoters include promoter DNA obtained from plants, plant viruses and bacteria such as Agrobacterium and Bradyrhizobium bacteria. Examples of promoters under develoμmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, or seeds. Such promoters are referred to as "tissue preferred". Promoters that initiate transcription only in certain tissues are referred to as "tissue specific". A "cell type" specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An "inducible" or "repressible" promoter is a promoter which is under environmental control. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions, or certain chemicals, or the presence of light. Tissue specific, tissue preferred, cell type specific, and inducible promoters constitute the class of "non- constitutive" promoters. A "constitutive" promoter is a promoter which is active under most conditions. Promoters useful in the present invention are not specifically limited. Those skilled in the art may select suitable promoters according to their knowledge.

[0136] Protein: As used herein, the term “protein” refers to a polypeptide (i.e., a string of at least two amino acids linked to one another by peptide bonds). Proteins may include moieties other than amino acids (e.g, may be glycoproteins, proteoglycans, etc.) and/or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a “protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence), or can be a characteristic portion thereof. Those of ordinary skill will appreciate that a protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means. Polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term “peptide” is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, proteins are antibodies, antibody fragments, biologically active portions thereof, and/or characteristic portions thereof.

[0137] Pure As used herein, an agent or entity is “pure” if it is substantially free of other components. For example, a preparation that contains more than about 90% of a particular agent or entity is typically considered to be a pure preparation. In some embodiments, an agent or entity is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure.

[0138] Recombinant, as used herein, is intended to refer to polypeptides that are designed, engineered, prepared, expressed, created, manufactured, and/or or isolated by recombinant means, such as polypeptides expressed using a recombinant expression vector transfected into a host cell; polypeptides isolated from a recombinant, combinatorial human polypeptide library; polypeptides isolated from an animal (e.g., a mouse, rabbit, sheep, fish, etc.) that is transgenic for or otherwise has been manipulated to express a gene or genes, or gene components that encode and/or direct expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof; and/or polypeptides prepared, expressed, created or isolated by any other means that involves splicing or ligating selected nucleic acid sequence elements to one another, chemically synthesizing selected sequence elements, and/or otherwise generating a nucleic acid that encodes and/or directs expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof. In some embodiments, one or more of such selected sequence elements is found in nature. In some embodiments, one or more of such selected sequence elements is designed in silico. In some embodiments, one or more such selected sequence elements results from mutagenesis (e.g., in vivo or in vitro) of a known sequence element, e.g., from a natural or synthetic source such as, for example, in the germline of a source organism of interest (e.g., of a human, a mouse, etc.).

[0139] Reference: As used herein describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and/or comparison to a particular possible reference or control.

[0140] Sample: As used herein, the term “sample” typically refers to an aliquot of material obtained or derived from a source of interest, as described herein. In some embodiments, a source of interest is a biological or environmental source. In some embodiments, a source of interest may be or comprise a cell or an organism, such as a microbe, a plant, or an animal (e.g., a human). In some embodiments, a source of interest is or comprises biological tissue or fluid. In some embodiments, a biological tissue or fluid may be or comprise amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, cerumen, chyle, chime, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secreations, vitreous humour, vomit, and/or combinations or component(s) thereof. In some embodiments, a biological fluid may be or comprise an intracellular fluid, an extracellular fluid, an intravascular fluid (blood plasma), an interstitial fluid, a lymphatic fluid, and/or a transcellular fluid. In some embodiments, a biological fluid may be or comprise a plant exudate. In some embodiments, a biological tissue or sample may be obtained, for example, by aspirate, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, washing or lavage (e.g., brocheoalvealar, ductal, nasal, ocular, oral, uterine, vaginal, or other washing or lavage). In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, a sample is a “primary sample” obtained directly from a source of interest by any appropriate means. In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane. Such a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to one or more techniques such as amplification or reverse transcription of nucleic acid, isolation and/or purification of certain components, etc.

[0141] Small molecule: As used herein, the term “small molecule” means a low molecular weight organic and/or inorganic compound. In general, a “small molecule” is a molecule that is less than about 5 kilodaltons (kD) in size. In some embodiments, a small molecule is less than about 4 kD, 3 kD, about 2 kD, or about 1 kD. In some embodiments, the small molecule is less than about 800 daltons (D), about 600 D, about 500 D, about 400 D, about 300 D, about 200 D, or about 100 D. In some embodiments, a small molecule is less than about 2000 g/mol, less than about 1500 g/mol, less than about 1000 g/mol, less than about 800 g/mol, or less than about 500 g/mol. In some embodiments, a small molecule is not a polymer. In some embodiments, a small molecule does not include a polymeric moiety. In some embodiments, a small molecule is not and/or does not comprise a protein or polypeptide (e.g., is not an oligopeptide or peptide). In some embodiments, a small molecule is not and/or does not comprise a polynucleotide (e.g., is not an oligonucleotide). In some embodiments, a small molecule is not and/or does not comprise a polysaccharide; for example, in some embodiments, a small molecule is not a glycoprotein, proteoglycan, glycolipid, etc.). In some embodiments, a small molecule is not a lipid. In some embodiments, a small molecule is a modulating agent (e.g., is an inhibiting agent or an activating agent). In some embodiments, a small molecule is biologically active. In some embodiments, a small molecule is detectable (e.g., comprises at least one detectable moiety). In some embodiments, a small molecule is a therapeutic agent. Those of ordinary skill in the art, reading the present disclosure, will appreciate that certain small molecule compounds described herein may be provided and/or utilized in any of a variety of forms such as, for example, crystal forms, salt forms, protected forms, pro-drug forms, ester forms, isomeric forms (e.g., optical and/or structural isomers), isotopic forms, etc. Those of skill in the art will appreciate that certain small molecule compounds have structures that can exist in one or more stereoisomeric forms. In some embodiments, such a small molecule may be utilized in accordance with the present disclosure in the form of an individual enantiomer, diastereomer or geometric isomer, or may be in the form of a mixture of stereoisomers; in some embodiments, such a small molecule may be utilized in accordance with the present disclosure in a racemic mixture form. Those of skill in the art will appreciate that certain small molecule compounds have structures that can exist in one or more tautomeric forms. In some embodiments, such a small molecule may be utilized in accordance with the present disclosure in the form of an individual tautomer, or in a form that interconverts between tautomeric forms. Those of skill in the art will appreciate that certain small molecule compounds have structures that permit isotopic substitution (e.g., ²H or ³H for H, ⁿC, ¹³C or ¹⁴C for 12C; , ¹³N or ¹⁵N for 14N; ¹⁷O or ¹⁸O for 160; ³⁶C1 for XXC; ¹⁸F for XXF; 1311 for XXXI; etc.). In some embodiments, such a small molecule may be utilized in accordance with the present disclosure in one or more isotopically modified forms, or mixtures thereof. In some embodiments, reference to a particular small molecule compound may relate to a specific form of that compound. In some embodiments, a particular small molecule compound may be provided and/or utilized in a salt form (e.g., in an acid-addition or base-addition salt form, depending on the compound); in some such embodiments, the salt form may be a pharmaceutically acceptable salt form. In some embodiments, where a small molecule compound is one that exists or is found in nature, that compound may be provided and/or utilized in accordance in the present disclosure in a form different from that in which it exists or is found in nature. Those of ordinary skill in the art will appreciate that, in some embodiments, a preparation of a particular small molecule compound that contains an absolute or relative amount of the compound, or of a particular form thereof, that is different from the absolute or relative (with respect to another component of the preparation including, for example, another form of the compound) amount of the compound or form that is present in a reference preparation of interest (e.g., in a primary sample from a source of interest such as a biological or environmental source) is distinct from the compound as it exists in the reference preparation or source. Thus, in some embodiments, for example, a preparation of a single stereoisomer of a small molecule compound may be considered to be a different form of the compound than a racemic mixture of the compound; a particular salt of a small molecule compound may be considered to be a different form from another salt form of the compound; a preparation that contains only a form of the compound that contains one conformational isomer ((Z) or (E)) of a double bond may be considered to be a different form of the compound from one that contains the other conformational isomer ((E) or (Z)) of the double bond; a preparation in which one or more atoms is a different isotope than is present in a reference preparation may be considered to be a different form; etc.

[0142] Stable: The term “stable,” when applied to compositions herein, means that the compositions maintain one or more aspects of their physical structure and/or activity over a period of time under a designated set of conditions. In some embodiments, the period of time is at least about one hour; in some embodiments the period of time is about 5 hours, about 10 hours, about one (1) day, about one (1) week, about two (2) weeks, about one (1) month, about two (2) months, about three (3) months, about four (4) months, about five (5) months, about six (6) months, about eight (8) months, about ten (10) months, about twelve (12) months, about twenty-four (24) months, about thirty-six (36) months, or longer. In some embodiments, the period of time is within the range of about one (1) day to about twenty-four (24) months, about two (2) weeks to about twelve (12) months, about two (2) months to about five (5) months, etc. In some embodiments, the designated conditions are ambient conditions (e.g., at room temperature and ambient pressure). In some embodiments, the designated conditions are physiologic conditions (e.g., in vivo or at about 37 °C for example in serum or in phosphate buffered saline). In some embodiments, the designated conditions are under cold storage (e.g., at or below about 4 °C, -20 °C, or -70 °C). In some embodiments, the designated conditions are in the dark.

[0143] Subject: As used herein, the term “subject” or “test subject” refers to any organism to which a provided compound or composition is administered in accordance with the present disclosure e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, chickens, goats, cows, cattle, non-human primates, and humans; insects; worms; etc.} and plants. In some embodiments, a non-human animal may be a monogastric animal, for example, swine, poultry, or horses. In some embodiments, a non-human animal may be a ruminant animal, for example, cattle, sheep, and/or goats.

[0144] Substantial identity: as used herein refers to a comparison between amino acid or nucleic acid sequences. As will be appreciated by those of ordinary skill in the art, two sequences are generally considered to be "substantially identical" if they contain identical residues in corresponding positions. As is well known in this art, amino acid or nucleic acid sequences may be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLAS TN for nucleotide sequences and BLASTP, gapped BLAST, and PSLBLAST for amino acid sequences. Exemplary such programs are described in Altschul et al., Basic local alignment search tool, J. Mol. Biol., 215(3): 403-410, 1990; Altschul et al., Methods in Enzymology; Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis et al., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener, et al, (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying identical sequences, the programs mentioned above typically provide an indication of the degree of identity. In some embodiments, two sequences are considered to be substantially identical if at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are identical over a relevant stretch of residues. In some embodiments, the relevant stretch is a complete sequence. In some embodiments, the relevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues.

[0145] Substantially: As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0146] Suffering from'. An individual who is “suffering from” a disease, disorder, and/or condition has been diagnosed with and/or displays one or more symptoms of a disease, disorder, and/or condition.

[0147] Susceptible to: An individual who is “susceptible to” a disease, disorder, and/or condition is one who has a higher risk of developing the disease, disorder, and/or condition than does a member of the general public. In some embodiments, an individual is an animal of a particular species or breed of animal (e.g., a cow, chicken, goat, or sheep) that has a higher risk of developing a certain disease or disorder. In some embodiments, an individual who is susceptible to a disease, disorder and/or condition may not have been diagnosed with the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may not exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.

[0148] Systemic: The phrases “systemic administration,” “administered systemically,” “peripheral administration,” and “administered peripherally” as used herein have their art-understood meaning referring to administration of a compound or composition such that it enters the recipient’s system. [0149] Therapeutic agent: As used herein, the phrase “therapeutic agent” refers to an agent that, when administered to a subject, has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect (e.g., induces an immunogenic response in a subject).

[0150] Therapeutically effective amount: As used herein, the term “therapeutically effective amount” means an amount of a substance (e.g., a therapeutic agent, composition, and/or formulation) that elicits a desired biological response (e.g., induces an immunogenic response in a subject) As will be appreciated by those of ordinary skill in this art, the effective amount of a substance may vary depending on such factors as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc. In some embodiments, a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.

[0151] Transformation: as used herein, refers to any process by which exogenous DNA is introduced into a host cell. Transformation may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. In some embodiments, a particular transformation methodology is selected based on the host cell being transformed and may include, but is not limited to, viral infection, electroporation, mating, lipofection, or using a chemical and/or nano- or micro-particle aid. In some embodiments, a "transformed" cell is stably transformed in that the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome (e.g., in a nucleus or chloroplast). In some embodiments, a transformed cell transiently expresses introduced nucleic acid for limited periods of time.

[0152] Transgenic plant: “Transgenic plant” as used herein, refers to a plant which comprises within its genome (e.g., chloroplast genome) a heterologous polynucleotide. Preferably, the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant DNA construct.

[0153] Trait. As used herein, the term “trait” refers to a detectable attribute of an individual. Typically, expression of a particular trait may be fully or partially influenced by an individual’s genetic constitution. In some embodiments, a trait is characteristic of a particular individual, line, breed or crossbreed, for example in that it can be relied upon (individually or as part of a set) to distinguish that individual, line, breed, or crossbreed from others.

[0154] Vaccination or Vaccine: As used herein, the term “vaccination” refers to the administration of a composition intended to generate an immune response, for example to a diseasecausing agent. For the purposes of the present invention, vaccination can be administered before, during, and/or after exposure to a disease-causing agent, and in certain embodiments, before, during, and/or shortly after exposure to the agent. In some embodiments, vaccination includes multiple administrations, appropriately spaced in time, of a vaccinating composition. As used herein, the term “vaccine” refers to any composition intended to generate an immune response. In some embodiments a vaccine includes a transgene organism, engineered to express and antigen.

[0155] Variant: As used herein in the context of molecules, e.g., nucleic acids, proteins, or small molecules, the term “variant” refers to a molecule that shows significant structural identity with a reference molecule but differs structurally from the reference molecule, e.g., in the presence or absence or in the level of one or more chemical moieties as compared to the reference entity. In some embodiments, a variant also differs functionally from its reference molecule. In general, whether a particular molecule is properly considered to be a “variant” of a reference molecule is based on its degree of structural identity with the reference molecule. As will be appreciated by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. A variant, by definition, is a distinct molecule that shares one or more such characteristic structural elements but differs in at least one aspect from the reference molecule. To give but a few examples, a polypeptide may have a characteristic sequence element comprised of a plurality of amino acids having designated positions relative to one another in linear or three-dimensional space and/or contributing to a particular structural motif and/or biological function; a nucleic acid may have a characteristic sequence element comprised of a plurality of nucleotide residues having designated positions relative to on another in linear or three-dimensional space. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and/or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalently components of the polypeptide or nucleic acid (e.g., that are attached to the polypeptide or nucleic acid backbone). In some embodiments, a variant polypeptide or nucleic acid shows an overall sequence identity with a reference polypeptide or nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In some embodiments, a reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, a variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid shows a reduced level of one or more biological activities as compared to the reference polypeptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered to be a “variant” of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence that is identical to that of the reference but for a small number of sequence alterations at particular positions. Typically, fewer than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues in a variant are substituted, inserted, or deleted, as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residues as compared to a reference. Often, a variant polypeptide or nucleic acid comprises a very small number (e.g., fewer than about 5, about 4, about 3, about 2, or about 1) number of substituted, inserted, or deleted, functional residues (i.e., residues that participate in a particular biological activity) relative to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises not more than about 5, about 4, about 3, about 2, or about 1 addition or deletion, and, in some embodiments, comprises no additions or deletions, as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and commonly fewer than about 5, about 4, about 3, or about 2 additions or deletions as compared to the reference. In some embodiments, a reference polypeptide or nucleic acid is one found in nature. In some embodiments, a reference polypeptide or nucleic acid is a human polypeptide or nucleic acid.

[0156] Vector as used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked.

Such vectors are referred to herein as "expression vectors."

[0157] Wild-type: As used herein, the term “wild-type” has its art-understood meaning that refers to an entity having a structure and/or activity as found in nature in a “normal” (as contrasted with mutant, diseased, altered, etc.) state or context. Those of ordinary skill in the art will appreciate that wild-type genes and polypeptides often exist in multiple different forms (e.g., alleles).

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0158] The present description encompasses, inter alia, methods of modifying plants to express an exogenous nucleic acid sequence, for example, encoding one or more proteins of interest. In some embodiments, methods of introducing one or more exogenous nucleic acid sequence(s) into a host plant cell, including e.g., via transformation. In some embodiments, transformation of a plant cell includes transformation of the exogenous nucleic acid sequence into a plastome (e.g., a chloroplast genome) of the host plant cell.

[0159] Methods disclosed herein include strategies for transforming a plant at a specific location within its plastome. For example, the methods include strategies for targeting regions of the chloroplast (e.g., the 16S or 23S ribosomal gene).

[0160] In general, successful chloroplast transformation relies on, inter alia, detailed plastomic sequence information to identify regions of the chloroplast genome suitable for homologous recombination and safe transgene incorporation, for example. However, in addition to the raw sequence information, considerable work must be done in order to determine optimal, or even viable, sites for the integration of an exogenous nucleic acid (e.g., a transgene of interest). Other challenges associated with chloroplast transformation include develoμment of target-specific nucleic acid constructs that can successfully integrate into a particular location within the chloroplast genome of, for example, sorghum, millet, alfalfa, barley, wheat, maize, and triticale species, and lead to expression of an exogenous protein.

[0161] In some embodiments, the provided methods allow for targeted transformation in the chloroplast genome of a plant, and the transformation of the plant introduces one or more mutations that result in the plant having superior properties (e.g., antibiotic resistance). In some embodiments, the provided methods allow for targeted transformation in the chloroplast genome of a plant, and the transformation of the plant introduces a mutation (or mutations) that results in the plant having properties that allow for simple identification that the plant has been transformed (e.g., antibiotic resistance). In some embodiments, the provided methods allow for targeted transformation in the chloroplast genome of a plant, and the targeted transformation of the plant introduces an exogenous nucleic acid sequence in a position that results in a plant that is homoplasmic in its chloroplast genome for the exogenous sequence.

[0162] For example, an expression cassette may be designed so that it includes one or more targeting sequences that correspond to a region within or adjacent to 16S or 23 S ribosomal gene DNA sequences. In some embodiments, a targeting sequence can include a 16S or 23S ribosomal gene DNA sequence, where the targeting sequence comprises at least one mutation (e.g., two mutations) with respect to the native 16S or 23 S ribosomal DNA sequence. In some embodiments, the one or more mutations are introduced to confer properties such as antibiotic resistance to a host plant cell.

[0163] Many challenges remain with producing transgenic plants comprising transformed chloroplasts, include low chloroplast transformation rates. Accordingly, methods for transformation described herein also include various strategies to improve delivery of a DNA expression cassette (e.g., including a transgene) to the chloroplast genome of a host plant cell.

[0164] Recently, chloroplast targeting peptides (CTPs) were used to transform plant chloroplasts (see Yoshizumi et al., 2018), and subsequent studies showed that coupling CTP-DNA complexes with cell-penetrating proteins (CPPs) further optimizes delivery (Thagun et al., 2019). However, to date, success with these methods have been limited to particular plant species where CTPs have been identified. [0165] Methods described herein include efficient transformation using CTP-DNA, CPP-DNA, and CTP-CPP-DNA complexes in e.g., millet, sorghum, wheat, maize, barley, triticale, and alfalfa plant species. In some embodiments, methods include utilizing an OEP34 CTP to target the chloroplast of a host plant cell (e.g., sorghum or millet). Additionally, the sequence analysis described herein suggests that the OEP34 amino acid sequence identified in millet and sorghum (same in both) may also be utilized in other grass/cereal species, and suggests a canonical grass chloroplast targeting sequence.

[0166] Chloroplast proteins are mostly encoded by the nuclear genome and are post- translationally imported into the chloroplast via the action of N-terminal extensions commonly referred to as targeting peptides. OEP34 (outer-envelope membrane protein 34), is unique among OEP proteins in that it likely uses an ATP-mediated proteinaceous receptor to import itself into the chloroplast (Li and Chen, 1997). However, prior to the present disclosure, these CTPs have not yet been identified in species other than Nicotiana and Arabidposis (see Yoshizumi et al. 2018).

[0167] The present disclosure identifies a roster of peptides that alone or together have the capacity bind to chloroplast transformation DNA constructs, penetrate plant cells, and deliver DNAs to the chloroplast of various plant species. Also described herein are the methods used to identify novel CTPs for sorghum and millet, among other species, and the combinations of CTP and/or CPPs that allow for efficient transformation of a chloroplast genome with a transgene of interest (e.g., included in an expression cassette).

[0168] Also described herein are methods of using other carriers (e.g., nanotubes) to facilitate delivery of an exogenous nucleic acid sequence (i.e., a transgene) to a plant chloroplast. Carriers described herein may also be used in combination with a targeting peptide to deliver and exogenous nucleic acid material to the chloroplast of a host plant cell.

[0169] Subcellular trafficking of genetic materials by carbon nanotubes has been demonstrated (see Kwak et al. (2019), demonstrated in Eruca sativa, Nasturtium officinale, Nicotiana tabacum, Spinacia oleracea, plants and Arabidopsis thaliana) and offers a potential solution for transforming crops known to be challenging for transgenics. To overcome the transformation barriers in recalcitrant cereal crops sorghum and millet, the present disclosure describes nanobiotechnology approaches to courier transgenic DNA constructs to the respective chloroplasts of each species (e.g., millet, sorghum, and alfalfa). Also described herein are methods that tailor delivery in order to obtain transformation of chloroplasts in other species (e.g., sorghum, millet, and alfalfa) not previously demonstrated. [0170] In some embodiments, an exogenous nucleic acid sequence delivered to (e.g., integrated into the genome of) a plant is passed on to progeny of that plant.

[0171] Another feature of the methods and compositions encompassed by the present disclosure is the ability to monitor the degree and nature of successful transformation and/or expression of exogenous nucleic acid sequence(s) in plants. For example, one approach to monitoring expression of exogenous genes in plants is to co-express one or more markers (“selection markers”), for example, those which emit fluorescence under appropriate conditions. Such markers include green fluorescent proteins (GFP) which has been identified in the jellyfish Aequorea victoria (Ormo et al., 1996), along with A. victoria mutants that result in cyan fluorescent proteins (Goedhard et al., 2012) and yellow fluorescent proteins (YFP, Nagai et al., 2002), as well as red fluorescent protein identified in the mushroom anemone Discosoma species (DsRED, Bevis et al., 2002). In some embodiments, the present disclosure provides for the use of one or more of such proteins to confirm incorporation and/or expression of transgenic material. In some embodiments, properties such as antibiotic resistance can be conferred in a plant upon successful transformation in order to select for successful transformants.

[0172] In order to properly express foreign proteins, it is necessary to equip the genes coding for these proteins with appropriate DNA signatures to facilitate normal cellular processing of genetic material. In general, three major classes of DNA signatures are necessary for foreign protein expression; two at the 5’ end of the coding regions, and one at the 3’ end. At the 5’ end are: the promoter - a DNA signature that serves as an RNA binding site, and the 5’ untranslated region (also called a leader sequence) which assists the newly produced RNA in binding to the ribosome. At the 3’ end, a transcription terminator sequence is necessary to disengage the transcriptional complex and mark the end of transcription.

[0173] Taken together, in some embodiments, a nucleic acid material is designed to deliver an expression cassette comprising an exogenous nucleic acid sequence(s) encoding e.g., an antigen of interest, to a plastome (e.g., a chloroplast genome) for homologous recombination integration and may comprise 1) DNA signatures that complement the host specie’s chloroplast, 2) one or more transgenes encoding one or more antigens of interest, and 3) one or more genetic markers, along with 4) the genetic machinery to properly to translate and express the transgenes. In some embodiments, such machinery may be exogenously supplied and/or under the control of a non-native control mechanism, in whole or in part. In some embodiments, such machinery may be endogenous to the plant and/or plant organelle, in whole or in part.

Plants

[0174] In accordance with various embodiments, any of a wide variety of plants may be used in accordance with methods encompassed by the present disclosure, for example, to integrate and express an exogenous nucleic acid (e.g., encoding an exogenous protein of interest). A plant of the present disclosure may include, without limitation, whole plants, mature plants, plant organs, plant tissues, seeds and plant cells and progeny of same. Plant cells may include, without limitation, one or more of cells from seeds, seedlings, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. Plants of the present disclosure may include, without limitation, food crops, economic crops, vegetable crops, legumes, fruits, flowers, grasses, trees, industrial raw material crops, feed crops or medicine crops.

[0175] Food crops, such as cereal crops, can include millet, triticale, alfalfa, and sorghum. Vegetable crops can include, but are not limited to, radish, Chinese cabbage, tomato, cucumber, onion, corn, pea, leafy greens (e.g., spinach, kale, collard, chard, and lettuce), mustard, sweet potato, cabbage, celery, beet, beets, radish, turnip, hot pepper, carrot, asparagus, broccoli, cabbage, cauliflower, eggplant, pepper, and potato.

[0176] Feed crops can include any plant used to feed domesticated livestock, such as cattle, rabbits, sheep, horses, chickens and pigs, for example, for livestock grazing, or the foodstuff for livestock. Examples include, but are not limited to, millet (Panicum miliaceum), sorghum, wheat, and alfalfa (Medicago saliva).

[0177] In some embodiments, a plant species of the present disclosure may be a cross of any of plants described herein, including any sub-species. For example, in some embodiments, a cereal species can include a cross of two sorghum species. In some embodiments, a sorghum species includes sorghum sudangrass, resultant from a cross of (Sorghum bicolor ((L.) Moench) × (Sorghum × drummondii) (Nees ex. Steud.)). [0178] In some embodiments, plants used in accordance with methods encompassed by the present disclosure may be of the Leguminosae plant family, the Poaceae plant family, or a combination thereof.

[0179] Examples of plants in the Leguminosae plant family include, but are not limited to, alfalfa, peas, beans, and lentils.

[0180] Examples of plants in the Poaceae plant family include, but are not limited to, corn, wheat, rice, sorghum, and millet.

Nucleic Acid Material

[0181] Nucleic acid material of the present disclosure may include nucleic acids alone or in combination with one or more other agents or compositions. In some embodiments, a nucleic acid material can be referred to as or include an expression cassette, where an expression cassette comprises an exogenous nucleic acid sequence and one or more components that allow for or enhance expression of an exogenous nucleic acid sequence.

[0182] In accordance with various embodiments, components of an expression cassette can include, without limitation, one or more targeting sequence(s), selection sequence(s), exogenous DNA sequence(s), enhancer sequence(s), promoter sequence(s), and termination sequence(s).

[0183] In some embodiments, an expression cassette comprises, in 5’ to 3’ orientation, a first (5’) targeting sequence, a promoter sequence, an exogenous nucleic acid sequence, and a second (3’) targeting sequence.

[0184] In some embodiments, a nucleic acid material is or comprises a RNA oligonucleotide, a DNA oligonucleotide, a plasmid, or any combination thereof. A DNA oligonucleotide can be a singlestranded DNA oligonucleotide, a double-stranded DNA oligonucleotide. In some embodiments, a DNA oligonucleotide can be from any DNA source, including, but not limited to, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence, or any other appropriate source of DNA. In some embodiments, an RNA oligonucleotide may comprise one or more of mRNA, snRNA, siRNA, or miRNA oligonucleotide. [0185] In some embodiments, a nucleic acid material may include a DNA construct that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to a DNA sequence including elements as described above, and shown, e.g., in constructs 1-3 in FIGs. 1-3 (SEQ ID NOs: 15-16, and 19) and constructs represented in SEQ ID NOs: 121, 122, and 131. A DNA construct of the present disclosure can include a DNA construct that includes any combination of the components shown in constructs 1-3 in FIGs. 1-3 and the constructs represented in SEQ ID NOs: 121, 122, and 131.

Exogenous Nucleic Acid Sequence

[0186] An exogenous nucleic acid sequence, as the term is used herein, refers to any nucleic acid that is non-native to an organism or host cell (i.e., is not normally expressed in a particular organism, also referred to as a “transgene”).

[0187] In some embodiments, an exogenous nucleic acid sequence may encode one or more proteins that impart an enhanced trait when expressed in a transgenic plant (as compared to a control plant). For example, an exogenous nucleic acid sequence, when expressed in a transgenic plant, e.g., millet or sorghum, may increase the yield of the transgenic plant (e.g., measure by weight, seed number per plant, seed weight, seed number per unit area). Increased yield may result from improved utilization of key biochemical compounds, such as nitrogen, phosphorous and carbohydrate, or from improved responses to environmental stresses, such as cold, heat, drought, salt, and attack by pests or pathogens. Exogenous nucleic acid sequences can also be used to provide transgenic plants having improved growth and develoμment, and ultimately increased yield, as the result of modified expression of plant growth regulators or modification of cell cycle or photosynthesis pathways. In some embodiments, an enhanced trait may be e.g., enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil.

