US20020046416A1

US20020046416A1 - Plant proteins

Info

Publication number: US20020046416A1
Application number: US09/770,657
Authority: US
Inventors: Crisanto Gutierrez-Armenta; Andres Sanz-Burgos
Original assignee: Consejo Superior de Investigaciones Cientificas CSIC
Current assignee: Consejo Superior de Investigaciones Cientificas CSIC
Priority date: 1996-06-13
Filing date: 2001-01-29
Publication date: 2002-04-18
Also published as: JP2001502522A; US6384299B1; NZ333100A; US20020133847A1

Abstract

The present invention is based on the isolation and characterization of a plant cell DNA sequence encoding for a retinoblastoma protein. Such finding is based on the structural and functional properties of the plant retinoblastoma protein as possible regulator of the cellular cycle, of the cellular growth and of the plant cellular differentiation. For this reason, among other aspects, it is claimed the use of retinoblastoma protein or the DNA sequence which encodes for it in the growing control of vegetable cells, plants and/or vegetable virus, as well as the use of vectors, cells, plants or animals, or animal cells modified through the manipulation of the control route based on plant retinoblastoma protein.

Description

DESCRIPTION

The present invention relates the proteins having biological activity in plant and animal systems, to polynucleotides encoding for the expression of such proteins, to oligonucleotides for use in identifying and synthesizing these proteins and polynucleotides, to vectors and cells containing the polynucleotides in recombinant form and to plants and animals comprising these, and to the use of the proteins and polynucleotides and fragments thereof in the control of plant growth and plant vulnerability to viruses.

Cell cycle progression is regulated by positive and negative effectors. Among the latter, the product of the retinoblastoma susceptibility gene (Rb) controls the passage of mammalian cells through G1 phase. In mammalian cells, Rb regulates G1/S transit by inhibiting the function of the E2F family of transcription factors, known to interact with sequences in the promoter region of genes required for cellular DNA replication (see eg Weinberg, R. A. Cell 81,323 (1995); Nevins, J. R. Science 258,424 (1992)). DNA tumor viruses that infect animal cells express oncoproteins that interact with the Rb protein via a LXCXE motif, disrupting Rb-E2F complexes and driving cells into S-phase (Weinberg ibid; Ludlow, J. W. FASEB J. 7, 866 (1993); Moran, E. FASEB J. 7, 880 (1993); Vousden, K. FASEB J. 7, 872 (1993)).

The present inventors have shown that efficient replication of a plant geminivirus requires the integrity of an LXCXE amino acid motif in the viral RepA protein and that RepA can interact with members of the human Rb family in yeast (Xie, Q., Suárez-López, P. and Gutiérrez, C. EMBO J. 14, 4073 (1995). The presence of the LXCXE motif in plant D-type cyclins has also been reported (Soni, R., Carmichael, J. P., Shah, Z. H. and Murray, J. A. H. Plant Cell 7, 85-103 (1995)).

The present inventors have now identified characteristic sequences of plant Rb proteins and corresponding encoding polynucleotides for the first time, isolated such a protein and polynucleotide, and particularly have identified sequences that distinguish it from known animal Rb protein sequences. The inventors have determined that a known DNA sequence from the maize encoding a vegetable Rb plant protein and is hereinafter called ZmRb1. ZmRb1 has been demonstrated by the inventors to interact in yeasts with RepA, a plant geminivirus protein containing LXCXE motif essential for its function. The inventors have further determined that geminivirus DNA replication is reduced in plant cells transfected with plasmids encoding either ZmRb1 or human p130, a member of the human Rb family.

Significantly the inventors work suggests that plant and animal cells may share fundamentally similar strategies for growth control, and thus human as well as plant Rb protein such as ZmRb1 will be expected to have utility in, inter alia, plant therapeutics, diagnostics, growth control or investigations and many such plant proteins will have similar utility in animals.

In a first aspect of the present invention there is provided the use of retinoblastoma protein in controlling the growth of plant cells and/or plant viruses. Particularly, the present invention provides control of viral infection and/or growth in plant cells wherein the virus requires the integrity of an LXCXE amino acid motif in one of its proteins, particularly, e.g., in the viral RepA protein, for normal reproduction. Particular plant viruses so controlled are Geminiviruses.

A preferred method of control using such proteins involves applying these to the plant cell, either directly or by introduction of DNA or RNA encoding for their expression into the plant cell which it is desired to treat. By over expressing the retinoblastoma protein, or expressing an Rb protein or peptide fragment thereof that interacts with the LXCXE motif of the virus but does not affect the normal functioning of the cell, it is possible to inhibit normal virus growth and thus also to produce infection spreading from that cell to its neighbours.

Alternatively, by means of introducing anti-sense DNA or RNA in plant cells in vectors form that contain the necessary promoters for the DNA or RNA transcription, it will be possible to exploit the well known anti-sense mechanism in order to inhibit the expression of the Rb protein, and thus the S-phase. Such plants will be of use, among other aspects to replicate DNA or RNA until high levels, e.g. in yeasts. The methods to introduce anti-sense DNA in cells are very well known for those skilled in the art: see for example Principles of gene manipulation—An introduction to Genetic Engineering (1994) R. W. Old & S. B. Primrose; Oxford-Blackwell Scientific Publications Fifth Edition p398.

