WO2001000840A1

WO2001000840A1 - Insecticidal and nematicidal proteins

Info

Publication number: WO2001000840A1
Application number: PCT/GB2000/002453
Authority: WO
Inventors: Didier Fournier; Laurent Paquereau; Alain Klaebe; Louis Chavant
Original assignee: Syngenta Limited
Priority date: 1999-06-24
Filing date: 2000-06-23
Publication date: 2001-01-04
Also published as: WO2001000840A9; AU5553000A; AR024483A1

Abstract

Pesticidal proteins obtainable from mushroom including the protein depicted as SEQ ID No. 1. In particular, these proteins are obtainable from Xerocomus chrysenteron. Such proteins exhibit insecticidal and/or nematicidal properties. The present invention also provides polynucleotide sequences encoding said proteins in particular sequences depicted as SEQ ID No. 2 and 3.

Description

INSECTICIDAL AND NEMATICIDAL PROTEINS

The present invention relates inter alia, to insecticidal and nematicidal proteins, DNA sequences encoding the proteins and transformed plants containing them. More specifically the proteins according to the present invention may be isolatable from mushrooms such as Xerocomus chrysenteron.

Some insecticidal small molecules including amatoxins and phallotoxins, which are known to be toxic to insects, have been isolated from mushrooms such as Amanita phalloides (Jaenike et al, 1983 ; Ying et al, 1987). Other insecticidal small molecules include Ibotenic acid found in the fungi A. muscaria, A. pantherina and A. stroberiformis (Takemoto et al, 1964); L-DOPA from Strobilomyces floccopus and Hygrocybe conica (Steglich & Esser, 1973; Steglich & Preuss, 1974) and the nucleoside named clitocine from Lepista inversa (K bo et al, 1986).

The term "insecticidal small molecules" includes molecules and compounds such as those referred to above and which are less than 10 kDa in size but does not include proteins and macromolecules.

It has surprisingly been found that most of the insecticidal properties of mushrooms originate from proteins and not from insecticidal small organic molecules. It has also unexpectedly been found that such proteins also exhibit nematicidal properties as well as other pesticidal properties. Such proteins include lectins, serpins and hemolysins.

Many lectins have been isolated from species such as plants, animals and microorganisms and the mechanisms of action of lectins having some insecticidal activity has been studied in some detail. For example, Powell et al (1998) suggested that lectin bound to cell surface carbohydrate moieties in the gut, crossed the midgut epithelial barrier, passed into the insect circulatory system and induced a toxic effect. Zhu-Salzman et al. (1998) showed that the carbohydrate binding of the G. simplicifolia lectin was involved in insecticidal activity. In addition to this, EP 0542 833 B l describes the mannose binding ability and the insecticidal nature of specific plant lectins.