[0188] In some embodiments, an exogenous nucleic acid sequence imparts an enhanced trait such as increased resistance to stress conditions such as water deficit, nitrogen deficiency, cold growing conditions, pathogen or insect attack or light deficiency, or increased plant density. In some embodiments, an enhanced trait is a morphology trait such or an enhanced agronomic trait such as taller, thicker, and/or greater number of leaves. In some embodiments, an enhanced trait is a decrease in days to pollen shed, days to silking, or an increase in leaf extension rate, chlorophyll content, leaf temperature, stand, seedling vigor, internode length, plant height, leaf number, leaf area, tillering, brace roots, stay green, stalk lodging, root lodging, plant health, barreness/prolificacy, green snap, and/or pest resistance.

[0189] In some embodiments, an exogenous nucleic acid sequence is a sequence from an organism that is not a plant. In some embodiments, an exogenous nucleic acid sequence is a sequence from an organism that is a different species of plant than the host plant species.

[0190] In some embodiments, an exogenous nucleic acid sequence may be or comprise a nucleic acid sequence encoding more than one transgene of interest.

[0191] In some embodiments, an exogenous nucleic acid sequence may encode a polypeptide of interest, for example, an antibody or antibody agent, (e.g., monoclonal antibodies, fragment antigen binding (Fab) fragments), cytokines, receptors, antigens, human vaccines, animal vaccines, and plant polypeptides. In some embodiments, a transgene is an immunogenic portion of an antigen of interest.

Antigens

[0192] In some embodiments, an exogenous nucleic acid sequence may encode a particular antigen or antigenic fragment. In some embodiments, an exogenous nucleic acid sequence encoding an antigen or antigenic fragment, when introduced into a plant cell, may function as a vaccine when consumed by a subject, such as a human or animal. In some embodiments, an exogenous nucleic acid sequence of interest may include, without limitation, a sequence encoding a virus (e.g., a pathogenic virus, for example, including a virulence factor) or portion such as a fragment or variant thereof, a bacteria (e.g., a pathogenic bacteria) or portion such as a fragment or variant thereof, or a fungi (e.g., a pathogenic fungi) or portion such as a fragment or variant thereof, or protozoa (e.g., a pathogenic protozoa) or portion such as a fragment or variant thereof. In some embodiments, an antigen may be or comprise an immunogenic portion or fragment of a full-length protein or peptide provided by or otherwise associated with a pathogenic virus (including a virulence factor), a pathogenic bacteria, pathogenic fungi, and/or a pathogenic protozoa.

[0193] Examples of pathogenic viruses may include, without limitation, single stranded RNA viruses (with and without envelope), double stranded RNA viruses, and single and double stranded DNA viruses such as (but not limited to) tobacco mosaic virus, tobacco rattle virus, pea enation mosaic virus, barley stripe mosaic virus, potato viruses X and Y, carnation latent virus, beet yellows virus, maize chlorotic virus, tobacco necrosis virus, turnip yellow mosaic virus, tomato bushy stunt virus, southern bean mosaic virus, barley yellow dwarf virus, tomato spotted wilt virus, lettuce necrotic yellows virus, wound tumor virus, maize streak virus, and cauliflower mosaic virus.

[0194] In some embodiments, an antigen is or comprises a bacterium or portion such as a fragment or variant thereof, for example, a virulence factor produced from a bacterium, or a fragment or variant thereof. In some embodiments, a virulence factor could be produced from bacterium that commonly infects ruminant livestock, or another non-human animal. In some embodiments, a bacterium can include, without limitation, Fusobacterium necrophorum (including e.g. one of its subspecies F. necrophorum subsp. necrophorum and F. necrophorum subsp. Funduliforme), Mannheimia (Pasteurella) haemolytica, Actinobacillus actinomycetemcomitans, P. haemolytica, A. actinomycetemcomitans, Examples of bacterial pathogens include bacteria from the following genera and species: Chlamydia (e.g, Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis), Legionella (e.g, Legionella pneumophila), Listeria (e.g, Listeria monocytogenes), Rickettsia (e.g, R. australis, R rickettsii, R. akari, R. conorii, R. sibirica, R. japonica, R. africae, R. typhi, R. prowazekii), Actinobacter (e.g., Actinobacter baumannii), Bordetella (e.g, Bordetella pertussis), Bacillus (e.g, Bacillus anthracis, Bacillus cereus), Bacteroides (e.g, Bacteroides fragilis), Bartonella (e.g, Bartonella henselae), Borrelia (e.g, Borrelia burgdorferi), Brucella (e.g, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis), Campylobacter (e.g, Campylobacter jejuni), Clostridium (e.g., Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani), Corynebacterium (e.g, Corynebacterium diphtheriae, Corynebacterium amycolatum), Enterococcus (e.g., Enterococcus faecalis, Enterococcus faecium), Escherichia (e.g, Escherichia coli), Francisella (e.g., Francisella tularensis), Haemophilus (e.g, Haemophilus influenzae), Helicobacter (e.g., Helicobacter pylori), Klebsiella (e.g, Klebsiella pneumoniae), Leptospira (e.g, Leptospira interrogans), Mycobacteria (e.g, Mycobacterium leprae, Mycobacterium tuberculosis), Mycoplasma (e.g., Mycoplasma pneumoniae), Neisseria (e.g, Neisseria gonorrhoeae, Neisseria meningitidis), Pseudomonas (e.g, Pseudomonas aeruginosa), Salmonella (e.g, Salmonella typhi, Salmonella typhimurium, Salmonella enterica), Shigella (e.g, Shigella dysenteriae, Shigella sonnei), Staphylococcus (e.g, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus), Streptococcus (e.g, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes), Treponoma (e.g., Treponoma pallidum), Vibrio (e.g, Vibrio cholerae, Vibrio vulnificus), and Yersinia (e.g., Yersinia pestis). [0195] In some embodiments, a virulence factor can include generally, without limitation, an endotoxin and/or an exotoxin. In some embodiments, a virulence factor can include, without limitation, Cholera toxin, Tetanus toxin, Botulinum toxin, Diphtheria toxin, Streptolysin, Pneumolysin, Alphatoxin, Alpha-toxin, Phospholipase C, Beta-toxin, Streptococcal mitogenic exotoxin, Streptococcal pyrogenic toxins, Leukotoxin A, hemagglutinin, hemolysin, hyaluronidase, protease, coagulase, lipases, deoxyribonucleases and enterotoxins, M protein, lipoteichoic acid, hyaluronic acid capsule, destructive enzymes (including streptokinase, streptodornase, and hyaluronidase), streptolysin, alin A, internalin B, lysteriolysin O, actA, and Cytolethal distending toxin.

[0196] Examples of protozoal pathogens include the following organisms: Cryptosporidium parvum, Entamoeba (e.g., Entamoeba histolytica), Giardia (e.g., Giardia lambda), Leishmania (e.g., Leishmania donovani), Plasmodium spp. (e.g, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae), Toxoplasma (e.g., Toxoplasma gondii), Trichomonas (e.g., Trichomonas vaginalis), and Trypanosoma (e.g., Trypanosoma brucei, Trypanosoma cruzi). Libraries for other protozoa can also be produced and used according to methods described herein.

[0197] Examples of fungal pathogens include the following: Aspergillus, Candida (e.g., Candida albicans), Coccidiodes (e.g., Coccidiodes immitis), Cryptococcus (e.g., Cryptococcus neoformans), Histoplasma (e.g., Histoplasma capsulatum), and Pneumocystis (e.g., Pneumocystis carinii).

[0198] In some embodiments, a transformed plant cell, for example functioning as or producing a plant-based vaccine, may be used to treat and/or prevent a common disease in ruminant livestock including, but not limited to Acetonaemia, acidosis, Acorn Poisoning, Anaplasmosis, Anthrax, Blackleg, Bloat, Bluetongue, Botulism, Bovine Anaemia, Bovine Babesiosis, Bovine Respiratory Disease Complex (BRDC), Bovine spongiform encephalopathy (BSE), Bovine Trichomoniasis, Bracken Poisoning, BRSV (Bovine Respiratory Syncytial Virus), Brucellosis, BVD (Bovine Viral Diarrhea), Calf Diphtheria, Calf Pneumonia, Calf Scour, Clostridial Disease, Cocci diosis, Cold Cow Syndrome, Copper Poisoning, Cryptosporidiosis, Cystic ovaries, Digital Dermatitis, Displaced Abomasum, Epizootic Hemorrhagic Disease, Fatty Liver, Fog Fever, Foot and Mouth, Foot Rot, foot thrush, Gut Worms, Haemophilus Somnus, Hypermagnesaemia, IBR (Infectious Bovine Rhinotracheitis), Infectious Bovin Rhinotracheitis (IBR), Johnes, Joint Ill, Lead Poisoning, Leptospirosis, Lice, Listeriosis, liver abscess, Liver Fluke, Mange, Mastitis, Molybdenum Toxicity, Necrotic Enteritis, Neosporosis, New Forest Eye, Nitrate poisoning, Pasteurella Haemolytica Pasteurella Multocida , Peri-Weaning Diarhheoa, Photosensitisation, PI3 (Parainfluenza Type 3), Pruritus/Pyrexia/Haemorrhagic Syndrome, pseudocowpox, Rabies, Ragwort Poisoning, Rain Scald, Repeat Breeding Syndrome, Retained Fetal Membranes, Rift Valley Fever, Ringowrm, Rotaviral Diarrhoea, Rumen Acidosis, rumenitis, Samonella, Schmallenberg, Selenium Deficiency, Sole Ulcer, Summer Mastitis, Tetanus, Thrombosis, Traumatic Reticuliti, Trypanosomosis, Tuberculosis (TB), Ulcerative Mammillitis, Vibriosis, and Wooden Tongue.

[0199] In some embodiments, an antigen may include an immunogenic fragment, variant, or truncation of a sequence encoding any one of the above-identified antigens and/or antigens from any of the above-identified organisms. In some embodiments, truncations of leukotoxin A (e.g., as identified in Sun et al. 2009 Vet Res Commun. Oct;33(7):749-55. doi: 10.1007/sl l259-009-9223-6) can be used to elicit immunoprotective effects in organisms challenged with Fusobacterium infection. In some embodiments, an exogenous nucleic acid sequence encodes a peptide comprising a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to a leukotoxin A (ItkA) protein represented by GenBank: DQ672338.1, or a fragment or variant thereof. In some embodiments an immunogenic fragment of ItkA can include a sequence encoding a region of ItkA selected from the group consisting of PL1 (GenBank: DQ672338.1 1-501), PL4 (DQ672338.1 5637-6606, and a combination of Pl and PL4 (as shown in the DNA constructs 1-2 in FIGs. 1-2), or any fragment or variant thereof. In some embodiments an immunogenic fragment of ItkA can include a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to a sequence encoding at least one region of ItkA selected from the group consisting of PL1 (DQ672338.1 1-498), PL2 (DQ672338.1 946 - 1911), PL3(DQ672338.1 3950- 6052), PL4 (DQ672338.1 5637-6606), PL5 (DQ672338.1 9226-9721) (e.g., as shown in the DNA constructs 1-2 in FIGs. 1-2), or any fragment or variant thereof. In some embodiments, an exogenous nucleic acid sequence comprises the PL1 sequence SEQ ID NO: 51 encoding an amino acid sequence SEQ ID NO: 52, or a fragment or variant thereof. In some embodiments, an exogenous nucleic acid sequence comprises the PL4 sequence SEQ ID NO: 53 encoding an amino acid sequence SEQ ID NO: 54, or a fragment or variant thereof.

[0200] In some embodiments, an exogenous nucleic acid sequence may comprise a sequence that encodes an immunogenic fragment, variant, or truncation of a full native antigen sequence. In some embodiments, an exogenous nucleic acid sequence may include a sequence that encodes an immunogenic fragment variant, or truncation of a native antigen sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to a native antigen sequence, or a fragment thereof.

[0201] In some embodiments, an exogenous nucleic acid sequence can include a sequence of one or more different transgenes, encoding e.g., one or more proteins, e.g., one or more antigens. In some embodiments, an exogenous nucleic acid sequence can include a sequence of one or more immunogenic fragments from one antigen. In some embodiments, an exogenous nucleic acid sequence can include a sequence of one or more immunogenic fragments from multiple antigens.

[0202] In addition to the exogenous nucleic acid sequence, a nucleic acid material may include one or more control elements operably linked to an exogenous nucleic acid in a manner that permits and/or enhances its transcription, translation and/or expression in a cell transformed with a nucleic acid material. Expression control sequences can include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (poly A) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. A number of expression control sequences, including promoters that are native, constitutive, inducible and/or tissue-specific, are known in the art and may be included in a vector described herein.

Promoters

[0203] In addition to an exogenous nucleic acid sequence encoding a transgene of interest, an expression cassette, may include one or more promoters in proximity (upstream) to the exogenous nucleic acid sequence, to initiate transcription of a protein encoded by the exogenous nucleic acid sequence (e.g., an antigen). A promoter may be “operably linked,” e.g., associated with one or more DNA fragments (e.g., an exogenous nucleic acid) in a nucleic acid material so that the function of one or more DNA fragments, e.g. protein-encoding DNA, are controlled by the promoter.

[0204] In some embodiments, a promoter is naturally occurring in the genome of a host cell, also referred to as an endogenous promoter. In some embodiments, an endogenous promoter may be used to control a gene that is not normally associated with that promoter (e.g., a transgene). In some embodiments, a promoter sequence may have at least 70%, 80%, 85%, 90%, 95%, 98% or 99% identity to a native or endogenous promoter. In some embodiments, a promoter is a non-natural or exogenous promoter.

[0205] In some embodiments, a nucleic acid material may include a constitutive promoter. In some embodiments, a constitutive promoter can comprise a native or non-native promoter that is operably linked to an exogenous nucleic acid sequence, for example, encoding a transgene of interest. In some embodiments, a constitutive promotor is part of a constitutive expression construct and may include a recombinant expression vector described herein.

[0206] In some embodiments, a nucleic acid material may include a regulated promoter. In some embodiments, a regulated promoter can comprise a native or non-native promoter that is operably linked to an exogenous nucleic acid sequence encoding a transgene of interest. In some embodiments, a regulated promotor is part of a regulatable expression construct and may include a recombinant expression vector described herein.

[0207] In some embodiments, a promoter can be a plant promoter, capable of initiating transcription in a host plant. In some embodiments, promoters can include any promoter DNA obtained from plants, plant viruses and/or bacteria such as Agrobacterium and Bradyrhizobium bacteria.

Examples of promoters under develoμmental control can include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, or seeds, i.e., “tissue preferred” promoters. In some embodiments, a promoter can be a “tissue specific”, i.e. promoters that initiate transcription only in certain tissues are referred to as “tissue specific”. In some embodiments, a promoter can be a “cell type” specific promoter, i.e., a promoter that primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves.

[0208] Example promoters include, without limitation, common CMV, E1F, VAV, TCRvbeta, MCSV, PGK, PpsbA, Prrn, Prna, psaA, PrbcL, CaMV35S, rbcS, Patpl and PatpB, or an A3 or RS324 promoter. In some embodiments, a promoter comprises a Prrn promoter comprising SEQ ID NO: 1 (GenBank: MF580999.1, 73-201). Additional types of promoter may be used, and may depend, for example, on the species of the host plant. In some embodiments, a plant promoter can be derived from any known plant including for example, food crops, economic crops, vegetable crops, legumes, fruits, flowers, grasses, trees, industrial raw material crops, feed crops or medicine crops. [0209] In some embodiments, where, e.g., an expression cassette includes more than one exogenous nucleic acid sequence, a promoter can be operably linked to each exogenous nucleic acid sequence. In some embodiments where an expression cassette includes multiple promoters, each of the promoters may be the same or different promoters.

Tarsetins Sequences

[0210] A nucleic acid material may include one or more targeting sequences, e.g., in order to be integrated into a particular location within the host genome. In some embodiments, more than one targeting sequence may be used, for example, a first and a second targeting sequence. Targeting sequences, in some embodiments, are nucleic acid sequences that are complementary, e.g., at least 80, 85, 90, 95, 98 or 99% complementary, e.g., fully complementary, to a target sequence on a nucleic acid of interest in, for example, a plant e.g., a sequence that is complementary to an endogenous nucleic acid sequence to the host cell (e.g., a sequence that is adjacent to a desired integration point).

[0211] In some embodiments, a first and/or second targeting sequence are designed to be complementary to regions of a host genome that flank (e.g., are adjacent to) a target endogenous nucleic acid sequence and/or target integration site for a transgene. In some embodiments, the host is a plant cell and the endogenous nucleic acid sequence is a sequence that is an endogenous sequence within a host genome (e.g., a plastome). In some embodiments, a plant cell is from any of the plants described above. In some embodiments, targeting sequences are complementary to sequences within a nuclear genome. In some embodiments, targeting sequences are complementary to sequences within a chloroplast genome. In some embodiments, a chloroplast genome can be the chloroplast genome of sorghum plant species (as represented by sorghum (Sorghum bicolor (L.) Moench, Genbank:

NC 008602.1 or NC 008602.1), the chloroplast or plastid of millet (e.g., “Broomcorn Millet” Panicum miliaceum L., GenBank: KU343177.1; “Little millet” Panicum sumatrense, NCBI accession number KX756177; “Pearl millet” Cenchrus americanus/Pennisetum americanum/ P. glaucum, NCBI accession number KJ490012; “Foxtail millet” Setaria italic, NCBI accession number NC_022850) or the chloroplast genome of any Triticeae species (e.g., as described in Middleton et al. 2013 PLoS One 9.3 (2014): e85761; e.g, Triticum aestivum, Genbank: FN645450.1, KC912694.1, or NC_002762.1 ), maize (Genbank: NC_001666.2), wheat (Genbank: NC_002762.1), barley (Genbank: NC_056985.1), or the chloroplast or plastid genome of alfalfa plant (e.g., GenBank Accession No. NC 042841.1). [0212] In some embodiments, targeting sequences may flank a target region (e.g., a site of desired transgene integration) or endogenous region that is between two genes within a nuclear genome.

[0213] In some embodiments a target region comprises a region that is at least 100 (e.g., at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 1000, 2kb, 3kb, 4kb or greater) nucleotides in length.

[0214] In some embodiments, a target region includes a region within the chloroplast or plastid genome. For example, in some embodiments, a target region is within the 16S ribosomal gene DNA sequence. In some embodiments, targeting sequences flank a target region that comprising a portion of the 16S ribosomal gene DNA sequence. In some embodiments a portion of the 16S ribosomal gene DNA sequence comprises a region that is at least 100 (e.g., at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 1000, 2kb, 3kb, 4kb or greater) nucleotides in length. In some embodiments a portion of the 16S ribosomal gene DNA sequence comprises SEQ ID NOs: 20, 21, 22, or 135.

[0215] In some embodiments, a target region is within the 23 S ribosomal gene DNA sequence. In some embodiments, targeting sequences flank a target region that comprising a portion of the 23 S ribosomal gene DNA sequence. In some embodiments a portion of the 23 S ribosomal gene DNA sequence comprises a region that is at least 20 (e.g., at least 25, 28, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 1000, 2kb, 3kb, 4kb or greater) nucleotides in length. In some embodiments a portion of the 23 S ribosomal gene DNA sequence comprises SEQ ID NOs: 80, 82, 89, 94, or 99.

[0216] In some embodiments, targeting sequences of a nucleic acid material as disclosed herein, can include sequences that have at least 70%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NOs: 11, 12, 13, 14, 17, 18, 20, 21, 22, 79, 80, 81, 82, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 132, 133, 136, or a fragment thereof.

[0217] In some embodiments, targeting sequences of an expression cassette as disclosed herein, can include sequences that have at least 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to a target region DNA sequence. In some embodiments, or both of the targeting sequence described herein include at least one nucleotide that is mutated with respect to the endogenous nucleic acid sequence. In some embodiments, a mutation in one or both of the targeting sequences, once transformed within a plant genome confer superior properties to the host plant. [0218] For example, in some embodiments, a mutation in one or both of the targeting sequences, once transformed within a plant genome, confers antibiotic resistance in the host plant (e.g., resistance to one or more of spectinomycin, streptomycin, kanamycin, gentamycin, neomycin, Beta lactam resistance, lincomycin, and any combination thereof). Mutations that confer antibiotic resistance have been demonstrated m ' Nicotiana species (see e.g., Svab et al (1991 Mol Gen. Genet. 1991 228: 316-319), Dix and Kavanagh (1995 Euphytica 85: 29-34), Craig et al (2008 Transgenic Res 7:769-782), and Cseplo, Agnes, et al. (1988 Molecular and General Genetics MGG 214: 295-299), which are herein incorporated by reference in their entirety.

[0219] In some embodiments, a targeting sequence (e.g., a first (5’) targeting sequence and/or a second (3’) targeting sequence) corresponds to region within the 16S ribosomal gene DNA sequence of the host plant genome. In some embodiments, the targeting sequence (e.g., a first (5’) targeting sequence) corresponds to a region that includes a core sequence, where the core sequence includes at least the region including and between at least two mutated nucleotides with respect to the native plant 16S ribosomal gene DNA sequence. In some embodiments, the at least two mutated nucleotides confer antibiotic resistance in the host plant (e.g., resistance to spectinomycin and/or streptomycin). In some embodiments, a core sequence includes a region including and between two mutated nucleotides with respect to the native plant 16S sequence, and also includes additional sequence adjacent (5’ and/or 3’) to the two mutated nucleotides. In some embodiments, a first (5’) targeting sequence comprises a 16S ribosomal gene DNA sequence and a second (3’) targeting sequence does not comprises 16S ribosomal gene DNA sequence. In some embodiments, a first (5’) targeting sequence does not comprises a 16S ribosomal gene DNA sequence and a second (3’) targeting sequence comprises 16S ribosomal gene DNA sequence. In some embodiments, both a first (5’) targeting and a second (3’) targeting sequence comprise 16S ribosomal gene DNA sequence.

[0220] In some embodiments, a targeting sequence (e.g., a first (5’) targeting sequence) includes a core sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity to a sequence corresponding to positions 95,395-95,672 of the millet plastid genome sequence (GenBank Accession No. KU343177.1). In some embodiments, a core sequence includes at least the region corresponding to positions 95,395-95,672 of the millet plastid genome sequence (GenBank Accession No. KU343177.1). In some embodiments, mutations in a targeting sequence (e.g., a first (5’) targeting sequence) include a nucleotide mutation at position 95,395 and/or a nucleotide mutation at position 95,672 of the millet plastid genome sequence (GenBank Accession No. KU343177.1). In some embodiments, mutations in a targeting sequence (e.g., a first (5’) targeting sequence) include a C to A nucleotide substitution at position 95,395 and/or an A to a C nucleotide substitution at position 95,672 of the millet plastid genome sequence (GenBank Accession No. KU343177.1). In some embodiments, the first and second targeting sequences comprise SEQ ID NOs: 13 and 14, respectively.

[0221] In some embodiments, a targeting sequence (e.g., a first (5’) targeting sequence) includes a core sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity to a sequence corresponding to positions 96,895-97,172 of the sorghum chloroplast genome sequence (GenBank Accession No. NC 008602.1). In some embodiments, a core sequence includes at least the region corresponding to positions 96,895-97,172 of the sorghum chloroplast genome sequence (GenBank Accession No. NC 008602.1). In some embodiments, mutations in a targeting sequence (e.g., a first (5’) targeting sequence) include a nucleotide mutation at position 96,895 and/or a nucleotide mutation at position 97,172 of the sorghum chloroplast genome sequence (GenBank Accession No. NC 008602.1). In some embodiments, mutations in a targeting sequence (e.g., a first (5’) targeting sequence) include a C to A nucleotide substitution at position 96,895 and/or an A to a C nucleotide substitution at position 97,172 of the sorghum chloroplast genome sequence (GenBank Accession No. NC 008602.1). In some embodiments, such a mutation confers resistance to streptomycin. In some embodiments, such a mutation confers resistance to spectinomycin. In some embodiments, the first and second targeting sequences comprise SEQ ID NOs: 11 and 12, respectively, or a fragment thereof.

[0222] In some embodiments, a targeting sequence (e.g., a first (5’) targeting sequence) includes a core sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity to a sequence corresponding to positions 99,019-99,297 of the alfalfa plastid genome sequence (GenBank Accession No. NC 042841.1). In some embodiments, a core sequence includes at least the region corresponding to positions 99,019-99,297 of the alfalfa plastid genome sequence (GenBank Accession No. NC 042841.1). In some embodiments, mutations in a targeting sequence (e.g., a first (5’) targeting sequence) include a nucleotide mutation at position 99,019 and/or a nucleotide mutation at position 99,297 of the alfalfa plastid genome sequence (GenBank Accession No. NC_042841.1). In some embodiments, mutations in a targeting sequence (e.g., a first (5’) targeting sequence) include a C to A nucleotide substitution at position 99,019 and/or an A to a C nucleotide substitution at position 99,297 of the alfalfa plastid genome sequence (GenBank Accession No. NC 042841.1). In some embodiments, the first and second targeting sequences comprise SEQ ID NOs: 17 or 136, and 18, respectively, or a fragment thereof.

[0223] In some embodiments, a targeting sequence (e.g., a first (5’) targeting sequence) includes a core sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity to a sequence corresponding to positions 33,201-33,479 of the alfalfa plastid genome (i.e., Medicago) sequence (GenBank Accession No. KU 321683.1). In some embodiments, a core sequence includes at least the region corresponding to positions 33,201-33,479 of the alfalfa plastid genome sequence (GenBank Accession No. KU 321683.1). In some embodiments, a mutation in a targeting sequence (e.g., a first (5’) targeting sequence) includes a nucleotide mutation at position 33,201 of the alfalfa plastid genome sequence (GenBank Accession No. KU 321683.1). In some embodiments, a mutation in a targeting sequence (e.g., a first (5’) targeting sequence) includes a nucleotide mutation at position 33,479 of the alfalfa plastid genome sequence (GenBank Accession No. KU 321683.1). In some embodiments, a mutation in a targeting sequence (e.g., a first (5’) targeting sequence) includes a C to A nucleotide substitution at position 33,201 of the alfalfa plastid genome sequence (GenBank Accession No. KU 321683.1). In some embodiments, a mutation in a targeting sequence (e.g., a first (5’) targeting sequence) includes an A to C nucleotide substitution at position 33,479 of the alfalfa plastid genome sequence (GenBank Accession No. KU 321683.1).

[0224] In some embodiments, a targeting sequence (e.g., a first (5’) targeting sequence and/or a second (3’) targeting sequence) corresponds to region within the 23 S ribosomal gene DNA sequence of the host plant genome. In some embodiments, the targeting sequence (e.g., a second (3’) targeting sequence) corresponds to a region that includes a core sequence, where the core sequence includes at least the region including and between at least two (e.g., three) mutated nucleotides with respect to the native plant 23S ribosomal gene DNA sequence. In some embodiments, the at least two (e.g., three) mutated nucleotides confer antibiotic resistance in the host plant (e.g., resistance to lincomycin). In some embodiments, a core sequence includes a region including and between three mutated nucleotides with respect to the native plant 23 S sequence. In some embodiments, a targeting sequence also includes additional sequence adjacent (5’ and/or 3’) to the two or three mutated nucleotides of the core sequence. [0225] In some embodiments, a first (5’) targeting sequence comprises a 23S ribosomal gene DNA sequence (e.g., a core 23S ribosomal sequence) and a second (3’) targeting sequence does not comprises 23 S ribosomal gene DNA sequence. In some embodiments, a first (5’) targeting sequence does not comprises a 23S ribosomal gene DNA sequence and a second (3’) targeting sequence comprises 23S ribosomal gene DNA sequence (e.g., a core 23S ribosomal sequence). In some embodiments, both a first (5’) targeting and a second (3’) targeting sequence comprise 23S ribosomal gene DNA sequence (e.g., a core 23 S ribosomal sequence).

[0226] In some embodiments, a first (5’) targeting sequence comprises a 16S ribosomal gene DNA sequence and a second (3’) targeting sequence comprises a 23S ribosomal gene DNA sequence. In some embodiments, a first (5’) targeting sequence comprises a 23S ribosomal gene DNA sequence and a second (3’) targeting sequence comprises a 16S ribosomal gene DNA sequence.

