WO1996022372A2 - Antibodies for the control of cyst nematodes and transgenic plants expressing them - Google Patents
Antibodies for the control of cyst nematodes and transgenic plants expressing them Download PDFInfo
- Publication number
- WO1996022372A2 WO1996022372A2 PCT/NL1996/000033 NL9600033W WO9622372A2 WO 1996022372 A2 WO1996022372 A2 WO 1996022372A2 NL 9600033 W NL9600033 W NL 9600033W WO 9622372 A2 WO9622372 A2 WO 9622372A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- protein
- antibody
- kda
- nucleotide sequence
- ser
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
- C07K14/4354—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8285—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for nematode resistance
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
Definitions
- 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.
- Globodera 13 species, including G. rostochiensis and G. pallida (potato), G. solanacearum and G. tabacum (tobacco); and Punctodera, 3 species, including P. punctata (grass).
- 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.
- 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; ⁇ ussey, 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 ( ⁇ ussey, 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).
- 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).
- the invention is primarily related to antibodies directed against a salivary protein of the nematode or at an immunogenic part or equivalent thereof.
- "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.
- 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.
- 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.
- the antibody may comprise the variable part of the light chain (V j and the variable part of the heavy chain (V ⁇ ), preferably fused by a linker peptide.
- V j variable part of the light chain
- V ⁇ variable part of the heavy chain
- 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.
- 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.
- 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 immunoge ⁇ ic 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.
- 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.
- CDR complementarity determining regions
- 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.
- 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.
- 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.
- 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.
- T-DNA includes the gene to be expressed.
- 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.
- Tissue electroporation (D'Hallui ⁇ 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
- 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 ⁇ m 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.
- ME 2-mercaptoethanol
- 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 ⁇ l 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 ⁇ l aliquots for successive immunisations.
- mice 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).
- 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).
- 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 ⁇ m. (MultiScreen-HV, Millipore Corp. Bedford, U.S.A.). To each well 20 ⁇ l of nematode suspension (containing approximately 200 sections) was added, followed by 80 ⁇ l of hybridoma culture supernatant or mouse antiserum diluted 1:1000 (v:v) in PBS.
- 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 ⁇ l of distilled water, and transferred to 24 well microscope slides (Cat Nr.
- 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).
- PVDF polyvinylidene difluoride
- 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 ⁇ g/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.
- 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.
- 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.
- 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.
- the 49 kDa fraction of H. glycines proteins was purified by immunoadso ⁇ tion 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 p ⁇ 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 J 2 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 p ⁇ 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 ⁇ g) 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- ⁇ Cl (p ⁇ 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- ⁇ Cl (p ⁇ 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 cm 2 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.
- 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.
- 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 mo ⁇ hology 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.
- Stylet secretion of J2 of G. rostochiensis was induced by incubation with the serotonin antagonist DMT (5-methoxy- ⁇ , ⁇ -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.
- 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.
- MAbs MGR 46 to 60
- 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.
