WO1996022372A2

WO1996022372A2 - Antibodies for the control of cyst nematodes and transgenic plants expressing them

Info

Publication number: WO1996022372A2
Application number: PCT/NL1996/000033
Authority: WO
Inventors: Jacob Bakker; Arjen Schots; Wilhelmus Johannes Stiekema
Original assignee: LANDBOUWUNIVERSITEIT WAGENINGEN; De Rijkslandbouwhogeschool
Current assignee: LANDBOUWUNIVERSITEIT WAGENINGEN; De Rijkslandbouwhogeschool
Priority date: 1995-01-17
Filing date: 1996-01-17
Publication date: 1996-07-25
Anticipated expiration: 1998-07-17
Also published as: CA2241977A1; WO1996022372A3; EP0871731A2

Abstract

The invention concerns antibodies directed against an antigenic subventral salivary protein of a cyst nematode, e.g. of the genera Heterodera, Globodera and Punctodera, or at an immunogenic part or equivalent thereof, said antigenic protein having an amino acid of 30-50 kDa and having an isoelectric point (pI) of about 6.8 to 8.7. Furthermore provided are expression systems comprising at least a nucleotide sequence encoding an antibody or antibody fragment against a cyst nematode salivary protein, and a sequence regulating expression of said nucleotide sequence, for transforming plants so as to protect them against cyst nematodes.

Description

Antibodies for the control of cyst nematodes and transgenic plants expressing them

The present invention is in the field of crop protection. More in particular, the invention relates to methods and means for controlling cyst nematodes in agriculture.

Cyst nematodes are nematodes of the Heteroderidae family. They represent an important group of pest organisms in agriculture. Subfamilies are the Heteroderinae, Meloidoderinae (one genus, one species) and Ataloderinae (4 genera, 12 species). The Heteroderinae are divided into 85 species in 7 genera, three of which comprise economically important species: Heterodera, 56 species, including H. schachtii (beet), H. avenae (oat), H. bifenestra (grass), H. cruciferae (cabbage), H. glycines (soybean), H. goettingiana (pea), H. oryzae (rice), H. sacchari (sugarcane), H. trifolii (clover) and H. zeae (maize); Globodera, 13 species, including G. rostochiensis and G. pallida (potato), G. solanacearum and G. tabacum (tobacco); and Punctodera, 3 species, including P. punctata (grass).

Especially potato cyst nematodes (Globodera rostochiensis) are a menace of the European potato industry. Control measures often include rotation with non-host plants, growing of resistant varieties and species, use of certified, nematode-free plant material (seed potatoes), and use of soil fumigants and other nematicides, such as methyl isothio- cyanate and dichloropropene, as pre-emergence or post-emergence treatments. Neither of these control methods is fully effective, and furthermore the use of chemicals is_# unwanted, since it results in the introduction of large amounts of toxic substances into the environment.

Sedentary plant parasitic nematodes feed from their host by transforming root cells into multinucleate feeding cells. In the case of cyst nematodes this feeding site consists of a large syncytiu , which results from a fusion of adjacent root cells. The successful formation and exploitation of these feeding cells involves a complex interaction between nematode and host plant, in which nematode secretions of both polypeptide and polysaccharide nature are considered to play an important role (Zuckerman & Jansson, 1984; Kaplan & Davis. 1987; Ηussey, 1989a).

Sedentary plant parasitic nematodes have three large esophageal gland cells, one dorsal and two subventral. They inject secretions from these esophageal (also referred to herein as salivary) glands via their stylet into the cells of their host plant. These saliva proteins are presumably involved in feeding site induction (Ηussey, 1989a), and they are also necessary for feeding itself, as follows from the formation of intracellular feeding tubes (Rumpenhorst, 1984; Wyss & Zunke, 1986; Hussey & Mi s, 1991).

It has been found now that cyst nematodes can be effectively controlled by means of antibodies which are directed against the (salivary) esophageal proteins of the nematode, especially against the subventral esophageal proteins (svp's). Thus, the invention is primarily related to antibodies directed against a salivary protein of the nematode or at an immunogenic part or equivalent thereof. According to the present invention, "directed against" means that the antibody interacts with the particular protein; in particular it means that the antibody inhibits the functioning of said protein. Salivary proteins of cyst nematodes that are most suitable as a target of antibody inhibition have an apparent molecular weight of about 28-52, more in particular 30-50 kDa. These proteins are produced by second stage juveniles (J2) of the cyst nematode and occur especially in the subventral salivary glands of the nematode. Individual proteins have molecular weights of about 30 kDa (svp30), 31 kDa, (svp31a), 31 kDa (svp31b), 32 kDa (svp32), 39 kDa (svp39), and 49 kDa (svp49), in case of G. rostochiensis salivary proteins, with corresponding isoelectric points (pi) of 7.6, 7.6, 7.9, 6.8, 7.3 and 8.7, respectively. In other species, such as H. glycines, these characteristics are similar. T ese proteins are related, probably in that the lower molecular weight proteins are processed forms of the 49 kDa protein, and they share at least one epitope. At least one of the svp3\ appears to be glycosylated, and it is assumed that they are glycosylated variants of svp3 ). The other four proteins are probably not glycosylated, since they are not affected by enzymic de- glycosylation.

The antibodies according to the invention are preferably directed against proteins derived from second-stage juveniles of the particular nematode. Preferably, the antibody reacts with more than one of the six proteins referred to above, in that it has an epitope shared by two or more up to six of the proteins. A very useful antibody was found to be the antibody denoted herein as MGR 48, which recognises the six proteins svp3Q, .svp31a, svp3\ i, svp32, svp39 and svp49, as confirmed by Western blotting.