The present invention seeks to provide inter alia, proteins which are isolatable from mushroom and have particularly strong activity against insects, nematodes and other pests. According to the present invention there is provided a protein comprising the amino acid sequence depicted as SEQ ID No. 1 or a variant having at least about 60% identify therewith wherein said protein or variant is insecticidal and/or nematicidal. In a further embodiment of the present invention the variant insecticidal and/or nematicidal protein has at least 61% identity to the protein depicted as SEQ ID No. 1. In a still further embodiment of the present invention the variant insecticidal and/or nematicidal protein has at least 65% identity to the protein depicted as SEQ ID No. 1. In a still further embodiment of the present invention the variant insecticidal and/or nematicidal protein has at least 70% identity to the protein depicted as SEQ ID No. 1. In a still further embodiment of the present invention the variant insecticidal and/or nematicidal protein has at least 75% identity to the protein depicted as SEQ ID No. 1. In a still further embodiment of the present invention the variant insecticidal and/or nematicidal protein has at least 80% identity to the protein depicted as SEQ ID No. 1. In a still further embodiment of the present invention the variant insecticidal and/or nematicidal protein has at least 85% identity to the protein depicted as SEQ ID No. 1. In a still further embodiment of the present invention the variant insecticidal and/or nematicidal protein has at least 90% identity to the protein depicted as SEQ ID No. 1. In a still further embodiment of the present invention the variant insecticidal and/or nematicidal protein has at least 91% identity to the protein depicted as SEQ ID No. 1. In a still further embodiment of the present invention the variant insecticidal and/or nematicidal protein has at least 92% identity to the protein depicted as SEQ ID No. 1. In a still further embodiment of the present invention the variant insecticidal and/or nematicidal protein has at least 93% identity to the protein depicted as SEQ ID No. 1. In a still further embodiment of the present invention the variant insecticidal and/or nematicidal protein has at least 94% identity to the protein depicted as SEQ ID No. 1. In a still further embodiment of the present invention the variant insecticidal and/or nematicidal protein has at least 95% identity to the protein depicted as SEQ ID No. 1. In a still further embodiment of the present invention the variant insecticidal and/or nematicidal protein has at least 96% identity to the protein depicted as SEQ ID No. 1. In a still further embodiment of the present invention the variant insecticidal and/or nematicidal protein has at least 97% identity to the protein depicted as SEQ ID No. 1. In a still further embodiment of the present invention the variant insecticidal and/or nematicidal protein has at least 98% identity to the protein depicted as SEQ ID No. 1. In a still further embodiment of the present invention the variant insecticidal and/or nematicidal protein has at least 99% identity to the protein depicted as SEQ ID No. 1. The percentage of sequence identity for proteins is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the amino acid sequence in the comparison window may comprise additions or deletions (i.e. gaps) as compared to the initial reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of match positions, dividing the number of match positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. When calculating the percentage sequence identity the sequences may be aligned allowing for up to 3 gaps with the proviso that in respect of the gaps, a total of not more than 15 amino acid residues is affected. Optimal alignment of sequences for comparison may also be conducted by computerised implementations of known algorithms. In a particular embodiment of the present invention the sequence identity is calculated using the FASTA version 3 algorithm which uses the method of Pearson and Lipman (Lipman, D.J. and Pearson, W.R. (1985) Rapid and sensitive protein similarity searches and Science. 227:1435- 1441 and Pearson, W.R. and Lipman, D.J. (1988) Improved tools for biological sequence comparison. PNAS. 85:2444-2448) to search for similarities between the reference sequence (also termed the query sequence) and any group of sequences (termed further sequences). There are also further algorithms available to the person skilled in the art which enable a calculation of the percentage sequence identity between polynucleotide sequences. The protein variant may differ from the basic insecticidal/nematicidal protein sequence (such as SEQ ID No. 1) by conservative or non-conservative amino acid substitutions. A conservative substitution is to be understood to mean that the amino acid is replaced with an amino acid with broadly similar chemical properties. In particular conservative substitutions may be made between amino acids with the following groups: (i) Alanine and Glycine;

(ii) Serine and Threonine;

(ii) Glutamic acid and Aspartic acid;

(iii) Arginine and Lysine;

(iv) Asparagine and Glutamine; (v) Isoleucine and Leucine,

(vi) Valine and Methionine;

(vii) Phenylalanine and Tryptophan. In general, more conservative than non-conservative substitutions will be possible without destroying the insecticidal and/or nematicidal properties of the proteins. Suitable variants may be determined by testing insecticidal and/or nematicidal properties of the peptide using routine methods which are well known to the person skilled in the art. The present invention further provides a polynucleotide encoding a protein or variant as described above.

The present invention still further provides a polynucleotide sequence which is the complement of one which hybridises to a polynucleotide as described above at a temperature of about 65°C in a solution containing 6xSSC, 0.01% SDS and 0.25% skimmed milk powder, followed by rinsing at the same temperature in a solution containing 0.2xSSC and 0.1% SDS wherein the said polynucleotide sequence still encodes a protein having insecticidal and/or nematicidal properties. In a further embodiment of the present invention the polynucleotide comprises the sequence depicted as SEQ ID No. 2 or SEQ ID No. 3. The present invention still further provides a method of evolving a polynucleotide which encodes a protein having insecticidal and/or nematicidal properties comprising:

(a) providing a population of variants of said polynucleotide and further polynucleotides which encode further proteins, at least one of which is in cell free form; and

(b) shuffling said variants and further polynucleotides to form recombinant polynucleotides; and (c) selecting or screening for recombinant polynucleotides which have evolved towards the said insecticidal and/or nematicidal properties; and (d) repeating steps (b) and (c) with the recombinant polynucleotides according to step (c) until an evolved polynucleotide which encodes a protein having insecticidal and/or nematicidal properties has been acquired wherein said population of variants in part (a) contains at least one polynucleotide as described in the preceding paragraph. The methods for evolving a polynucleotide as described above are well known to the person skilled in the art and are described inter alia, in US Patent No. 5,811,238.