[0227] In some embodiments, a targeting sequence (e.g., a second (3’) targeting sequence) includes a core sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity to a sequence corresponding to positions 100,566-100,594 of the millet plastid genome sequence (GenBank Accession No. KU343177.1). In some embodiments, a core sequence includes at least the region corresponding to positions 100,566-100,594 of the millet plastid genome sequence (GenBank Accession No. KU343177.1). In some embodiments, mutations in a targeting sequence (e.g., a second (3’) targeting sequence) include a nucleotide mutation at position 100,566, position 100,593, and/or position 100,594 of the millet plastid genome sequence (GenBank Accession No. KU343177.1). In some embodiments, mutations in a targeting sequence (e.g., a second (3’) targeting sequence) include one or more of: (i) a Gto A nucleotide substitution at position 100,566; (ii) an A to a G nucleotide substitution at position 100,593; and (iii) an A to G nucleotide substitution at position 100,594 of the millet plastid genome sequence (GenBank Accession No. KU343177.1).

[0228] In some embodiments, the first and second targeting sequences together include all or a portion of SEQ ID NO: 79. In some embodiments, the first and/or second targeting sequences include a portion of SEQ ID NO: 79. In some embodiments, a first targeting sequence includes SEQ ID NO: 80. In some embodiments, a second targeting sequence includes SEQ ID NO: 80. In some embodiments, the first and second targeting sequences together include all or a portion of SEQ ID NO: 89. In some embodiments, the first and/or second targeting sequences include a portion of SEQ ID NO: 89. [0229] In some embodiments, the first and second targeting sequences comprise a sequence having at least at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity to SEQ ID NOs: 85 and 87, respectively, or a fragment or variant thereof. In some embodiments, the first and second targeting sequences comprise SEQ ID NOs: 85 and 87, respectively, or a fragment or variant thereof. In some embodiments, a targeting sequence (e.g., a second (3’) targeting sequence) includes a core sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity to a sequence corresponding to positions 102,072-102,099 of the sorghum chloroplast genome sequence (GenBank Accession No. NC 008602.1). In some embodiments, a core sequence of a targeting sequence includes at least the region corresponding to positions 102,072-102,099 of the sorghum chloroplast genome sequence (GenBank Accession No. NC 008602.1). In some embodiments, mutations in a targeting sequence (e.g., a second (3’) targeting sequence) include a nucleotide mutation at position 102,072, at position 102,098, and/or at position 102,099 of the sorghum chloroplast genome sequence (GenBank Accession No. NC 008602.1). In some embodiments, mutations in a targeting sequence (e.g., a second (3’) targeting sequence) include one or more of: (i) a G to A nucleotide substitution at position 102,072; (ii) an A to a G nucleotide substitution at position 102,098; and (iii) an A to G nucleotide substitution at position 102,099 of the sorghum chloroplast genome sequence (GenBank Accession No. NC 008602.1). In some embodiments, such mutations confer resistance to lincomycin. In some embodiments, the first and second targeting sequences together include all or a portion of SEQ ID NO: 81. In some embodiments, the first and/or second targeting sequences include a portion of SEQ ID NO: 81. In some embodiments, a first targeting sequence includes SEQ ID NO: 82. In some embodiments, a second targeting sequence includes SEQ ID NO: 82. In some embodiments, a first targeting sequence includes SEQ ID NO: 94. In some embodiments, a second targeting sequence includes SEQ ID NO: 94.

[0230] In some embodiments, the first and second targeting sequences comprise a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity to SEQ ID NOs: 90 and 92, respectively, or a fragment thereof. In some embodiments, the first and second targeting sequences comprise SEQ ID NOs: 90 and 92, respectively, or a fragment or variant thereof.

[0231] In some embodiments, a targeting sequence (e.g., a second (3’) targeting sequence) includes a core sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity to a sequence corresponding to positions 38,069-38,097 of the alfalfa plastid genome sequence (GenBank Accession No. KU 321683.1). In some embodiments, a core sequence includes at least the region corresponding to positions 38,069-38,097 of the alfalfa plastid genome sequence (GenBank Accession No. KU 321683.1). In some embodiments, a mutation in a targeting sequence (e.g., a second (3’) targeting sequence) includes a nucleotide mutation at position 38,069 of the alfalfa plastid genome sequence (GenBank Accession No. KU 321683.1). In some embodiments, a mutation in a targeting sequence (e.g., a second (3’) targeting sequence) includes a nucleotide mutation at position 38,096 of the alfalfa plastid genome sequence (GenBank Accession No. KU 321683.1). In some embodiments, a mutation in a targeting sequence (e.g., a second (3’) targeting sequence) includes a nucleotide mutation at position 38,097 of the alfalfa plastid genome sequence (GenBank Accession No. KU 321683.1). In some embodiments, a mutation in a targeting sequence (e.g., a second (3’) targeting sequence) includes a Gto A nucleotide substitution at position 38,069 of the alfalfa plastid genome sequence (GenBank Accession No. KU 321683.1), to confer lincomycin resistance. In some embodiments, a mutation in a targeting sequence (e.g., a second (3’) targeting sequence) includes an A to G nucleotide substitution at position 38,096 of the alfalfa plastid genome sequence (GenBank Accession No. KU 321683.1), to confer lincomycin resistance. In some embodiments, a mutation in a targeting sequence (e.g., a second (3’) targeting sequence) includes an A to G nucleotide substitution at position 38,097 of the alfalfa plastid genome sequence (GenBank Accession No. KU 321683.1), to confer lincomycin resistance.

[0232] In some embodiments, the first and second targeting sequences comprise a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity to SEQ ID NOs: 95 and 97, respectively, or a fragment thereof. In some embodiments, the first and second targeting sequences comprise SEQ ID NOs: 95 and 97, respectively, or a fragment or variant thereof.

[0233] A targeting sequence may be described by the position (i.e., coordinates) of the complementary region it targets within a host chloroplast genome (i.e., sorghum chloroplast genome, millet chloroplast genome, etc.). One of skill in the art will appreciate that the same targeting sequence may be described by different coordinates dependent upon the particular version of the sequenced genome obtained. For many plant species, their chloroplast genome has been sequenced by different groups, and there exists several versions that vary to some degree, be it from species variation or even local variations within a particular species due to known rearrangements of genetic material over time. In this case, a targeting sequence may be described based on its sequence or the sequence it aims to target, rather than the particular position (i.e., coordinates) within the host genome that it targets. It is contemplated that one of skill in the art could ascertain the coordinates within a particular version of the sequenced chloroplast genome based on the unique targeting sequence.

Enhancer Elements

[0234] In various aspects of the disclosure, a nucleic material of the present disclosure may include one or more enhancer sequences, for example, to increase transcription of an exogenous nucleic acid. For example, in some embodiments, one or more enhancer sequences can be included at the 5’ untranslated region (also called a leader sequence) which may assist the newly produced RNA in binding to the ribosome.

[0235] In some embodiments, an enhancer sequence can include one or more enhancer sequences selected from: ggagg, rrn 5’UTR, T7genel0 5’ UTR (e.g., GenBank: EU520588.1:5627- 5689, SEQ ID NO: 113 or 125), LrbcL 5’UTR (e.g., Genbank EU224430.1 : 1456-1512; SEQ ID NO: 115 or 127), LatpB 5’UTR (e.g., Genbank: EU224425.1: 2006-2095; SEQ ID NO: 117), Tobacco mosaic virus omega prime 5’UTR (GenBank: KM507060.1), Lcry9Aa2 5’UTR, atpl 5’UTR, psbA 5’UTR, cry2a, rrnB, rpsl6, petD, psbA, pabA, and any combination or variant thereof.

[0236] In some embodiments, a nucleic acid material of the present disclosure may include one or more termination sequences. In some embodiments, a termination sequence can include tobacco Trpsl6 (GenBank accession MF580999), TpsbA, TrbcL, TrpL32, and TpetD.

[0237] Various enhancers may be used for each exogenous nucleic acid sequence component. In some embodiments, a different enhancer sequence may be used for different exogenous nucleic acid sequence components. For example, in some embodiments, a t7genel0 may be the enhancer for a “PL1 ” LeukotoxinA fragment and LrbcL may be the enhancer for a “PL4” LeukotoxinA fragment in an exogenous nucleic acid sequence. In other embodiments, each component of an exogenous nucleic acid sequence may contain the same enhancer sequences.

[0238] In some embodiments, the one or more enhancers included in a nucleic acid material can include any one of the enhancer sequences identified in SEQ ID NOs: 2, 4, and 6 (e.g., as shown in the DNA constructs of FIGs. 1-3 and constructs represented in SEQ ID NOs: 121, 122 and 131). Selection Sequences

[0239] In accordance with various embodiments, nucleic acid materials, e.g., DNA constructs as described herein, can include one or more selection sequences. In some embodiments, selection sequences may be used to provide an efficient system for identification of those cells that have been successfully transformed and transiently and/or stably express an exogenous nucleic acid sequence, for example, after receiving and integrating a DNA construct into their genomes. In some embodiments, a selection sequence may provide (e.g., facilitate or allow the expression of) one or more selection markers which confer resistance to a selection agent, such as an antibiotic or herbicide. Then, for example, potentially transformed cells may be exposed to the selection agent, and the population of surviving cells will be those cells where, generally, the resistance-conferring gene is integrated and expressed at sufficient levels to permit cell survival. In some embodiments, cells may be tested further to confirm stable integration of the exogenous DNA. Commonly used selection sequences may encode genes conferring resistance to antibiotics such as kanamycin and paromomycin (nptll), hygromycin B (aph IV) and gentamycin (aac3 and aacC4), spectinomycin and streptomycin resistance gene (aadA) or resistance to herbicides such as glufosinate (bar or pat) and glyphosate (aroA or EPSPS). In some embodiments, a gene conferring resistance to antibiotics is a 16S or 23 S rRNA gene, e.g., a 16SrRNA or 23SrRNA gene with one or more mutations. In some embodiments, resistance to antibiotics is passive resistance. In some embodiments, resistance to antibiotics is “binding-type” resistance.

Examples of such selection sequences and/or selection agents are illustrated in U.S. Patents 5,550,318; 5,633,435; 5,780,708 and 6,118,047, all of which are incorporated herein by reference. In some embodiments, an antibiotic selection sequence can include a nucleic acid sequence encoding a lincomycin gene, a spectinomycin resistance gene, a gentamycin resistance gene, a streptomycin resistance gene, a Kanamycin resistance gene, a neomycin resistance gene, a Beta lactam resistance gene, or any combination thereof. In some embodiments, antibiotic resistance comprises resistance to lincomycin, spectinomycin, and/or streptomycin. In some embodiments a mutation in a host cell 16S rRNA gene confers resistance to spectinomycin and streptomycin. In some embodiments a mutation in a host cell 23 S rRNA gene confers resistance to lincomycin. In some embodiments, a mutation refers to a mutation in a first (5’) and/or second (3’) targeting sequence as described herein.

[0240] In some embodiments, a selection sequence may also provide an ability to visually identify transformants (e.g., by encoding an observable moiety), for example, a nucleic acid sequence encoding a colored or fluorescent protein such as a luciferase or green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), a green fluorescent protein (eGFP), a His tag, GUS uidA lacz, or a gene expressing a beta glucuronidase or uidA gene (GUS) for which various chromogenic substrates are known, or any combination thereof.

[0241] In some embodiments, a selection sequence comprises a His tag comprising the sequence HIS-Tag: CATCACCATCACCATCAC-TAA (SEQ ID NO: 100), CATCATCATCATCATCAT (SEQ ID NO: 101), CATCACCATCACCATCAC (SEQ ID NO: 8), or a fragment or variant thereof.

[0242] In some embodiments, a selection sequence can be or include one or more of the selection sequences encoding yellow fluorescent protein (YFP, GenBank: GQ221700.1), red fluorescent protein (DsRED, GenBank: KY426960.1 or SEQ ID NO: 7), a green fluorescent protein (eGFP, GenBank: AAB02572.1), or cyan fluorescent protein (CFP, GenBank: HQ993060.1) (e.g., as shown in the constructs of Figs. 1-3 and constructs represented in SEQ ID NOs: 121, 122, and 131).

Vectors

[0243] In some embodiments, a vector is used for expression and/or integration of a nucleic acid material (i.e., DNA construct) in a host cell. In some embodiments, a vector has a copy number that is more than 25, 50, 75, 100, 150, 200, or 250 copies per cell. In accordance with various embodiments, useful vectors for polypeptide expression in plants include viral vectors or plasmids. Examples, without limitation include lentiviral vectors, adenoviral vectors, adeno-associated viral vectors (AAVs), pET vectors (Novagen), Gateway® pDEST vectors (Invitrogen), pGEX vectors (Amersham Biosciences), pPRO vectors (BD Biosciences), pBAD vectors (Invitrogen), pLEX vectors (Invitrogen), pMAL™ vectors (New England BioLabs), pGEMEX vectors (Promega), and pQE vectors (Qiagen). Vector systems for producing phage libraries are known and include Novagen T7Select® vectors, pMX vector plasmid (Invitrogen’ s GeneArt Gene Synthesis), and New England Biolabs Ph.D.™ Peptide Display Cloning System. In some embodiments, a vector may be or comprise a plantspecific vector. In some embodiments, a plant-specific vector can be or include Ti plasmid of Agrobacterium tumefaciens, tobacco mosaic virus (TMV), potato virus X, cauliflower mosaic virus (CaMV) 35S promoter, Bean yellow dwarf virus, geminiviruses, Wheat dwarf virus (WDV), Wheat streak mosaic virus (WSMV), Barley stripe mosaic virus (BSMV), Cabbage leaf curl virus (CaLCuV), Tobacco rattle virus (TRV), and cowpea mosaic virus.

Methods for Introducing Nucleic Acid Material

[0244] Various methods may be used for introducing (i.e., transforming, transducing and/or transfecting) a nucleic acid material into a plant cell. The introduction of a nucleic acid material into a plant may occur via any suitable technique, including, but not limited to, direct DNA uptake, chemical treatment, electroporation, microinjection, cell fusion, infection, vector mediated DNA transfer, bombardment (e.g., gene gun), nanoparticle-guided biomolecule delivery, liposome, protoplast, callus, silicon carbide fiber, and pollen tube transformation, or Agrobacterium mediated transformation. Methods including some form of bombardment can include, without limitation, methods known in the art, including using the biolistic device PDSIOOO/He (Bio-Rad) as described in U.S. Patent Publication No.: US20060117412A1, and Daniell 1997 (Nature Biotech, (16):345-348).

[0245] In some embodiments, methods include targeted insertion of the nucleic acid material in order to achieve site-specific integration, for example to replace an existing gene in the genome, to use an existing promoter in the plant genome, or to insert a recombinant polynucleotide at a predetermined site known to be active for gene expression. Several site specific recombination systems exist which are known to function in plants include cre-lox as disclosed in U.S. Patent 4,959,317 and FLP-FRT as disclosed in U.S. Patent 5,527,695, both incorporated herein by reference.

[0246] In some embodiments, an exogenous nucleic acid sequence is introduced (e.g., transformed, transduced, and/or transfected) into a plastome. In some embodiments, an exogenous nucleic acid sequence is introduced into a chloroplast genome of a plant cell. In some embodiments, an exogenous nucleic acid sequence is introduced into a region within the 16S ribosomal gene DNA sequence in a chloroplast genome of a plant cell. In some embodiments, an exogenous nucleic acid sequence is introduced into a region within the 23 S ribosomal gene DNA sequence in a chloroplast genome of a plant cell.

[0247] In some embodiments, an exogenous nucleic acid sequence is introduced into a nuclear genome of a plant cell. In some embodiments, introducing an exogenous nucleic acid sequence is performed such that the plant cell is stably, that is, permanently transformed with the exogenous nucleic acid sequence (e.g., through site-specific homologous recombination), including the progeny thereof. In some embodiments, a stably transformed exogenous nucleic acid material is capable of autonomous expression of a nucleotide coding region in a plant cell to produce at least one polypeptide (e.g., antigen). In such instances, introducing an exogenous nucleic acid sequence into a plant cell is performed so that the plant cell may transiently express an exogenous nucleic acid sequence (i.e., an antigen). In some embodiments, a transformed plant cell is homoplastic in its genome for the exogenous nucleic acid sequence.

[0248] In some embodiments, transformation methods encompassed by this disclosure may be practiced in vitro and/or in a controlled environment. Recipient cell targets can include, but are not limited to, meristem cells, callus, immature embryos and gametic cells such as microspores, pollen, sperm and egg cells. In accordance with various embodiments, it is contemplated that any cell from which a fertile plant may be regenerated is useful as a recipient cell. Callus may be initiated from tissue sources including, but not limited to, immature embryos, seedling apical meristems, microspores and the like. Cells capable of proliferating as callus are also recipient cells for genetic transformation. Practical transformation methods and materials for making transgenic plants of this invention, for example various media and recipient target cells, transformation of immature embryo cells and subsequent regeneration of fertile transformed plants are disclosed in U.S. Patents 6,194,636 and 6,232,526, which are incorporated herein by reference.

[0249] In some embodiments, plants comprising one or more nucleic acid materials in accordance with the present disclosure may be self-pollinated to provide homozygous transformed plants. In other embodiments, pollen obtained from a plant comprising one or more nucleic acid materials is crossed to seed-grown plants of agronomically important lines. In still other embodiments, pollen from plants comprising one or more nucleic acid materials may be used to pollinate naturally occurring plants. A transformed plant of the present invention comprising an exogenous nucleic acid sequence encoding, e.g., an antigen, may be cultivated using methods known to one skilled in the art.

[0250] Methods described herein for transforming a chloroplast of a host plant cell include contacting the plant cell with the complex comprises the carrier conjugated to a nucleic acid material or solution comprising a nucleic acid material. In some embodiments, contacting comprises applying a vacuum and/or compression. In some embodiments, a method of infusing a nucleic acid material into a plant material or host plant cell includes usage of a vacuum chamber. In some embodiments, live plant materials, sometimes in the form of mature plants, adolescent plants, or calli, are housed within a glass bell-jar greased with a rubber ring. In such embodiments, air within the chamber is pumped out of chamber, drawing out the air within the interstitial tissue of the plant material. Removal of the air within the plant allows the nucleic acid material to be suspended in liquid to better penetrate deep within the plant and improve contact between the host plant cell or plant material and nucleic acid material.

[0251] In some embodiments, plant materials or plant cells are cultured directly in a solution containing nucleic acid material to be introduced. Such an embodiment may or may not include the usage of a vacuum chamber in order to improve the interaction between plant material and nucleic acid materials and assist transformation efficiency.

[0252] In some embodiments, nucleic acid material is delivered to a host plant cell or plant material via infusion. In some embodiments, a method of infusing includes the usage of needless syringe and pressure to transform the plant material. In some embodiments, a needleless syringe is pressed directly onto the adaxial leaf surface and gentle pressure is applied to the syringe to pass nucleic acid material into plant material. In some embodiments, a leaf of a host plant material is first wounded e.g., by scraping using a scalpel or micro punctures to promote the infiltration.

[0253] In some embodiments, infusing nucleic acid material is via syringe injection. In some embodiments, the method of introducing nucleic acid material into a chloroplast of a plant host cell involves the use of a syringe directly into or onto the plants themselves. For example, this may include the injection of nucleic acid materials directly into the vasculature system of a plant host cell, or deposition of the nucleic acid materials onto the surface of a host plant cell. In some embodiments, infusing nucleic acid material into a host plant material comprises macro-injection techniques using a needled syringe.

[0254] In some embodiments, contacting comprises culturing the nucleic acid material, including or not included a carrier complex, in a solution comprising the plant for at least 5 min (e.g., at least 5, at least 10, at least 15, at least 30, at least 45, at least 60 min, or longer). In some embodiments, contacting comprises culturing the nucleic acid material and carrier complex in a solution comprising the plant for at least 1 hour (e.g., at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 8 hours, at least 12 hours, at least 24 hours, at least 2 days, at least 3 days, or longer). Nanoparticles and Nanotubes

[0255] In some embodiments, nucleic acid materials as described herein may be delivered to and/or transformed into a host cell (e.g., a plant cell) via a nanoparticle.

[0256] In some embodiments, a nanoparticle is a particle having a diameter of less than 1000 nanometers (nm), less than 300 nm, or less than 100 nm (e.g., between 1-2 nm). In some embodiments, nanoparticles are micelles in that they comprise an enclosed compartment, separated from the bulk solution by a micellar membrane, typically comprised of amphiphilic entities which surround and enclose a space or compartment (e.g., to define a lumen). In some embodiments, a micellar membrane is comprised of at least one polymer, such as for example a biocompatible and/or biodegradable polymer.

[0257] In some embodiments, a nanoparticle may have or comprise a nanoparticle membrane or boundary or interface between a nanoparticle outer surface and a surrounding environment. In some embodiments, the nanoparticle membrane is a polymer membrane having an outer surface and bounding lumen.

[0258] In some embodiments, a nanoparticle is conjugated to the nucleic acid material. In some embodiments, a nanoparticle is conjugated to an exogenous nucleic acid sequence to be delivered to a host cell (e.g., plant cell).

[0259] In some embodiments, a nanoparticle can be a nanotube. It has been demonstrated that certain nanotubes have the ability to traverse rigid cell walls in plant cells, including the double lipid bilayers of chloroplasts. In some embodiments, a nanoparticle, such as a nanotube, is sized and dimensioned so that the nanoparticle can penetrate the cell membrane and, for example, a chloroplast envelope in a plant cell. In some embodiments, nanoparticle size and surface charge are selected based on the where an exogenous nucleic acid is integrated in a plant cell (e.g., using the lipid exchange envelope penetration (LEEP) model described in Kwak, Seon-Yeong, et al. (2019 Nature nanotechnology (14.5): 447)). In some embodiments, a nanotube is a carbon nanotube. In some embodiments, structure of nanoparticles include 6-membered carbon rings in the shape of a cylindrical tube (see e.g., Ijima et al., 1991). In some embodiments, a nanotube is a single-walled nanotube (SWNT). SWNT material allow for small particle size, structural integrity, and electrical conductivity, providing considerable benefit as a carrier for exogenous nucleic acid materials into the chloroplast of plant cells for genetic engineering. In some embodiments, a nanotube is a single-walled nanotube or a single-walled carbon nanotube (SWCNT). Methods of conjugating an exogenous nucleic acid sequence can be any known method including, but not limited to, those described in Kwak, Seon- Yeong, et al. (2019 Nature nanotechnology (14.5): 447). Conjugating a nucleic acid material to a nanoparticle (e.g., SWCNT) can include incubation of the nanoparticle with the nucleic acid material (e.g., in a dialysis cartridge).

[0260] In some embodiments, nucleic acid materials may be delivered to a particular organelle within a plant host genome. In accordance with various embodiments, an organelle may be any organelle within a plant host cell, including a nucleus or chloroplast. In some embodiments, a nanotube may be modified to promote delivery to a particular organelle and/or to promote efficient delivery. In some embodiments, a nanotube or nanoparticle may be covalently modified. In some embodiments, a nanotube or nanoparticle may be non-covalently modified. In some embodiments, a nanotube may be a chitosan-wrapped nanotube and/or a chitosan-wrapped single-walled nanotube (CS-SWNT). In some embodiments, a chitosan-nanotube complex is further modified through the addition of PEG (Poly ethylene glycol). In some such embodiments, HO-PEG5k-NHS is added to the CS-SWCNT in order to crosslink chitosan strands and enhance nanoparticle colloidal stability. Such a process assists a complex’s efficacy in transformation of host plant materials by preventing clumping, and also improving the stability of the chitosan-SWCNT complex. In some embodiments, a nanoparticle (e.g., a nanotube) may be PEGylated. In some embodiments, a nanotube may be non-covalently bonded to a 5,000 Mw PEG. In some embodiments, a nanoparticle (e.g., a nanotube) may be modified such that the modifications protect the exogenous nucleic acid from nuclease degradation. In some embodiments, a modified nanoparticle (e.g., a nanotube) has a radius of less than 200 nm, less than 150 nm, less than 100 nm, or less than 50 nm.

[0261] One of skill in the art will recognize that the methods described for the delivery of nucleic acid material to the chloroplast of one plant species may not be effective in the delivery in another plant species due to many factors. Provided herein are materials and methods designed for various plant species not previously demonstrated (e.g., millet, sorghum, and alfalfa). Size of a nanotube (i.e., length/diameter), charge/zeta potential, amount of DNA carried, and other types of modifications and parameters are described herein. [0262] In some embodiments, a nanotube (e.g, a single walled carbon nanotube [SWCNT]) described herein has a length of less than 100μm (e.g., less than 90 μm, less than 80 μm, less than 70 gm, less than 60 μm, less than 50 μm, less than 40 μm, less than 30 μm, less than 20 μm, less than 10 μm, less than 9 μm, less than 8 μm, less than 7 μm, less than 6 μm, less than 5 μm, less than 4 μm, less than 3 μm, less than 2 μm, less than 1 μm, less than 100nm, less than 10nm, less than 1.0nm). In some embodiments, a nanotube (e.g., a SWCNT) described herein has a length of at least 1.0nm (e.g., at least

10.0nm, at least 100.0nm, at least 1μm, at least 2μm, at least 3 μm, at least 4 μm, at least 5 μm, at least 6 μm, at least 7 μm, at least 8 μm, at least 9 μm, at least 10.0 μm, at least 20 μm, at least 30 μm, or more). In some embodiments, a nanotube (e.g., a SWCNT) described herein has a length of between about 1.0nm and 100μm (e.g., between about 10.0nm and 100 μm, between about 100nm and 100 μm, between about 1 μm and 100 μm, between about 1 μm and 90 μm, between about 1 μm and 80 μm, between about 1 μm and 70 μm, between about 1 μm and 60 μm, between about 1 μm and 50 μm, between about 1 μm and 40 μm, between about 1 μm and 30 μm, between about 1 μm and 20 μm, between about 1 μm and 10 μm, between about 2 μm and 9 μm, or between about 3 μm and 8 μm).

[0263] In some embodiments, a nanotube described herein (e.g., a SWCNT) has a diameter of less than 200 nm, less than 150 nm, less than 100 nm, less than 50 nm, or less than 10 nm (e.g, between 1-2nm). In some embodiments, a nanotube described herein (e.g., a SWCNT) has a dimeter of at least about 1.0nm (e.g., at least about 0.0001, 0.001, 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9nm or more). In some embodiments, a nanotube (e.g., a SWCNT) described herein has a dimeter of less than about 10.0nm (e.g., less than about 9.0, 8.0, 7.0, 6.0, 5.0, 4.0, 3.0, 2.0 or 1.0nm). In some embodiments, a nanotube (e.g., a SWCNT) described herein has a diameter of between about 1.0-

10.0nm (e.g., between about 1.0-2.0nm).

[0264] In some embodiments, a nanoparticle is designed and constructed (e.g., using chitosan) so that the nucleic acid material is conjugated to a nanoparticle in one location within a plant cell (e.g., within the plant cytosol) and can be released from the nanoparticle in another location (e.g., within the chloroplast stroma). In some embodiments, a nanoparticle (e.g., carbon nanotubes) are complexed with chitosan, where strands of chitosan wrap around the nanotubes and then are cross-linked or modified with PEG. In some embodiments, a nanoparticle (e.g., CS-SWCNT) is complexed a nucleic acid material (e.g., a DNA cargo) through electrostatic interactions between the positively charged chitosan (at certain pH), and the negatively charged DNA. [0265] In some embodiments, a nanoparticle is designed and constructed so that the nanoparticle is released from the nucleic acid material upon exposure to an environment that has a pH of greater than 6.0, greater than 6.5, greater than 7.0, greater than 7.5, or greater than 8.0. In some embodiments, such a DNA-CS-SWCNT complex utilizes electrostatic interactions between the functionalized carbon nanotubes and the nucleic acid material to form a delivery complex. The electrostatic interaction results from association between positive charges present on the amide of the chitosan and negative charges present on the phosphate backbone of the nucleic acid materials. In some embodiments, such a complex (DNA-CS-SWCNT) comprises a pKa value of ~6.5. Positive charges of the amide group found on the chitosan are present predominantly at lower pH values. Once a DNA-CS-SWCNT complex enters a host plant cell chloroplast, where the pH is slightly elevated, the charge of the amide groups will decrease electrostatic interactions binding the nucleic acid material to the CS-SWCNT is reduced and leads to dissociation between the CS-SWCNT and the exogenous nucleic acid material upon entry into a chloroplast. In some embodiments, a nanoparticle (e.g., a SWCNT) described herein, when conjugated to a nucleic acid material, exhibits a zeta potential that is at least 10mV. A zeta potential is a net charge of a positively charged nanoparticle (e.g., SWCNT) more positive than the negatively charged DNA cargo. In some embodiments, a nanoparticle (e.g., a SWCNT) described herein, when conjugated to a nucleic acid material, exhibits a zeta potential that is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40mV or more. In some embodiments, a nanoparticle (e.g., a SWCNT) described herein, when conjugated to a nucleic acid material, exhibits a zeta potential that less than 50mV (e.g., less than 49, 45, 40, 35, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 5mV or less). In some embodiments, a nanoparticle (e.g., a SWCNT) described herein, when conjugated to a nucleic acid material, exhibits a zeta potential of less than 29mV. In some embodiments, a nanoparticle (e.g., a SWCNT) described herein, when conjugated to a nucleic acid material, exhibits a zeta potential of between about 5-30mV (e.g., between about 5-29mV, e.g., between about 10-29mV, e.g., between about 10-20mV, e.g., between about 10-15 mV, e.g., about 13.5mV).In some embodiments, a nanoparticle (e.g., a SWCNT) is combined with a nucleic acid material in a ratio of less than 1 : 1 nucleic acid material: nanoparticle in order to achieve a desirable zeta potential. A ratio 1 : 1 (nucleic acid material manoparticle) ratio may result in a zeta potential at or near 0, and, therefore, may not be sufficient to target the chloroplast of a host plant that is negatively charged. In some embodiments, a nucleic acid material is combined with a nanoparticle (e.g., a SWCNT) in a ratio of less than 1 : 1 , 1: 1.1, 1: 1.2, 1:1.3, 1 :1.4, 1 :1.5, 1 :1.6, 1 :1.7, 1: 1.8, 1: 1.9, 1:2.0, 1:3.0, 1:5.0, or 1:6.0 or less (nucleic acid material: nanoparticle).