A salivary protein (svp49) of H. glycines recognised by an antibody according to the invention (MGR 48) induced by

of G. rostochiensis has the N-terminal amino acid sequence Xaa-Xaa-Ala-Val-Ala-Pro-Pro-Phe-Gly-Gln-Leu-Ser-Val-Ser-Gly- Ser-Asn-Lys-Gln-Pro-Val-Gln-Leu-Ile-Ser-Asn-Ser-Leu-Phe-Glu-His (SEQ ID No. 1). Antibodies according to the invention can be directed against peptide or protein comprising an amino acid sequence having a homology of at least 50% with the corresponding part of the amino acid sequence of SEQ ID No. 1. Said homology is preferably at least 60%, more preferably at least 75%. They can also be directed at other parts of the proteins defined above. They are preferably directed at a continuous poly- peptide epitope, not at a carbohydrate moiety, nor at a disulphide moiety.

The cyst nematodes to be controlled by the present invention are those of the Heteroderidae family as mentioned above, especially of the Heteroderinae subfamily, including the genera Heterodera, Punctodera and Globodera. Examples are the soybean cyst nematode H. glycines, the potato cyst nematodes G. rostochiensis and G. pallida and the tobacco cyst nematode G. tabacum.

The antibody to be used according to the invention may be complete, multimeric antibodies, i.e. antibodies which contain all four subunits. The antibody may also be partial antibodies which comprise one or more variable parts of the complete antibody. In particular, the antibody may comprise the variable part of the light chain (V_j and the variable part of the heavy chain (V_Η), preferably fused by a linker peptide. Such a single chain antibody (scFv) also requires one gene for expression, whereas a complete antibody requires at least two genes.

The antibodies according to the invention may be monovalent, i.e. contain one antigen-binding site, or bivalent (two binding sites). Bivalent antibodies may be obtained e.g. by coupling two single-chain (scFv) antibodies by means of a linker, or by shortening the linker peptide between the variable parts of an scFv so as to induce dimerisation of scFv's, or by providing scFv's with a tail sequence leading to oligomerisation of antibody chains. They may be monospecific or bispecific, i.e. contain variable parts reacting with a single antigen or with two different antigens. The amino acid sequence of two variable regions of an antibody specifically reacting with a salivary protein of cyst nematodes, fused by a 15-mer linker peptide, as well as the nucleotide sequence encoding this amino acid sequence are depicted in SEQ ID No. 2. Instead of the Gly-Ser linker peptide shown in SEQ ID No. 2 (amino acids 113- 127), another linker such as the 16-mer linker 202, depicted in SEQ ID No. 3, may be used. The amino acid and nucleotide sequences of a signal peptide that can be used for excretion of an antibody according to the invention is depicted in SEQ ID No. 4.

The invention also relates to polypeptides corresponding to the salivary (esophageal) proteins as mentioned above or to an immunogeπic part thereof having at least 5, preferably at least 8 amino acids, as well as to nucleotide sequences of at least 15, preferably at least 24 nucleotides, encoding such a polypeptide. Such polypeptides can comprise an amino acid sequence having a degree of homology of at least 50%, preferably at least 60%, more preferably at least 75% with the amino acid sequence depicted in SEQ ID No. 1.

The invention also relates to peptides corresponding to the antibodies described above or to parts thereof, especially to variable domains thereof, such as depicted in SEQ ID No. 2 and equivalents and parts thereof. Suitable parts include the complementarity determining regions (CDR) corresponding to amino acids 25-39, 55-61 and 94-102 (light chain), and 158-162, 177-193 and 226-232 (heavy chain), and combinations thereof. Also covered by the invention are nucleotide sequences encoding such peptides, such as given in SEQ ID No. 2. Variable domains are e.g. comprised by the partial nucleotide sequences 1-336 and 382-819 of SEQ ID No. 2.

The invention is further concerned with expression systems comprising at least such an antibody-encoding nucleotide sequence and a sequence regulating expression of said nucleotide sequence. These expression systems may be constructed by first producing antibodies against a salivary protein or immunogenic part thereof, followed by isolating the antibody and determining the nucleotide sequence of the gene encoding the antibody peptide, and then synthesising a polynucleotide corresponding to that antibody gene or part thereof and combining it with one or more regulatory sequences. The expression system according to the invention may also comprise other genes, which other genes may encode other peptides having a plant protecting or regulating function.

Also comprised by the invention is a process for protecting a plant against the action of a cyst nematode, wherein the plant is transformed using a nucleotide sequence encoding an antibody or part thereof or an expression system as described above. Furthermore, transgenic plants containing in their genome such a nucleotide sequence and capable of expressing said sequence are part of the invention. Such transgenic plants are resistant to cyst nematode attack. Antibodies against a salivary protein of a cyst nematode, when expressed in plants, are also part of the invention. Gene constructs to be used may comprise nucleotide sequences encoding the complete antibody molecule, the Fab part, scFv part or any other part (like complemen¬ tarity determining regions) which shows binding to the target proteins.

The desired cellular location of the proteins can be achieved using the appropriate signal sequences. The signal sequence is responsible for targeting the protein from the cytoplasm to another subcellular compartment. An example of a targeting route is the secretion via endoplasmic reticulum and golgi apparatus. Examples of signal sequences for secretion are described in Briggs and Gierasch (1986), Firek et al. (1993), During et al. (1990) and Shirasu et al. (1988). SEQ NO. 4 shows the nucleotide and amino acid sequences of a suitable signal peptide; the particular signal peptide shown is that of the kappa light chain of antibody CEA 66E3'CL (see Kabat et al. 1991 and Cabilly et al. 1984) with Glu at position 23 substituted by Asp.