The present invention still further provides a polynucleotide obtained or obtainable by the method of the preceding paragraph and a protein encoded by any such polynucleotide. In a further aspect of the present invention there is provided a method of isolating a polynucleotide encoding an insecticidal and/or nematicidal protein from a Xerocomus sp. DNA library comprising: (a) constructing a DNA library of Xerocomus sp. or a particular strain thereof preferably Xerocomus chrysenteron; and (b) probing said library with at least one oligonucleotide probe which is capable of detecting said polynucleotide present in said library wherein said probe is derived from at least one peptide sequence selected from the group consisting of SEQ ID Nos. 4-8; and (c) identifying and isolating the thus detected polynucleotide. Once in possession of the sequences depicted as SEQ ID Nos. 4 to 8 the skilled man may generate oligonucleotide probes which are capable of identifying the insecticidal and nematicidal proteins from a DNA library of Xerocomus sp. General techniques for DNA library construction, library screening, identification and cloning of polynucleotides encoding the insecticidal and/or nematicidal proteins according to the present invention are all known to the person skilled in the art. The present invention still further provides an isolated polynucleotide obtainable by a method of the preceding paragraph and an insecticidal and/or nematicidal protein encoded by said isolated polynucleotide.

The present invention still further provides a protein or variant as described above which has a molecular weight of greater than or equal to 10 kDa. Preferably the protein or variant is a lectin. Lectins are generally accepted in the art as being proteins which bind carbohydrates.

The present invention still further provides a DNA construct comprising in sequence a plant operable promoter operably linked to a polynucleotide encoding a protein as described above operably linked to a transcription termination region. In a further embodiment of the present invention the DNA construct may further comprise a region which provides for the targeting of the protein product to a particular location. For example if it is desired to provide the protein outside of the cell then an extracellular targeting sequence may be ligated to the polynucleotide encoding the protein of the present invention. Other examples of targeting include targeting to a specific intracellular organelle or compartment such as a chloroplast, vacuole, mitochondrion or lipoxisome.

The present invention still further provides a DNA construct as described above which further comprises a region which provides for the production of a selectable marker. The selectable marker may, in particular, confer resistance to kanamycin; hygromycin or gentamycin. Further suitable selectable markers include genes which confer resistance to herbicides such as glyphosate based herbicides or resistance to toxins such as eutypine.

Other forms of selection are also available such as hormone based selection systems such as the Multi Auto Transformation (MAT) system of Hiroyrasu Ebinuma et al. 1997. PNAS Vol. 94 pp21 17-2121; visual selection systems which use the known green fluorescence protein, β glucoronidase and any other selection system such as mannose isomerase, xylose isomerase and 2-DOG.

The present invention still further provides a DNA construct as described above wherein the plant operable promoter is selected from the group consisting of: CaMV35S, FMV35S, NOS, OCS, Patatin, E9, alcA/alcR switch, GST switch, RMS switch, oleosin, ribulose bisphosphate carboxylase-oxygenase small sub-unit, actin 7 or root specific promoters including MR7 promoter (maize), Gos 9 (rice), GOS2 promoters, MasOcs (or super promoter) and the Agrobacteήum rhizogenes RolD promoter. Terminators which can be used in the constructs according to the present invention include Nos, proteinase inhibitor II and the terminator of a gene of alpha-tubulin (EP-A 652,286). It is equally possible to use, in association with the promoter regulation sequence, other regulation sequences which are situated between the promoter and the sequence encoding the protein according to the present invention, such as transcriptional or translational enhancers, for example, tobacco etch virus (TEV) translation activator described in International Patent application, PCT publication number WO87/07644. The polynucleotide encoding the insecticidal and/or nematicidal protein according to the invention may also be codon-optimised, or otherwise altered to enhance for example, transcription once it is incorporated into plant material.