In a second aspect of the present invention there is provided recombinant nucleic acid, particularly in the form of DNA or cRNA (mRNA), encoding for expression of Rb protein that is characteristic of plants. This nucleic acid is characterised by one or more characteristic regions that differ from known animal Rb protein nucleic acid and is exemplified herein by SEQ ID No 1, bases 31-2079.

The DNA or RNA can have a sequence that contains the degenerated substitution in the nucleotides of the codons in SEQ ID No. 1, and in where the RNA the T is U. The most preferred DNA or RNA are capable of hybridate with the polynucleotide of the SEQ ID No. 1 in conditions of low stringency, preferably being the hybridization produced in conditions of high stringency.

The expressions “conditions of low stringency” and “conditions of high stringency” are understood by those skilled, but are conveniently exemplified in U.S. Pat. No. 5,202,257, Col-9-Col 10. If some modifications were made to lead to the expression of a protein with different amino acids, preferably of the same kind of the corresponding amino acids to the SEQ ID No 1; that is, are conservative substitutions. Such substitutions are known by those skilled, for example, see U.S. Pat. No. 5,380,712, and it is only contemplated when the protein has activity with retinoblastoma protein.

Preferred DNA or cRNA encodes for a plant Rb protein having A and B pocket sub-domains having between 30% and 75% homology with human Rb protein, particularly as compared with p130, more preferably from 50% to 64% homology. Particularly the plant Rb protein so encoded has the C706 amino acid of human Rb conserved. Preferably the spacer sequence between the A and B pockets is not conserved with respect to animal Rb proteins, preferably being less than 50% homologous to the same region as found in such animal proteins. Most preferably the protein so encoded has 80% or more homology with that of SEQ NO 2 of the sequence listing attached hereto, still more preferably 90% or more and most preferably 95% or more. Particularly provided is recombinant DNA of SEQ ID No 1 bases 31 to 2079, or the entire SEQ ID No 1, or corresponding RNAs, encoding for maize cDNA clone encoding ZmRb1 of SQ ID No 2.

In a third aspect of the present invention there is provided the protein expressed by the recombinant DNA or RNA of the second aspect, novel proteins derived from such DNA or RNA, and protein derived from naturally occurring DNA or RNA by mutagenic means such as use of mutagenic PCR primers.

In a fourth aspect there are provided vectors, cells and plants and animals comprising the recombinant DNA or RNA of correct sense or anti-sense, of the invention.

In a particularly preferred use of the first aspect there is provided a method of controlling cell or viral growth comprising administering the DNA, RNA or protein of the second or third aspects to the cell. Such administration may be direct in the case of proteins or may involve indirect means, such as electroporation of plant seed cells with DNA or by transformation of cells with expression vectors capable of expressing or over expressing the proteins of the invention or fragments thereof that are capable of inhibiting cell or viral growth.

Alternatively, the method uses an expression vector capable of producing anti-sense RNA of the cDNA of the invention.

Another one of the specific characteristics of the plants protein and of the nucleic acids includes a N-terminal domain corresponding in sequence to the amino acids 1 to 90 of the SEQ ID No. 2 and a nucleotides sequence corresponding to the basis 31 to 300 of the SEQ ID No. 1. These sequences are characterized by possessing less than 150 and less than 450 units that the animal sequences which possess more than 300 amino acids and 900 pairs of more bases.

The present invention will now be illustrated further by reference to the following non-limiting Examples. Further embodiments falling within the scope of the claims attached hereto will occur to those skilled in the light of these.

Figures. [0019]
FIG. 1. The sub-figure A shows the relative lengths of the present ZmRb1 protein and the human retinoblastoma proteins. The sub-figure B shows the alignment of the amino acids sequences of the Pocket A and Pocket B of the ZmRb1 with that of the Xenopus, chicken, rat and three human protein (Rb, p107 and p130). [0020]
FIG. 2. This figure is a map of the main characteristics of the WDV virus and the pWori vector derived from WDV and the positions of the deletions and mutations used in order to establish that the LXCXE motif is required for its replication in plants cells.[0021]