The antibody-encoding genes can be expressed in plants under the control of any type of promoter which is active in plants. Examples are: a) constitutive promoters such as the CaMV-35S (Kay et al. 1987); b) tissue specific promoters such as described by Nap et al. (1993) (leave), De Almeida et al. (1989) (leave, SSU-promoter), Nap et al. (1992) (potato tuber, patatin promoter), Hendriks et al. (1991) (potato tuber), Guerche et al. (1990) (seed); c) inducible promoters such as the TR2' promoter (Langridge et al. 1994), wound inducible promoters (Logemann et al. 1989; Suh et al. 1991) or chemically induced promoters (Williams et al. 1992). Promoters may be used singly, in tandem or as divergent promoters.

Transformation can be done using any method which ensures a stable integration of the antibody encoding gene in the plant genome in such a way that it can still be transcribed. Examples of transformation methods are: a) Agrobacterium tumefaciens mediated transformation (Horsch et al. 1985): based on a natural transformation system in which the bacterium stably incorporates part of a plasmid DNA (T-DNA) into the plant genome. The T-DNA includes the gene to be expressed. b) Microprojectile bombardment (Vasil et al. 1992): particles coated with DNA penetrate the plant cell nuclei at high velocity where the DNA is integrated into the genome by host recombination processes. c) Tissue electroporation (D'Halluiπ et al. 1992): under the influence of a strong electric field DNA penetrates the plant cells and, after being transported to the nuclei, it is in¬ corporated into the plant genome by host recombination processes. Examples

Materials and methods

Nematodes: Second stage juveniles (J2) of Globodera rostochiensis pathotype Rol, and of G. pallida pathotype Pa2 were hatched for 7 days by soaking cysts on a 100 μm sieve in potato root diffusate (Clarke & Perry, 1977). The J2 suspensions were mixed with an equal volume of 70% (w/v) sucrose in a centrifuge tube. The 35% sucrose mixture was covered with a layer of demineralised water, and centrifuged briefly at 1000 g. Purified juveniles were collected from the sucrose-water interface with a pasteur pipette, washed with tap water and used for further processing in the various experiments. Preparative electrophoresis: In total 2.75 million J2 of G. rostochiensis were homogenised in 208 M Tris-HCI pH 6.8 supplemented with 8.33% (v/v) 2-mercaptoethanol (ME) at 5 °C using a small glass mortar and pestle. The J2 were homogenised in aliquots of approximately 300,000 individuals each. Following homogenisation the samples were pooled and mixed in a ratio of 3:2 (v:v) with a solution of 5% SDS / 25% glycerol / 0.1% Bromophenol Blue, thus producing standard SDS-sample buffer (O'Farrell 1975) The homogenate was heated for 5 minutes in boiling water, centrifuged for 5 min. at 10000 g, and the supernatant was stored at -80 °C until use. The nematode sample (2 ml, approximately 13 mg of protein) was fractionated by preparative SDS-PAGE using a Model 491 Prep Cell apparatus (Bio-Rad, Richmond, California, USA), essentially according to the instruction manual, with the correction that 0.1% SDS was added to the buffers of the acrylamide gels. The cylindrical separating gel was 50 x 28 mm (height x diameter) and contained 10% acrylamide. The stacking gel contained 4% acrylamide and was 15 mm in height. The proteins were separated with a current of 40 mA, and 1.5 ml fractions were collected using an elution buffer flow rate of 1 ml/min. The fractions were concentrated by freeze drying, washed three times in phosphate buffered saline pH 7.4 (PBS) using 1.5 ml microcentrifuge filter concentration units with molecular weight cut¬ off of 5 kDa (Ultrafree-MC, Millipore Corp. Bedford, USA), taken up in 50 μl PBS and stored at -20 °C. The 50 original fractions were pooled in groups of 2 to 5 fractions to form a representative set of 16 samples covering the entire molecular weight range of the fractionation experiment. Each pooled sample was diluted in PBS to obtain three 200 μl aliquots for successive immunisations.

Immunisations and cell fusions: Sixteen mice were immunised intraperitoneally with the isolated protein fractions, which were mixed 1:1 with Freund's incomplete adjuvant. After four weeks a second immunisation was given, also with incomplete adjuvant. Two weeks later antiserum samples were collected for immunofluorescence microscopy. One mouse that was selected for monoclonal antibody production received a final booster injection 12 weeks after the first immunisation. Three days later the mouse was sacrificed, and MAb- producing hybridoma cell lines were obtained by fusing spleen cells with SP 2/0 myeloma cells (Goding 1983; Schots et al. 1992b).