The present invention still further provides a method of providing a plant or plant part with an insecticidal and/or nematicidal protein comprising: (a) inserting into the genome of plant material, a polynucleotide encoding a protein or a DNA construct as described above; and (b) regenerating plants or plant parts therefrom; and (c) selecting those plants or plant parts having said protein. The said polynucleotide/DNA construct may be incorporated into the cells by plant transformation techniques which are well known to the person skilled in the art. Such techniques include but are not limited to particle mediated biolistic transformation, Agrobacterium- ediatcd transformation, protoplast transformation (optionally in the presence of polyethylene glycols); sonication of plant tissues, cells or protoplasts in a medium comprising the polynucleotide or vector; micro-insertion of the polynucleotide or vector into totipotent plant material (optionally employing the known silicon carbide "whiskers" technique), electroporation and the like.

The present invention still further provides plants or plant parts obtained according to the method of the preceding paragraph. Preferably the plants or plant parts of the present invention are selected from the group consisting of: melons, mangoes, soybean, cotton, tobacco, sugarbeet, oilseed rape, canola, flax, sunflower, potato, tomato, alfalfa, lettuce, maize, wheat, sorghum, rye, bananas, barley, oat, turf grass, forage grass, sugar cane, pea, field bean, rice, pine, poplar, apple, peaches, grape, strawberries, carrot, lettuce, cabbage, onion, citrus, cereal or nut plants or any other horticultural crops.

The present invention still further provides plants or plant parts as described above which comprise a further agronomic trait selected from the group consisting of: herbicide resistance; insect resistance; nematode resistance; altered stress tolerance; altered yield; altered nutritional content or any desired agronomic trait. In a further embodiment of the present invention the further agronomic trait provides resistance to a herbicide which comprises glyphosate acid or agriculturally acceptable salt thereof.

In a further aspect of the present invention there is provided the use of a polynucleotide encoding a protein as described above or a DNA construct as described above in a method of producing plants which are resistant and/or tolerant to insects and/or nematodes.

In a further aspect of the present invention there is provided the use of a Xerocomus sp. protein extract as an active ingredient in the production of an insecticide and/or nematicide. In a still further embodiment of the present invention the Xerocomus sp. is Xerocomus chrysenteron and the protein extract comprises a protein or variant as described above. In a still further embodiment of the present invention the extract further comprises an agriculturally acceptable carrier and/or a diluent and may be formulated for use as a spray.

In a still further aspect of the present invention there is provided the use of a protein or variant as described above as an insecticide and/or nematicide.

In a still further aspect of the present invention there is provided a method of controlling insects and/or nematodes comprising providing at a locus where said insects and/or nematodes feed a protein, variant or an extract as described above.

The present invention still further provides an insecticidal and/or nematicidal protein obtained or obtainable from the mushroom Xerocomus sp. preferably Xerocomus chrysenteron. The present invention still further provides a composition comprising an insecticidally and/or nematicidally effective amount of a protein, variant or an extract as described above and optionally an agriculturally acceptable carrier and/or a diluent and/or an insect/nematode attractant.

The present invention still further provides a plant cell comprising a protein or variant as described above. The present invention still further provides an insecticidal and/or nematicidal synergistic combination comprising a protein or variant as described above and a further protein. In a further embodiment of the invention said protein comprises the sequence depicted as SEQ ID No 1 and said further protein is insecticidal and/or nematicidal. In a still further embodiment of the present invention said further protein is a variant as described above. In a still further embodiment of the invention the synergistic combination comprises a first protein and a further protein both of which are different variant proteins as described above. In a still further embodiment of the present invention the said further protein comprises an insecticidal CRY protein or a vegetative insecticidal protein (VIPs). Preferably said further protein is selected from the group consisting of: cry Hal (Embl. Accession No. X62821); crylla2 (Embl. Accession No. M98544); crylla3 (Embl. Accession No. L36338); crylla4 (Embl. Accession No. L49391); crylla5 (Embl. Accession No. Y08920) and cryllbl (Embl. Accession No. U07642).