EXAMPLE 1

Isolation of DNA and Protein Expressing Clones. [0022]
Total RNA was isolated from maize root and mature leaves by grinding the material previously frozen in liquid nitrogen essentially as described in Soni et al (1995). The major and minor p75ZmRb1 mRNAs were identified by hybridization to a random-primed 32P-labelled PstI internal fragment (1.4 kb). [0023]
A portion of a maize cDNA library (106 pfu) in IZAPII (Stratagene) was screened by subsequent hybridization to 5′-labelled oligonucleotides designed to be complementary to a known EST sequence of homologue maize of p130. These oligonucleotides were 5′-AATAGACACATCGATCAA/G (M.5m, nt positions 1411-1438) and 5′-GTAATGATACCAACATGG (M.3c, nt positions 1606-1590) (Isogen Biosciences). [0024]
After the second round of screening, pBluescript SK-(pBS) phagemids from positive clones were isolated by in vivo excision with ExAssist helper phage (Stratagene) according to protocols recommended by the manufacturer. DNA sequencing was carried out using a SequenaseTM Kit (USB). [0025]
The 5′-end of the mRNAs encoding p75ZmRb1 was determined by RACE-PCR. Poly-A+mRNA was purified by chromatography on oligo-dT-cellulose (Amersham). The first strand was synthesized using oligonucleotide DraI35 (5′-GATTTAAAATCAAGCTCC, nt positions 113-96). After denaturation at 90° C. for 3 min, RNA was eliminated by RNase treatment, the cDNA recovered and 5′-tailed with terminal transferase and DATP. Then a PCR fragment was amplified using primer DraI35 and the linker-primer (50 bp) of the Stratagene cDNA synthesis kit. [0026]
One of the positive clones so produced contained a ˜4 kb insert that, according to restriction analysis, extended both 5′ and 3′ of the region contained in the Expressed Sequence Tag used. The nucleotide sequence corresponding to the longest cDNA insert (3747 bp) is shown in SEQ ID No. 1. This ZmRb1 cDNA contains a single open reading frame capable of encoding a protein of 683 amino acids (predicted Mr 75247, p75ZmRb1) followed by a 1646 bp 3′-untranslated region. Untranslated regions of similar length have been also found in mammalian Rb cDNAs (Lee, W. -L. et al, Science 235, 1394 (1987); Bernards, R. et al, Proc. Natl. Acad. Sci. USA 86, 6474 (1989)). Northern analysis indicates that maize cells derived from both root meristems and mature leaves contain a major message, ˜2.7±0.2 kb in length. In addition, a minor ˜3.7±0.2 kb message also appears. Heterogeneous transcripts have been detected in other species (Destrée, O. H. J. et al, Dev. Biol. 153, 141 (1992)). [0027]
Plasmid pWoriΔΔ was constructed by deleting in pWori most of the sequences encoding WDV proteins (Sanz and Gutierrez, unpublished). Plasmid p35S.Rb1 was constructed by inserting the CaMV 35S promoter (obtained from pWDV3:35SGUS) upstream of the ZmRb1 cDNA in the pBS vector. Plasmid p35S.130 was constructed by introducing the complete coding sequence of human p130 instead of ZmRb1 sequences into p35S.Rb1. Plasmid p35.A+B was constructed by substituting sequences encoding the WDV RepA and RepB ORFs instead of ZmRb1 in p35S.Rb1 plasmid. (See Soni, R. and Murray, J. A. H. Anal. Biochem. 218, 474-476 (1994)). [0028]
The sequence around the methionine codon at nucleotide position 31 contains a consensus translation start (Kozak, M. J. Mol. Biol. 196, 947 (1987)). To determine whether the cDNA contained the full-length Zmb1 coding region, the 5′-end of the mRNAs was amplified by RACE-PCR using an oligonucleotide derived from a region close to the putative initiator AUG, which would produce a fragment of ˜150 bp. The results are consistent with the ZmRb1 cDNA clone containing the complete coding region. [0029]
The ZmRb1 protein contains segments homologous to the A and B subdomains of the “pocket” that is present in all members of the Rb family. These subdomains are separated by a non-conserved spacer. ZmRb1 also contains non-conserved N-terminal and C-terminal domains. Overall, ZmRb1 shares ˜28-30% amino acid identity (˜50% similarity) with the Rb family members (Hannon, G. J., Demetrick, D. & Beach, D. Genes Dev. 7, 2378 (1993); Cobrinik, D., Whyte, P., Peeper, D. S., Jacks, T. & Weinberg, R. A. ibid., p. 2392 (1993). Ewen, M. E., Xing, Y. Lawrence, J. B. and Livingston, D. M. Cell 66, 1155 (1991)) (Lee W. L. et al, Science 235, 1394 (1987) Bernards et al, Proc. Natl. Acad. Sci. USA 86, 6974 (1989)), with the A and B subdomains exhibiting the highest homology (˜50-64%). Interestingly, amino acid C706 in human Rb, critical for its function (Kaye, F. J., Kratzke R. A., Gerster, J. L. and Horowitz, J. M. Proc. Natl. Acad. Sci. USA 87, 6922 (1990)), is also conserved in maize p75ZmRb1. [0030]
Note: The 561-577 amino acids encompass a proline-rich domain. [0031]
ZmRb1 contains 16 consensus sites, SP or TP for phosphorilation by cyclins dependant kinases (CDKs) with one of the 5′-tail of the sub-domain A and several in the C-terminal area which are potential sites of phosphorilation. A nucleic acid preferred group which encodes proteins in which one or more of these sites are changed or deleted, making the protein more resistant to the phosphorilation and thus, to its functionality, for example linking to E2F or similar. This can be easily carried out by means of mutagenesis conducted by means of PCR. [0032]