Immunofluorescence microscopy: Preparation of J2 for indirect immunofluorescence testing of MAbs and antisera was essentially according to Atkinson et al. (1988) and Hussey (1989). J2 of G. rostochiensis were fixed in 2% paraformaldehyde in PBS pH 7.4 for 3 days. The nematodes were then washed in distilled water, and drops of concentrated suspension were spread evenly onto aluminium dishes (diameter 2 cm), which were glued to microscope slides for easy manipulation. The drops were allowed to dry at room temperature in a box with silica gel, after which the dishes with nematodes were stored dry at -20 °C. For screening of the MAbs, the dried J2 were cut into small pieces on their aluminium dish using a razor blade. By cutting parallel lines in three different directions, it was assured that most of the nematodes were cut in 2 or more pieces. Then the nematodes were taken up in 1 ml of PBS containing 1 mg/ml proteinase K (Merck, Darm¬ stadt, Germany) in a 1.5 ml micro-centrifuge tube and incubated for 20 minutes with agitation at room temperature. After this, the nematodes were pelleted (2 minutes 2000 g, swing out rotor) and subsequently taken up in cold methanol (1 in; -20 °C) and cold acetone (2 min: -20 °C). After removal of the acetone, the nematode sections were resuspended in blocking buffer containing PBS pH 7.2, 10% horse serum, and 1 mM phenylmethylsulphonyl fluoride. Labelling of the J2 was done in 96 well filtration plates with a pore size of 0.45 μm. (MultiScreen-HV, Millipore Corp. Bedford, U.S.A.). To each well 20 μl of nematode suspension (containing approximately 200 sections) was added, followed by 80 μl of hybridoma culture supernatant or mouse antiserum diluted 1:1000 (v:v) in PBS. After incubation overnight in a moist atmosphere, the nematode sections were washed three times with PBS / 0.1% Tween 20 by applying vacuum to the filtration plates, and they were next incubated for 2 hours with FITC-conjugated rat-anti-mouse IgG (Cat. nr. 415-095-100; Jackson Immuno Research Laboratories Inc, West Grove, USA), diluted 1:100 in PBS containing 0.1% BSA and 0.1% Tween-20. After three washes with PBS / 0.1% Tween-20, the nematode sections were taken up in 20 μl of distilled water, and transferred to 24 well microscope slides (Cat Nr. 10-448-S, Cel-Line Associates Inc., New Field, U.S.A.) precoated with 0.1% poly-L-lysine (2 μl/well). After drying in the dark 2 μl of anti-quenching solution, consisting of 0.5M sodium carbonate buffer pH 8.6 mixed 1:1 with Citifluor Antifadent 2 (Agar Scientific Ltd., Stansted, England), was applied to the wells and the slides were covered with a large coverslip, which was fixed with dots of nail polish. Specimens were viewed with the 50x water immersion objective of a Leitz epifluorescence microscope, using an L 02.1 or I filter block. Hybridoma cell lines producing antibodies to the subventral glands of G. rostochiensis, were subcloned to stability, and stored in liquid nitrogen. Isotyping of the light and heavy chains of the MAbs was essentially as described by Schots et al. (1992b). Immunofluorescence testing of J2 from G. pallida and M. hapla followed the same procedure as described for G. rostochiensis, with the exception that the initial fixations in paraformaldehyde were different: two days for G. pallida, and one day for M. hapla. Immunofluorescence testing of J2 from G. tabacum, M. incognita and H. glycines, H. schachtii was as described by Davis et al. (1992). SDS-PAGE and Western blotting: Analytical mini SDS-PAGE was performed essentially as described by De Boer et al. (1992). For the examination of the protein fraction's that were obtained with preparative electrophoresis, 4 μl of SDS-sample buffer was added to 2 μl from the concentrated fractions in PBS, and these samples applied to 20 μl slots in the stacking gel. Following electrophoresis, the gels were stained with Coomassie Blue G-250 (Neuhoff et al. 1988). For Western blot testing of MAbs, J2 of G. rostochiensis were homogenised as described above, and per minigel approximately 10000 J2 were added to a single 73 mm wide slot in the stacking gel. An adjacent reference well (3 mm wide) was filled with prestained molecular weight markers (Bio-Rad, Richmond, USA). Following electrophoresis (13% separating gel), the proteins were transferred to polyvinylidene difluoride (PVDF) membrane (Immobilon-P, Millipore Corp., Bedford, USA) using a semi-dry blotting apparatus. A continuous transfer buffer system was used containing 39 mM glycine, 48 M Tris and 20% (v/v) methanol. Transfer was carried out with 0.8 mA cm" for 1 hour. The blots were cut into strips, which were blocked overnight in PBS pH 7.4 / 0.1% (v/v) Tween-20 (PBST) supplemented with 5% (w/v) defatted milk powder. Following a wash in PBST / 1% milk powder, the strips were incubated for 2 hours in hybridoma culture fluid, diluted 1:6 in PBST / 1% milk powder. For MAb MGR 48 affinity-purified antibody (1 μg/ml) was used instead of culture fluid. After washing three times in PBST / 1% milk powder, the strips were next incubated individually in alkaline phosphatase conjugated rat-anti-mouse IgG (H+L) (Cat. Nr. 415-055-100, Jackson Immuno Research Laboratories Inc, West Grove, USA) diluted 1:5000 in PBST / 1% milk powder for 1 hour. After washing in PBST / 0.1% milk powder (lx) and PBST (3x), the strips were stained individually in 0.1 M ethanolamine-HCl pH 9.6, supple- mented with 4 mM MgCl₂, 5-bromo-4-chloro-3-indolyl phosphate (0.06 mg/ml) and nitro blue tetrazolium (0.1 mg/ml).