- ORGANISM Heterodera glycines
- ORGANISM Mouse (Mus musculus)
- 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
- ORGANISM Mouse (Mus musculus)
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002241977A CA2241977A1 (en) | 1995-01-17 | 1996-01-17 | Antibodies for the control of cyst nematodes and transgenic plants expressing them |
EP96902500A EP0871731A2 (en) | 1995-01-17 | 1996-01-17 | Antibodies for the control of cyst nematodes and transgenic plants expressing them |
PCT/NL1996/000033 WO1996022372A2 (en) | 1995-01-17 | 1996-01-17 | Antibodies for the control of cyst nematodes and transgenic plants expressing them |
JP8522176A JPH11511644A (en) | 1995-01-17 | 1996-01-17 | Antibodies for controlling cyst nematodes and transgenic plants expressing them |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP95200110 | 1995-01-17 | ||
CA002241977A CA2241977A1 (en) | 1995-01-17 | 1996-01-17 | Antibodies for the control of cyst nematodes and transgenic plants expressing them |
PCT/NL1996/000033 WO1996022372A2 (en) | 1995-01-17 | 1996-01-17 | Antibodies for the control of cyst nematodes and transgenic plants expressing them |
Publications (2)
Publication Number | Publication Date |
---|---|
WO1996022372A2 true WO1996022372A2 (en) | 1996-07-25 |
WO1996022372A3 WO1996022372A3 (en) | 1996-09-19 |
Family
ID=27170748
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/NL1996/000033 WO1996022372A2 (en) | 1995-01-17 | 1996-01-17 | Antibodies for the control of cyst nematodes and transgenic plants expressing them |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP0871731A2 (en) |
CA (1) | CA2241977A1 (en) |
WO (1) | WO1996022372A2 (en) |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998001569A1 (en) * | 1996-07-08 | 1998-01-15 | Rijkslandbouwuniversiteit Wageningen | Cellulases |
US5990385A (en) * | 1997-11-10 | 1999-11-23 | Her Majesty The Queen In Right Of Canada, As Represented By The Minister Of Agriculture And Agri-Food | Protein production in transgenic alfalfa plants |
WO2000006754A2 (en) * | 1998-07-31 | 2000-02-10 | Centrum Voor Plantenveredelings-En Reproductieonderzoek (Cpro-Dlo) | Engineering nematode resistance in solanacae |
WO2002016625A2 (en) | 2000-08-25 | 2002-02-28 | Basf Plant Science Gmbh | Plant polynucleotides encoding prenyl proteases |
WO2017212127A1 (en) * | 2016-06-08 | 2017-12-14 | Denis Baron | Product for treating plants and combating certain pests, in particular aphids, and method for producing same |
FR3052336A1 (en) * | 2016-06-08 | 2017-12-15 | Philippe Baron D | PLANT TREATMENT PRODUCT AND FOR CONTROLLING CERTAIN BIOAGRESSORS, IN PARTICULAR SUCKS, AND MANUFACTURING METHODS |
FR3065621A1 (en) * | 2017-04-28 | 2018-11-02 | Denis Philippe Baron | PLANT TREATMENT AND CONTROL AGENT FOR CERTAIN BIOAGRESSERS, IN PARTICULAR PUCERONS, AND METHODS OF MAKING |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
NL8601642A (en) * | 1986-06-24 | 1988-01-18 | Rijkslandbouwhogeschool | New potato root eel-worm proteins - and antibodies useful for determn. of eel-worm populations in soil |
WO1992004453A1 (en) * | 1990-09-10 | 1992-03-19 | The University Of Leeds | Plant parasitic nematode control |
-
1996
- 1996-01-17 EP EP96902500A patent/EP0871731A2/en not_active Withdrawn
- 1996-01-17 WO PCT/NL1996/000033 patent/WO1996022372A2/en not_active Application Discontinuation
- 1996-01-17 CA CA002241977A patent/CA2241977A1/en not_active Abandoned
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
NL8601642A (en) * | 1986-06-24 | 1988-01-18 | Rijkslandbouwhogeschool | New potato root eel-worm proteins - and antibodies useful for determn. of eel-worm populations in soil |
WO1992004453A1 (en) * | 1990-09-10 | 1992-03-19 | The University Of Leeds | Plant parasitic nematode control |
Non-Patent Citations (13)
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998001569A1 (en) * | 1996-07-08 | 1998-01-15 | Rijkslandbouwuniversiteit Wageningen | Cellulases |
US5990385A (en) * | 1997-11-10 | 1999-11-23 | Her Majesty The Queen In Right Of Canada, As Represented By The Minister Of Agriculture And Agri-Food | Protein production in transgenic alfalfa plants |