EXAMPLE 2

In vivo Activity. [0033]
Replication of wheat dwarf geminivirus (WDV) is dependent upon an intact LXCXE motif of the viral RepA protein. This motif can mediate interaction with a member of the human Rb family, p130, in yeasts. Therefore, the inventors investigated whether p75ZmRb1 could complex with WDV RepA by using the yeast two-hybrid system (Fields, S. and Song, O. Nature 340, 245-246 (1989)). Yeast cells were co-transformed with a plasmid encoding the fusion GAL4BD-RepA protein and with plasmids encoding different GAL4AD fusion protein. The GAL4AD-p75ZmRb1 fusion could also complex with GAL4BD-RepA to allow growth of the recipient yeast cells in the absence of histidine. This interaction was slightly stronger than that seen with the human p130 protein. RepA could also bind to some extent to a N-terminally truncated form of p75ZmRb1. The role of the LXCXE motif in RepA-p75ZmRb1 interaction was assessed using a point mutation in WDV RepA (E198K) which we previously showed to destroy interaction with human p130. Co-transformation of ZmRb1 with a plasmid encoding the fusion GAL4BD-RepA(E198K) indicated that the interaction between RepA and p75ZmRb1 occurred through the LXCXE motif. [0034]
In this respect, the E198K mutant of WDV RepA behaves similarly to analogous point mutants of animal virus oncoproteins (Moran, E., Zerler, B., Harrison, T. M. and Mathews, M. B. Mol. Cell Biol. 6, 3470 (1986); Cherington, V. et al., ibid., p. 1380 (1988); Lillie, J. W., Lowenstein, P. M., Green, M. R. and Green, M. Cell 50, 1091 (1987); DeCarpio, J. A. et al., ibid., p. 275 (1988)). [0035]
Specific interaction between maize p75ZmRb1 and WDV RepA in the yeast two-hybrid system (Fields et al) relied on the ability to reconstitute a functional GAL4 activity from two separated GAL4 fusion proteins containing the DNA binding domain (GAL4BD) and the activation domain (GAL4AD). Yeast HF7c cells were co-transformed with a plasmid expressing the GAL4BD-RepA or the GAL4BD-RepA(E198K) fusions and the plasmids expressing the GAL4AD alone (Vec) or fused to human p130, maize p75 (p75ZmRb1) or a 69 amino acids N-terminal deletion of p75 (p75ZmRb1-DN). Cells were streaked on plates with or without histidine according to the distribution shown in the upper left corner. The ability to grow in the absence of histidine depends on the functional reconstitution of a GAL4 activity upon interaction of the fusion proteins, since this triggers expression of the HIS3 gene which is under the control of a GAL4 responsive element. The growth characteristics of these yeast co-transformants correlate with the levels of b-galactosidase activity. [0036]
Procedures for two-hybrid analysis are described in Xie et al (1995). The GAL4AD-ZmRb1 fusions were construed in the pGAD424 vector. [0037]