[0266] In some embodiments, a nanoparticle conjugated to a nucleic acid material is delivered to a host plant cell using localized infiltration. In some embodiments, a nanoparticle (e.g., a SWCNT) is further conjugated to a peptide (e.g., a chloroplast-targeting peptide (CTP) and/or a chloroplastpenetrating peptide (CPP)) to aid delivery into a host plant cell. In some embodiments, a solution containing a nanoparticle conjugated to a nucleic acid material is infused into a part or parts of a plant. In some embodiments, a nanoparticle is conjugated to an exogenous nucleic acid sequence contained within plasmid DNA. In some embodiments, a nanoparticle is conjugated to a DNA sequence that comprises 100% exogenous nucleic acid sequence (e.g., not contained in plasmid DNA).

[0267] In some embodiments, a solution is infused in an amount of about 1-1,000 μl, 20-1,500 μl, 30-1,000 μl, 40-750 μl, 50-500 μl, 100 pl-10ml. In some embodiments, a solution is infused in an amount of at least 1 μl,10 μl, 100 μl, 1000 μl, 2ml, 3ml, 4ml, 5ml, 6ml, 7ml, 8ml, 9ml, or 10ml. In some embodiments, the amount of nucleic acid material that is delivered to a plant cell is about 1 ng, 5 ng, 10 ng, 20ng, 50 ng, 100ng or greater. In some embodiments, the amount of nucleic acid material that is delivered to a plant cell is about 1 μg, 5 μg, 10 μg, 20 μg, 50 μg, or greater. In some embodiments, the ratio of nanoparticle to nucleic acid material is at least 1 :1, 3: 1, or 6: 1 (w/w).

[0268] In some embodiments, a nanoparticle conjugated to a nucleic acid material is delivered to a plant cell with or without the use of biolistic force. In some embodiments, nanoparticle conjugated to a nucleic acid material is delivered to a plant cell using methods that include, e.g., surface leaf infusion through a needleless syringe and/or stem injection through a needled syringe. Additional delivery methods are described in, e.g., Kwak et al., 2019 and Demerier et al., 2019.

Peptides

[0269] In some embodiments, nucleic acid materials as described herein may be delivered to and/or transformed into a host cell (e.g., a plant cell) via one or more peptide carriers. In some embodiments, one or more peptides allow for targeted delivery of an exogenous nucleic acid material to the chloroplast of a host plant cell genome. [0270] Described herein are peptides and peptide complexes that alone or together have the capacity bind to chloroplast transformation DNA constructs, penetrate plant cells, and deliver DNA to the chloroplast (e.g., within the 16S or 23S ribosomal gene). In some embodiments, a peptide carrier is coupled to (e.g., complexed with) an exogenous nucleic acid sequence to be delivered. In some embodiments, coupled to refers to a coupling that occurs between a negatively charged exogenous nucleic acid material and a positively charged peptide carrier. As used herein, the term “complexed” refers to a carrier and an exogenous nucleic acid sequence or two carriers that are somehow associated (e.g., linked, interacted, grafted, condensed, and/or combined) with each other, whether it be through fusion, conjugation, a covalent or non-covalent bond, or through electrostatic interaction. In some embodiments, a peptide carrier is also complexed with a nanoparticle (e.g., a SWCNT).

[0271] In some embodiments, a peptide carrier is coupled to an exogenous nucleic acid sequence as described herein, that includes, among other things, targeting sequences that introduce the exogenous nucleic acid material to a particular region with a host plant genome. Such targeting sequences, as described herein, include sequences that target a region within the 16S and 23 S ribosomal sequence of a host plant chloroplast genome (e.g., in a sorghum, millet, or alfalfa host plant chloroplast genome).

[0272] In some embodiments, a peptide carrier is native to a host cell plant. In some embodiments, a peptide carrier is a fragment of a native protein. In some embodiments, a peptide carrier is chosen by its ability to target the chloroplast genome of a host plant cell. In some embodiments, a peptide carrier is chosen by its ability to target the chloroplast genome of a host plant cell (e.g., Chloroplast-targeting peptides (CTPs)). In some embodiments, a peptide carrier is chosen by its ability to penetrate host plant cell (e.g., Chloroplast-penetrating peptides (CPPs)).

[0273] In some embodiments, a peptide carrier is not native to a host cell plant.

[0274] In some embodiments, a peptide is a synthetic peptide. In some embodiments, a peptide carrier is a fragment of a native protein of the host cell plant that is conjugated to a synthetic peptide.

Chloroplast-Targeting Peptides

[0275] Chloroplasts are organelles found in plant cells and eukaryotic algae that conduct photosynthesis. The chloroplast is a complex cellular organelle composed of three membranes: the inner envelope membrane, the outer envelope membrane, and the thylakoid membrane. The membranes together enclose three aqueous compartments termed the intermediate space, the stroma, and the thylakoid lumen. While chloroplasts contain their own circular genome, many constituent chloroplast proteins are encoded by the nuclear genes and are cytoplasmically-synthesized as precursor forms which contain N-terminal extensions known as chloroplast transit peptides or chloroplast targeting peptides (CTPs). CTPs are important for specific recognition of the chloroplast surface and in mediating the post-translational translocation of pre-proteins across the chloroplast envelope and into the various different subcompartments within the chloroplast (e.g., stroma, thylakoid and thylakoid membrane).

[0276] Genes reported to have naturally encoded transit peptide sequences at their N- terminus include the chloroplast small subunit of ribulose-l,5-bisphosphate carboxylase (RuBisCo), de Castro Silva Filho et al. (1996) Plant Mol. Biol. 30: 769- 780; Schnell, D. J. et al. (1991) J. Biol. Chem. 266 (5): 3335-3342; 5-(enolpyruvyl) shikimate-3 -phosphate synthase (EPSPS), Archer et al. (1990) J. Bioenerg. and Biomemb. 22 (6):789-810; tryptophan synthase. Zhao, J. et al. (1995) J. Biol. Chem. 2 70 (1 1):6081-6087; plastocyamn, Lawrence et al. (1997) J. Biol. Chem. 272 (33):20357-20363; chorismate synthase, Schmidt et al. (1993) J. Biol. Chem. 268 (36):27477-27457; and the light harvesting chlorophyll a/b binding protein (LHBP), Lamppa et al. (1988) J. Biol. Chem. 263: 14996- 14999. Although several CTPs have been described, only a few have been utilized successfully in attempts to target chimeric molecules to chloroplasts in higher plants. The present disclosure provides, among other things, the use of CTPs derived from cereal plant species such as sorghum, millet, and alfalfa, that can be utilized for targeted delivery of exogenous nucleic acid material.

[0277] CTPs disclosed herein are useful for targeting an exogenous nucleic acid sequence to the chloroplast of a host plant cell. In some embodiments, CTPs disclosed herein provide improved translocation compared to delivery of an exogenous nucleic acid sequence without a peptide carrier or with a CTP from a different host plant species. In some embodiments, CTP carriers disclosed herein result in an at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, or greater, or at least about 2-fold, at least about 3 -fold, at least about 4-fold, or greater improvement in translocation of an exogenous nucleic acid material into the chloroplast compared to delivery without a peptide carrier or with a CTP from a different host plant species. An improvement can be measured in terms of the amount of exogenous nucleic acid material that is actually translocated into the chloroplast, the amount of active exogenous nucleic acid material that is translocated into the chloroplast, or both. An improvement can also be measured in terms of an improvement in the phenotype of an organism transformed with the chloroplast-targeted protein of interest. For example, where the CTP of the disclosure is used to target an herbicide resistance protein to the chloroplast of the plant, an improvement in activity can be measured in terms of an improvement in herbicide resistance.

[0278] Chloroplast proteins are mostly encoded by the nuclear genome and are post- translationally imported the chloroplast via the action of N-terminal extensions commonly referred to as targeting peptides. An example CTP is OEP34 (outer-envelope membrane protein 34), which is unique among OEP proteins in that it likely uses an ATP-mediated proteinaceous receptor to import itself into the chloroplast (Li and Chen, 1997). Alignments of OEP34 of Pisum sativum with AtOEP34 of the model species Arabidopsis, along with deletion studies, strengthened support for C-terminal hydrophobic core of these proteins as responsible for importation into the chloroplast (Li and Chen, 1997). Yoshizumi et al. (2018) describes methods utilizing 10 amino acids of the C-terminal hydrophobic core of AtOEP34 and transformed Arabidopsis seedlings.

[0279] In some embodiments, a CTP comprises a protein derived from one or more of Arabidopsis thaliana outer envelope membrane protein; molecular weight of 34 d OEP34 (Genbank accession no. NP 850768.1), Pisium sativum functional homologue translocase of chloroplasts 34 (TOC34; Genbank accession no. Q41009.1), TOC34 proteins of sorghum (Genbank accession no. XP 021306533.1), millet (Genbank accession no. RLN39229.1), and Medicago truncatula (Genbank accession no. XP 003624825.1), or a fragment or variant thereof.

[0280] Methods described herein include CTPs identified in other plant species, e.g., in cereal plant species such as sorghum, millet and alfalfa, and the characterization of these CTPs to function as carriers of exogenous nucleic acid materials for targeted delivery to a site within the host chloroplast genome (e.g., within the 16S or 23S ribosomal gene).

[0281] In some embodiments, a CTP used in the methods and compositions disclosed herein is derived from any one of the plant species described herein. In some embodiments, a CTP used in the methods and compositions disclosed herein is derived from millet. In some embodiments, a CTP used in the methods and compositions disclosed herein is derived from sorghum. In some embodiments, a CTP used in the methods and compositions disclosed herein is derived from alfalfa. In some embodiments, a CTP used in the methods and compositions disclosed herein is derived from wheat. In some embodiments, a CTP used in the methods and compositions disclosed herein is derived from maize. In some embodiments, a CTP used in the methods and compositions disclosed herein is derived from barley. In some embodiments, a CTP used in the methods and compositions disclosed herein is derived from triticale.

[0282] In some embodiments, a CTP comprises an outer envelope membrane protein; molecular weight of 34 d (OEP34) or a fragment or variant thereof. In some embodiments, a CTP comprises the hydrophobic core region of the OEP34 protein. In some embodiments, a CTP comprises an OEP34 protein encoded by the amino acid sequence SEQ ID NO: 23 [ILAVEYFLVV], SEQ ID NO: 24 [IFALQYLFLA], SEQ ID NO: 25 [LFALEFLLIM], SEQ ID NO: 26 [ILAVQYFFW], or SEQ ID NO: 27 [MFAFQYLLVM], or a fragment or variant thereof.

[0283] In some embodiments, a CTP is conjugated to the nucleic acid material via positively charged amino acids at the N-terminus of the CTP. In some embodiments, a positively charged amino acids comprise a KH9 sequence (SEQ ID NO: 28 In some

embodiments, a CTP and KH9 sequence comprises one of SEQ ID NO: 83 (Sorghum/millet KH9-

OR SEQ ID NO: 84 (Medicago KH9-OEP34

Cell-Penetrating Peptides

[0284] Other peptides that facilitate penetration through the cell wall of a host plant cell may be utilized to deliver exogenous nucleic acid material to a host plant cell genome (e.g., within the chloroplast). For example, cell-penetrating peptides (CPPs) are known to have the function of transporting complexes containing such peptides and other substances (e.g, proteins, nucleic acids, etc.) through biological membranes in mammalian and human cell lines. Use of CPP in plant cells is more limited, because, unlike animal cells, plant cells have a double hindrance by the cell wall and cell membrane against internalization of the complex containing CPP.

[0285] Described herein are compositions comprising an exogenous nucleic acid material and a carrier peptide that is characterized by comprising a cell-permeable sequence and optionally a polycationic sequence. In some embodiments, a CPP may contain a sugar chain, a lipid, and/or a phosphate residue in addition to the peptide component. [0286] By cell permeable sequence is meant the sequence of a cell permeable peptide (CPP). Examples of cell-penetrating peptides include BP100 (Appl Environ Microbiol 72 (5), 3302, 2006), HIV Tat (Journal Biological Chemistry, 272, pp.16010-16017, 1997), Tat.₂ (Biochim Biophys Acta 1768 (3), 419, 2007), Penetratin, pVEC, pAntp (Journal Biological Chemistry, 269, pp.10444-10450, 1994), HSV-1 VP22 (Cell, 8823, p.2223).1997), MAP (Model amphiphilic peptide) (Biochimica Biophysica Acta, 1414, pp.127-139, 1998), Transportan (FEBS Journal, 12, pp.67-77, 1998), R7 (Neur, ed.1253-1257, 2000), MPG (Nucleic Acid Research 25) Pp.2730-2736, 1997), and Pep-1 (Nature Biotechnology, 19, pp.1173-1176, 2001). In some cases, peptide sequences in which one to several amino acid residues contained in these peptide sequences are substituted, inserted, and/or deleted may be preferably used. In some embodiments, two or more types of CPPs may be used in combination. In some embodiments, a CPP may contain two or more cell permeable sequences. In some embodiments, it is preferred to select a CPP that is specific for the particular cell of interest. [0287] Examples of cell permeable sequences included in a CPP include any of the following sequences (or fragments or variants thereof): KKLFKKILKYL (SEQ ID NO: 31), RKKRRRQRRRRKKRRQRRRR (SEQ ID NO: 32), RKKRRQRRR (SEQ ID NO: 33), PLSSIFSRIGDP (SEQ ID NO: 34), PISSIFSRTGDP (SEQ ID NO: 35), AISSILSKTGDP (SEQ ID NO: 36), PISSIFKIGDP (SEQ ID NO: 37), PLSSIFSHIGDP (SEQ ID NO: 38), PLSSIFSSIGDP (SEQ ID NO:39), RQKIKIWFQNRRMKWKK (SEQ ID NO: 40), DATATRGRSAASRPTERPRAPSASRPRRPPVD (SEQ ID NO: 41), AAVALLPAVLLLALLAP (SEQ ID NO: 42) AALPLP (SEQ ID NO: 43), GALFLGWLGAAGSTMGA (SEQ ID NO: 44), MGLGLHLLVLAAALQGA (SEQ ID NO: 45), LGTYTQDFNKFHTFPQTAIGVGAP (SEQ ID NO: 46), GWTLNSAGYLLKINLKALAALAKKIL (SEQ ID NO: 47), KLALKLALKALKAALKLA (SEQ ID NO: 48). [0288] In embodiments, a CPP includes one of the peptides described in Numata et al., 2018. In some embodiments, a CPP comprises BP100 (sequence: SEQ ID NO: 29 [KKLFKKILKYL]-amide), K9 (SEQ ID NO: 30 [KKKKKKKKK]), or HPV33L2-DD447 (SEQ ID NO: 102 [SYDDLRRRRKRFPYFFTDVRVAA]) Peptide Complexes

[0289] In some embodiments, a peptide carrier comprises one or more peptides complexed with an exogenous nucleic acid material for targeted delivery to the host plant cell genome (e.g., within the chloroplast). For example, coupling CTP-DNA complexes with cell-penetrating proteins (CPPs) has been demonstrated resulting in a DNA-CTP-CPP complex (see e.g., Thagun et al., 2019). Previous studies have described using these complexes for chloroplast delivery in a limited number of plant species. The methods and compositions described herein describe peptide mediated chloroplast transformation that is effective in delivery of exogenous nucleic acid materials in various crop species, such as sorghum, millet and alfalfa, among others.

[0290] In some embodiments, a CPP or a CTP includes or is conjugated or complexed to a polycationic sequence. In some embodiments, a polycationic sequences is preferably 4 or more, more preferably 5 or more, still more preferably 7 or more, preferably 30 or less, more preferably 25 or less, even more preferably 20 or less lysine, arginine and and/or histidine residues. In some embodiments, a polycationic sequence has a series of 3 or more consecutive lysine, arginine and/or histidine residues, and preferably has a series of 5 or more consecutive lysine, arginine and/or histidine residues. In some embodiments, a cationic sequence has a series of 7 or more consecutive lysine, arginine and/or histidine residues. Among the cationic amino acid residues, when the proportion of arginine is high, introduction into the cell tends to be quick, and when the proportion of histidine and lysine is high, introduction into the cell tends to be slow. For example, depending on the purpose of use of the complex of the present invention such as the following organelle-specific introduction, the introduction rate into the cell can be controlled by appropriately selecting the polycationic sequence. Preferable examples of the polycationic sequence include a KH repetitive sequence, for example, a KH repetitive sequence of 3-20, more preferably a KH repetitive sequence of 5-15, and even more preferably a repetitive sequence of 7-12. Arginine (R) continuous sequence, for example, R 3-20 continuous sequence, preferably R 5-15 continuous sequence, more preferably R 7-12 continuous sequence, lysine (K) continuous sequence, for example, 3 to 20 continuous sequences of K, preferably 5 to 15 continuous sequences of K, more preferably 7 to 12 continuous sequences of K, continuous sequences of histidine (H), for example 3 to 20 of H. Examples of polycationic sequences include a continuous array, preferably a 5-15 continuous array of H, and more preferably a 7-12 continuous array of H. Specific examples of the polycation sequence include the following sequences: RRRRRRR (SEQ ID NO: 49),

[0291] In some embodiments, a protein complex includes a CTP and KH9 sequence comprising one of SEQ ID NO: 83 (Sorghum/millet KH9-OEP34 KHKHKHKHKHKHKHKHKHILAVEYFLVV) OR SEQ ID NO: 84 (Medicago KH9-OEP34 KHKHKHKHKHKHKHKHKHLFALEFLLIM).

[0292] In some embodiments, a carrier peptide described herein includes a CPP (including a cell permeable sequence) and/or a CTP and a poly cationic sequence. In some embodiments, a polycationic sequence is attached to the N-terminus and/or C-terminus of the CTP and/or CPP. In some embodiments, conjugation may be performed chemically according to a normal peptide bond reaction, or may be performed biologically using an enzyme such as ligase. For example, it can be performed according to a general peptide synthesis method such as a solid phase method. In binding the CPP, an appropriate oligopeptide linker or the like can be interposed between the two. For example, a linker consisting of one to several amino acids can be interposed, and the amino acid residues constituting the linker can be appropriately selected. Since cell-penetrating peptides exhibit their properties at the N- terminus, it is preferable that the CPP is bound to the N-terminal side of the polycationic sequence. A carrier peptide described herein can also be obtained by recombinant DNA technology. For example, a DNA fragment encoding a polycationic sequence is ligated to one or both ends of a DNA fragment encoding a CPP by ligation reaction with an appropriate DNA adapter or by in vitro mutagenesis. Such genetic manipulation methods are well known to those skilled in the field of molecular biology.

[0293] In some embodiments, a peptide complex described herein includes a CPP complexed with a CTP-DNA complex. In some embodiments, a CPP-CTP-DNA complex comprises the CTP comprises an OEP34 protein encoded by any of the amino acid sequences SEQ ID NO: 25-27, or a fragment or variant thereof conjugated to a nucleic acid material via positively charged amino acid at its N-terminus (e.g., KH9) and further comprises a CPP (e.g., BP100 [SEQ ID NO: 29] or K9 [SEQ ID NO: 30]).

[0294] In some embodiments, a CPP-CPT-DNA complex includes DNA comprising any one of SEQ ID NOs: 1-22 and 79-82. In some embodiments, DNA comprises a 16S ribosomal sequence. In some embodiments, DNA comprises a 23S ribosomal sequence. In some embodiments, DNA comprises a sequence that targets the chloroplast genome of a plant (e.g., the genome of sorghum, millet, or alfalfa). In some embodiments, a peptide complex is further coupled to a nanoparticle (e.g., a SWCNT).

[0295] Methods described herein for transforming a chloroplast of a host plant cell include contacting the plant cell with the complex comprises the carrier conjugated to the nucleic acid material.

[0296] In some embodiments, carrier peptides described herein target the chloroplast of a host plant cell genome such that a transformed exogenous nucleic acid sequence is expressed in the chloroplast of the plant cell. In some embodiments, a transformed exogenous nucleic acid sequence is integrated in the chloroplast genome of the plant cell. In some embodiments, a transformed exogenous nucleic acid sequence is stably integrated in the chloroplast genome of the plant cell.

[0297] In some embodiments, an exogenous nucleic acid sequence encodes an exogenous protein, and wherein the transformed plant expresses the exogenous protein. In some embodiments, methods of transforming a chloroplast genome results in expression of an exogenous protein that is capable of binding a natural target.

Viral Vectors

[0298] As is described herein, various methods of delivering nucleic acid material to a host cell may be used. In some embodiments, an exogenous nucleic acid sequence as described herein can be introduced into a plant cell in a viral vector.

Vector Design

[0299] In some embodiments, a viral vector can be derived from any known plant-based or plant-compatible viral vector. A viral vector may be chosen based on a number of factors, for example, the plant species being transformed, size of the exogenous nucleic acid and location targeted within the host genome. Viral DNA of a viral vector for modifying plants is, for example, designed and constructed to optimize infectivity, movement throughout the plant host cell, and high multiplication.

[0300] In some embodiments, an exogenous nucleic acid sequence as described herein can be cloned into a number of types of vectors. For example, a nucleic acid can be cloned into a plasmid, a phagemid, a phage derivative, an animal virus, a plant virus, or a cosmid. [0301] In some embodiments, a virus can include, for example, Ti plasmid of Agrobacterium tumefaciens, tobacco mosaic virus (TMV), potato virus X, cauliflower mosaic virus (CaMV) 35S promoter, Bean yellow dwarf virus, geminiviruses, Wheat dwarf virus (WDV), Wheat streak mosaic virus (WSMV), Barley stripe mosaic virus (BSMV), Cabbage leaf curl virus (CaLCuV), Tobacco rattle virus (TRV), Tomato golden mosaic virus (TGMV), Alfalfa Mosaic Virus (A1MV), ilarviruses, cucumoviruses such as Cucumber Green Mottle Mosaic virus (CGMMV), Tobacco Etch Virus (TEV), Cowpea Mosaic virus (CMV), and viruses from the brome mosaic virus group such as Brome Mosaic virus (BMV), broad bean mottle virus, cowpea chlorotic mottle virus, Rice Necrosis virus (RNV), Cassaya latent virus (CLV) and maize streak virus (MSV). Alternative vectors can include expression vectors, replication vectors, probe generation vectors, and sequencing vectors, and non-plant derived viral vectors.

[0302] In some embodiments, vectors may have one or more transcription termination regions. A transcription termination region is a sequence that controls formation of the 3' end of the transcript, e.g., polyadenylation sequences and self-cleaving ribozymes. Termination signals for expression in other organisms are well known in the literature. Sequences for accurate splicing of the transcript may also be included. Examples are introns and transposons.

[0303] Viral vector design and technology is well known in the art as described in Sambrook et al, (Molecular Cloning: A Laboratory Manual, 2001), and in other virology and molecular biology manuals.

Viral transduction

[0304] Viruses are highly efficient at nucleic acid delivery to specific cell types, while often avoiding detection by the infected host immune system. These features make certain viruses attractive candidates as vehicles for introduction of nucleic acid material into target cells (e.g., plant cells). A number of viral based systems have been developed for gene transfer into mammalian and plant cells. In general, a suitable vector comprises an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. A viral vector described herein can be in DNA or RNA form. [0305] In some embodiments, a viral vector can be used to deliver exogenous nucleic acid sequences of various sizes to a host cell (e.g., a plant cell). In some embodiments, a viral vector can accommodate an exogenous nucleic acid sequence that is greater than 50, 100, 200, 400, 500, 1000 nucleotides in length.

[0306] In some embodiments, an exogenous nucleic acid sequence can be cloned into a viral vector and then introduced into a host cell (e.g., a plant cell). In some embodiments, viral vectors can be introduced into a plant host cell using bombardment (e.g., gene gun), Agrobacterium mediated transformation, or any other method encompassed by the present disclosure.

[0307] Any of a variety of methods for facilitating infection of a target plant can be applied to cell(s) of the plant according to any technique known to those skilled in the art. For example, in some embodiments, suitable techniques include, but are not limited to, hand inoculations such as abrasive inoculations (leaf abrasion, abrasion in a buffer solution), mechanized spray inoculations, vacuum infiltration, particle bombardment and/or electroporation.

[0308] In some embodiments, a viral vector can be delivered to a plant at different growth stages such as seedling stage, leaf stage, flowering, seed formation and maturation stages through roots, cotyledons, leaves, seed coat, seeds, pods, stem inoculations, etc. In some embodiments, a viral vector can be applied at one or more locations of a host plant. For example, a viral vector can be applied on leaves and roots either simultaneously or successively. In some embodiments, a viral vector can be applied at the same location (e.g., on a given leaf) more than once at successive intervals. The time intervals can depend on the experimental conditions and the target gene to be silenced. Two types of vectors (e.g. local and systemic) capable of introducing two different genes can be mixed and applied at a given location or more than one location. Once applied, samples can be collected and screened for virus infection.

[0309] In some embodiments, a viral vector may be designed and constructed for systemic infection. In some embodiments, a viral vector can also be engineered in a manner that initiation of target gene silencing also initiates destruction and elimination of the vector from plant (approximately 15-20 days after inoculation). In some embodiments, a viral vector may be designed and constructed for localized infection, e.g., if a leaf is infected, the infection does not spread beyond said leaf. Expression of Exogenous Nucleic Acid Sequence(s)

[0310] In some embodiments, the present disclosure includes a plant that has been transformed such that the plastome (e.g., chloroplast genome) of the plant or plant cell has been stably, that is, permanently transformed in accordance with methods of the invention (e.g., through site-specific homologous recombination, for example in the 16S ribosomal gene), including the progeny thereof. In some embodiments, a nucleic acid material comprises one or more cloning or expression vectors; for instance, a vaccine comprising one or more of the compositions or transformed plants as described herein may comprise a plurality of expression vectors each capable of autonomous expression of a nucleotide coding region in a plant cell to produce at least one immunogenic polypeptide. In such instances, a transformed plant may transiently express an exogenous nucleic acid sequence (i.e., an antigen). In some embodiments, a transformed plant contains an exogenous nucleic acid sequence where the expression of the sequence (i.e., an antigen) is driven by a promoter that is constitutively expressed. In some embodiments, a transformed plant contains an exogenous nucleic acid sequence where the expression of the sequence (i.e., an antigen) is driven by a promoter that is differentially expressed, e.g., in the absence or presence of light, a selection agent, or another control mechanism.

[0311] In some embodiments, expression of exogenous nucleic acid material is detectable 1 hour after transformation/inoculation of the host species. In some embodiments, expression of exogenous nucleic acid material remains detectable for at least 1, 2, 3, 4, 5, 6, 7, 14, or 21 days after transformation/inoculation of the host species. In some embodiments, expression of exogenous nucleic acid material remains detectable for at least 1, 2, 3, 4, 5, 6, or 12 months after transformation/ inoculation of the host species.

[0312] In some embodiments, detecting transformation of a plant cell can be determined when the expression of an exogenous nucleic acid sequence is greater than the expression in a control cell (i.e., a non-transformed cell). In some embodiments, detecting transformation of a plant cell can be determined when the expression of an exogenous nucleic acid sequence is greater than at least 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or greater than the expression in a control cell (i.e., a non-transformed cell).