Electron microscopy: For ultrastructural examination, J2 of G. rostochiensis were fixed at 4 °C with 4% paraformaldehyde in 0.1 M HEPES-buffer pH 7.5 for 2 days. The suspension of fixed J2 was spread on a microscope slide and the nematodes were chopped into pieces with a razor blade. The nematode sections were then fixed with subsequently 2% glutaraldehyde and 1% osmium tetraoxide (both in 0.1 M HEPES pH 7.5), and stained with 1% aqueous uranyl acetate. Following stepwise dehydration in ethanol, the J2 were infiltrated for 1 day with Spurr epoxy resin (Spurr, 1969). The nematode fragments were then transferred to fresh epoxy resin in a BEEM capsule, centrifuged to the bottom, and polymerised at 60°C. Thin sections were cut with an LKB ultra-microtome, collected on formvar-coated 100 mesh copper grids, poststained with 2% uranyl acetate in 50% methangl. For immunolabelling, J2 of G. rostochiensis were fixed for 2 days at 4°C in 2% paraformaldehyde in PBS pH 7.4. After chopping them into pieces, the J2 were washed 2 times in distilled water, dehydrated in 30%-50%-70%-96% ethanol, infiltrated at room temperature for 1 hour in a 1:1 mixture of 96% ethanol and LR-White acrylic resin (London Resin Co. Ltd., Basingstoke, GB) and subsequently in pure LR-White resin for 4 hours. The nematode fragments were transferred to fresh resin in a gelatin capsule, centri¬ fuged to the bottom, and polymerised at 60 °C. Following ultramicrotomy, thin sections were collected on formvar-coated copper grids and immunolabelled with colloidal gold according to the following protocol (Aurion Immuno Gold Reagents, Wageningen, NL) 10 min. PBS pH 7.6 / 0.05 M glycine, 2 hours affinity purified MAb MGR 48 diluted to 1 μg/ml in incubation buffer (PBS pH 7.6 / 0.2% BSA-C / 20 mM NaN₃), 6x 5 min. wash with incubation buffer, 2 hours colloidal gold solution (10 πm particle size, conjugated with goat-anti-mouse-IgG immunoglobulins; Cat. nr 810.022, Aurion Immuno Gold Reagents, Wageningen, NL) diluted 1:10 in incubation buffer, 6x 5 min. wash with incubation buffer, 3x wash with PBS, and 3x wash with distilled water. The sections were not post-stained. Electron micrographs were taken with a Zeiss EM 109 transmission electron microscope. Sequencing of variable parts of MAb MGR 48: cDNA to mRNA from the hybridoma cell-line producing MGR 48 was produced and cloned into an E. coli vector. The single chains were sequenced according to standard methods. Sequencing of N-terminal part of H. glycines svp49:

The 49 kDa fraction of H. glycines proteins was purified by immunoadsoφtion using a protein A column to which MGR 48 was covalently bound. The purified protein was sequenced according to standard methods. Two-dimensional gel electrophoresis Proteins were separated in two-dimensional gel electrophoresis using an immobilised pΗ gradient and a 8-18% (w/v) acrylamide SDS-PAGE gradient gel (IPG- Dalt), essentially according to the manufacturers' protocol (Immobiline Dry Strips 3-10L, and ExcelGel, Pharmacia LKB Biotechnology, Uppsala, SE). Freshly hatched J₂ of G. rostochiensis were homogenised in 1% (v/v) Nonidet P-40 and 2% (v/v) ME. Protein concentration was estimated according to Bradford (1976) using BSA as a standard. Short¬ ly before gel electrophoresis, five volumes of 8 M urea, 0.5% (v/v) Nonidet P-40, 2% (v/v) ME, 2% (v/v) carrier ampholytes pΗ 3-10 (BioRad), 1 mM Pefabloc (Boehringer Mannheim, D), and a trace of bromophenol blue were added to the sample. This solution was thoroughly mixed, incubated for 1 h at room temperature and centrifuged for 10 min at 14,000 x g. The IPG strips (4x110 mm) were rehydrated in 8 M urea, 0.25% (v/v) Nonidet P-40, 10 mM dithiothreitol (DTT), 2 mM acetic acid, and a trace of Orange G for 7 h. The samples (15 μg) were applied 1 cm from the anodic side of the IPG strips. The isoelectric focusing conditions were as follows: 3 h at 300 V, a linear increase from 300 to 2,000 V in 5 h, 8 h at 2,000 V. The temperature was maintained at 14°C. The IPG strips with the focused proteins were equilibrated for 15 min in freshly prepared 10 ml 50 mM Tris-ΗCl (pΗ 6.8), 4.17 M urea, 30% (v/v) glycerol, 1% (w/v) SDS, 16.2 mM DTT, and subsequently for 15 min in 10 ml 50 mM Tris-ΗCl (pΗ 6.8), 4.17 M urea, 30% (v/v) glycerol, 1% (w/v) SDS, 240 mM iodoacetamide, with a trace of bromophenol blue. Pairs of IPG strips were transferred to a precast 8-18% (w/v) acrylamide SDS-PAGE gradient gel (ExcelGel 110x245x0.5 mm, Pharmacia) and second dimension electrophoresis was performed at 20 mA for 30 min and at 40 mA for another 75 min. The SDS-PAGE gel was used for semi-dry Western blotting on PVDF membrane at 0.8 mA cm² for 1 h. Six protein spots were detected on Western blot using MGR48. Svp49 focused at pi 8.7, whereas svp39 focused at pi 7.3 in the applied pH range. A new protein species (svp32) which was not separated on one-dimensional Western blotting before, appeared at pi 6.8 and 32 kDa. Svp31 focused as two isoelectric point variants (pi 7.6 and 7.9), while svp3Q focused at 7.6. The results are shown in figure 2.

Results

Antibody production: Fractionation of homogenised J2 of G. rostochiensis by preparative continuous flow electrophoresis in the presence of sodium dodecyl sulphate (SDS) yielded 50 protein fractions with average apparent molecular weights ranging from 30 to 52 kDa. Examination of successive fractions by analytical SDS polyacrylamide gel electrophoresis (SDS-PAGE) revealed very narrow protein zones, with a very gradual increase in average molecular weight. Therefore the original fractions were pooled to form a representative set of 16 protein samples of increasing molecular weight, which were used for immunising 16 mice. Antisera collected after the second immunisations with these samples were screened with immunofluorescence microscopy for reaction with the salivary glands of J2 of G. rostochiensis. It was found that four mice had produced antisera that reacted with the salivary glands. Because of simultaneous strong staining of additional structures within the J2, it was at this stage not yet possible to determine with certainty whether these antisera labelled the dorsal or the subventral gland. The mouse that was immunised with the 38- 40.5 kDa protein sample was chosen for monoclonal antibody production. Screening of hybridoma's: Screening of the hybridoma culture supernatants by immuno¬ fluorescence microscopy resulted in 12 MAbs that bound specifically to the subventral glands of J2 from G. rostochiensis. The staining effectiveness of the glands by the antibodies was found to be variable, both between different MAbs in a single experiment, between individual J2 stained with the same antibody, and between repeated experiments. Ten MAbs (MGR 46, 47, 48, 49, 50, 53, 54, 55, 56, 59) were able to stain the entire contents of subventral gland cells including the gland ducts. Two MABs (MGR 57 and 60) persistently stained only the cut surfaces of the gland cells, and failed to penetrate sufficiently to obtain an even staining. Immunofluorescence microscopy was also used to test the cross reactivity of the MAbs with J2 of other species of plant parasitic nematodes (Table 1). It was found that all MAbs also bind to the subventral glands of G. pallida, and that all MAbs, except MGR 49. also bind to the subventral glands of G. tabacum. In H. schachtii, H. glycines, M. incognita and M. hapla no binding to salivary glands was observed, except for MGR 48, which reacted with the subventral glands of H. glycines.