EXAMPLE 3

In vivo Activity. [0038]
Geminivirus DNA replication requires the cellular DNA replication machinery as well as other S-phase specific factors (Davies, J. W. and Stanley, J. Trends Genet. 5, 77 (1989); Lazarowitz, S. Crit. Rev. Plant Sci. 11, 327 (1992)). Consistent with this requirement, geminivirus infection appears to drive non-proliferating cells into S-phase, as indicated by the accumulation of the proliferating cell nuclear antigen (PCNA), a protein which is not normally present in the nuclei of differentiated cells (Nagar, S., Pedersen, T. J., Carrick, K. M., Hanley-Bowdoin, L. and Robertson, D. Plant Cell 7, 705 (1995)). The inventors finding that efficient WDV DNA replication requires an intact LXCXE motif in RepA coupled with the discovery of a plant homolog of Rb supports the model that, as in animal cells, sequestration of plant Rb by viral RepA protein promotes inappropriate entry of infected cells into S-phase. Therefore, one way to investigate the function of p75ZmRb1 was to measure geminivirus DNA replication in cells transfected with a plasmid bearing the ZmRb1 sequences under a promoter functional in plant cells, an approach analogous to that previously used in human cells (Uzvolgi, E. et al., Cell Growth Diff 2, 297 (1991)). Accumulation of newly replicated viral plasmid DNA was impaired in wheat cells transfected with plasmids expressing p75ZmRb1 or human p130, when expression of WDV replication protein(s) is directed wither by the WDV promoter or by the CaMV 35S promoter. [0039]
Since WDV DNA replication requires an S-phase cellular environment, interference with viral DNA replication by p75ZmRb1 and human p130 strongly evidences a role for retinoblastoma protein in the control of the G1/S transition in plants. The existence of a plant Rb homolog implies that despite their ancient divergence, plant and animal cells use, at least in part, similar regulatory proteins and pathways for cell cycle control. [0040]
Two lines of evidences reinforce this model. First, a gene encoding a protein that complements specifically the G1/S, but not the G2/M transition of the budding yeast cdc28 mutant has been identified in alfalfa cells (Hirt, H., Páy, A., Bögre, L., Meskiene, I. and Heberle-Bors, E. Plant J. 4, 61 (1993)). Second, plant homologs of D-type cyclins have been isolated from Arabidopsis and these, like their mammalian relatives, contain LXCXE motifs. In concert with plant versions of CDK4 and CDK6, plant D-type cyclins may regulate passage through G1 phase by controlling the phosphorylation state of Rb-like proteins. [0041]
In animal cells, the Rb family has been implicated in tumor suppression and in the control of differentiation and development. Thus, p75ZmRb1 could also play key regulatory roles at other levels during the plant cell life. One key question that is raised by the existence of Rb homologs in plant cells in whether, as in animals disruption of the Rb pathway leads to a tumor-prone condition. In this regard, the inventors have noted that the VirB4 protein encoded by the Ti plasmids of both [0042] Agrobacterium tumefaciens and A. rhyzogenes contains an LXCXE motif. Although the VirB4 protein is required for tumor induction (Hooykas, P. J. J. and Beijersbergen, A. G. M. Annu. Rev. Phytopathol. 32, 157 (1994), the function of its LXCXE motif in this context remains to be examined. Geminivirus infection is not accompanied by tumor development in the infected plant, but in some cases an abnormal growth of enactions has been observed (G. Dafalla and B. Gronenborn, personal communication).
Inhibition of wheat dwarf geminivirus (WDV) DNA replication by ZmRb1 or human p130 in cultured wheat cells was carried out as follows. A. Wheat cells were transfected, as indicated, with pWori (Xie et al. 1995) alone (0.5 g), a replicating WDV-based plasmid which encodes WDV proteins required for viral DNA replication, and with control plasmid pBS (10 g) or p35S.Rb1 (10 g), which encodes ZmRb1 sequences under the control of the CaMV 35S promoter. Total DNA was purified one and two days after transfection, equal amounts fractionated in agarose gels and ethidium bromide staining and viral pWori DNA identified by Southern hybridization. Plasmid DNA represents exclusively newly-replicated plasmid DNA since it is fully resistant to DpnI digestion and sensitive to Mbol. Note that the MboI-digested samples were run for about half of the length than the undigested samples. B. To test the effect of human p130 on WDV DNA replication, wheat