[0313] Methods of measuring expression may include, without limitation, southern blot analysis using probes that can detect a particular nucleotide sequence, or amplification of a transgene by PCR. Methods of measuring/detecting expression of an exogenous protein (e.g., an antigen) produced by a transformed plant as encompassed by the present disclosure include, without limitation, ELISA (enzyme-linked immunosorbent assay), Western blotting, competition assay, and spot-blot. Means of detection may be or include, for instance, chemiluminesce, fluorescence, or colorimetric detection. One suitable method for measuring binding of the antigen, using a known antibody, is the Luminex xMAP system, where peptides are conjugated to a dye- containing microsphere. In some embodiments, other systems are used to assay a plurality of markers, for example, profiling may be performed using any of the following systems: antigen microarrays, bead microarrays, nanobarcodes particle technology, arrayed proteins from cDNA expression libraries, protein in situ array, protein arrays of living transformants, universal protein array, lab-on-a-chip microfluidics, and peptides on pins. Another type of clinical assay is a chemiluminescent assay to detect antigen-antibody binding.

Expression of Exogenous Proteins

[0314] In some embodiments, the present disclosure includes a plant that has been transformed such that the plastome (e.g., chloroplast genome) of the plant or plant cell has been stably, that is, permanently transformed in accordance with methods of the such that the plant is able to express an exogenous protein and the expressed protein is able to bind its natural targets.

[0315] In some embodiments, other exogenous protein sequences are contemplated. Methods for confirming the structures of synthesized proteins is by conducting protein-protein binding assays where the expressed proteins are incubated with their native ligands in vitro.

Production

[0316] In accordance with various embodiments, any of a variety of methods for growing/producing transformed plants, selecting and/or formulating said transformed plants into immunogenic compositions (e.g., plant-based vaccines) may be used. As used herein, the term “plantbased vaccine” or “plant-based vaccine composition” includes compositions comprising one or more parts of a plant or one or more components produced in a plant (e.g., an exogenous nucleic acid sequence). Method of production, selection and/or formulation may depend e.g., on the species of the subject the immunogenic composition is being administered to, the type of plant, or the antigen of interest to be expressed in the transformed plant. [0317] Various methods of growing and propagating transformed plants may include any systems or procedures used in farming and agriculture, and may depend on the plant species used in a particular application. In some embodiments, seeds of a transformed plant can be harvested from fertile transformed plants, and can be used to grow progeny generations of transformed plants. In some embodiments, a selection sequence is used to select the plants that have been transformed with the exogenous nucleic acid sequence. In addition to direct transformation of a plant with a nucleic acid material, transformed plants can be prepared by crossing a first transformed plant with a second nontransformed plant. For example, an exogenous nucleic acid sequence encoding an antigen protein can be introduced into first plant line that is amenable to transformation to produce a transgenic plant, which can be crossed with a second plant line to introduce the exogenous nucleic acid into the second plant line.

Selection Methods

[0318] Once a host plant has been transformed with and is expressing an exogenous nucleic acid sequence, various methods may be used in order to select a successfully transformed plant from with a population of transgenic plants.

[0319] Selection methods are helpful in that within a population of progeny from a transgenic plant, there can be many plants that do not have in their genomes or do not express the exogenous nucleic acid material. In some embodiments, where an expression cassette comprising an exogenous nucleic acid sequence imparts an enhanced trait in the plant, selection from the population may be determined by measuring said enhanced trait. Transgenic plants having enhanced traits are selected from populations of plants regenerated or derived from plant cells transformed as described herein by evaluating the plants in a variety of assays to detect an enhanced trait, e.g. enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil. These assays also may take many forms including, but not limited to, direct screening for the trait in a greenhouse or field trial or by screening for a surrogate trait. Such analyses can be directed to detecting changes in the chemical composition, biomass, physiological properties, morphology of the plant. Changes in chemical compositions such as nutritional composition of grain can be detected by analysis of the seed composition and content of protein, free amino acids, oil, free fatty acids, starch or tocopherols. Changes in biomass characteristics can be made on greenhouse or field grown plants and can include plant height, stem diameter, root and shoot dry weights; and, for corn plants, ear length and diameter. Changes in physiological properties can be identified by evaluating responses to stress conditions, for example assays using imposed stress conditions such as water deficit, nitrogen deficiency, cold growing conditions, pathogen or insect attack or light deficiency, or increased plant density. Changes in morphology can be measured by visual observation of tendency of a transformed plant with an enhanced agronomic trait to also appear to be a normal plant as compared to changes toward bushy, taller, thicker, narrower leaves, striped leaves, knotted trait, chlorosis, albino, anthocyanin production, or altered tassels, ears or roots. Other selection properties include days to pollen shed, days to silking, leaf extension rate, chlorophyll content, leaf temperature, stand, seedling vigor, internode length, plant height, leaf number, leaf area, tillering, brace roots, stay green, stalk lodging, root lodging, plant health, barreness/prolificacy, green snap, and pest resistance. In addition, phenotypic characteristics of harvested grain may be evaluated, including number of kernels per row on the ear, number of rows of kernels on the ear, kernel abortion, kernel weight, kernel size, kernel density and physical grain quality.

[0320] Assays for screening for a desired trait are readily designed by those practicing in the art.

[0321] In some embodiments, transformed plants may be selected based on their antibiotic resistance. For example, a plant may include a sequence or a mutation in its genome that confers antibiotic resistance. For example, a transformed plant may be resistant to lincomycin, spectinomycin, streptomycin, kanamycin, gentamycin, neomycin, Beta lactam resistance, and any combination thereof.

[0322] In some embodiments, transformed plants may be selected based on a selection sequence. In some embodiments, a selection sequence is or comprises a nucleic acid sequence encoding: a His tag, GUS uidA lacz, green fluorescent protein, yellow fluorescent protein, red fluorescent protein, cyan fluorescent protein, and any combination thereof. Example fluorescent proteins include yellow fluorescent protein (YFP, GenBank: GQ221700.1), red fluorescent protein (DsRED, GenBank: KY426960.1), or cyan fluorescent protein (CFP, GenBank: HQ993060.1). Formulation

[0323] In some embodiments, a transformed plant expressing an exogenous nucleic acid sequence encoding a protein of interest (or fragment thereof) is grown to a certain confluence and/or maturity, and then subsequently harvested. In some embodiments, a transformed plant is cut and harvested wet (e.g., containing about 65% moisture). In some embodiments, harvested plant material is treated and/or preserved, e.g., by sun-drying (to cure the plant material). In some embodiments, the harvested plant material is processed (e.g., by dehydration and e.g. further baled). In some embodiments, the harvested plant material is baled and used as dry (e.g., sun-cured) feed for livestock animals.

[0324] In some embodiments, a transformed plant is harvested as hay (e.g., air dried 85-90% dry matter). In some embodiments, a transformed plant is harvested as hay is ground through a screen (e.g., a 2-3” screen). In some embodiments, harvested hay is mixed into a ration to be feed to a nonhuman animal, e.g., to be 35-45% of the total roughage in the ration.

[0325] In some embodiments, when the harvested plant material is processed, e.g., by dehydration, the dried material is further processed, e.g., compressed into pellet form or into a larger block so that it can be fed to livestock animals. The pellets can be administered as supplements, e.g., by a trained professional. In some embodiments, the plant material in compressed, block form, can be placed in a living area of one or more livestock animals so that they can access the block and ingest the plant material by licking the block throughout the day (e.g., have free access to the plant material).

[0326] In some embodiments, harvested plant material is processed into silage (crop ensiled). In some embodiments, the harvested plant material is ensiled without drying and the harvested, wet (e.g., containing about 65% moisture) plant material may be fed to livestock animals e.g., daily, every other day, weekly, monthly, or intermittently. In some embodiments, harvested plant material is not ensiled before it is fed to a livestock animal e.g., daily, every other day, weekly, monthly, or intermittently. In some embodiments, the transformed plants are harvested and then directly fed to a livestock animal (e.g., without further processing, e.g., a “green chop”).

[0327] In some embodiments, a plant cell producing a protein of interest (i.e., has been transformed with an exogenous nucleic acid sequence), can be administered to livestock animals by allowing the livestock animal to graze on the live plant cell line producing the protein. As such, delivery to the animal via grazing is constant, i.e., throughout the day, several times per day, at regular or irregular intervals as grazing of the live plant occurs.

[0328] In some embodiments, a transformed plant cell line can be used to grow and expand the plant population expressing a particular protein (e.g., antigen), so that it can be harvested and the protein (e.g., antigen) can be purified from the transformed plant cells, and further processed into a different form, e.g, in the form of a conventional vaccine. In some embodiments, a protein purified from transformed plant cells can be a fragment of the protein, such as an immunogenic fragment. In some embodiments, a protein purified from transformed plant cells can be concentrated to a particular concentration and purity of protein, depending, for example, on the use of the composition.

[0329] In some embodiments, a transformed plant is cultivated to produce a particular protein of interest and can be compared with a control plant. As used herein a “control plant” means a plant that does not contain the exogenous nucleic acid sequence encoding a particular protein of interest or a “non-transformed” plant. A control plant may be used to identify and select a transformed plant that is producing (e.g., expressing) a particular protein of interest. In some embodiments, a suitable control plant can be a non-transformed plant of the parental line used to generate a transformed plant, i.e. devoid of the exogenous nucleic acid sequence encoding a particular protein of interest. A suitable control plant may, in some embodiments, be a progeny of a transformed plant line that does not contain an exogenous nucleic acid encoding a particular protein of interest, known as a negative segregant. Cultivated transformed plants can be harvested and quantified in order to prepare a specific concentration of protein for a composition (e.g., dosage) to be provided to a non-human animal for treatment.

Immunogenic Compositions

[0330] In some embodiments, one or more plants (e.g., a mixture of plants) may be formulated into an immunogenic composition (e.g., a plant-based vaccine) and administered to a subject. By way of a further non-limiting example, specified amounts of a transformed plant (e.g., transgenic plant) can be diluted with a non-transformed plant, for example, to achieve a particular ratio of transformed plant mass to non-transformed plant mass to achieve, inter alia, a desired concentration (or concentration range) of an antigen in the immunogenic composition. In some embodiments, a desired concentration will depend on any of several factors, for example, the timing of use of an immunogenic composition (i.e., whether used prophylactically or for therapeutic treatment), the particular subject (e.g., species, age, size), the progression of the disease or infection being treated, and also the particular dosing regimen desired.

[0331] In some embodiments, immunogenic compositions (e.g., plant-based vaccines) may include a delivery system for use in administering a provided immunogenic composition to a subject (e.g., a ruminant animal). In some embodiments a delivery system may comprise a material and/or coating that will resist degradation due to gastric and enteric environments. In some embodiments, a delivery system may include, but is not limited to, a liposome, a proteasome, cochleates, virus-like particles, immune-stimulating complexes, microparticles and nanoparticles (e.g., nanotubes).

[0332] In some embodiments, immunogenic compositions may include a transformed plant produced using a system and/or method described herein and an application-appropriate carrier or excipient.

[0333] Formulations of immunogenic compositions described herein may be prepared by any method known or hereafter developed in the art. In general, such preparatory methods include the step of bringing a transformed plant into association with a diluent (e.g., a non-transformed plant), a carrier, and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit (e.g., into a pellet or block).

[0334] An immunogenic composition in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a "unit dose" is discrete amount of a composition comprising a predetermined amount of at least one plant-based product produced using a system and/or method described herein.

[0335] Relative amounts of transformed plant produced using a system and/or method described herein, a carrier, and/or any additional ingredients in a immunogenic composition can vary, depending upon the subject to be treated (e.g., species of non-human animal, age, size), target cells, diseases or disorders, and may also further depend upon the route by which the composition is to be administered.

Pharmaceutical Compositions

[0336] According to some embodiments, a composition can include a protein purified from transformed plant cells that is concentrated to a particular concentration and purity. A purified and/or concentrated protein may be combined with an additional component e.g., a pharmaceutically effective carrier or excipient into a pharmaceutical composition (e.g., a vaccine).

[0337] Pharmaceutical compositions may comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface-active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this disclosure.

[0338] In some embodiments, a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, an excipient is approved for use in humans and for veterinary use. In some embodiments, an excipient is approved by the United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.

[0339] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in pharmaceutical formulations. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/or perfuming agents can be present in the composition.

[0340] Pharmaceutical compositions may be formulated such that they are suitable for administration to a human and/or non-human animal subject. In some embodiments, a pharmaceutical composition is substantially free of either endotoxins or exotoxins. Endotoxins include pyrogens, such as lipopolysaccharide (LPS) molecules. A pharmaceutical composition may also be substantially free of inactive protein fragments. In some embodiments, a pharmaceutical composition has lower levels of pyrogens than industrial water, tap water, or distilled water. Other components of a pharmaceutical composition may be purified using methods known in the art, such as ion-exchange chromatography, ultrafiltration, or distillation. In other embodiments, the pyrogens may be inactivated or destroyed prior to administration to a subject. Raw materials for a pharmaceutical composition, such as water, buffers, salts and other chemicals may also be screened and depyrogenated. A pharmaceutical composition may be sterile, and each lot of the pharmaceutical composition may be tested for sterility. Thus, in certain embodiments the endotoxin levels in the a pharmaceutical composition fall below the levels set by the USFDA, for example 0.2 endotoxin (EU)/kg of product for an intrathecal injectable composition; 5 EU/kg of product for a non-intrathecal injectable composition, and 0.25-0.5 EU/mL for sterile water. It is preferred that a pharmaceutical composition has low or no toxicity, within a reasonable risk-benefit ratio.

[0341] The formulations suitable for introduction of a pharmaceutical composition vary according to route of administration. Formulations suitable for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, intranasal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials.

[0342] Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

[0343] Formulations suitable for oral administration of a pharmaceutical composition can include (a) liquid solutions, such as an effective amount of the polypeptides or packaged nucleic acids suspended in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, tragacanth, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art. In some embodiments, a pharmaceutical composition can be encapsulated, e.g., in liposomes, or in a formulation that provides for slow release of the active ingredient.

[0344] A pharmaceutical composition can be made into aerosol formulations (e.g., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

[0345] Suitable formulations for vaginal or rectal administration of a pharmaceutical composition can include, for example, suppositories, which consist of the pharmaceutical composition with a suppository base. Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons. In addition, it is also possible to use gelatin rectal capsules, which consist of a combination of the pharmaceutical composition with a base, including, for example, liquid triglycerides, polyethylene glycols, and paraffin hydrocarbons.

Components of Immunogenic Compositions

[0346] In certain embodiments, immunogenic compositions, including e.g, one or more transformed plants or a pharmaceutical composition comprising an antigen purified from transformed plant cells, may be formulated as described above and/or additionally with one or more additional components. In some embodiments an additional component may be or comprise one or more of the following: an adjuvant, stabilizer, buffer, surfactant, controlled release component, salt, preservative, and an antibody specific to said antigen.

Adjuvants

[0347] In some embodiments, an immunogenic composition and/or a transformed plant can include or be administered with an adjuvant. In some embodiments where the immunogenic composition comprises one or more transformed plants, the transformed plant cells, containing lignins and HSPs (heat shock proteins) can act as an adjuvant in a subject (e.g., a non-human animal) being administered the immunogenic composition. For example, plant species such as sorghum and millet contain high quantities in saponins, and can act as an adjuvant in a subject being administered an immunogenic composition comprising transformed sorghum or millet.

[0348] In some embodiments, immunogenic compositions may additionally include or be administered with a biological adjuvant. Examples of biological adjuvants can include cholera toxin subunit B (CTB), hepatitis B virus core antigen (HBcAg), Escherichia coli heat labile enterotoxin subunit B (LTB), and monophosphoryl lipid A.

[0349] In some embodiments, an adjuvant can include inorganic adjuvants. Examples of inorganic adjuvants include alum salts such as aluminum phosphate, amorphous aluminum hydroxyphosphate sulfate, and aluminum hydroxide.

[0350] In some embodiments, an adjuvant can include a saponin. Typically, a saponin is a triterpene glycoside, such as those isolated from the bark of the Quillaja saponaria tree. A saponin extract from a biological source can be further fractionated (e.g., by chromatography) to isolate the portions of the extract with the best adjuvant activity and with acceptable toxicity. Typical fractions of extract from Quillaja saponaria tree used as adjuvants are known as fractions A and C. An exemplary saponin adjuvant is QS-21, which is available from Antigenics. QS-21 is an oligosaccharide- conjugated small molecule. Optionally, QS-21 may be admixed with a lipid such as 3D-MPL or cholesterol.

[0351] A particular form of saponins that may be used in immunogenic compositions described herein is immunostimulating complexes (ISCOMs). ISCOMs are an art-recognized class of adjuvants, that generally comprise Quillaja saponin fractions and lipids (e.g., cholesterol and phospholipids such as phosphatidyl choline).

[0352] In some embodiments, an adjuvant can include a TLR (Toll-like receptor) ligand. TLRs are proteins that may be found on leukocyte membranes, and recognize foreign antigens (including microbial antigens). An exemplary TLR ligand is IC-31, which is available from Intercell. IC31 comprises an anti-microbial peptide, KLK, and an immunostimulatory oligodeoxynucleotide, ODNla. IC31 has TLR9 agonist activity. Another example is CpG-containing DNA, and different varieties of CpG-containing DNA are available from Prizer (Coley): Vaxlmmune is CpG 7909 (a (CpG)-containing oligodeoxy-nucleotide), and Actilon is TLR9 agonist, CpG 10101 (a (CpG)-containing oligodeoxynucleotide).

[0353] In some embodiments, an immunogenic composition (e.g., a pharmaceutical composition as described above) may include adjuvants that are covalently bound to antigens (e.g., purified from transformed plants, as described above). In some embodiments, an adjuvant can be recombinantly fused with an antigen. Other exemplary adjuvants that may be covalently bound to an antigen include, without limitation, polysaccharides, synthetic peptides, lipopeptides, and nucleic acids.

[0354] In some embodiments, an adjuvant can be co-expressed and part of the exogenous nucleic acid sequence encoding an antigen. In some embodiments, an adjuvant, can be co-expressed in a transformed plant cell with any antigen of interest (e.g., using a 2A sequence).

[0355] An adjuvant can be included in or administered with an immunogenic composition alone or in combination with another adjuvant. Adjuvants may be combined to increase the magnitude of the immune response to the antigen. In some embodiments, the same adjuvant or mixture of adjuvants is present in each dose of immunogenic composition. In some embodiments, an adjuvant may be administered with the first dose of immunogenic composition and not with subsequent doses. In some embodiments, a strong adjuvant may be administered with the first dose of immunogenic composition and a weaker adjuvant or lower dose of the strong adjuvant may be administered with subsequent doses. An adjuvant can be administered before the administration of an immunogenic composition, concurrent with the administration of an immunogenic composition or after the administration of an immunogenic composition to a subject (sometimes within 1, 2, 6, or 12 hours, and sometimes within 1, 2, or 5 days). Certain adjuvants are appropriate for human patients, non-human animals, or both.

Additional components of compositions

[0356] In some embodiments, a composition, including e.g., pharmaceutical compositions, may include one or more optional additional components.

[0357] In some embodiments, a composition can include one or more stabilizers such as sugars (such as sucrose, glucose, or fructose), phosphate (such as sodium phosphate dibasic, potassium phosphate monobasic, dibasic potassium phosphate, or monosodium phosphate), glutamate (such as monosodium L-glutamate), gelatin (such as processed gelatin, hydrolyzed gelatin, or porcine gelatin), amino acids (such as arginine, asparagine, histidine, L-histidine, alanine, valine, leucine, isoleucine, serine, threonine, lysine, phenylalanine, tyrosine, and the alkyl esters thereof), inosine, or sodium borate.

[0358] In some embodiments, a composition can include one or more buffers such as a mixture of sodium bicarbonate and ascorbic acid. In some embodiments, the composition may be administered in saline, such as phosphate buffered saline (PBS), or distilled water. In certain embodiments, a composition includes one or more salts such as sodium chloride, ammonium chloride, calcium chloride, or potassium chloride. In certain embodiments, a preservative is included in the composition. In other embodiments, no preservative is used. In certain embodiments, a preservative is 2-phenoxyethanol, methyl and propyl parabens, benzyl alcohol, and/or sorbic acid.

[0359] In certain embodiments, a composition or pharmaceutical composition is a controlled- release formulation.

Administration

[0360] Various methods of administering a transformed plant and/or a particular composition (e.g., a plant-based vaccine) to a subject, (e.g., a non-human animal such as a ruminant livestock) can be used.

Routes of administration

[0361] In some embodiments, a transformed plant (e.g, a plant expressing an exogenous nucleic acid sequence encoding a protein of interest) or a composition (e.g, a plant-based vaccine) is fed to a non-human animal (e.g., a livestock animal).

[0362] In some embodiments, compositions herein can be delivered by administration to an individual, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, intradermal, subcutaneous, transdermal, subdermal, intracranial, intranasal, mucosal, anal, vaginal, oral, sublingual, buccal route or they can be inhaled) or they can be administered by topical application.

[0363] In some embodiments, a composition can be administered via the intramuscular route. Typically, in this route, the vaccine is injected into an accessible area of muscle tissue. Intramuscular injections are, in some embodiments, given in the deltoid, vastus lateralis, ventrogluteal or dorsogluteal muscles. The injection is typically given at an approximately 90° angle to the surface of the skin, so the vaccine penetrates the muscle.

[0364] A composition may also be administered subcutaneously. The injection is typically given at a 45° angle to the surface of the skin, so the vaccine is administered to the subcutis and not the muscle.

[0365] In some embodiments, a composition is administered intradermally. Intradermal administration is similar to subcutaneous administration, but the injection is not as deep and the target skin layer is the dermis. The injection is typically given at a 10-15° angle to the surface of the skin, so the vaccine is delivered just beneath the epidermis.

Timins of Administration

[0366] In some embodiments, a transformed plant is harvested and included in a formulation or feed composition before administration. In some embodiments, a transformed plant may be produced to stably express a protein of interest, and is then harvested and further cultivated in order to generate progeny expressing the protein of interest.

[0367] In some embodiments, a non-human animal self-administers a transformed plant and/or composition, e.g., is subject to grazing the transformed plant and/or composition. In some embodiments, where a transformed plant is transiently expressing a protein of interest, expression of the protein sequence may be tested before administration.

[0368] In some embodiments, administration may be or comprise one or more doses of a transformed plant and/or composition. By way of specific example, a non-human animal may be administered (e.g., fed) the transformed plant multiple time over an extended period of time. In some embodiments, an extended period of time may be a period of time that is greater than 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hour, or 1, 2, 3, 4, 5, 6, or 7 days. In some embodiments, administration of the transformed plant and/or composition occurs over a period of 1, 2, 3, 4, 5, 6, 7 days, or more.

[0369] In some embodiments, a non-human animal is administered (e.g., fed) a transformed plant and/or composition over a period time of greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks (e.g., consecutive weeks). In some embodiments, a non-human animal is administered (e.g. fed) a transformed plant and/or composition hourly, daily, multiple times a day (e.g., 2-4), weekly, monthly, or yearly. In some embodiments, a non-human animal is administered a transformed plant and/or composition for 1 or 2 days per week. In some embodiments, a non-human animal is administered (e.g. fed) a transformed plant and/or composition at least 1, 2, 3, 4, 5, 6, or 7 days per month (e.g., consecutive days). In some embodiments, only one dose of the transformed plant and/or composition (e.g., plant-based vaccine) is administered to achieve the results described above. In other embodiments, following an initial dosing, subjects receive one or more additional doses, for a total of two, three, four or five doses. A second or additional dose may be administered, for example, about 1 month, 2 months, 4 months, 6 months, or 12 months after the initial dose, for example, one dosing regimen can involve administration at day 0, between 0.5-2 months, and between 4-8 months. It may be advantageous to administer split doses of a composition by the same or different routes.

[0370] In some embodiments, a non-human animal is administered (e.g., fed) a transformed plant and/or composition continuously (e.g., allowed to graze continually). In some embodiments, a non-human animal is (e.g., fed) a transformed plant and/or composition continuously for at least 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hour, or 1, 2, 3, 4, 5, 6, or 7 days. In some embodiments, a non- human animal is (e.g., fed) a transformed plant and/or composition continuously for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks. As used herein, the term “continuously” means each meal of a particular hour, day, or week.

[0371] In some embodiments, a dose is administered (i.e., fed) to a non-human animal in a specified amount of feed or a non-human animal is allowed to feed for a specified period of time (i.e., “pulse” feeding). In some embodiments, a pulse feeding regimen includes weekly one-day pulses (e.g., at day 0, day 7, and day 14).

[0372] In some embodiments, a treatment regimen comprises a first dose of transformed plant and/or composition (e.g., a plant-based vaccine) followed by a second, third or fourth dose. In some embodiments, a first dose of composition comprises a composition that contains one or more proteins of interest, or nucleic acids encoding one or more proteins of interest, or a combination of one or more proteins of interest and nucleic acids encoding the same or other proteins of interest. In some embodiments, a dose is formulated with the same proteins of interest, nucleic acids encoding the same, or a combination as the first dose. In some embodiments, a second or additional dose is formulated with different proteins of interest, nucleic acids encoding the same, or a combination with different proteins from the first dose. In some embodiments, an adjuvant is delivered concurrently or sequentially with one or more doses of transformed plant and/or composition (e.g., a plant-based vaccine).

Dosins

[0373] In some embodiments, the appropriate amount of protein to be delivered will depend on the age, weight, and health (e.g., immunocompromised status) of a subject (e.g., a non-human animal such as a ruminant livestock).

[0374] Compositions as described herein may take on a variety of dosage forms. In certain embodiments, the composition is provided in solid or powdered (e.g., lyophilized) form; it also may be provided in solution form. In certain embodiments, a dosage form is provided as a dose of lyophilized composition and at least one separate sterile container of diluent.

[0375] In some embodiments, a dose of composition is calculated based on the amount of exogenous protein desired to be delivered to a subject (i.e., a non-human animal). In some embodiments, a protein is formulated in an amount of 1 μmol per dose. In some embodiments, the protein is delivered at a dose ranging from 10 nmol to 100 nmol per dose. The appropriate amount of protein to be delivered may be determined by one of skill in the art. In some embodiments, the appropriate amount of protein to be delivered will depend on the age, weight, and health (e.g., immunocompromised status), and species of a non-human animal subject.

[0376] Compositions disclosed herein (e.g., immunogenic compositions) are, in some embodiments, administered in amounts sufficient to elicit production of antibodies as part of an immunogenic response. In some embodiments, a composition may be formulated to contain 5 μg /0.5 ml or an amount ranging from 10 μg /1 ml to 200 μg /1 ml of an antigen. In other embodiments, a composition may comprise a combination of antigens. A plurality of antigens may each be the same concentration, or may be different concentrations. In some embodiments, immunogenic compositions formulated as plant- based vaccines will include a higher amount and/or concentration of antigen than an immunogenic composition formulated as a conventional vaccine or pharmaceutical composition. In some embodiments, immunogenic compositions formulated as plant-based vaccines will include at least 2X, 3X, 4X, or 5X the amount and/or concentration of antigen than an immunogenic composition formulated as a conventional vaccine or pharmaceutical composition. In some embodiments, the composition may be formulated as a ration of feed to be administered (i.e., fed) to a non-human animal. In some embodiments, the antigen(s) concentration to be included in the ration is based on antigen concentration as a percentage of total soluble protein in the ration. In some embodiments, a ration or composition includes an amount of antigen that is at least about 0.1% of the total soluble protein in the ration or composition. In some embodiments, a ration or composition includes an amount of antigen that is at least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1,0%, 2.0%, 3.0%, 4.0%, or 5.0% or more of the total soluble protein in the ration or composition. In some embodiments, the amount of antigen in a ration or composition is within the range of about 0.5% to about 2% of the total soluble protein in the ration or composition.

[0377] In some embodiments, an immunogenic composition will be administered in a dose escalation manner, such that successive administrations of the immunogenic composition contain a higher concentration of composition than previous administrations. In some embodiments, an immunogenic composition will be administered in a manner such that successive administrations of an immunogenic composition contain a lower concentration of composition than previous administrations.

[0378] In some embodiments, only one dose (administration) of an immunogenic composition is administered. In other embodiments, the immunogenic composition is administered in multiple doses and/or multiple times. In various embodiments, the immunogenic composition is administered once, twice, three times, or more than three times. The number of doses administered to a subject can be dependent upon, for example, the antigen in the immunogenic composition, the extent of the disease or the expected exposure to the disease, and the response of a subject (e.g., a non-human animal) to the composition.

[0379] Other features of the invention will become apparent in the course of the following descriptions of exemplary embodiments, which are given for illustration of the invention and are not intended to be limiting thereof. EXAMPLES

[0380] The following examples disclose exemplary methods of transforming plant cells (e.g., from sorghum, millet, and alfalfa plant species) with a nucleic acid material (e.g., an expression cassette) including an exogenous nucleic acid sequence encoding one or more exogenous proteins

Example 1: Nucleic Acid Constructs targeting 16S Ribosomal gene DNA

[0381] The examples below utilize two immunodominant regions of leukotoxin, namely PL1 and PL4 as an exemplary exogenous nucleic acid sequence, to develop selected crop species (i.e., sorghum, millet and alfalfa) that are able to synthesize these proteins. For the purposes of this example, the chloroplast of sorghum (Sorghum bicolor (L.) Moench, Genbank: NC 008602.1), the chloroplast of millet (Panicum miliaceum L., GenBank: KU343177.1), and the chloroplast of alfalfa (Medicago sativa plastid genome NC 042841.1) were selected as host plastomes. Immunogenic proteins to be produced by the plant plastomes include PL1 and PL4. Described below are the details of the DNA construct in order express an antigen in each the host species’ plastome.