Table 1

List of monoclonal antibodies to the subventral salivary glands of Globodera rostochiensis and their immunofluorescence cross-reactivity with other species of sedentary plant parasitic nematodes. "+" reaction with subventral glands, "-" no reaction with subventral glands.

Antibody Isotype Globodera Globodera Heterodera Heterodera Meloidogyne Meloidogyne

10 (a) heavy light pallida tabacum schachtii glycines incognita hapla

MGR 46 IgG2a λ + +

MGR 47 IgGl K + +

MGR 48 IgGl + + (b) - (b)

. ._ ₊

MGR 50 IgGl K +

MGR 53 IgGl K + +

MGR 54 IgG2a K + +

MGR 55 IgGl K + + 0 M MGGRR 5566 I IggGGll K K + + +

MGR 57 IgGl λ + +

MGR 59 IgGl K + +

MGR 60 IgGl λ + +

5 (a) Affinily-purified antibody (MGR 48) or hybridoma culture fluid (all other MABs) (b) Background staining

Western blotting: The MAbs were tested for reactivity with protein homogenates of J2 which were separated by SDS-PAGE and electroblotted onto PVDF membrane. Figure 1 shows that 11 of the 12 MAbs stained protein bands in the electrophoresis pattern. 0 Together these MAbs identified 4 (6) different polypeptides with apparent molecular weights of about 30-31 (svp30; SV-lb), 31-32 (svp3la, ,svp31b, sv_P32 SV-la). 38-39 (svp39. SV-2) and 49-50 kDa (svp49; SV-3) respectively. All reactive MAbs bound to the SV-2 band, with 10 showing additional binding to one or more of the other bands. MGR 47, 53, 54, 55, and 59 all reacted strongly with the SV-la, SV-lb and SV-2 kDa 5 bands, while MGR 46, 57 and 60 showed moderate binding to SV-2 and SV-3. Reactivity with all six protein bands was found only with MGR 48. The binding specificity of MGR 56 most resembled that of MGR 48 with the difference that no binding was observed with bands SV-la and SV-3. Both MGR 48 and MGR 56 stained additional smaller bands in the gel region below SV-lb.

Electron microscopy: Because ultrastructural information about the subventral glands in potato cyst-nematodes was completely lacking, and could only be inferred from studies with related plant-parasitic nematode species (Endo 1984; Endo 1988; Endo 1993), we first examined subventral gland moφhology in hatched J2 of G. rostochiensis. Thin sections showed that both the cell body and the cell extension are packed with secretory vesicles. It was only in the immediate vicinity of the nucleus that parts of the cytoplasm were free from secretory vesicles. The secretory granules invariably possessed an electron translucent halo which surrounded an electron dense core. This characteristic made it easy to identify the subventral glands in the low-contrast formaldehyde fixed specimens that were used for immunolabelling. Immunogold labelling of formaldehyde-fixed J2 with MAb MGR 48 was localised to the subventral salivary glands. Binding of the gold particles occurred both to the electron dense granule matrix and also to the patches of cytoplasm surrounding the granules. Induced antigen production

Stylet secretion of J2 of G. rostochiensis was induced by incubation with the serotonin antagonist DMT (5-methoxy-Ν,Ν-dimethyltryptamine-hydrogen-oxalate; Research Bio- chemicals Inc., Natick, MA, US), as described previously for preparasitic J2 of Heterodera glycines (Goverse et al. 1994). Salivary proteins were collected by sieving to remove the nematodes. Antibody MGR 48 was used for probing Western blots of collected stylet secretions. Secretion of Ag48 (32, 39 and 49 kDa) appeared in samples from DMT and control solutions. At pH below 5.5 the protein pattern varied indicating high sensitivity for acidic hydrolysis. In solutions up to pH 8.0 Ag48 could be detected resulting in a highly stable secretory pattern when the secretions were obtained in standard pore water (0.1 mM H⁺, 0.1 M K⁺, 0.2 M Na⁺, 0.35 mM Ca²⁺, 0.3 mM Mg²⁺, 0.3 mM NH₄ ⁺, 1.7 mM N0₃ ^~, and 0.3 mM Cl^", pH 6), with ionic concentrations simulating soil fluid. The secretion of Ag48 in control solutions was less for spontaneously hatched J2 than for J2 exposed to potato root diffusate. The enhancing effect of DMT on the secretion of Ag48 was significant. DMT did not harm the nematodes since they were still able to infect potato roots and develop into females as also described for H. glycines (Goverse et al, 1994). Description of the figures

Figure 1 shows a Western blot of second stage juveniles (J2) of Globodera rostochiensi stained with MAbs (MGR 46 to 60) specific to the subventral salivary glands. Four majo protein bands are identified, labelled SV-la, SV-lb, SV-2, and SV-3. MW Mark prestained molecular weigh markers. The arrow indicates a characteristic major protei band (presumably actin).