cells were co-transfected with pWori (0.5 g) and plasmids pBS (control), p35S.Rb1 or p35S.130 (10 g in each case). Replication of the test plasmid (pWori) was analyzed two days after transfection and was detected as described in part A using ethidium bromide staining; and Southern hybridization. C. To test the effect of ZmRb1 or human p130 on WDV DNA replication when expression of viral proteins was directed by the CaMV 35S promoter, the test plasmid pWoriΔΔ (which does not encode functional WDV replication proteins but replicates when they are provided by a different plasmid, i.e. pWori) was used. Wheat cells were co-transfected, as indicated, with pWoriΔΔ (0.25 g), pWori (0.25 g), p35S.A+B (6 g), a p35S.Rb1 (10 g) and/or p35S.130 (10 g). Replication of the test plasmid (pWoriΔΔ) was analyzed 36 hours after transfection and was detected as described in part A using ethidium bromide staining; Southern hybridization. Plasmids pWori (M1) and pWoriΔΔ (M2; Sanz and Gutiérrez, unpublished), 100 pg in each case, were used as markers. Suspension cultures of wheat cells, transfection by particle bombardment and analysis of viral DNA replication were carried out as described in (Xie et al. 1995), except that DNA extraction was modified as in (Soni and Murray. Arnal. Biochem. 218, 474-476 (1995). [0043]
1 2 1 3747 DNA Zea mays 1 gaattcggca cgagcaaagg tctgattgat atggaatgtt tccagtcaaa tttggaaaaa 60 atggagaaac tatgtaattc taatagctgt aaaggggagc ttgattttaa atcaattttg 120 atcaataatg attatattcc ctatgatgag aactcgacgg gggattccac caatttagga 180 cattcaaagt gtgcctttga aacattggca tctcccacaa agacaataaa gaacatgctg 240 actgttccta gttctccttt gtcaccagcc accggtggtt cagtcaagat tgtgcaaatg 300 acaccagtaa cttctgccat gacgacagct aagtggcttc gtgaggtgat atcttcattg 360 ccagataagc cttcatctaa gcttcagcag tttctgtcat catgcgatag ggatttgaca 420 aatgctgtca cagaaagggt cagcatagtt ttggaagcaa tttttccaac caaatcttct 480 gccaatcggg gtgtatcgtt aggtctcaat tgtgcaaatg cctttgacat tccgtgggca 540 gaagccagaa aagtggaggc ttccaagttg tactataggg tattagaggc aatctgcaga 600 gcggagttac aaaacagcaa tgtaaataat ctaactccat tgctgtcaaa tgagcgtttc 660 caccgatgtt tgattgcatg ttcagcggac ttagtattgg cgacacataa gacagtcatc 720 atgatgtttc ctgctgttct tgagagtacc ggtctaactg catttgattt gagcaaaata 780 attgagaact ttgtgagaca tgaagagacc ctcccaagag aattgaaaag gcacctaaat 840 tccttagaag aacagctttt ggaaagcatg gcatgggaga aaggttcatc attgtataac 900 tcactgattg ttgccaggcc atctgttgct tcagaaataa accgccttgg tcttttggct 960 gaaccaatgc catctcttga tgacttagtg tcaaggcaga atgttcgtat cgagggcttg 1020 cctgctacac catctaaaaa acgtgctgct ggtccagatg acaacgctga tcctcgatca 1080 ccaaagagat cgtgcaatga atctaggaac acagtagtag agcgcaattt gcagacacct 1140 ccacccaagc aaagccacat ggtgtcaact agtttgaaag caaaatgcca tccactccag 1200 tccacatttg caagtccaac tgtctgtaat cctgttggtg ggaatgaaaa atgtgctgac 1260 gtgacaattc atatattctt ttccaagatt ctgaagttgg ctgctattag aataagaaac 1320 ttgtgcgaaa gggttcaatg tgtggaacag acagagcgtg tctataatgt cttcaagcag 1380 attcttgagc aacagacaac attatttttt aatagacaca tcgatcaact tatcctttgc 1440 tgtctttatg gtgttgcaaa ggtttgtcaa ttagaactca cattcaggga gatactcaac 1500 aattacaaaa gagaagcaca atgcaagcca gaagtttttt caagtatcta tattgggagt 1560 acgaaccgta atggggtatt agtatcgcgc catgttggta tcattacttt ttacaatgag 1620 gtatttgttc cagcagcgaa gcctttcctg gtgtcactaa tatcatctgg tactcatcca 1680 gaagacaaga agaatgctag tggccaaatt cctggatcac ccaagccatc tcctttccca 1740 aatttaccag atatgtcccc gaagaaagtt tcagcatctc ataatgtata tgtgtctcct 1800 ttgcggcaaa ccaagttgga tctactgctg tcaccaagtt ccaggagttt ttatgcatgc 1860 attggtgaag gcacccatgc ttatcagagc ccatctaagg atttggctgc tataaatagc 1920 cgcctaaatt ataatggcag gaaagtaaac agtcgattaa atttcgacat ggtgagtgac 1980 tcagtggtag ccggcagtct gggccagata aatggtggtt ctacctcgga tcctgcagct 2040 gcatttagcc ccctttcaaa gaagagagag acagatactt gatcaattat aaatggtggc 2100 ctctctcgta tatagctcac agatccgtgc tccgtagcag tctattcttc tgaataagtg 2160 gattaactgg agcgatttaa ctgtacatgt atgtgttagt gagaagcagc agtttttagg 2220 cagcaaactg tttcaagtta gcttttgagc tatcaccatt tctctgctga ttgaacatat 2280 ccgctgtgta gagtgctaat gaatctttag ttttcattgg gctgacataa caaatcttta 2340 tcctagttgg ctggttgttg ggaggcattc atcagggtta tatttggttg tcaaaaagta 2400 ctgtacttaa ttcacatctt tcacattttt cactagcaat agcagcccca aattgctttc 2460 ctgactagga acatattctt tacaggtata agcatgccaa ctctaaacta tatgaatcct 2520 ttttatattc tcatttttaa gtacttctct gtttctgcta cttttgtact gtatatttcc 2580 agcttctcca tcagactgat gatcccatat tcagtgtgct gcaagtgatt tgaccatatg 2640 tggcttatcc ttcaggtatg tctcatgttg tgacttcatt gctgattgct tttgtaatgg 2700 tactgttgag ttcatttctg