Targeting Sequences

[0382] In order to provide a successfully transformed and productive plant, several variables must be considered. By way of non-limiting example, selection of a proper chromosomal location is critical, inter alia, to ensure normal gene expression occurs with minimal or no disruption, and also to ensure that desired levels of the protein(s) encoded by the exogenous nucleic acid are produced.

[0383] In this example, the targeting sequences are localized to the 16S ribosomal gene DNA and contain certain mutations that confer antibiotic resistance, which is useful for the selection of successfully transformed plants. FIG. 4 shows an exemplary targeting strategy used in this example for integrating an exogenous nucleic acid material into a host plant cell chloroplast genome using a first (5’) targeting sequence that includes 16S ribosomal gene sequence DNA. The sorghum chloroplast (Genbank: NC_008602.1, Saski et al., 2007), and specifically the 16S ribosomal gene was analyzed. Chloroplast bases 95,658-97,657 (including bases 96,034-97,525 of 16S ribosomal DNA sequence) and 97,658-99,657 were designated as the first (5’) targeting sequence and the second (3’) targeting sequence for the nucleic acid construct, respectively, such that the construct will be inserted at coordinates 97,657-97,658 of the Sorghum bicolor chloroplast (Genbank NC 008602.1). Additionally, mutations were introduced at the following locations: a C to A nucleotide substitution at position 96,895 and an A to a C nucleotide substitution at position 97,172 of the sorghum chloroplast genome sequence (GenBank Accession No. NC 008602.1) to confer antibiotic resistance to streptomycin and spectinomycin, respectively.

[0384] The millet chloroplast (GenBank: KU343177.1) was analyzed and specifically the 16S ribosomal gene. Millet chloroplast bases 94,158-96,157 (including bases 94,534-96,025 of 16S ribosomal sequence), and 96,158-98,157 were designated as the first (5’) targeting sequence and second (3’) targeting sequence, respectively, such that the construct will be inserted at coordinates 96,157 and 96,158 of the Panicum miliaceum plastid genome (Genbank KU343177.1). Additionally, mutations were introduced at the following locations: a C to A nucleotide substitution at position 95,395 and an A to a C nucleotide substitution at position 95,672 of the millet plastid genome sequence (GenBank Accession No. KU343177.1) to confer antibiotic resistance to streptomycin and spectinomycin, respectively.

[0385] The alfalfa plastid genome (Medicago sativa GenBank: NC_042841.1) was analyzed and specifically the 16S ribosomal gene. Alfalfa plastid genome bases 97,807-99,806 (including 16S ribosomal DNA at positions 98,158-99,649) and 99,807-101,806 were designated as the first (5’) targeting sequence and second (3’) targeting sequence, respectively, such that the construct will be inserted at coordinates 99,806-99,807 of the Medicago sativa voucher I.S. Choi MD003 plastid, complete genome (NC 042841.1. Additionally, mutations were introduced at the following locations: a C to A nucleotide substitution at position 99,019 and an A to a C nucleotide substitution at position 99,297 of the alfalfa plastid genome sequence (GenBank Accession No. NC_042841.1) to confer antibiotic resistance to streptomycin and spectinomycin, respectively.

[0386] These flanking regions facilitate the homologous recombination to maneuver the exogenous nucleic acid sequence into the chloroplast genome.

Exogenous Nucleic Acid Sequence

[0387] As is known in the art, fusobacterium infection in ruminant livestock can lead to a number of symptoms, including ruminal acidosis, rumenitis, and liver abscess, and presents a costly problem for the livestock industry. Immunodominant Fusobacterium leukotoxin (Genbank: DQ672338) regions PL1 and PL4 (SEQ ID NOs: 51 (corresponding to Genbank DQ672338.1 : 1-498 and 53 corresponding to Genbank DQ672338.1:5641 -6606, respectively) were selected as the exogenous nucleic acid sequence encoding PL1 and PL4 antigens of interest (SEQ ID NOs: 52 and 54, respectively). PL1 and PL4 DNA sequences were translated separately in silico and their respective sequences were amended to include in-frame start (ATG) and stop (TAA) codons, to ensure correct genetic transcription of these sequences.

Selection Sequence

[0388] In order to assess whether and how plants are transformed, as well as to assess the level of expression of the exogenous nucleic acid sequence, a selection sequence is used in this example. For ease of assessment, a fluorescent selection sequence is used in this example, though this need not always be the case. Specifically, the fluorescent proteins selected to discretely confirm the expression the immunogenic protein operons is: Red fluorescence protein (DsRED, GenBank: KY426960.1 or SEQ ID NO: 7). Additionally, in this example, a His Tag was utilized for detection and purification purposes (HIS-Tag CATCACCATCACCATCAC; SEQ ID NO: 8).

Enhancer Sequence

[0389] In order to increase transcription of the exogenous nucleic acid sequence, certain enhancer sequences were selected. Enhancer sequences are positioned relative to a promoter sequence and the antigen of interest to be expressed (in this example, PL1+PL4). Each antigen to be expressed will be equipped with its own leader sequence:

• Enhancer: G10L; T7phage gene10 leader sequence; (GenBank:EU520588.1; SEQ ID NO: 2)

• Enhancer: LrbcL; leader from EU224430.1 (SEQ ID NO: 4)

• Enhancer: LatpB; leaderfromEU224425.1 (SEQ ID NO: 6) Promoter Sequence

[0390] In addition to an enhancer sequence, the DNA constructs used in this example include a promoter sequence in proximity (upstream) of the 5’ end of the exogenous nucleic acid sequence, to initiate transcription of the antigen (in this example, PL1+PL4). A single constitutively expressed rRNA promoter Prrn (GenBank: MF580999.1 ; SEQ ID NO. 1 ) was selected.

Termination Sequence

[0391] In the DNA construct of this example, a single terminator sequence was selected to cease transcription of the transgenic operon and to be placed within the DNA construct in a position relative to the exogenous nucleic acid sequence encoding the antigen (at the 3’ end of the sequence encoding PL1, PL4). Specifically, the tobacco gene rpsl6 “Trps16” (GenBank: MF580999.1; SEQ ID NO: 9) was selected as it has been successfully used in many chloroplast transformation vectors.

Sorshum Nucleic Acid Constructs

[0392] The elements described above in this example were arranged and incorporated to form a DNA expression cassette to be introduced (e.g., by transformation) into a host plant (in this particular example, the host plants are sorghum).

[0393] In this example, and in accordance with the above, the following sorghum-targeted expression cassette was made:

Millet Nucleic Acid Constructs

[0394] The elements described above in this example were arranged and incorporated to form a DNA construct to be introduced (e.g., by transformation) into a host plant (in this particular example, the host plants are millet). [0395] In this example, and in accordance with the above, a millet-targeted construct was made:

Example 2: Methods of Generating Nucleic Acid Constructs

[0396] In order to obtain sufficient copies of the DNA construct to be introduced into a host plant genome, the DNA expression cassettes in Example 1 were obtained and then copied using standard PCR reactions, and purified from the product and formulated to be delivered to a host plant genome.

[0397] The nucleic acid constructs were generated within the pMX vector plasmid through Invitrogen’s GeneArt Gene Synthesis (www.thermofisher.com/ca/en/home/life-science/cloning/gene- synthesis/geneart-gene-synthesis) service. The dry DNAs supplied by the manufacturer were resuspended to 100 ng DNA / μL 10mM Tris, 1 mM EDTA pH 8.0.

[0398] An abundance of copies of each DNA construct were generated by polymerase chain reaction (PCR) using specific forward and reverse primers (synthesized by Eurofins Genomics (Brussels, Belgium)) aligned to the 5’ end of the respective second (3’) targeting sequence and the 3’ of the first (5’) targeting sequence, respectively. The reaction components were assembled as described below:

[0399] Each PCR reaction targeting templates >1 kb were thermocycled as described below in a BioRad CFX96 Optical Thermocycler (BioRad):

[0400] The resulting reactions were size fractionated in 2% agarose and amplicons of appropriate sizes were excised and cleaned using a QIAquick Gel Extraction Kit (Qiagen, Venlo, Netherlands) according to manufacturer’s instructions. Cleaned amplicons were sequence confirmed using the Applied Biosystems (AB) 3500XL capillary sequencer and analyzed using Sequence Analysis v5.4 software (ThermoFisher Scientific, Waltham, Massachusetts). At least 10 μg of each sequence- confirmed amplicons were stored at 4°C until processing.

Example 3: Transformation of Chloroplasts using Single-Walled Carbon Nanotubes (SWCNTs) [0401] Once the DNA constructs were isolated and purified in amounts of, for example, about 100 ng of each sequence, the DNA constructs described above, were formulated with a carrier. The carrier, in this example, aids in the efficiency and accuracy of the transformation into the host plant cell.

[0402] In this example, single-walled carbon nanotubes (SWCNTs, Sigma) were used to guide the construct to chloroplasts of sorghum, millet, and alfalfa leaves.

CS-PEG-SWCNT preparation

[0403] Low molecular weight deacetylated chitosan (0.03 g) was dissolved in 30 mL 0.3% acetic acid and water solution in a 50 mL glass beaker, and stirred with a magnetic stir bar for 60 seconds. High-pressure carbon monoxide (HiPco)-synthesized single- walled carbon nanotubes (SWCNTs; Nanoshel (Punjab, India), 1 - 2 nm diameter and 3 - 8 um in length) (0.015 g) were then mixed into the chitosan & acetic acid solution to achieve 2: 1 chitosan to SWNCT ratio. [0404] In this example, the chitosan was deacetlyated. 0.3g chitosan in 30ml of 0.3% acetic acid was found to be the best ratio to obtain the ideal consistency. Multiple amounts were tested to determine the correct amount of chitosan to dissolve into the 0.3% acetic acid. It was found that lower volumes of acetic acid to higher amounts chitosan resulted in a highly viscous solution or the chitosan would not dissolve. Using 0.3g chitosan resulted in the weight of HiPCO SWNTs being 0.015g. Both chitosan and HiPCO SWNTs have quite the electrostatic charge and despite using the static gun on these products, loss due to static attraction occurred when using the weigh boat. However, loss on the weighboat not measurable.

[0405] This mixture was probe sonicated for 30 minutes using a 6 mm probe tip at 40% amplitude and subsequently dialyzed overnight using a 5 mL 100 kDa dialysis kit (SpectrumLabs), changing out water in 12 hour intervals.

[0406] The solution was probe sonicated on ice in the fume hood for 30 mins using a 6mm probe tip at 40% amplitude. The end-product being a black solution (CS-SWNTs). Due to reasons such as temperature control, sonication was kept to 30mins, which is shorter than other methods previously described (see Kwak et al. 2019).

[0407] Dialyzed mixtures were centrifuged for 1.5 hours at 16,100 × g at room temperature twice by placing the mixtures in fresh Eppendorf tubes each time to remove unbound chitosan and SWCNT aggregates.

[0408] The chitosan complexed SWCNTs were PEGylated by mixing 0.1 equivalent PEG^5K and allowing the mixture to stand at room temperature for 6 hours: about 0.005g (0.1 equivalent is 0.0045g) was used and added carefully to the tube containing the CS-SWNTs.

[0409] PEG^5K -CS-SWNTS were dialyzed using two 5mL volume 100kDa dialysis kits (SpectrumLabs) overnight, changing the water three times, to remove free PEG chains, followed by centrifugation at 16,100 × g for 1.5 hours and transferring the supernatant to one 15 mL falcon tube.

[0410] Determining the concentration PEG^5K -CS-SWNTs is required prior to adding DNA. This will enable to us to create different mass ratios of DNA: PEG^5K -CS-SWNTs. The concentration of PEG5K-CS-SWCNTs were performed by measuring the spectral absorbance of the solution and using the Beer’s law equation:

A = € × C × 1 where A is the absorbance of carbon SWNTs (632nm), 1 is the path length, (standard 1 cm on spectrometer), € is the extinction coefficient for CS-SWNTs (0.036 L mg^-1cm^-1; Demerier et al., 2019), and C is the unknown concentration. Nanoparticle surface charge was characterized by Phase Analysis Light Scattering Zeta Potential by NanoComposix using a Zetasizer Ver. 7.02 (Malvern Instruments).

[0411] Using a zeta potential machine, the zeta potential was. Expected zeta potential for the PEG^5K-CS-SWNT based on Kwak et al. 2019 is 32.2mV. The resulting mixture was stored in the fridge (for up to 1 month). Not all preparations of the PEG^5K-CS-SWNTs were sent for analysis. A preparation was sent to nanoComposix. This preparation had a zeta potential of 19.9(±9.83) mV. FIG.

6 shows size of exemplary individual carbon nanoparticles to have an average diameter between 1.0 to 2.0 um confirmed using by TEM.

DNA-CS-PEG^5K -SWNT preparation

[0412] PEG^5K -CS-SWNT solutions were diluted in MES Buffer to achieve a final concentration of 2.5 mg/L and aliquoted in 1 mL volumes that will be subsequently conjugated to construct DNA.

[0413] Construct DNA was amplified from plasmid templates by PCR, cleaned with Exo-S AP, and blended with PEG^5K -CS-SWNTs in 2-(N-Morpholino) ethanesulfonic acid (MES) to achieve a ratio of 1 :3 DNA:SWCNTs. DNA-SWCTNs were shaken for 30 minutes at 500 rpm to allow conjugation. Plants were treated with 80 ng DNA.

Example:

[0414] Concentration of SWNTs is 2.5mg/L = 2.5ng/μl a) 2.5ng/ul ÷ 3 = 0.833ng/μl b) Minimum treatment of plant is 80ng so x100 = 83.3ng/100μl

[0415] PCR Product concentration is 4ng/μl. For 1ml of DNA-PEG-CS-SWNTs:

4ng/μl × X = 83.3ng/100μl (1000μl)

X = 800÷4= 208.25μl of PCR Product [0416] The appropriate volume (e.g., 208.25 pl) of PCR Product to the MES + PEG-CS- SWNTs labeled for that DNA treatment was added. Double stranded linear DNA was used, while other groups have previously used plasmid DNA (see Kwak et. a. (2019)).

[0417] One preparation that was sent to nanoComposix for analysis had zeta potential of 13.5 (±.4.36) mV, which is 13.1 mV lower than the expected and lower than the threshold of >25mV to cross the chloroplast (Kwak et al., 2019).

[0418] The appropriate amount for treating the number of plants selected, 100-300ul per plant was reserved. Using needle syringes, 100 uL of DNA-SWCNTs MES was infused into the plants via the stem. When infusing the needle will go inside the stem but not through the stem. The needle method was necessary due to the waxy nature of sorghum and millet leaves. It was not possible to infuse with a needless syringe.

[0419] As described above, the methods including conjugating the SWCNTs with construct DNA, and in this example, the SWCNTs are conjugated to 100% construct DNA and not a plasmid containing the DNA construct (as described in Kwak et al. 2019). Additionally, the methods of preparing the DNA-SWCNT complexes were shown to result in the complex having a zeta potential of 13.5mV. Prior groups have noted that the zeta potential should be approximately 26.6mV, to have the correct suspension and charge for crossing the plant cell wall and the membrane of the chloroplast (see Kwak et al., 2019).

[0420] The present example seeks to demonstrate nanotube carries that are differently dimensioned than prior groups and that contain different (i.e., lower) zeta potentials, are able to carry a DNA construct to the chloroplast and successfully integrate therein in millet, sorghum, and alfalfa.

[0421] Confirmation of DNA adsorption to SWCNTs will be conducted by comparing the infrared fluorescence of the DNA-conjugated SWCNTs to a control sample, being the SWCNTs dialyzed in the absence of the DNA constructs. DNA adsorption to the SWCNTs is observed by higher infrared fluorescence than the control sample. DNA-CS-PEG-SWCNT transfection

[0422] Other methods of infusion into the host plant were also tested in this example. Mature leaves of sorghum plant were infused via evaporation of the nucleic acid material (DNA-CS-PEG- SWCNT complex) on the leaf adaxial surface. Observation of cyan fluorescence within the surface of leaf exposed to DNA-CS-PEG-SWCNT complex. No fluorescence was observed on areas exposed to CS-PEG-SWCNT without DNA and areas exposed to water only (see FIG. 7).

[0423] After 24 - 72 hours of homologous recombination, the success of transformation will be evaluated using: o Level of transgenic gene expression will be evaluated using quantitative real-time PCR, by extracting total RNA using the RNeasy plant mini kit (Qiagen), iScript cDNA synthesis kit (Bio-Rad) and Powerup SyBR green master mix (Applied Biosystems), and comparing quantification thresholds between reactions with gene specific primers to reactions with ‘housekeeping’ gene(s). o Fluorescence will be observed using a confocal microscope by excising a small section of the infiltrated leaf, placing it between glass slide and coverslip, and exposing slides to appropriate excitation wavelengths to observe the fluorescence of the selection sequence (YFP, DsRED, and CFP, depending on the construct).

[0424] The amount of protein production (encoded by the transformed expression cassette) from the transformed plant will be quantified using ELISA. Methods for quantifying the amount of antigen produced from the transformed plant include the following :

1. Fresh leaf tissue (100 mg) will be ground by motor and pistil;

2. Ground tissues will be resuspended in 500 μL of extraction buffer (100 mM NaH₂PO₄, 8 M Urea, and 0.5 M NaCl; pH 8);

3. A standard curve (1-10 pg) of pure recombinant PL1 and PL4 antigens, provided by ThermoFisher Scientific (www.thermofisher.com/ca/en/home/life- science/antibodies/primary-antibodies/polyclonal-antibodies), diluted in carbonate buffer (pH 9.6), will also be plated;

4. Samples will be placed in a microfuge tube and centrifuged at 14,000 rpm at 4 °C for 10 minutes. 5. Protein extractions (diluted in carbonate buffer) will be incubated in select ELISA plate wells overnight at 4 °C;

6. Wash plate with PBST (3.2 mM Na₂HPO₄, 0.5 mM KH₂PO₄, 1.3 mM KCl, 135 mM NaCl, 0.05% Tween 20, pH 7.4.);

7. Block plate with 2% fat-free dry milk in carbonate buffer for 60 minutes;

8. Wash plate once with PBST;

9. Incubate plate with anti-PLl and -PL4 polyclonal antibodies (1 :5002% fat-free dry milk) for 60 minutes;

10. Wash with PBST;

11. Incubate plate with secondary monoclonal antibodies (1: 100002% fat- free dry milk) for 60 minutes;

12. Add 0.3 mg/L 2-20 Azino-bis-3 etilbenztiasoline-6-sulphuric acid (ABTS; Sigma, Missouri, USA) and 0.1 M citric acid, pH 4.35;

13. Using a Multiskan Ascent (Thermo Scientific, Massachusetts, USA) microplate reader, record the optical density at 405 nm; and

14. Expressed PL1 and PL4 will be quantified by comparing OD₄₀₅ in 100 mg of total protein to OD₄₀₅ of standard curve.

Confirmation of Transformation by Sequencing of PCR products

[0425] Successful integration of the DNA-carrier (i.e., DNA-SWCNT) complexes into host cell chloroplasts was evidenced by the resulting PCR products.

[0426] FIG. 8 shows PCR confirmation of the integration of nucleic acid material into the plastid genome in millet. DNA was extracted from treated millet microcalli, and long PCR was performed using integrated forward/reverse primer pairs (-one inside the native plastid genome, and one within various parts of the exogenous nucleic acid sequence (strategy shown in Panel B). The primer sequences used are as follows: outside Left FP1 : TGTAAAACGACGGCCAGT CGA CTC GAC CCG TGC (SEQ ID NO: 103), Rpl CCA TTG ACA AAG TTA AAA AGA TTA TTT ACC (SEQ ID NO: 104), Rp2: ACT TTA TCT ACT TGC CCT TGA GTA G (SEQ ID NO: 105), Rp 3: CGCCCTCGAACTTCACC (SEQ ID NO: 106), Fp2 GTT TTA ATA GAT TTG CTT TAA CAG AAA ATA TAG C (SEQ ID NO: 107), Fp3 GGA TCT ACA AAA GCA TAT GTA AAA GAT TC

(A) shows that all bands were observed (left), and matched the expected band sizes when properly integrated (right).

[0427] Figure 9 shows PCR confirmation of the integration of nucleic acid material into the plastid genome in sudangrass (sorghum). DNA was extracted from treated sudangrass calli, and long PCR was performed using integrated forward/reverse primer pairs - one inside the native plastid genome, and one within various parts of the exogenous nucleic acid sequence. All bands observed (left), matched the expected band sizes if integrated (right). The entirety of the sequence was further validated via sequencing from the excised bands of the gel.

[0428] The entirety of the sequence was further validated via sequencing from the excised bands of the gel.

[0429] Sequencing of the millet plastid genome revealed that the millet plant contained a sequence with high similarity to the core millet sequence (as shown in SEQ ID NO: 21, 135, or 95,395- 95,672 of the millet plastid genome sequence GenBank Accession No. KU343177.1), as shown in the alignment below:

[0430] Sequencing of the sorghum plastid genome revealed that the sorghum plant contained a sequence with high similarity to the core sorghum sequence (as shown in SEQ ID NO: 20 or 96,895 - 97,172 of the sorghum core plastid genome sequence GenBank Accession No. NC_008602.1), as shown in the alignment below:

[0431] When sequenced, the both the transformed sorghum and millet plants contained the complete integrated sequence encoding the terminator rpsl6 (SEQ ID 9), as shown in the alignment below:

[0432] Sequencing of the millet plastid genome revealed that the millet plant contained a sequence with fragments of leukotoxin A (ItkA) sequence according to Genbank: DQ672338.1, as shown in the alignment below:



Example 4: Exemplary Delivery Methods using CTPs and CPPs

[0433] Once the DNA constructs are isolated and purified in amounts of, for example, about 100 ng of each sequence, the expression cassettes (each of the three described above), are formulated with a carrier. The carrier, in this example, aids in the efficiency and accuracy of the transformation into the host plant cell.

[0434] The aim of this experiment is to develop a set of peptides that alone or together have the capacity to bind to (e.g., complex with) chloroplast transformation DNA expression cassettes, penetrate plant cells, and deliver DNAs to the chloroplast. Described below are the methods by which chloroplast targeting peptides (CTPs) for sorghum, millet, and Medicago species were identified. Another aim of this experiment is to test various combinations of identified CTPs and/or cellpenetrating proteins (CPPs) in their ability to bring DNA expression cassettes to the specific locations within the plant chloroplast genome of particular species.

[0435] In this example, CTPs were identified in sorghum and millet species and the CTPs are complexed with the expression cassettes described in Example 1. Additionally, the CTP-DNA complexes are coupled with a CPP to aid in targeted delivery to a site within the plant chloroplast genome.

[0436] In this example, the CTP protein identified and used in subsequent experiments is an outer envelope membrane protein, molecular weight of 34 d (OEP34).

Materials and Methods

Identification of the CTPs for sorghum, millet, and alfalfa

[0437] The OEP34 of Arabidopsis thaliana (AtEOP34), Pisum sativum (PsOEP34), Sorghum bicolor (SbEOP34), Panicum miliaceum (PmOEP34), and Medicago truncatula (MtOEP34) have the GenBank accessions NP_850768.1 , Q41009.1 , XP_021306533.1, RLN39229.1 , XP_003624825.1 , respectively. Below shows an alignment of a portion of the C-terminal of each OEP34 sequence, with the hydrophobic cores (identified by Li and Chen (1997)) underlined. The sequence of AtOEP34 used by Yoshizumi et al., (2018) and Thagun et al., (2019) that successfully targeted the peptide the Arabidopsis chloroplast is bolded.

[0438] Based on an alignment of the OEP34 proteins across species, the hydrophobic core sequences of sorghum/millet (which are identical) and Medicago truncatula were predicted, and are shown underlined in subsequent rows.

[0439] As shown by the above alignment, the OEP34 hydophobic core sequence are specific to a particular species and vary in sequence. As there is variability in sequence, this experiment aims to test various OEP34 CTPs for chloroplast delivery of a DNA expression cassette in their respective plant species.

[0440] Additionally, based on the sequence analysis among other species of plants, no corresponding OEP34 protein was identified in Medicago sativum. As such, this experiment additionally examined whether the OEP34 chloroplast targeting sequence identified in the model legume Medicago truncatula could be used for chloroplast delivery of an expression cassette in Medicago sativum.

[0441] The two new CTPs identified, ILAVEYFLVV and LFALEFLLIM were synthesized and tested for their ability to shuttle an expression cassette to the chloroplasts of sorghum/millet and Medicago species (e.g., M. sativa), respectively. [0442] Each CTP was linked to a DNA-binding KH9 sequence. The two peptides were synthesized by Genscript (Genscript USA, Piscataway NJ) and are shown in Table 1 below.

Table 1 .

Selection of CPP

[0443] To fully equip the DNA-KH9CTP complex with cell penetrating abilities, the complex is further complexed with a CPP. Nurimata et al., (2018) profiled 55 CPPs for their ability to traverse leaves of Arabidopsis, tobacco, tomato, and poplar, along with rice callus; however, none have been demonstrated in sorghum, millet, or alfalfa. The cationic K9 sequence is used as the CPP in this experiment because of its particularly strong cell penetrating properties in rice callus, and because of the sequence similarity, may also have cell similar cell penetrating efficiencies to the KH9 sequence. The K9 sequence (Table 2; SEQ ID NO: 30) is synthesized by Genscript so that it can be used in combination with KH9-CTP peptides.

Table 2.

Plant materials

[0444] Sorghum bicolor seeds were grown on 0.5 M × urashige and Skoog (MS) plates lacking sugar under continuous light at 22 °C.

Preparation of DNA-KH9-CTP and DNA-KH9-CTP-CPP complexes

[0445] In order to prepare DNA-KH9-CTP and DNA-KH9-CTP-CPP complexes, 1 μL of 1 mg/mL Sorghum/millet KH9-OEP34 peptide stock was added to 3 μL of a 1 mg/mL chloroplast transformation DNA stock, and subsequently diluted with autoclaved water to a 100 μL final volume. This solution was mixed by pipette and allowed to stabilize for 30 minutes at 25°C, allowing the DNA- KH9-CTP complex to form.

[0446] In order to optimize particle size and complex charge, N/P ratios (ratio of the moles of amine groups from the peptide to that of phosphate groups from the DNA) between 0.5 and 2.5 are tested based on transfection rate. Transfection rates of DNA-KH9-CTPs are further monitored upon complexing with the K9 peptide (CPP) sequence, where the CPP is added to the DNA-KH9-CTP complexes over a N/P ratio range of 0.1 - 10.

Treating seedlings with DNA-KH9-CTP and DNA-KH9-CTP-CPP complexes

[0447] 3 -day old seedlings are individually placed into 1.5 mL Eppendorf tubes containing 100 pL of DNA-KH9-CTP and DNA-KH9-CTP-CPP complexes. This tube was then placed under vacuum pressure of 20 mm/Hg for one minute. Formation of DNA-KH9-CTP-CPP complexes will be formulated Seedlings were then transferred to hormone free MS media and allowed to rest prior to nucleic acid extraction 24, 48, or 72 hours later.

Nucleic acid extraction, cDNA synthesis, and realtime PCR

[0448] In order to assess whether the DNA-KH9-CTP-CPP complexes were able to successfully transform the plant chloroplast, realtime PCR is performed using primer sequences that span sequences within the host chloroplast genome and the expression cassettes (from Example 1) in a successful target location.

[0449] Total RNA is extracted from non-treated and treated seedlings using the RNeasy plant mini kit (Qiagen) in combination with RNase-Free DNase Set (Qiagen). Complimentary DNAs (cDNAs) are generated from extracted RNAs using iScript cDNA synthesis kit (Bio-Rad). Realtime PCR reactions are prepared using the following recipe:

[0450] Primer and probe sequences for each assay set used in these experiments are documented below in Table 3:

Table 3:

[0451] Realtime PCR reactions are conducted on a BioRad CFX96 and analyzed using BioRad

Maestro analysis software (version 3.1.1517.0823) using the cycling conditions below:

References:

Mayfield, S. P., Franklin, S. E., & Lerner, R. A. (2003). Expression and assembly of a fully active antibody in algae. Proceedings of the National Academy of Sciences of the United States of America, 100(2), 438-442.