The present invention still further provides a polynucleotide having a first region encoding a protein or variant as described above and a second region encoding a further protein. The regions may be separated by a region which provides for a self processing polypeptide which is capable of separating the proteins such as the self processing polypeptide described in US5,846,767 or any similarly functioning element. Alternatively the regions may be separated by a sequence which acts as a target site for an external element which is capable of separating the protein sequences. Alternatively the polynucleotide may provide for a polyprotein which comprises a plurality of protein functions. In a further embodiment of the present invention the proteins of the polyprotein may be arranged in tandem. These polyproteins may comprise the proteins and/or variants according to the present invention and optionally further proteins such as those encoding any desired agronomic trait. In a further aspect of the present invention there is provided an insecticidal and/or nematicidal protein having a FASTA opt score greater than 489 when compared with SEQ ID No. 1 calculated using FASTA V.3. In a further embodiment of the present invention the insecticidal and/or nematicidal protein has a FASTA opt score greater than 490 when compared with SEQ ID No. 1 calculated using FASTA V.3. In a still further embodiment of the present invention the insecticidal and/or nematicidal protein has a FASTA opt score greater than 495 when compared with SEQ ID No. 1 calculated using FASTA V.3. In a still further embodiment of the present invention the insecticidal and/or nematicidal protein has a FASTA opt score greater than 500 when compared with SEQ ID No. 1 calculated using FASTA V.3. In a still further embodiment of the present invention the insecticidal and/or nematicidal protein has a FASTA opt score greater than 515 when compared with SEQ ID No. 1 calculated using FASTA V.3. The FASTA opt score may be calculated using the FASTA algorithm as described above. After computing the initial scores, FASTA determines the best segment of similarity between the query sequence and the further sequences using a the Smith- Waterman algorithm (Smith, T.F. and Waterman, M.S. (1981) Comparison of biosequences. Adv. Appl. Math. 2:482-489). The output is presented in the form of an opt score and this procedure is well known to the person skilled in the art. The present invention still further provides an insecticidal and/or nematicidal protein or variant as described above having a specific galactose and/or lactose binding ability. In a further embodiment of the present invention the protein or variant can bind galactose at levels greater than said protein or variants ability to bind mannose. In a still further embodiment of the present invention the protein or variant can bind lactose at levels greater than said protein or variants ability to bind mannose. In a still further embodiment of the present invention the protein or variant can bind lactose at levels greater than said protein or variants ability to bind galactose.

The present invention still further provides an insecticidal and/or nematicidal protein obtainable from mushroom and having a specific galactose and/or lactose binding ability. The mushroom may be selected from the group consisting of: Xerocomus chrysenteron; Clitocybe geotropa; Tricholoma rutilans; Tryomyces sulfureus; Lepista nuda; Amanita ovoidea; Amanda phalloides; Clitocybe inversa; Cortinarius venetus; Entoloma lividum; Ganderma lucidium; Laccaria amethystea; Lacca a laccata and Leucopaxillus giganteus. The present invention still further provides a recombinant micro-organism which provides for production of a protein or variant as described above. In a further embodiment of the invention the recombinant micro-organism is an endophyte or a Pseudomonas sp. An endophyte is generally accepted within the art as a micro-organism having the ability to enter into non-pathogenic endosymbiotic relationships with a plant host. A method of endophyte-enhanced protection of plants has been described in a series of patent applications by Crop Genetics International Corporation (for example, International Application Publication Number WO90/13224, European Patent Publication Number EP-125468-B 1, International Application Publication Number WO91/10363, International Application Publication Number WO87/03303). International Patent Application Publication Number WO94/16076 (ZENECA Limited) describes the use of endophytes which have been genetically modified to express a plant-derived insecticidal peptide.

The present invention still further provides a recombinant baculovirus which provides for production of a protein or variant as described above.

In a further aspect of the present invention there is provided the use of a recombinant micro-organism or a baculovirus as described above in a method of controlling insects.

In a further aspect of the present invention there is provided an insecticidal and/or nematicidal protein which is capable of reacting with a monoclonal antibody raised to the protein depicted as SEQ ID No. 1. The present invention still further provides an insecticidal and/or nematicidal protein which is capable of reacting with a polyclonal antibody raised to the protein depicted as SEQ ID No. 1. Such antibodies may be generated and used to identify other proteins within the ambit of the present invention according to well known techniques within the art. The present invention still further provides for the use of a protein or variant as described above in a method of detection, isolation and characterisation of specific glycans. The present invention still further provides an insecticidal and/or nematicidal protein obtainable from mushroom which protein has a molecular weight greater than or equal to 10 kDa and comprises at least one amino acid selected from the group consisting of: Asparagine; Cysteine; Glutamine; Histidine and Tryptophan. In a further embodiment of the present invention the insecticidal and/or nematicidal protein is obtainable from Xerocomus sp. preferably Xerocomus chrysenteron.