gttacaatca gcctttactg ctttatattg ttctactaat 2760 tttggcttgc acagccagga cgattggttt tctgcatcaa tcaatctttt ttaggacaag 2820 atatttttgt atgctacact tcccaaattg caattaatcc agaagtctac cttgttttat 2880 tctattagtt ctcagcaaca gtgaatgaat atgaatcagt catgctgata gatgttcatc 2940 tggttattcc aaacaatctg acatcgcatc tctttctgca agtgagatga agaaaacctg 3000 aaatgctatc accatttaaa acattggctt ctggaagttc aggtgattag caggagacgt 3060 tctgacattg ccattgacat gtacggtagt gatggcagga gacgttctta aacagcagct 3120 gctccttcag cttgtaatgt ctgattgtat tgaccaagag catccacctt gccttatggt 3180 actaactgaa tgagctggtg acgctgactc atctgcataa tggcagatgc ttaaccatct 3240 ttaggagctc atgtcatgat tccagctgca ccgtgtcaaa tgtgaaggcc ctgcaaggct 3300 ttccaggccg caccaatcct gcttgcttct tgaagataca tatggtgcca cctaaataaa 3360 agctgtttct ggttatgtct gtccttgaca tgtcaacaga ttagtgttgg gttgcagtca 3420 tgtggtgttt aagtcttgga gaaggcgaga agtcattgct gccagcattg tgatcgtcag 3480 gcacagaagt actcaaaagt gagagctact tgttgcgagc aaacggaggg cgatataggt 3540 tgatagccaa tttcagttct ctatatacaa gcagcggatt ttgtttagag ttagcttttg 3600 agatgcatca tttctttcac atctgattct gtgtgttgta actcggagtc gcgtagaagt 3660 tagaatgcta actgacctta attttcaccg aataatttgc tagcgttttt cagtatgaaa 3720 tccttgtctt aaaaaaaaaa aaaaaaa 3747 2 683 PRT Unknown Description of Unknown Organism protein 2 Met Glu Cys Phe Gln Ser Asn Leu Glu Lys Met Glu Lys Leu Cys Asn 1 5 10 15 Ser Asn Ser Cys Lys Gly Glu Leu Asp Phe Lys Ser Ile Leu Ile Asn 20 25 30 Asn Asp Tyr Ile Pro Tyr Asp Glu Asn Ser Thr Gly Asp Ser Thr Asn 35 40 45 Leu Gly His Ser Lys Cys Ala Phe Glu Thr Leu Ala Ser Pro Thr Lys 50 55 60 Thr Ile Lys Asn Met Leu Thr Val Pro Ser Ser Pro Leu Ser Pro Ala 65 70 75 80 Thr Gly Gly Ser Val Lys Ile Val Gln Met Thr Pro Val Thr Ser Ala 85 90 95 Met Thr Thr Ala Lys Trp Leu Arg Glu Val Ile Ser Ser Leu Pro Asp 100 105 110 Lys Pro Ser Ser Lys Leu Gln Gln Phe Leu Ser Ser Cys Asp Arg Asp 115 120 125 Leu Thr Asn Ala Val Thr Glu Arg Val Ser Ile Val Leu Glu Ala Ile 130 135 140 Phe Pro Thr Lys Ser Ser Ala Asn Arg Gly Val Ser Leu Gly Leu Asn 145 150 155 160 Cys Ala Asn Ala Phe Asp Ile Pro Trp Ala Glu Ala Arg Lys Val Glu 165 170 175 Ala Ser Lys Leu Tyr Tyr Arg Val Leu Glu Ala Ile Cys Arg Ala Glu 180 185 190 Leu Gln Asn Ser Asn Val Asn Asn Leu Thr Pro Leu Leu Ser Asn Glu 195 200 205 Arg Phe His Arg Cys Leu Ile Ala Cys Ser Ala Asp Leu Val Leu Ala 210 215 220 Thr His Lys Thr Val Ile Met Met Phe Pro Ala Val Leu Glu Ser Thr 225 230 235 240 Gly Leu Thr Ala Phe Asp Leu Ser Lys Ile Ile Glu Asn Phe Val Arg 245 250 255 His Glu Glu Thr Leu Pro Arg Glu Leu Lys Arg His Leu Asn Ser Leu 260 265 270 Glu Glu Gln Leu Leu Glu Ser Met Ala Trp Glu Lys Gly Ser Ser Leu 275 280 285 Tyr Asn Ser Leu Ile Val Ala Arg Pro Ser Val Ala Ser Glu Ile Asn 290 295 300 Arg Leu Gly Leu Leu Ala Glu Pro Met Pro Ser Leu Asp Asp Leu Val 305 310 315 320 Ser Arg Gln Asn Val Arg Ile Glu Gly Leu Pro Ala Thr Pro Ser Lys 325 330 335 Lys Arg Ala Ala Gly Pro Asp Asp Asn Ala Asp Pro Arg Ser Pro Lys 340 345 350 Arg Ser Cys Asn Glu Ser Arg Asn Thr Val Val Glu Arg Asn Leu Gln 355 360 365 Thr Pro Pro Pro Lys Gln Ser His Met Val Ser Thr Ser Leu Lys Ala 370 375 380 Lys Cys His Pro Leu Gln Ser Thr Phe Ala Ser Pro Thr Val Cys Asn 385 390 395 400 Pro Val Gly Gly Asn Glu Lys Cys Ala Asp Val Thr Ile His Ile Phe 405 410 415 Phe Ser Lys Ile Leu Lys Leu Ala Ala Ile Arg Ile Arg Asn Leu Cys 420 425 430 Glu Arg Val Gln Cys Val Glu Gln Thr Glu Arg Val Tyr Asn Val Phe 435 440 445 Lys Gln Ile Leu Glu Gln Gln Thr Thr Leu Phe Phe Asn Arg His Ile 450 455 460 Asp Gln Leu Ile Leu Cys Cys Leu Tyr Gly Val Ala Lys Val Cys Gln 465 470 475 480 Leu Glu Leu Thr Phe Arg Glu Ile Leu Asn Asn Tyr Lys Arg Glu Ala 485 490 495 Gln Cys Lys Pro Glu Val Phe Ser Ser Ile Tyr Ile Gly Ser Thr Asn 500 505 510 Arg Asn Gly Val Leu Val Ser Arg His Val Gly Ile Ile Thr Phe Tyr 515 520 525 Asn Glu Val Phe Val Pro Ala Ala Lys Pro Phe Leu Val Ser Leu Ile 530 535 540 Ser Ser Gly Thr His Pro Glu Asp Lys Lys Asn Ala Ser Gly Gln Ile 545 550 555 560 Pro Gly Ser Pro Lys Pro Ser Pro Phe Pro Asn Leu Pro Asp Met Ser 565 570 575 Pro Lys Lys Val Ser Ala Ser His Asn Val Tyr Val Ser Pro Leu Arg 580 585 590 Gln Thr Lys Leu Asp Leu Leu Leu Ser Pro Ser Ser Arg Ser Phe Tyr 595 600 605 Ala Cys Ile Gly Glu Gly Thr His Ala Tyr Gln Ser Pro Ser Lys Asp 610 615 620 Leu Ala Ala Ile Asn Ser Arg Leu Asn Tyr Asn Gly Arg Lys Val Asn 625 630 635 640 Ser Arg Leu Asn Phe Asp Met Val Ser Asp Ser Val Val Ala Gly Ser 645 650 655 Leu Gly Gln Ile Asn Gly Gly Ser Thr Ser Asp Pro Ala Ala Ala Phe 660 665 670 Ser Pro Leu Ser Lys Lys Arg Glu Thr Asp Thr 675 680