Figure 2 shows twodimensional electrophoresis and electroblotting on a PVDF membran of svp's using MAb MGR 48.

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Davis, E.L., Aron. L.M., Pratt, L.H. & Hussey, R.S. (1992). Phytopathology 82, 1244-50. De Almeida, E.R.P., Gossele, V., Muller C.G., Dockx, J., Reynaerts, A., Bottermarj, J.,

Krebbers, E. and Timko, M.P. (1989). Mol. Gen. Genet. 218, 78-86. De Boer, J.M., Overmars, H.A., Bakker, J. & Gommers,^* F.J. (1992a). Parasitology 105, 461-74.

De Boer, J.M., Overmars, H., Bouwman-Smits, L., De Boevere, M., Gommers, F.J., &

Bakker, J. (1992b). Fundamental and Applied Nematology 15, 495-501. D'Halluin, K.. Bonne, E., Bossut, M., DeBeuckeleer, M., and Leemans, J. (1992). Plant Cell 4, 1495-1506. Endo, B.Y. (1984). Proc. Helminthological Soc. Washington 51, 1-24. Endo, B.Y. (1987). Journal of Nematology 19, 469-483. Endo, B.Y. (1988). Proc. Helminthological Soc. Washington 55, 117-31. Firek, S., Draper, J., Owen, M.R.L., Gandecha, A., Cockburn, B., and Whitelam, G.C. (1993). Plant Mol. Biol. 23, 861-870. Goding, J.W. (1983). Monoclonal antibodies: principles and practice. London: Academic Press. Goverse, A., Davis, E.L. and Hussey, R.S. (1994). J. Nematology 26, 251-259. Guerche, P., Tire. C, Grossi De Sa, F., De Clercq, A., Van Montagu, and Krebbers, E. (1990). Plant Cell 2, 69-478. Hendriks, T.. Vreugdenhil, D., Stiekema, W.J. (1991). Plant Mol. Biol., 385-394. Horsch, R.B., Fry, J.E., Hoffmann, N.L., Eichholtz, N.L., Rogers, S.G., and Fraley, R.T.

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Zuckerman, B.M. & Jansson, H.-B. (1984). Ann. Rev. Phytopathology 22, 95-113. SEQUENCE LISTINGS

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Rijkslandbou universiteit Wageningen

(B) P.O. BOX: 9101

(C) CITY: Wageningen

(E) COUNTRY: The Netherlands

(F) POSTAL CODE: 6700 HB

(G) TELEPHONE: 0317--.89111

(ii) TITLE OF INVENTION: Antibodies for the control of cyst nematod and transgenic plants expressing them

(iii) NUMBER OF SEQUENCES: 4

(iv) COMPUTER-READABLE FORM:

(A) MEDIUM TYPE: Diskette

(B) COMPUTER: IBM-compatible

(C) OPERATING SYSTEM: MS-DOS

(D) SOFTWARE: WordPerfect/MS-DOS

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 31 amino acid residues

(B) TYPE: amino acid

^■(C) STRANDEDNESS: single '

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vi) ORIGINAL SOURCE: ^*

(A) ORGANISM: Heterodera glycines

(ix) FEATURE

(A) KEY: unknown amino acid

(B) LOCATION: 1

(ix) FEATURE

(A) KEY: unknown amino acid

(B) LOCATION: 2

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Xaa Xaa Ala Val Ala Pro Pro Phe Gly Gin Leu Ser Val Ser Gly Ser

5 ιo 15

Asn Lys Gin Pro Val Gin Leu lie Ser Asn Ser Leu Phe Glu His 20 25 30

(3) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 819 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Mouse (Mus musculus)

(ix) FEATURE:

(A) KEY: Antibody variable domain light chain

(B) LOCATION: 1-336

(ix) FEATURE:

(A) KEY: Complementarity Determining Region

(B) LOCATION: 73-117

(ix) FEATURE:

(A) KEY: Complementarity Determining Region

(B) LOCATION: 163-183

(ix) FEATURE:

(A) KEY: Complementarity Determining Region

(B) LOCATION: 280-306

(ix) FEATURE:

(A) KEY: Gly-Ser linker

(B) LOCATION: 337"38l

(ix) FEATURE:

(A) KEY: Antibody variable domain heavy chain

(B) LOCATION: 382-819

(ix) FEATURE:

(A) KEY: Complementarity Determining Region

(B) LOCATION: 472-486

(ix) FEATURE:

(A) KEY: Complementarity Determining Region

(B) LOCATION: 29- 79

(ix) FEATURE:

(A) KEY: Complementarity Determining Region

(B) LOCATION: 676-696

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

GAT GTT GTG ATG ACC CAA ACT CCA CTC ACT TTG TCG GTT ACC ATT GGA 48 Asp Val Val Met Thr Gin Thr Pro Leu Thr Leu Ser Val Thr He Gly

5 10 15

CAA CCA GCC TCC ATC TCT TGC AAG TCC AGT CAG AGC CTC TTA CAT AGT 96 Gin Pro Ala Ser He Ser Cys Lys Ser Ser Gin Ser Leu Leu His Ser 20 25 30 GAT GGA AAG ACA TAT TTG AGT TGG TTG TCA CAG AGG CCA GGC CAG TCG 1 Asp Gly Lys Thr Tyr Leu Ser Trp Leu Ser Gin Arg Pro Gly Gin Ser 35 40 45

CCA AAG CGC CTT ATC TAT CTG GTG TCT AAA CTG GAC TCT GGA GTC CCT 1 Pro Lys Arg Leu He Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60