The nematodes to be controlled by the proteins and variants of the present invention include but are not limited to: Heterodera sp.; H. schachtii; Meliodogyne sp.; M. incognita; Globodera sp. including G rostochiensis; G. pallida; Tylenchulus sp.; Rotylenchulus sp.; Xiphinema sp.; Longidorus sp.; Trichodorus sp.; Paratrichodorus sp.; Scutellonema sp.; Helicotylenchus sp.; Pratylenchus sp.; Ditylenchus sp.; Radolpholus sp.; The insects ro be controlled by the proteins and variants of the present invention include the plant chewing insects and the plant chewing stages of insects including: Coleoptera, Lepidoptera, Orthoptera and Drosophila, including, but not limited to: Ac anthosc elides obtectus, Bruchus sps., Callosobruchus sps. (bruchid beetles), Agriotes sps. (wireworms), Amphimallon sps. (chafer beetles), Anthonomus grandis (cotton boll weevil), Ceutorhynchus assimilis (cabbage seed weevil), Cylas sps. (sweet potato weevils), Diabrotica sps. (corn root worms), Epicauta sps. (black blister beetles), Epilachna sps. (melon beetles etc.), Leptinotarsa decemlineata (Colorado potato beetle) Meligisthes sps. (blossom beetles), Melolontha sps. (cockchafers), Phyleotreta sps., Psylliodes sps. (flea beetles), Popilliajaponica (Japanese beetle), Scolytus sps. (bark beetles), Sitophilus sps. (grain weevils), Tenebrio molitor (yellow mealworm),

Tribolium sps. (flour beetles), Trogoderma granarium (Khapra beetle), Acleris sps. (fruit tree tortrixs), Acraea acerata (sweet potato butterfly), Agrotis sps. (cutworms), Autographa gamma (silver-Y moth), Chilo sps. (stalk borers), Cydia pomonella (codling moth), Diparopsis sps. (red bollworms), Ephestia sps. (warehouse moths), Heliothis sps., Helicoverpa sps. (budworms, bollworms), Mamestra brassicae (cabbage moth), Manduca sps. (hornworms), Maruca testulalis (mung moth), Mythimna sps. (cereal army worms), Ostrinia nubilalis (European corn borer), Pectinophora gossypiella (pink bollworm), Phthorimaea operculella (potato tuber moth), Pieris brassicae (large white butterfly), Pieris rapae (small white butterfly), Plodia interpunctella (Indian grain moth), Plutella xylostella (diamond-back moth), Sitatroga cerealella (Angoumois grain moth), Spodoptera sps.

(armyworms), Trichoplusia ni (cabbage semilooper), Acheta sps. (field crickets), Gryllotalph sps. (mole crickets), Locusta migratoria (migratory locust), Schistocerca gregaria (desert locust), Acrythosiphon pisum and Drosophila melanogaster.

The invention will now be described by way of the following non-limiting examples in combination with the following figures and sequence listing of which:

FIGURE 1 - Shows an SDS-PAGE showing the effect of pronase digestion on proteins of some crude extracts.

FIGURE 2 - Shows a resulting band on a gel after purifying a lectin from X. chrysenteron by affinity chromatography. Reference proteins were lysozyme (M_r 14,300), soybean trypsin inhibitor (M_R20 100), carbonic anhydrase (M_r 29,000), ovalbumin (M_r 45,000) and bovine serum albumin (M_r 66 000) FIGURE 3 - Shows X. chrysenteron lectin toxicity on D. melanogaster. (■) X. chrysenteron

LDso: 0.38 mg/ml, (^A) G. « vα/w LD₅₀: 0.72 mg/ml, (▼) . ochrus LD₅₀: 8.51 mg/ml.

FIGURE 4 - shows the percentage of growth inhibition of X chrysenteron lectin on A. pisum.

FIGURE 5 - shows the percentage of M. incognita immobilised larvae by X. chrysenteron lectin. LD₅₀:0.014 mg/ml.

FIGURE 6 - Shows the genomic, cDNA and protein sequences.

FIGURE 7 - Shows the vector pMOG1467.

SEQ ID No. l - Insecticidal/nematicidal protein obtainable from Xerocomus chrysenteron.

SEQ ID No. 2 - Genomic DNA encoding SEQ ID No. 1 SEQ ID No. 3 - cDNA encoding SEQ ID No. 1.