Claims

1. A method of controlling the growth of a plant cell or a plant virus within that cell comprising increasing or decreasing the level and/or activity of retinoblastoma protein in that plant cell by incorporation therein of a recombinant nucleic acid.

2. A method as claimed in claim 1 characterised in that the nucleic acid is such as to increase or inhibit expression of a retinoblastoma protein in the cell.

3. A method as claimed in claim 1 characterised in that the nucleic acid is such as to express a retinoblastoma protein or peptide fragment of a retinoblastoma protein that interacts with viral LXCXE motif without affecting the normal functioning of the cell.

4. A method as claimed in claim 3 characterised in that the retinoblastoma protein has been rendered resistant to phosphorylation by cyclin dependent kinases by change or deletion of one or more consensus SP or TP sites found in the SEQ ID No. 2.

5. A method as claimed in claim 2 characterised in that the DNA or RNA is antisense to retinoblastoma protein encoding DWA or RNA and inhibits retinoblastoma protein expression.

6. A method of transforming a plant cell such that it is directed into the S phase of the cell cycle comprising introducing a nucleic acid encoding antisense RNA to a plant retinoblastoma protein.

7. Recombinant nucleic acid encoding for expression of a retinoblastoma protein characterised in that the retinoblastoma protein has an amino acid sequence having 80% or more homology with that of SEQ No. 2 of the sequence listing attached hereto.

8. Recombinant nucleic acid as claimed in claim 7 characterised in that it comprises SEQ ID no. 1, bases 31-207, sequences only having degenerate substitutions thereof or sequences capable of hybridizing with a polynucleotide of SEQ ID No. 1 under conditions of high stringency.

9. Recombinant nucleic acid as claimed in claim 7 or 8 characterised in that it encodes for a retinoblastoma protein conservatively substituted with respect to SEQ ID No. 2.

10. Recombinant nucleic acid characterised in that it comprises antisense DNA or RNA to a plant retinoblastoma protein.

11. Recombinant nucleic acid as claimed in claim 10 characterised in that it comprises antisense DNA or RNA to a plant retinoblastoma protein comprising SEQ ID No. 2 or a sequence having at least 80% homology thereto.

12. Recombinant nucleic acid as claimed in claim 10 or 11 characterised in that it comprises antisense DNA or RNA to that of SEQ ID No. 1 or a sequence having at least 80% homology thereto.

13. Recombinant nucleic acid characterised in that it encodes for a retinoblastoma protein or a peptide fragment of a retinoblastoma protein that interacts with viral LXCXE motif without affecting the normal functioning of a plant cell.

14. Recombinant nucleic acid as claimed in claim 13 characterised in that it encodes for a plant retinoblastoma protein in which one or more consensus SP or TP sites found in the SEQ ID No. 2 have been changed or deleted.

15. A protein produced by the expression of a recombinant DNA or RNA as claimed in any one of claims 7 to 9, 13 and 14.

16. A protein as claimed in claim 15 characterised in that one or more consensus SP or TP sites found in the SEQ ID No. 2 have been changed or deleted.

17. A recombinant vector characterised in that it comprises a recombinant nucleic acid as claimed in any one of claims 7 to 9, 13 and 14.

18. A plant cell characterised in that it comprises a recombinant nucleic acid encoding for expression of a retinoblastoma protein.

19. A plant cell as claimed in claim 18 characterised in that it comprises a recombinant nucleic acid as claimed in any one of claims 7 to 9, 13 and 14.

20. A plant cell as claimed in claim 18 or 19 characterised in that it expresses a retinoblastoma protein from said nucleic acid.

21. A transgenic plant characterised in that it comprises a cell as claimed in any one of claims 18 to 20.