GAC AGG TTC ACT GGC AGT GGA TCA GGG ACA GAT TTC ACA CTG AAA ATC 2 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys He 65 70 75 80

AGC AGA GTG GAG GCT GTG GAT TTG GGA GTT TAT TAT TGT TGG CAA GGA 2 Ser Arg Val Glu Ala Val Asp Leu Gly Val Tyr Tyr Cys Trp Gin Gly 85 90 95

ACA CAT CTT CCG TAC ACG TTC GGA GGG GGG ACC AAG CTG GAA ATA AAA 3 Thr His Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu He Lys 100 105 110

GGA GGT GGT GGT TCT GGA GGT GGA GGA TCT GGT GGA GGC GGA TCC GAG 3 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu 115 120 125

GTT CAG CTG CAG CAG TCT GGG CCT GAG CTC GTG AGG CCT GGG GCT TCA 43 Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Arg Pro Gly Ala Ser 130 135 140

GTG AAG ATG TCC TGC AAG GCT TCA GGC TAT ACC TTC ACC GAC TAT TGG 48 Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Trp 145 150 155 160

ATG CAC TGG GTG AAA CAG AGG CCT GGA CAA GGC CTT GAG TGG ATT GGC 52 Met His Trp Val Lys Gin Arg Pro Gly Gin Gly Leu Glu Trp He Gly 165 170 175

ATG ATT GAT CCT TCC AAT GGT GGG ACT TGG TTA AAT CAG CAG TTC AAG 57 Met He Asp Pro Ser Asn Gly Gly Thr Trp Leu Asn Gin Gin Phe Lys 180 185 190

GGC AAA GCA ACA TTG AAT GTG GCC AAA TCC TCC AAC ACA GCC TAC ATG 62 Gly Lys Ala Thr Leu Asn Val Ala Lys Ser Ser Asn Thr Ala Tyr Met 195 200 205

CAG CTC AGC AGC CTG ACG TCT GAG GAC TCT GCA GTC TAT TAC TGT TCA 67 Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ser 210 215 220

AGA AAC TCC CCC TGG TTT GCT TAC TGG GGC CAA GGG ACT CTG GTC ACA 72 Arg Asn Ser Pro Trp Phe Ala Tyr Trp Gly Gin Gly Thr Leu Val Thr 225 230 235 240

GTC TCT GCA GCC AAA ACG ACA CCC CCA TCG GTC TAT CCA CTG GTT GGG 76 Val Ser Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Val Gly 245 250 255 GCG GCC GCA GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG AAT GAT ATC 8l6 Ala Ala Ala Glu Gin Lys Leu He Ser Glu Glu Asp Leu Asn Asp He 260 265 270

TAG 819

(4) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 48 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA

(ix) FEATURE:

(A) KEY: linker peptide 202

(B) LOCATION: 1-48

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

CTC GAG GGT AAA TCC TCA GGA TCC GGC TCC GAA TCC AAA CTC GAG TCT 48 Leu Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Leu Glu Ser

5 10 15

(5) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 72 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Mouse (Mus musculus)

(ix) FEATURE:

(A) KEY: Signal peptide

(B) LOCATION: 1-72

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

ATG GGC ATC AAG ATG GAG ACA CAT TCT CAG GTC TTT GTA TAC ATG TTG 48 Met Gly He Lys Met Glu Thr His Ser Gin Val Phe Val Tyr Met Leu

5 10 15

CTG TGG TTG TCT GGT GTC GAC GGT 72

Leu Trp Leu Ser Gly Val Asp Gly 20

Claims

1. Antibody directed against a subventral salivary protein of a cyst nematode or at an immunogenic part or equivalent thereof, said protein having a molecular weight of 30- 50 kDa and having an isoelectric point (pi) of about 6.8 to 8.7.

2. Antibody according to claim 1, wherein said protein is selected from a protein of about 30 kDa having a pi of 7.6, a protein of about 31 kDa having a pi of 7.6, a protein of about 31 kDa having a pi of 7.9, a protein of about 32 kDa having a pi of 6.8, a protein of about 39 kDa having a pi of 7.3, and a protein of about 49 kDa having a pi of 8.7.

3. Antibody according to claim 1 or 2, wherein said protein comprises an amino acid sequence having a homology of at least 50% with the amino acid sequence depicted in

SEQ ID No. 1.

4. Antibody according to any one of the preceding claims, wherein said cyst nematode belongs to one of the. genera Heterodera, Globodera and Punctodera.

5. Antibody according to any one of the preceding claims, wherein said protein is derived from a second-stage juvenile of said nematode.

6. Antibody according to any one of the preceding claims, which comprises one or more variable domains of the complete antibody, such as a single chain antibody.

7. Antibody according to claim 6, which comprises a peptide corresponding to an amino acid sequence or encoded by a nucleotide sequence as depicted in SEQ ID No.2.

8. Polypeptide corresponding to the protein according to any one of the preceding claims or to a part thereof, in substantially isolated form.

9. Nucleotide sequence encoding a polypeptide according to claim 8.

10. Nucleotide sequence encoding a peptide corresponding to an antibody according to any one of claims 1-7, or to a part thereof.

11. Nucleotide according to claim 10, comprising the nucleotide sequence 1-336 and/or 391-828 as depicted in SEQ ID No.2.

12. Expression system comprising at least a nucleotide sequence according to claim

10 or 11 and a sequence regulating expression of said nucleotide sequence.

13. Process for protecting a plant against the action of a cyst nematode, wherein the plant is stably transformed using a nucleotide sequence according to claim 10 or 11 or an expression system according to claim 12.

14. Transgenic plant containing in its genome a nucleotide sequence according to claim 10 or 11, and capable of expressing said sequence.