SEQ ID Nos. 4 to 8 - Peptide sequences horn Xerocomus chrysenteron.

SEQ ID No. 9 - polynucleotide codon optimised.

SEQ ID Nos. 10 to 12 - Oligonucleotide primers.

SEQ ID No. 13 - cDNA of region upstream of the sequence encoding SEQ ID No. 1. SEQ ID No. 14 - cDNA of region downstream of the sequence encoding SEQ ID No. 1.

EXAMPLES Example 1

Preparation of crude extract. Fruitbodies of 14 species studied were collected in South- western France and kept frozen at -20°C until used. Fruitbodies were crushed in distilled water (1/1.5, W/V) and centrifuged 20 minutes at 6500g to remove insoluble materials. In order to study the extraction of toxicity in water, the pellet was successively crushed in the same volume of water for three times.

Example 2

Water solubility. The pellets and supernatants which originated from the same amount of material homogenised in water, were compared for their toxicity against D. melanogaster development (see Table 1 below). Most of the insecticidal activities were recovered in the supernatants suggesting that toxic compounds were soluble in aqueous solutions. Toxicity found in the first pellet were extractable by additional dissolution in water showing that toxicity remaining in the pellet was usually co-precipitated. Proteins, lectins, hemolysins and serpins estimated in each samples are presented in Table 2. Table 1 : Water solubility of the active principles (TU : toxicity unit).

Name of species Toxicity in Toxicity in Toxicity in Percent of Percent of the I^s' the r¹ the 3^rd toxicity toxicity supernatant pellet pellet extracted extracted (TU in mf¹) (TU in mr¹) (TU in ml-¹) by one by three crush crushes

Amanda phalloϊdes 500 17.5 0.7 97 99.9 Xerocomus chrysenteron 278 16.7 16.7 94 94

Xerocomus subtomentosus 70 12.8 9.4 85 89

Lepista nuda 49 12.3 2.8 80 95 Xerocomus badius 23 13.3 3.7 63 90

Boletus speciocus 22 9.1 2.9 71 90 Hygrophoropsis aurantiaca 16 8.3 0.5 66 98 Clitocybe nebularis 13 0.8 0.3 94 98

Polyporus squamosus 10 1.4 1.4 88 88 Boletus aereus 9.7 2.0 1.8 83 85

Clitopilus prunulus 9.5 4.4 1.8 68 87 Albatrellus cristatus 7.6 2.2 1.7 78 83

Gyrophana lacrymans 6.9 1.6 0.5 81 94 Hygrophorus chrysodon 3.2 0.3 0.1 91 97

Table 2: Proteins extracted in each mushroom.

Name of species Proteins Lectins Hemolysins Serpins mg/ml UA (mf¹) UH (mf¹) UI (ml^"1)

Amanita phalloϊdes 1.6 <640 640 0

Xerocomus chrysenteron 5.3 1280 160 12

Xerocomus subtomentosus 10 640 0 0

Lepista nuda 3.8 320 20 0

Xerocomus badius 8.6 320 80 0

Boletus speciocus 1.4 640 40 0

Hygrophoropsis aurantiaca 3.9 <20 20 0

Clitocybe nebularis 4.8 320 20 3

Polyporus squamosus 5 <320 320 0

Boletus aereus 4.2 1280 20 3

Clitopilus prunulus 5.3 <40 40 3

Albatrellus cristatus 3.3 160 40 0

Gyrophana lacrymans 2.3 <10 10 0

Hygrophorus chrysodon 1.2 1280 40 0 Example 3

Toxicological test for D. melanogaster larvae. Determination of toxicity was performed by ingestion. Several amounts of each extract were added to 1ml rearing medium (10 % yeast, 2% agar, 0.5% p-hydroxybenzoic acid) before pouring in 5 ml tubes. 10 eggs of Canton S strain were deposited on the medium and the tubes were maintained at 23-25°C for two weeks to allow complete larval development. At the end, the numbers of pupae were recorded in each tube, corresponding to different concentrations of the extract. In the reference assay, without extract, 70 to 100 % eggs developed to pupae. Corrected mortality due to the added extract was estimated using the Abbott formula : mortality = (mortality in the sample - mortality in the reference) / (1 - mortality in the reference). LD50 values were determined by fitting log of concentration versus mortality data to sigmoid curves by nonlinear regression and was expressed in ml of crude extract. Toxicity units (TU) corresponds to the inverse of LD₅o, and indicates the number of LD₅₀ in one millilitre of crude extract. Protein concentrations of extracts were estimated using Bradford reagent and bovine serum albumin as reference.