Tran M, Van C, Barrera DJ, Pettersson PL, Peinado CD, Bui J, Mayfield SP. Production of unique immunotoxin cancer therapeutics in algal chloroplasts. Proc Natl Acad Sci U S A. 2013 Jan 2;110(l):E15-22. doi: 10.1073/pnas.l214638110. Epub 2012 Dec 10. PMID: 23236148; PMCID: PMC3538218. Example 5: Nucleic Acid Constructs targeting 16S and 23S Ribosomal gene DNA

[0452] This Example demonstrates successful chloroplast transformation of a host plant with two immunodominant regions of leukotoxin, namely PL1 and PL4 and 5-enolpyruvylshikimate-3- phosphate (EPSP) synthase (EPSPS) as an exemplary exogenous nucleic acid sequence, to develop selected crop species (i.e., sorgum and millet and alfalfa) that are able to synthesize these proteins. For the purposes of this example, the chloroplast of sorghum (Sorghum bicolor (L.) Moench, Genbank: NC 008602.1), the chloroplast of millet (Panicum miliaceum L., GenBank: KU343177.1), and the chloroplast of alfalfa (Medicago sativa plastid genome NC_042841.1) were selected as host plastomes. Immunogenic proteins to be produced by the plant plastomes include PL1 and PL4. Described below are the details of the DNA construct in order express an antigen in each the host species’ plastome. Methods of transformation include utilizing carries such as CTP/CPP complexes and SWCNTs as described herein.

[0453] Two individual SNPs are contained within the flanking region of the construct containing the 16s region were inserted in order to confer antibiotic resistance to streptomycin and spectinomycin in millet and sorghum respectively. Three unique SNPs within the flanking region of the construct containing the 23 s region were inserted in order to confer antibiotic resistance to lincomycin. FIG. 5 shows an exemplary targeting strategy for integrating an exogenous nucleic acid material into a host plant cell chloroplast genome using a first (5’) targeting sequence that includes 16S ribosomal gene sequence DNA and a second (3’) targeting sequence that includes 23 S ribosomal gene sequence DNA. The aforementioned antibiotic resistances may then be used for selection of putatively transformed plant materials during subsequent regeneration.

Materials and Methods

Chloroplast transformation DNA constructs

[0454] Sorghum, and millet DNA constructs used in the Example include the following components:

[0455] Sequences for immunogenic leukotoxin expression cassettes utilized in millet and sorghum constructs in this example: (Promoter: Prrn (GenBank: MF580999.1:73-201, SEQ ID NO: 112), Enhancer: T7 phage gene 10 leader sequence (GenBank: EU520588.1:5627-5689, SEQ ID NO: 113), LktA PLl subunit (GenBank: DQ672338.1: 1-498-TAA, SEQ ID NO: 114), Enhancer: LrbcL (Genbank EU224430.1: 1456-1512, SEQ ID NO: 115), LktA PL4 subunit (GenBank: DQ672338.1: ATG-5638-6606-TAA, SEQ ID NO: 116), Enhancer: LatpB (Genbank: EU224425.1: 2006-2095, SEQ ID NO: 117), eGFP (GenBank: AAB02572.1 or GenBank: U55761.1 : 97-816, SEQ ID NO: 118); HIS- Tag: CATCACCATCACCATCAC-TAA, SEQ ID NO: 119); Terminator: TRPS16 (GenBank: MF580999.1: 1769-1918, SEQ ID NO: 120)). The full expression cassette is represented in SEQ ID NO: 111. These sequences were inserted between flanking sequences of sorghum and millet targeting sequences:

[0456] Sorghum (left flanking sequence is Genbank: NC 008602.1: 96841-99873, 96895 C>A, 97172 A>C (SEQ ID NO: 90); right flanking sequence is NC_008602.1:99874-102180, 102072 G>A, 102099 A>G, 102100 A>G (SEQ ID NO: 92)). The full Sorghum construct is represented in SEQ ID NO: 121.

[0457] Millet (left flanking sequence is Genbank: KU343177.1, 95341-98372, 95395 C>A, 95672 A>C (SEQ ID NO 85); right flanking sequence is Genbank: KU343177.1: 98373-100620, 100566 G>A, 100593 A>G, 100594 A>G (SEQ ID NO: 87)). The full millet construct is represented in SEQ ID NO: 122.

[0458] Sequences were submitted for synthetic oligo construction in plasmid pUC57 (Genscript).

[0459] Alfalfa DNA constructs used in the Example include the following components:

[0460] The sequence for 5-enolpyruvylshikimate-3 -phosphate (EPSP) synthase (EPSPS) expression (Promoter: Prrn (GenBank: MF580999.1 :73-201, SEQ ID NO: 124), Enhancer: T7 phage gene 10 leader sequence (GenBank: EU520588.1 :5627-5689, SEQ ID NO: 125), EPSP (GenBank: AY086717.1: 136-1701, SEQ ID NO: 126), Enhancer: LatpB (Genbank: EU224425.1: 2006-2095) or Enhancer LrbcL (Genbank EU224430.1: 1456-1512), eGFP (GenBank: AAB02572.1 or GenBank: U55761.1 : 97-816, SEQ ID NO: 128); HIS-Tag: CATCACCATCACCATCAC-TAA (SEQ ID NO: 129); Terminator: TRPS16 (GenBank: MF580999.1 :1769-1918, SEQ ID NO: 130)). These sequences were inserted between flanking sequences of alfalfa targeting sequences:

[0461] Alfalfa: (left flanking sequence is GenBank: KU321683.1, 33181-35942, 33201 OA, 33479 A>C (SEQ ID NO: 95); right flanking sequence is Genbank: KU321683.1, 35943-38160, 38069 G>A, 38096 A>G, 38097 A>G (SEQ ID NO: 97)). The full alfalfa construct is represented in SEQ ID NO: 131.

[0462] Sequences were submitted for synthetic oligo construction in plasmid pUC57 (Genscript).

Plant material

[0463] Seeds from Sudan grass (Sorghum × drummondii (Nees ex. Steud.) Millsp. & Chasel, cv. Sugar Grazer), millet (Panicum miliaceum (L.)), and alfalfa (Medicago sativa (L.)) were surface sterilized using warm tap water and soap for 10 minutes, then rinsed with 95% ethanol for 1 minute before being rinsed with sterile water. Seeds were transferred to a new sterile container containing 5% NaOCl + 0.1% Tween 20 (Sigma- Aldrich) and sterilized for 20 minutes with 0.1% Tween 20 (Sigma- Aldrich) under agitation using a rotary shaker. After rinsing the seeds three times in sterile water using a strainer, seeds were plated into 100 mm diameter × 60 mm height tissue culture vessels containing solidified half-MS agar (2.2 g L^-1 Murashige and Skoog salts (Sigma-Aldrich), 1% [w/v] sucrose, 1% [w/v] agar) to a density of ~30 to 100 seeds per plate (depending on plant species). Seedlings were germinated in vitro under darkness overnight using a GEN1000 TC Growth Chamber [Conviron], After one day, seedlings were transferred to a second GEN1000 chamber under light conditions set to an 18-h-light/6-h-dark photoperiod with 35 μmol m^-2 s^-1 at 22°C.

Cell penetrating and chloroplast targeting peptides

[0464] Amphipathic cell penetrating peptides (CPPs) BP100 (SEQ ID NO: 31

; Badosa et al., 2007) and HPV33L2-DD447 (SEQ ID NO: 102

Kamper et al., 2008), along with cationic CPP K9 (SEQ ID NO: were synthesized as lyophilized powder (Genscript). Putative chloroplast targeting

peptides for Sudan grass and millet (SEQ ID NO: 23 ILAVEYFLW) and alfalfa (SEQ ID NO: 25 LFALEFLLIM) were used based on sequence alignments with CTPs from Arabidopsis (Arabidopsis thaliana) and pea (Pisium sativum) and were each conjugated to an N-terminal KH9 (SEQ ID NO: 28

peptide. These peptides were then synthesized as lyophilized powder (Genscript). Formation of DNA-peptide complexes

[0465] DNA/peptide complexes were formulated using the amine: phosphate (N:P) ratio, where of moles of positively charged amine groups (NH³⁺; N) of the peptides were blended with the negatively charged phosphate groups (PO⁴; P) of the DNA molecule.

[0466] To prepare peptide-DNA complexes, an appropriate volume of CTP peptide was added to 40 ug of plasmid DNA (1.0 mg/mL), and an appropriate volume of Img/ml stock solution of cell penetration protein (either K9, HPV33L2-DD447, or BP100) to achieve a molar N/P ratio between 1.0 and 2.5 (ratio of amine groups from the peptide vs. phosphate groups from the DNA). CTP was first added to the plasmid in a 1.5 mL tube, and the volume brought to 100 μ uLsing ultrapure water. The solution was briefly mixed then left unagitated for 30 minutes. CPP was then added, briefly mixed, and left unagitated for a further 30 minutes. The treatment mix was brought to up to 1 mL before inoculating the cells.

DNA/CTP/CPP transfection

[0467] Plants were inoculated with 500 μL DNA/CTP/CPP complexes containing 40.0 μg of DNA in 4 mL round bottom tubes and allowed to transfect overnight at room temperature.

Preparation of CS-PEG-SWCNTs (Chitosan-PEG Single Walled carbon Nanotubes)

[0468] In a separate experiment, CS-PEG-SWCNTs were used as carrier of the nucleic acid material to be delivered to the host cell chloroplasts, rather than the CTP/CPPs.

[0469] Low-molecular-weight deacetylated chitosan (0.03 g) (>75% deacetlyated Sigma Adrich, C3646-25g) was dissolved into 30ml of 0.3% acetic acid solution for a period of approximately 1 minute using a magnetic stirbar and hotplate. Once the chitosan dissolved, 0.015g of HiPCO SWCNTs (Nanolntegris, HP32-162, 0.8-1.2 nm in diameter with 1 nm mean diameter and 100 nm to 1 μm initial length) in 2: 1 (w/w) ratio were added to the chitosan acetic acid solution. The mixture was tip-sonicated with a 6-mm probe tip at 40% amplitude for 40 min in an ice bath, in order to break apart the agglomerated CNT mass, which then is able to physically wrap around the nanomaterials. The resulting solution was a uniform opaque black color with no visible particulates. Using a 100 kDA dialysis kit (Float- A-Lyzer G2, 100kDa 5mL, SpectraPor), which was first prepared using 10% ethanol and soaking for 20 minutes with deionized water prior to use, the CS-SWCNT solution was dialyzed overnight with at least 3 water changes.

[0470] The next day, the dialysis solution was drawn from the tubes and transferred to 2ml Eppendorf tubes, and centrifuged for 1.5 hours at 16,000g to remove aggregates. The supernatant was transferred to new tube and spun again for 2.5 hours at 16000g to remove unbound chitosan and SWCNT aggregates. The solution was then PEGylated by mixing 0.1 equivalent of HO-PEG5k-NHS (Aldrich, JKA5078-lg) to the CS-SWCNTs. After a period of manual agitation, the solution was allowed to rest at room temperature for a minimum of 6 hours to overnight.

[0471] The following day, the reaction solution was transferred to a new set of 100 kDA dialysis kits as previously described, and allowed to dialyze overnight deionized water to remove free PEG chains, N-hydroxysuccinimide (NHS), and any remaining unbound chitosan. The water was changed at least three times over the time period. The next day the dialysis solution was drawn from the tubes and transferred to 2ml Eppendorf tubes, and centrifuged for 1.5 hours at 16,000g to remove aggregates. The supernatant was transferred to new and spun again for 2.5 hours at 16000g. All of the supernatant from the tubes was transferred to one 15mL falcon tube.

[0472] To determine the concentration of the CS-PEG SWCNTs, a cuvette was filled with the reaction solution and passed through a sprectrometer (Spectra NanoStar), using Beer’s law equation. Absorbance was recorded at 632nm and the path length was set to 1cm on the spec, an aliquot of the same was also tested for Zeta potential using a zeta-potential machine from a third party laboratory (NanoComposix, USA).

Functionalization of CS-PEG-SWNTs

[0473] Chitosan-complexed SWCNTs in MES buffer (20 mM MES, 10 mM MgC12 , pH 5.7) were mixed with plasmid DNA (pDNA) solution in different mass ratios. The mixture was then shaken at 500 r.p.m. at room temperature for 30 min to allow for full condensation of the pDNA.

[0474] FIG. 6 shows size of exemplary individual carbon nanoparticles to have an average diameter between 1.0 to 2.0 um confirmed by transmission electron microscope visualization (TEM).

[0475] Following functionalization of CS-PG-SWCNTs, the nanocarrier is complexed with the nucleic acid material through electrostatic interaction between the positive charges resulting from the amide groups on the chitosan (at a pH below~6.5 dictated by the pKA value of the molecule) and the negative charges found on the phosphate backbone of the nucleic acid (DNA) molecules.

DNA-CS-PEG-SWCNT transfection

[0476] Mature leaves of sorghum plant are infused via evaporation of the nucleic acid material (DNA-CS-PEG-SWCNT complex) on the leaf adaxial surface. Fluorescence within the surface of leaf exposed to DNA-CS-PEG-SWCNT complex is observed.

EQUIVALENTS AND SCOPE

[0477] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims:

Claims

CLAIMS We Claim:

1. A method of transforming a plant comprising: providing a nucleic acid material comprising an expression cassette comprising, in 5’ to 3’ orientation a first 5’ targeting sequence; a promoter sequence; an exogenous nucleic acid sequence; and a second 3’ targeting sequence; and wherein the first (5’) targeting sequence and/or the second (3’) targeting sequence comprises 16S ribosomal gene DNA sequence of the plant containing at least one mutation relative to the native plant 16S ribosomal gene DNA sequence; and transforming a chloroplast in a plant cell with the nucleic acid material.

2. The method of claim 1, wherein the first (5’) targeting sequence comprises 16S ribosomal gene DNA sequence of the plant containing at least one mutation relative to the native plant 16S ribosomal gene DNA sequence.

3. The method of claim 1 or claim 2, wherein the second (3’) targeting sequence comprises 23S ribosomal gene DNA sequence of the plant.

4. The method of claim 3, wherein the second (3’) targeting sequence comprises 23S ribosomal gene DNA sequence of the plant comprises at least one mutation relative to the native plant 23 S ribosomal gene DNA sequence.

5. The method of any one of claims 1-4, further comprising expressing the exogenous nucleic acid sequence, wherein the expression occurs, at least in part, in a chloroplast.

6. The method of any one of claims 1-5, wherein transformation occurs, at least in part, through homologous recombination.

7. The method of any one of claims 1 -6, wherein transforming the chloroplast comprises contacting the plant cell with the nucleic acid material.

8. The method of claim 7, wherein contacting the plant cell comprises use of biolistics or gene gun, use of chloroplast targeting sequences/peptides, cell penetrating peptides, use of a carrier such as a functionalized nanoparticle, electroporation, chemical-mediated transfection (e.g. using polyethylene gylcol), or any combination thereof.

9. The method of claim 7, wherein contacting comprises culturing the nucleic acid material in a solution comprising the plant cell (e.g., for at least 1 minute).

10. The method of claim 7, wherein contacting comprises introducing the nucleic acid material into the plant via syringe injection.

11. The method of claim 7, wherein the syringe injection comprises surface leaf infusion through a needleless syringe.

12. The method of claim 10, wherein syringe injection comprises stem injection through a needled syringe.

13. The method of claim 7, wherein the contacting comprises applying a vacuum and/or compression to the plant cell.

14. The method of any one of claims 1-13, wherein the at least one mutation comprises a mutation that confers antibiotic resistance in the plant.

15. The method of claim 14, wherein the antibiotic resistance comprises resistance to one or more of lincomycin, spectinomycin, streptomycin, kanamycin, gentamycin, neomycin, Beta lactam resistance, and any combination thereof.

16. The method of claim 16, wherein the antibiotic resistance comprises resistance to lincomycin, spectinomycin, and streptomycin.

17. The method of any one of claims 1-16, wherein the plant is millet (Panicum miliaceum), sorghum (e.g., Sorghum × drum mondi /), wheat, maize, barley, triticale, or alfalfa (Medicago saliva).

18. The method of any one of claims 1-17, wherein the 16S ribosomal gene DNA sequence of the first (5’) targeting sequence comprises a core sequence that has 90% sequence identity to the sequence corresponding to positions 95,395-95,672 of the millet plastid genome sequence (GenBank Accession No. KU343177.1 or the sequence represented SEQ ID NO: 21 or 135).

19. The method of any one of claims 1-18, wherein the 16S ribosomal gene DNA sequence of the first (5’) targeting sequence comprises a core sequence corresponding to positions 95, 395-95, 672of the millet plastid genome sequence (GenBank Accession No. KU343177.1, or the sequence represented by SEQ ID NO: 21 or 135).

20. The method of any one of claims 1-19, wherein the at least one mutation comprises a C to A nucleotide substitution at position 95,395 and/or an A to a C nucleotide substitution at position 95,672 of the millet plastid genome sequence (GenBank Accession No. KU343177.1).

21. The method of any one of claims 3-20, wherein the 23 S ribosomal gene DNA sequence of the second (3’) targeting sequence comprises a core sequence that has at least 90% identity to the sequence corresponding to positions 100,566-100,594 (GenBank Accession No. KU343177.1 or SEQ ID NO: 89).

22. The method of any one of claims 3-21, wherein the 23 S ribosomal gene DNA sequence of the second (3’) targeting sequence comprises a core sequence corresponding to positions 100,566-100,594 (GenBank Accession No. KU343177.1 or SEQ ID NO: 89).

23. The method of any one of claims 4-22, wherein the at least one mutation relative to the native plant 23 S ribosomal gene DNA sequence comprises one or more of: (i) a Gto A nucleotide substitution at position 100,566; (ii) an A to a G nucleotide substitution at position 100,593; and (iii) an A to G nucleotide substitution at position 100,594 of the millet plastid genome sequence (GenBank Accession No. KU343177.1 OR SEQ ID NO: 89).

24. The method of any one of claims 1-23, wherein the first and second targeting sequences comprise SEQ ID NO: 13, 133, or 85 and SEQ ID NO: 14 or 87, respectively.

25. The method of any one of claims 1-17, wherein the 16S ribosomal gene DNA sequence of the first (5’) targeting sequence comprises a core sequence that has at least 90% identity to the sequence corresponding to positions 96,895-97,172 of the sorghum chloroplast genome sequence (GenBank Accession No. NC_008602.1 or SEQ ID NO: 20).

26. The method of any one of claims 1-17 and 25, wherein the 16S ribosomal gene DNA sequence of the first (5’) targeting sequence comprises a core sequence corresponding to positions 96,895-97,172 of the sorghum chloroplast genome sequence (GenBank Accession No. NC 008602.1 or SEQ ID NO: 20).

27. The method of any one of claims 1-17, and 25-26, wherein the at least one mutation comprises a C to A nucleotide substitution at position 96,895 and/or an A to a C nucleotide substitution at position 97,172 of the sorghum chloroplast genome sequence (GenBank Accession No. NC 008602.1 or SEQ ID NO: 20).

28. The method of any one of claims 3-17, and 25-27, wherein the 23 S ribosomal gene DNA sequence of the second (3’) targeting sequence comprises a core sequence that has at least 90% identity to the sequence corresponding to positions 102,072-102,100 of the sorghum chloroplast genome sequence (GenBank Accession No. NC_008602.1 or SEQ ID NO: 94).

29. The method of any one of claims 3-17, and 25-28, wherein the 23 S ribosomal gene DNA sequence of the second (3’) targeting sequence comprises a core sequence corresponding to positions 102,072-102,100 of the sorghum chloroplast genome sequence (GenBank Accession No. NC_008602.1 or SEQ ID NO: 94).

30. The method of any one of claims 4-17 and 25-29, wherein the at least one mutations relative to the native plant 23 S ribosomal gene DNA sequence comprises one or more of: (i) a G to A nucleotide substitution at position 102,072; (ii) an A to a G nucleotide substitution at position 102,098; and (iii) an A to G nucleotide substitution at position 102,099 of the sorghum chloroplast genome sequence (GenBank Accession No. NC_008602.1 or SEQ ID NO: 94).

31. The method of any one of claims 1-17, and 25-30, wherein the first and second targeting sequences comprise SEQ ID NO: 11, 132, or 90 and SEQ ID NO: 12 or 92, respectively.

32. The method of any one of claims 1-17, wherein the 16S ribosomal gene DNA sequence of the first (5’) targeting sequence comprises a core sequence that has at least 90% sequence identity to the sequence corresponding to positions 99,019-99,297 of the alfalfa plastid genome sequence (GenBank Accession No. NC_042841.1, positions 38,069-38,097 of the alfalfa plastid genome sequence GenBank KU321681.1, or SEQ ID NO: 22).

33. The method of any one of claims 1-17 and 32, wherein the 16S ribosomal gene DNA sequence of the first (5’) targeting sequence comprises a core sequence corresponding to positions 99,019 - 99,297 of the alfalfa plastid genome sequence (GenBank Accession No. NC_042841.1, positions 38,069-38,097 of the alfalfa plastid genome sequence GenBank KU321681.1 or SEQ ID NO: 22).

34. The method of any one of claims 1-17 and 32-33, wherein the at least one mutation comprises a C to A nucleotide substitution at position 99,019 and/or an A to a C nucleotide substitution at position 99,297 of the alfalfa plastid genome sequence (GenBank Accession No. NC_042841.1m positions 38,069-38,097 of the alfalfa plastid genome sequence GenBank KU321681.1 or SEQ ID NO: 22).

35. The method of any one of claims 3-17 and 32-34, wherein the 23S ribosomal gene DNA sequence of the second (3’) targeting sequence comprises a core sequence that has at least 90% identity to the sequence corresponding to positions 38,069-38,097 of the alfalfa plastid genome sequence GenBank Accession No. KU321683.1 or SEQ ID NO: 99.

36. The method of any one of claims 3-17 and 32-35, wherein the 23S ribosomal gene DNA sequence of the second (3’) targeting sequence comprises a core sequence corresponding to positions 38,069-38,097 of the alfalfa plastid genome sequence GenBank Accession No. KU321683.1 or SEQ ID NO: 99.

37. The method of any one of claims 4-17 and 32-36, wherein the at least one mutation relative to the native plant 23 S ribosomal gene DNA sequence comprises one or more of: (i) a G to A nucleotide substitution at position 38,069; (ii) an A to a G nucleotide substitution at position 38,096; and (iii) an A to G nucleotide substitution at position 38,097 of the alfalfa plastid genome sequence GenBank Accession No. KU321683.1 or SEQ ID NO: 99.

38. The method of any one of claims 1-17, and 32-37, wherein the first and second targeting sequences comprise SEQ ID NO: 17, 136, or 95 and SEQ ID NO: 18 or 97, respectively.

39. The method of any one of claims 1-38, wherein a promoter sequence is selected from PpsbA, Prrn, Prna, psaA, PrbcL, CaMV35S, rbcS, and any combination thereof.

40. The method of claim 39, wherein the promoter sequence is a Prrn promoter sequence comprising SEQ ID NO: 1 or GenBank: MF580999.1.

41. The method of any one claims 1-40, wherein the nucleic acid material further comprises at least one enhancer sequence.

42. The method of claim 41, wherein the at least one enhancer sequence is selected from a sequence encoding: ggagg, rrn 5’UTR, T7genel0 5’ UTR (GenBank: EU520588.1), LrbcL 5’UTR, LatpB 5’UTR, Tobacco mosaic virus omega prime 5’UTR (GenBank: KM507060.1), Lcry9Aa2 5’UTR, atpl 5’UTR, psbA 5’UTR, cry2a, rrnB, rpsl6, petD, psbA, pabA, and any combination thereof.

43. The method of claim 41 or claim 42, wherein the at least one enhancer sequences comprises a sequence selected from SEQ ID NOs: 2, 4, and 6.

44. The method of any one of claims 1-43, wherein the nucleic acid material further comprises a selection sequence.

45. The method of claim 44, wherein a selection sequence is or comprises a nucleic acid sequence encoding: a His tag, GUS uidA lacz, green fluorescent protein, yellow fluorescent protein, red fluorescent protein, cyan fluorescent protein, green fluorescent protein (eGFP), and any combination thereof.

46. The method of claim 44 or claim 45, wherein a selection sequence is or comprises a yellow fluorescent protein (YFP, GenBank: GQ221700.1), red fluorescent protein (DsRED, GenBank: KY426960.1), green fluorescent protein (eGFP, Genbank: AAB02572.1) or cyan fluorescent protein (CFP, GenBank: HQ993060.1).

47. The method of claim 45, wherein the His tag comprises the sequence CATCACCATCACCATCAC-TAA (SEQ ID NO: 100), CATCATCATCATCATCAT (SEQ ID NO: 101), CATCACCATCACCATCAC (SEQ ID NO: 8), or a fragment or variant thereof.

48. The method of any one claims 1-47, wherein the exogenous nucleic acid material is or comprises a RNA oligonucleotide, a DNA oligonucleotide, a plasmid, and any combination thereof.

49. The method of any one of claims 1 -48, wherein the nucleic acid material comprises two or more exogenous nucleic acid sequences.

50. The method of any one claims 1-49, wherein the exogenous nucleic acid sequence encodes a peptide comprising a sequence that is at least 90% identical to a leukotoxin A (ItkA) protein) according to Genbank: DQ672338.1, or a fragment or variant thereof.

51. The method of claim 50, wherein the exogenous nucleic acid sequence comprises a sequence encoding at least one region of ItkA selected from the group consisting of PL1, PL4, or a fragment or variant thereof.

52. The method of claim 51, wherein the exogenous nucleic acid sequence comprises a PL1 sequence comprising SEQ ID NO: 51 encoding an amino acid sequence SEQ ID NO: 52 and/or a nucleic acid sequence comprising SEQ ID NO: 53 encoding an amino acid sequence SEQ ID NO: 54, or a fragment or variant thereof.

53. The method of any one claims 1-52, wherein the exogenous nucleic acid sequence further comprises a termination sequence.

54. The method of claim 53, wherein the termination sequence comprises a sequence encoding rpsl6 (GenBank: MF580999.1) or a portion or fragment thereof.

55. A plant comprising a nucleic acid material comprising an expression cassette comprising, in 5’ to 3’ orientation a first (5’) sequence; a promoter sequence; an exogenous nucleic acid sequence; and a second (3’) sequence; and wherein the first (5’) sequence and/or the second (3’) sequence comprises 16S ribosomal gene DNA sequence of the plant containing at least one mutation relative to the native plant 16S ribosomal gene DNA sequence; and wherein at least one exogenous nucleic acid sequence is expressed, at least in part, in the chloroplast of the plant.

56. The plant of claim 55, wherein the first (5’) sequence comprises 16S ribosomal gene DNA sequence of the plant containing at least one mutation relative to the native plant 16S ribosomal gene DNA sequence.

57. The plant of claim 55 or claim 56, wherein the second (3’) sequence comprises 23S ribosomal gene DNA sequence of the plant.

58. The plant of claim 57, wherein the second (3’) sequence comprises 23S ribosomal gene DNA sequence of the plant comprises at least one mutation relative to the native plant 23 S ribosomal gene DNA sequence.

59. The plant of any one of claims 55-58, wherein the at least one mutation comprises a mutation that confers antibiotic resistance in the plant.

60. The plant of claim 59, wherein the antibiotic resistance comprises resistance to one or more of lincomycin, spectinomycin, streptomycin, kanamycin, gentamycin, neomycin, Beta lactam resistance, and any combination thereof.

61. The plant of claim 59 or claim 60, wherein the antibiotic resistance comprises resistance to lincomycin, spectinomycin, and streptomycin.

62. The plant of any one of claims 55-61, wherein the plant is millet (Panicum miliaceum), sorghum (Sorghum × drummondii), wheat, maize, barley, triticale, or alfalfa (Medicago sativa).

63. The plant of any one of claims 55-62, wherein the 16S ribosomal gene DNA sequence of the first (5’) sequence comprises a core sequence that has 85% sequence identity to the sequence corresponding to positions 95,395-95,672 of the millet plastid genome sequence (GenBank Accession No. KU343177.1 or the sequence represented SEQ ID NO: 21 or 135).

64. The plant of any one of claims 55-63, wherein the 16S ribosomal gene DNA sequence of the first (5’) sequence comprises a core sequence corresponding to positions 95,395-95,672 of the millet plastid genome sequence (GenBank Accession No. KU343177.1 or the sequence represented SEQ ID NO: 21 or 135).

65. The plant of any one of claims 55-64, wherein the at least one mutation comprises a C to A nucleotide substitution at position 95,395 and/or an A to a C nucleotide substitution at position 95,672 of the millet plastid genome sequence (GenBank Accession No. KU343177.1 or SEQ ID NO: 21 or 135).