WO2000006754A2 (en) * | 1998-07-31 | 2000-02-10 | Centrum Voor Plantenveredelings-En Reproductieonderzoek (Cpro-Dlo) | Engineering nematode resistance in solanacae |
WO2000006753A1 (en) * | 1998-07-31 | 2000-02-10 | Van Der Vossen, Edwin, Andries, Gerard | Engineering nematode resistance in solanacae |
WO2000006754A3 (en) * | 1998-07-31 | 2000-04-27 | Cpro Dlo | Engineering nematode resistance in solanacae |
WO2002016625A2 (en) | 2000-08-25 | 2002-02-28 | Basf Plant Science Gmbh | Plant polynucleotides encoding prenyl proteases |
WO2017212127A1 (en) * | 2016-06-08 | 2017-12-14 | Denis Baron | Product for treating plants and combating certain pests, in particular aphids, and method for producing same |
FR3052336A1 (en) * | 2016-06-08 | 2017-12-15 | Philippe Baron D | PLANT TREATMENT PRODUCT AND FOR CONTROLLING CERTAIN BIOAGRESSORS, IN PARTICULAR SUCKS, AND MANUFACTURING METHODS |
FR3065621A1 (en) * | 2017-04-28 | 2018-11-02 | Denis Philippe Baron | PLANT TREATMENT AND CONTROL AGENT FOR CERTAIN BIOAGRESSERS, IN PARTICULAR PUCERONS, AND METHODS OF MAKING |
Also Published As
Publication number | Publication date |
---|---|
CA2241977A1 (en) | 1996-07-25 |
WO1996022372A3 (en) | 1996-09-19 |
EP0871731A2 (en) | 1998-10-21 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
DE69926647T2 (en) | MOLECULAR PATHOGENICIDE-MEDIATED DISEASE RESISTANCE IN PLANTS | |
US6121436A (en) | Antifungal polypeptide and methods for controlling plant pathogenic fungi | |
JP2000508529A (en) | Antifungal polypeptides and methods for controlling phytopathogenic fungi | |
AU742709B2 (en) | Proteins having insecticidal activities and method of use | |
KR20000022313A (en) | Aatt promoter element and transcription factor | |
EP0871731A2 (en) | Antibodies for the control of cyst nematodes and transgenic plants expressing them | |
CA2337093A1 (en) | Receptor-like protein kinase, rkn, and methods of use for increasing growth and yield in plants | |
WO2011023522A1 (en) | Antibody fusion-mediated plant resistance against oomycota | |
US6586583B1 (en) | Soybean peroxidase gene family and an assay for detecting soybean peroxidase activity | |
CN114196651B (en) | New application of D6 protein kinase D6PKL2 | |
WO2001016144A2 (en) | Plant derived calcium-dependent calmodulin-binding polypeptides and polynucleotides encoding same | |
EP0818538A1 (en) | Novel cellulases | |
JPH11511644A (en) | Antibodies for controlling cyst nematodes and transgenic plants expressing them | |
US20030177526A1 (en) | Receptors for hypersensitive response elicitors and uses thereof | |
Smant | Endogenous cellulases in stylet secretions of cyst nematodes | |
MXPA01003718A (en) | Molecular pathogenicide mediated plant disease resistance | |
EP1711519A1 (en) | Proteins inducing multiple resistance of plants to phytopathogens and pests | |
MXPA99010882A (en) | Proteins having insecticidal activities and method of use | |
AU2005215825A1 (en) | Antifungal peptides |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A2 Designated state(s): CA JP US |
|
AL | Designated countries for regional patents |
Kind code of ref document: A2 Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE |
|
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
AK | Designated states |
Kind code of ref document: A3 Designated state(s): CA JP US |
|
AL | Designated countries for regional patents |
Kind code of ref document: A3 Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
WWE | Wipo information: entry into national phase |
Ref document number: 1996902500 Country of ref document: EP |
|
ENP | Entry into the national phase |
Ref document number: 2241977 Country of ref document: CA Ref country code: CA Ref document number: 2241977 Kind code of ref document: A Format of ref document f/p: F |
|
ENP | Entry into the national phase |
Ref country code: JP Ref document number: 1996 522176 Kind code of ref document: A Format of ref document f/p: F |
|
WWP | Wipo information: published in national office |
Ref document number: 1996902500 Country of ref document: EP |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 1996902500 Country of ref document: EP |