66. The plant of any one of claims 57-65, wherein the 23 S ribosomal gene DNA sequence of the second (3’) sequence comprises a core sequence that has at least 90% identity to the sequence corresponding to positions 100,566-100,594 of the millet plastid genome sequence GenBank Accession No. KU343177.1 or SEQ ID NO: 89.

67. The plant of any one of claims 57-66, wherein the 23 S ribosomal gene DNA sequence of the second (3’) sequence comprises a core sequence corresponding to positions 100,566-100,594 of the millet plastid genome sequence GenBank Accession No. KU343177.1 or SEQ ID NO: 89.

68. The plant of any one of claims 57-67, wherein the at least one mutation relative to the native plant 23 S ribosomal gene DNA sequence comprises one or more of: (i) a Gto A nucleotide substitution at position 100,566; (ii) an A to a G nucleotide substitution at position 100,593; and (iii) an A to G nucleotide substitution at position 100,594 of the millet plastid genome sequence (GenBank Accession No. KU343177.1 or SEQ ID NO: 89).

69. The plant of any one of claims 55-68, wherein the first and second sequences comprise SEQ ID NO: 13, 133, or 85 and SEQ ID NO: 14 or 87, respectively.

70. The plant of any one of claims 55-62, wherein the 16S ribosomal gene DNA sequence of the first (5’) sequence comprises a core sequence that has at least 90% identity to the sequence corresponding to positions 96,895-97,172 of the sorghum chloroplast genome sequence (GenBank Accession No. NC_008602.1 or SEQ ID NO: 20).

71. The plant of any one of claims 55-62 and 70, wherein the 16S ribosomal gene DNA sequence of the first (5’) sequence comprises a core sequence corresponding to positions 96,895-97,172 of the sorghum chloroplast genome sequence (GenBank Accession No. NC 008602.1 or SEQ ID NO: 20).

72. The plant of any one of claims 55-62 and 70-71, wherein the at least one mutation comprises a C to A nucleotide substitution at position 96,895 and/or an A to a C nucleotide substitution at position 97,172 of the sorghum chloroplast genome sequence (GenBank Accession No. NC 008602.1 or SEQ ID NO: 20).

73. The plant of any one of claims 57-62 and 70-72, wherein the 23S ribosomal gene DNA sequence of the second (3’) sequence comprises a core sequence that has at least 90% identity to the sequence corresponding to positions 102,072-102,100 of the sorghum chloroplast genome sequence (GenBank Accession No. NC_008602.1 or SEQ ID NO: 94).

74. The plant of any one of claims 57-62 and 70-73, wherein the 23S ribosomal gene DNA sequence of the second (3’) sequence comprises a core sequence corresponding to positions 102,072- 102,100 of the sorghum chloroplast genome sequence (GenBank Accession No. NC 008602.1 or SEQ ID NO: 94).

75. The plant of any one of claims 58-62 and 70-74, wherein the at least one mutations relative to the native plant 23 S ribosomal gene DNA sequence comprises one or more of: (i) a G to A nucleotide substitution at position 102,072; (ii) an A to a G nucleotide substitution at position 102,098; and (iii) an A to G nucleotide substitution at position 102,099 of the sorghum chloroplast genome sequence (GenBank Accession No. NC_008602.1 or SEQ ID NO: 94).

76. The plant of any one of claims 55-62 and 70-75, wherein the first and second sequences comprise SEQ ID NO: 1, 132, or 90 and SEQ ID NO: 12 or 92, respectively.

77. The plant of any one of claims 55-62, wherein the 16S ribosomal gene DNA sequence of the first (5’) sequence comprises a core sequence that has at least 90% sequence identity to the sequence corresponding to positions 99,019 -99,297 of the alfalfa plastid genome sequence (GenBank Accession No. NC_042841.1, positions 38,069-38,097 of the alfalfa plastid genome sequence GenBank KU321681.1, or SEQ ID NO: 22).

78. The plant of any one of claims 55-62 and 77, wherein the 16S ribosomal gene DNA sequence of the first (5’) sequence comprises a core sequence corresponding to positions 99,019 -99,297 of the alfalfa plastid genome sequence (GenBank Accession No. NC_042841.1 or positions 38,069-38,097 of the alfalfa plastid genome sequence GenBank KU321681.1 or SEQ ID NO: 22).

79. The plant of any one of claims 55-62 and 77-78, wherein the at least one mutation comprises a C to A nucleotide substitution at position 99,019 and/or an A to a C nucleotide substitution at position 99,297 of the alfalfa plastid genome sequence (GenBank Accession No. NC_042841.1 or positions 38,069-38,097 of the alfalfa plastid genome sequence GenBank KU321681.1 or SEQ ID NO: 22).

80. The plant of any one of claims 57-62 and 77-79, wherein the 23 S ribosomal gene DNA sequence of the second (3’) sequence comprises a core sequence that has at least 90% identity to the sequence corresponding to positions 38,069-38,097 of the alfalfa plastid genome sequence GenBank Accession No. KU321683.1 or SEQ ID NO: 99.

81. The plant of any one of claims 57-62 and 77-80, wherein the 23 S ribosomal gene DNA sequence of the second (3’) sequence comprises a core sequence corresponding to positions 38,069- 38,097 of the alfalfa plastid genome sequence GenBank Accession No. KU321683.1 or SEQ ID NO: 99.

82. The plant of any one of claims 58-62 and 77-81, wherein the at least one mutation relative to the native plant 23S ribosomal gene DNA sequence comprises one or more of: (i) a Gto A nucleotide substitution at position 38,069; (ii) an A to a G nucleotide substitution at position 38,096; and (iii) an A to G nucleotide substitution at position 38,097 of the alfalfa plastid genome sequence GenBank Accession No. KU321683.1 or SEQ ID NO: 99.

83. The plant of any one of claims 55-62 and 77-82, wherein the first and second sequences comprise SEQ ID NO: 17, 136, or 95 and SEQ ID NO: 18 or 97, respectively.

84. The plant of any one of claims 55-83, wherein a promoter sequence is selected from PpsbA, Prrn, Prna, psaA, PrbcL, CaMV35S, rbcS, and any combination thereof.

85. The plant of claim 84, wherein the promoter sequence is a Prrn promoter sequence comprising SEQ ID NO: 1 or GenBank: MF580999.1.

86. The plant of any one claims 55-85, wherein the nucleic acid material further comprises at least one enhancer sequence.

87. The plant of claim 86, wherein the at least one enhancer sequence is selected from a sequence encoding: ggagg, rrn 5’UTR, T7genel0 5’ UTR (GenBank: EU520588.1), LrbcL 5’UTR, LatpB 5’UTR, Tobacco mosaic virus omega prime 5’UTR (GenBank: KM507060.1), Lcry9Aa2 5’UTR, atpl 5’UTR, psbA 5’UTR, cry2a, rrnB, rpsl6, petD, psbA, pabA, and any combination thereof.

88. The plant of claim 86 or claim 87, wherein the at least one enhancer sequences comprises a sequence selected from SEQ ID NOs: 2, 4, and 6.

89. The plant of any one of claims 55-88, wherein the nucleic acid material further comprises a selection sequence.

90. The plant of claim 89, wherein a selection sequence is or comprises a nucleic acid sequence encoding: a His tag, GUS uidA lacz, green fluorescent protein, yellow fluorescent protein, red fluorescent protein, cyan fluorescent protein, green fluorescent protein (eGFP), and any combination thereof.

91. The plant of claim 89 or claim 90, wherein a selection sequence is or comprises a yellow fluorescent protein (YFP, GenBank: GQ221700.1), red fluorescent protein (DsRED, GenBank: KY426960.1), green fluorescent protein (eGFP, Genbank: AAB02572.1) or cyan fluorescent protein (CFP, GenBank: HQ993060.1).

92. The plant of claim 90, wherein the His tag comprises the sequence CATCACCATCACCATCAC-TAA (SEQ ID NO: 100), CATCATCATCATCATCAT (SEQ ID NO: 101), CATCACCATCACCATCAC (SEQ ID NO: 8), or a fragment or variant thereof.

93. The plant of any one claims 55-92, wherein the exogenous nucleic acid material, when integrated into the chloroplast genome of the plant, is or comprises cpDNA (chloroplast DNA), or RNA when transcribed within the chloroplast.

94. The plant of any one of claims 55-93, wherein the nucleic acid material comprises two or more exogenous nucleic acid sequences.

95. The plant of any one claims 55-94, wherein the exogenous nucleic acid sequence encodes a peptide comprising a sequence that is at least 90% identical to a leukotoxin A (ItkA) protein according to Genbank: DQ672338.1, or a fragment or variant thereof.

96. The plant of claim 95, wherein the exogenous nucleic acid sequence comprises a sequence encoding at least one region of ItkA selected from the group consisting of PL1, PL4, or a fragment or variant thereof.

97. The plant of claim 96, wherein the exogenous nucleic acid sequence comprises a PL1 sequence comprising SEQ ID NO: 51 encoding an amino acid sequence SEQ ID NO: 52 and/or a nucleic acid sequence comprising SEQ ID NO: 53 encoding an amino acid sequence SEQ ID NO: 54, or a fragment or variant thereof.

98. The plant of any one claims 55-97, wherein the exogenous nucleic acid sequence further comprises a termination sequence.

99. The plant of claim 98, wherein the termination sequence comprises a sequence encoding rpsl6 (GenBank: MF580999.1 or SEQ ID NO: 9), or a portion or fragment thereof.

100. A method of transforming a plant comprising providing a nucleic acid material conjugated (e.g., fused) to a carrier to form a complex; wherein the nucleic acid material comprises an expression cassette comprising in 5’ to 3’ orientation a first (5’) targeting sequence that corresponds to a region in the plant chloroplast genome; a promoter sequence, and an exogenous nucleic acid sequence; and a second (3’) targeting sequence that corresponds to a region in the plant chloroplast genome that is 3’ of the sequence targeted by the first (5’) targeting sequence; wherein the carrier comprises a chloroplast-targeting peptide (CTP); and transforming a chloroplast in a plant cell with the nucleic acid material.

101. The method of claim 100, wherein the first (5’) targeting sequence comprises 16S or 23S ribosomal gene DNA sequence of the plant containing at least one mutation relative to the native plant 16S or 23 S ribosomal gene DNA sequence.

102. The method of claim 101, wherein the first (5’) targeting sequence comprises 16S ribosomal gene DNA sequence of the plant containing at least one mutation relative to the native plant 16S ribosomal gene DNA sequence.

103. The method of claim 101 or claim 102, wherein the second (3’) targeting sequence comprises 23 S ribosomal gene DNA sequence of the plant containing at least one mutation relative to the native plant 23 S ribosomal gene DNA sequence.

104. The method of any one of claims 101-103, wherein the at least one mutation comprises a mutation that confers antibiotic resistance in the plant.

105. The method of claim 104, wherein the antibiotic resistance comprises resistance to one or more of lincomycin, spectinomycin, streptomycin, kanamycin, gentamycin, neomycin, Beta lactam resistance, and any combination thereof.

106. The method of claim 104 or claim 105, wherein the antibiotic resistance comprises resistance to lincomycin, spectinomycin, and streptomycin.

107. The method of any one of claims 100-106, wherein the plant is millet, sorghum, wheat, maize, barley, triticale, or alfalfa.

108. The method of any one of claims 100-107, wherein the CTP comprises a protein derived from one of more of: an Arabidopsis thaliana outer envelope membrane protein; molecular weight of 34 d (OEP34) ( Genbank accession no. NP 850768.1), Pisium sativum functional homologue translocase of chloroplasts 34 (TOC34; Genbank accession no. Q41009.1), TOC34 proteins of sorghum (Genbank accession no. XP_021306533.1), millet (Genbank accession no. RLN39229.1), and Medicago truncatula (Genbank accession no. XP 003624825.1), or a fragment or variant thereof.

109. The method of claim 108, wherein the CTP comprises the hydrophobic core region of the OEP34 protein.

110. The method of any one of claims 100-109, wherein the CTP comprises an OEP34 protein encoded by the amino acid sequence SEQ ID NO: 23 [ILAVEYFLVV], SEQ ID NO: 24 [IFALQYLFLA], or SEQ ID NO: 25 [LFALEFLLIM], or a fragment or variant thereof.

111. The method of any one of claims 100- 110, wherein the carrier further comprises one or more cell-penetrating peptides (CPP).

112. The method of claim 111, wherein the CTP is conjugated to positively charged amino acids at the N-terminus of the CPP to form a CTP-CPP complex.

113. The method of claim 111 or claim 112, wherein the CPP comprises KH9 (SEQ ID NO: 28 [KHKHKHKHKHKHKHKHKH]), BP100 (sequence: SEQ ID NO: 29 [KKLFKKILKYL] -amide), K9 (SEQ ID NO: 30 [KKKKKKKKK]), or HPV33L2-DD447 (SEQ ID NO: 102 [SYDDLRRRRKRFPYFFTDVRVAA]).

114. The method of claim 112 or claims 113, wherein the CTP-CPP complex comprises SEQ ID NO: 83 (Sorghum/millet KH9-OEP34 KHKHKHKHKHKHKHKHKHILAVEYFLVV) OR SEQ ID NO: 84 (Medicago KH9-OEP34 KHKHKHKHKHKHKHKHKHLFALEFLLIM).

115. The method of any one of claims 111-114, wherein the C-terminus of the CPP is conjugated to the nucleic acid material.

116. The method of any one of claims 100-115, wherein transforming the chloroplast comprises contacting the plant cell with the complex comprising the carrier conjugated to the nucleic acid material.

117. The method of claim 116, wherein contacting comprises culturing the nucleic acid material and carrier complex in a solution comprising the plant (e.g., for at least 1 minute).

118. The method of any one of claims 100-117, wherein the transformed exogenous nucleic acid sequence is expressed in the chloroplast of the plant cell.

119. The method of any one of claims 100-118, wherein the transformed exogenous nucleic acid sequence is integrated in the chloroplast genome of the plant cell.

120. The method of any one of claims 100-119, wherein the transformed exogenous nucleic acid sequence is stably integrated in the chloroplast genome of the plant cell.

121. The method of any one of claims 100-120, wherein the exogenous nucleic acid sequence encodes an exogenous protein, and wherein the transformed plant expresses the exogenous protein.

122. A method of transforming a plant comprising providing a nucleic acid material complexed with a carrier, wherein the nucleic acid material comprises an expression cassette comprising in 5’ to 3’ orientation a first (5’) targeting sequence that corresponds to a region in the plant chloroplast genome; a promoter sequence, and an exogenous nucleic acid sequence; and a second (3’) targeting sequence that corresponds to a region in the plant chloroplast genome that is 3’ of the sequence targeted by the first (5’) targeting sequence; wherein the carrier comprises a nanotube that is positively charged (i.e., has a zeta potential) and is sized and dimensioned such that it is able to pass through the chloroplast envelope of the plant; and transforming a chloroplast in a plant cell with the nucleic acid material.

123. The method of claim 122, wherein the nanotube comprises a single- walled nanotube.

124. The method of claim 122 or 123, wherein the nanotube is a single- walled carbon nanotube (SWCNT).

125. The method of claim 124, wherein the nanotube is complexed with chitosan (CS-SWCNT).

126. The method of claim 125, wherein the CS-SWCNT is PEGylated (CS^PEG-SWCNT).

127. The method of any one of claims 122-126, wherein the nanotube comprises a zeta potential that is at least 10 mV.

128. The method of any one of claims 122-127, wherein the nanotube comprises a zeta potential that less than 20 mV.

129. The method of any one of claims 122-128, wherein the dimension of the nanotube comprises a length of between about 1.0 and 10.0 μm.

130. The method of any one of claims 122-129, wherein the dimension of the nanotube comprises a diameter of between about 1.0 to 2.0 nm.

131. The method of any one of claims 122-130, wherein transforming the chloroplast comprises contacting the plant cell with the complex comprising the nanotube conjugated to the nucleic acid material.

132. The method of claim 131, wherein contacting comprises introducing the nucleic acid material and carrier complex into the plant via syringe injection.

133. The method of claim 132, wherein the syringe injection comprises surface leaf infusion through a needleless syringe.

134. The method of claim 132, wherein syringe injection comprises stem injection through a needled syringe.

135. The method of claim 131, wherein the contacting comprises applying a vacuum and/or compression to the plant cell.

136. The method of any one of claims 122-135, wherein once the exogenous nucleic acid material enters the chloroplast of the plant, it separates from the nanotube.

137. The method of any one of claims 122-136, wherein the transformed exogenous nucleic acid sequence is expressed in the chloroplast of the plant cell.

138. The method of any one of claims 122-137, wherein the transformed exogenous nucleic acid sequence is integrated in the chloroplast genome of the plant cell.

139. The method of claim 138, wherein the transformed exogenous nucleic acid sequence is stably integrated in the chloroplast genome of the plant cell.

140. The method of any one of claims 122-139, wherein the exogenous nucleic acid sequence encodes an exogenous protein, and wherein the transformed plant expresses the exogenous protein.

141. The method of any one of claims 122-140, the plant is millet (Panicum miliaceum), sorghum (Sorghum × drummondii), wheat, maize, barley, triticale, or alfalfa (Medicago sativa).

142. The method of claim 139, wherein the transformed exogenous nucleic acid sequence is stably integrated within a region of the 16S or 23 S ribosomal gene DNA sequence.

143. A method of transforming a plant comprising: providing a nucleic acid material comprising an expression cassette comprising, in 5’ to 3’ orientation a first 5’ targeting sequence; a promoter sequence; an exogenous nucleic acid sequence; and a second 3’ targeting sequence; and wherein the first (5’) targeting sequence and/or the second (3’) targeting sequence comprises 23 S ribosomal gene DNA sequence of the plant containing at least one mutation relative to the native plant 23 S ribosomal gene DNA sequence; and transforming a chloroplast in a plant cell with the nucleic acid material.

144. The method of claim 143, wherein the second (3’) targeting sequence comprises 23S ribosomal gene DNA sequence of the plant.

145. The method of claim 144, wherein the second (3’) targeting sequence comprises 23 S ribosomal gene DNA sequence of the plant comprising at least one mutation relative to the native plant 23 S ribosomal gene DNA sequence.

146. The method of any one of claims 142-145, wherein the first (5’) targeting sequence comprises 16S ribosomal gene DNA sequence of the plant containing at least one mutation relative to the native plant 16S ribosomal gene DNA sequence.

147. The method of any one of claims 143-146, further comprising expressing the exogenous nucleic acid sequence, wherein the expression occurs, at least in part, in a chloroplast.

148. The method of any one of claims 143-147, wherein transforming the chloroplast comprises contacting the plant cell with the nucleic acid material.

149. The method of claim 148, wherein contacting the plant cell comprises use of biolistics or gene gun, use of chloroplast targeting sequences/peptides, cell penetrating peptides, use of a carrier such as a functionalized nanoparticle, electroporation, chemical-mediated transfection (e.g., using polyethylene gylcol), or any combination thereof.

150. The method of any one of claims 143-149, wherein the at least one mutation comprises a mutation that confers antibiotic resistance in the plant.

151. The method of claim 150, wherein the antibiotic resistance comprises resistance to one or more of lincomycin, spectinomycin, streptomycin, kanamycin, gentamycin, neomycin, Beta lactam resistance, and any combination thereof.

152. The method of claim 151, wherein the antibiotic resistance comprises resistance to lincomycin, spectinomycin, and streptomycin.

153. The method of any one of claims 143-152, wherein the plant is millet, sorghum, wheat, maize, barley, triticale, or alfalfa.

154. The method of any one of claims 143-153, wherein the 23S ribosomal gene DNA sequence of the second (3’) targeting sequence comprises a core sequence that has 90% sequence identity to the sequence corresponding to positions 100,566-100,594 of the 23S ribosomal gene of the millet plastid genome sequence (GenBank Accession No. KU343177.1 or SEQ ID NO: 89).

155. The method of any one of claims 143-154, wherein the 23 S ribosomal gene DNA sequence of the second (3’) targeting sequence comprises a core sequence corresponding to positions 100,566- 100,594 of the 23 S ribosomal gene of the millet plastid genome sequence (GenBank Accession No. KU343177.1 or SEQ ID NO: 89).

156. The method of any one of claims 143-155, wherein the at least one mutation comprises one or more of: (i) a G to A nucleotide substitution at position 100,566; (ii) an A to a G nucleotide substitution at position 100,593; and (iii) an A to G nucleotide substitution at position 100,594 of the millet plastid genome sequence (GenBank Accession No. KU343177.1 or SEQ ID NO: 89).

157. The method of any one of claims 146-156, wherein the 16S ribosomal gene DNA sequence of the first (5’) targeting sequence comprises a core sequence that has 90% sequence identity to the sequence corresponding to positions 95,395-95,672 of the millet plastid genome sequence (GenBank Accession No. KU343177.1 or the sequence represented SEQ ID NO: 21 or 135).

158. The method of any one of claims 146-157, wherein the 16S ribosomal gene DNA sequence of the first (5’) targeting sequence comprises a core sequence corresponding to positions 95,395-95,672 of the millet plastid genome sequence (GenBank Accession No. KU343177.1 or the sequence represented by SEQ ID NO: 21 or 135).

159. The method of any one of claims 146-158, wherein the at least one mutation comprises a C to A nucleotide substitution at position 95,395 and/or an A to a C nucleotide substitution at position 95,672 of the millet plastid genome sequence (GenBank Accession No. KU343177.1 or SEQ ID NO: 21 or 135).

160. The method of any one of claims 143-159, wherein the first and/or second targeting sequences comprises all or a portion of SEQ ID NO: 79.

161. The method of any one of claims 143-160, wherein the first (5’) and second (3) targeting sequences comprise SEQ ID NO: 13, 133, or 85 and SEQ ID NO: 14 or 87, respectively.

162. The method of any one of claims 143-153, wherein the 23S ribosomal gene DNA sequence of the second (3’) targeting sequence comprises a core sequence that has at least 90% identity to the sequence corresponding to positions 102,072-102,100 of the sorghum 23 S ribosomal gene of the sorghum plastid genome sequence (GenBank Accession No. NC 008602.1 or SEQ ID NO: 94).

163. The method of any one of claims 143-153 and 162, wherein the 23S ribosomal gene DNA sequence of the second (3’) targeting sequence comprises a core sequence corresponding to positions 102,072-102,100 of the 23 S ribosomal gene of the sorghum plastid genome sequence (GenBank Accession No. NC_008602.1 or SEQ ID NO: 94).

164. The method of any one of claims 143-153 and 162-163, wherein the at least one mutation comprises one or more of: (i) a Gto A nucleotide substitution at position 102,072; (ii) an A to a G nucleotide substitution at position 102,098; and (iii) an A to G nucleotide substitution at position 102,099 of the sorghum chloroplast genome sequence (GenBank Accession No. NC 008602.1 or SEQ ID NO: 94).

165. The method of any one of claims 146-153, wherein the 16S ribosomal gene DNA sequence of the first (5’) targeting sequence comprises a core sequence that has at least 90% identity to the sequence corresponding to positions 96,895-97,172 of the sorghum chloroplast genome sequence (GenBank Accession No. NC_008602.1 or SEQ ID NO: 20).

166. The method of any one of claims 146-153 and 165, wherein the 16S ribosomal gene DNA sequence of the first (5’) targeting sequence comprises a core sequence corresponding to positions 96,895-97,172 of the sorghum chloroplast genome sequence (GenBank Accession No. NC_008602.1 or SEQ ID NO: 20).

167. The method of any one of claims 146-153 and 165-166, wherein the at least one mutation comprises a C to A nucleotide substitution at position 96,895 and/or an A to a C nucleotide substitution at position 97,172 of the sorghum chloroplast genome sequence (GenBank Accession No.

NC_008602.1 or SEQ ID NO: 20).

168. The method of any one of claims 143-153 and 162-167, wherein the first and/or second targeting sequences comprises all or a portion of SEQ ID NO: 81.

169. The method of any one of claims 143-153 and 162-168, wherein the first and second targeting sequences comprise SEQ ID NO: 11, 132, or 90 and SEQ ID NO: 12 or 92, respectively.

170. The method of any one of claims 143-153, wherein the 23S ribosomal gene DNA sequence of the second (3’) targeting sequence comprises a core sequence that has at least 90% identity to the sequence corresponding to positions 38,069-38,097 of the alfalfa plastid genome sequence GenBank Accession No. KU321683.1 or SEQ ID NO: 99.

171. The method of any one of claims 143-153 and 170, wherein the 23S ribosomal gene DNA sequence of the second (3’) targeting sequence comprises a core sequence corresponding to positions 38,069-38,097 of the alfalfa plastid genome sequence GenBank Accession No. KU321683.1 or SEQ ID NO: 99.

172. The method of any one of claims 143-153 and 170-171, wherein the at least one mutation relative to the native plant 23 S ribosomal gene DNA sequence comprises one or more of: (i) a G to A nucleotide substitution at position 38,069; (ii) an A to a G nucleotide substitution at position 38,096; and (iii) an A to G nucleotide substitution at position 38,097 of the alfalfa plastid genome sequence GenBank Accession No. KU321683.1.

173. The method of any one of claims 146-153, wherein the 16S ribosomal gene DNA sequence of the first (5’) targeting sequence comprises a core sequence that has at least 90% sequence identity to the sequence corresponding to positions 99,019-99,297 of the alfalfa plastid genome sequence (GenBank Accession No. NC_042841.1), positions 38,069-38,097 of the alfalfa plastid genome sequence GenBank KU321681.1 or SEQ ID NO: 22.

174. The method of any one of claims 146-153 and 173, wherein the 16S ribosomal gene DNA sequence of the first (5’) targeting sequence comprises a core sequence corresponding to positions 99,019 -99,297 of the alfalfa plastid genome sequence (GenBank Accession No. NC_042841.1) or positions 38,069-38,097 of the alfalfa plastid genome sequence GenBank KU321681.1 or SEQ ID NO: 22.

175. The method of any one of claims 146-153 and 173-174, wherein the at least one mutation comprises a C to A nucleotide substitution at position 99,019 and/or an A to a C nucleotide substitution at position 99,297 of the alfalfa plastid genome sequence (GenBank Accession No. NC_042841.1 or SEQ ID NO: 20).

176. The method of any one of claims 146-153 and 170-175, wherein the first and second targeting sequences comprise SEQ ID NO: 17, 136, or 95 and SEQ ID NO: 18 or 97, respectively.

177. The method of any one of claims 143-176, wherein a promoter sequence is selected from PpsbA, Prrn, Prna, psaA, PrbcL, CaMV35S, rbcS, and any combination thereof.

178. The method of claim 177, wherein the promoter sequence comprises Prrn (GenBank: MF580999.1:73-201).

179. The method of any one claims 143-178, wherein the nucleic acid material further comprises at least one enhancer sequence.

180. The method of claim 179, wherein the enhancer sequence comprises one or more of T7 phage gene 10 leader sequence (GenBank: EU520588.1 :5627-5689), LrbcL (Genbank EU224430.1: 1456- 1512), and LatpB (Genbank: EU224425.1 : 2006-2095).

181. The method of any one of claims 143-180, wherein the nucleic acid material further comprises a selection sequence.

182. The method of claim 181, wherein a selection sequence is or comprises a yellow fluorescent protein (YFP, GenBank: GQ221700.1), red fluorescent protein (DsRED, GenBank: KY426960.1), green fluorescent protein (eGFP; GenBank: AAB02572.1), or cyan fluorescent protein (CFP, GenBank: HQ993060.1).

183. The method of claim 181, wherein the selection sequence comprises a His tag that comprises the sequence CATCACCATCACCATCAC-TAA (SEQ ID NO: 100), SEQ ID NO: CATCATCATCATCATCAT (SEQ ID NO: 101), CATCACCATCACCATCAC (SEQ ID NO: 8) or a fragment or variant thereof.

184. The method of any one claims 143-183, wherein the exogenous nucleic acid material is or comprises a RNA oligonucleotide, a DNA oligonucleotide, a plasmid, and any combination thereof.

185. The method of any one claims 143-184, wherein the exogenous nucleic acid sequence encodes a peptide comprising a sequence that is at least 90% identical to a leukotoxin A (ItkA) protein) according to Genbank: DQ672338.1, or a fragment or variant thereof.

186. The method of claim 185, wherein the exogenous nucleic acid sequence comprises a sequence encoding at least one region of ItkA selected from the group consisting of PL1, PL4, or a fragment or variant thereof.

187. The method of claim 186, wherein the exogenous nucleic acid sequence comprises a PL1 sequence comprising SEQ ID NO: 51 encoding an amino acid sequence SEQ ID NO: 52 and/or a nucleic acid sequence comprising SEQ ID NO: 53 encoding an amino acid sequence SEQ ID NO: 54, or a fragment or variant thereof.