Example 4

Agglutination and haemolysis assays (from human blood). The erythrocytes were washed three times in 10 mM phosphate buffer pH 7, 145 mM NaCl (PBS) by centrifugation (3500 rpm, 20 min.). The pellet obtained was used to prepare a suspension of 4% (v/v) erythrocytes in PBS. Determination of the agglutination activity was carried out in a final volume of 225 μl. The sample was serially diluted in PBS, with two fold increments for obtaining a volume of 200 μl. 25 μl of the suspension of erythrocytes was then added. The tubes had been left for two hours at room temperature and the agglutination was monitored visually. Agglutination activity corresponds to the minimum extract amount needed to precipitate the erythrocytes and agglutination unit (AU) was noted as the number of agglutination activity found in one millilitre. The same procedure was followed to estimate the haemolysis activity noted in haemolysine unit (HU).

Example 5

Serpin activity. Different amounts of crude extracts were incubated with 0.1 mg/ml trypsin in 25mM Tris-HCl pH 7.6, 100 mM NaCl, 25mM CaCl₂ at 25°C for 30 min. Remaining trypsin activities were measured spectrophotometrically at 405 nm using 25 μM α-N- benzoyl-DL-arginin-p-nitroanilide in 25mM Tris-HCl pH 7.6, 100 mM NaCl, 25mM CaCl₂ (Ellant et al, 1985). As inhibition followed a pseudo first order kinetic, ID50 (inhibiting dose of 50%) were determined by fitting log of amount of extract against remaining activity in linear regression. Inhibition units (IU) correspond to the inverse of ID50, and indicates the number of ID50 in one millilitre of crude extract.

Example 6

Dialysis test. Crude extracts were dialysed against the same volume of 10 mM phosphate buffer pH 7 overnight at 4°C, using a 10 kDa cut-off membrane. Proteins, lectins, serpins and hemolysins were estimated in the two compartments together with the toxicity.

Effect of dialysis to insecticidal toxicity. The interior and exterior of the dialysis tubing were compared for their toxicity against D. melanogaster development. If toxicity were due to macromolecules, all of the toxicity should be found in the interior of dialysis tubing. On the other hand, if the toxicity is due to small molecules, the toxicity should equilibrate between the two compartments. The proteins remained inside (see Table 3 which shows the effect of dialysis on the toxicity of crude extracts). The percent of activity in interior of the dialysis tubing was recorded by a global staining method or by specific activity such as lectin, serpin or hemolysin activities. The majority of toxicity was recovered in the interior for all tested species suggesting that toxic compounds are the molecules greater than 10 kDa in size. For some species, such as Xerocomus chrysenteron or Lepista nuda, all of the toxicity is due to macromolecules. However, for some other species, for example for Amanita phalloides, some toxicity was recovered outside the dialysis tubing. This is in accordance with the presence of toxic cycle peptides smaller than 10 kDa in this species. In this instance, however, the toxicity was not evenly distributed suggesting that also in this species, some macromolecules may be involved in the insecticidal activity. Table 3: Effect of dialysis on the toxicity of crude extracts : percent of activities in the interior of dialysis tubing

Name of species Protein Toxicity Lectin Hemolysin Serpin

% % % % %

Amanita phalloides 100 63 - 100 -

Xerocomus chrysenteron 84 98 99 100 100

Xerocomus subtomentosus 99 99 100 - -

Lepista nuda 94 99 - 100 -

Boletus speciocus 100 88 100 100 -

Xerocomus badius 99 91 100 77 -

Hygrophoropsis aurantiaca 100 91 - 80 -

Clitocybe nebularis 95 82 91 100 100

Polyporus squamosus 100 92 - 100 -

Boletus aereus 100 88 100 100 100

Clitopilus prunulus 100 89 - 90 100

Albatrellus cristatus 95 83 100 77 -

Gyrophana lacrymans 96 84 - 100 -

Hygrophorus chrysodon 100 87 100 100 -

Example 7

Heating treatment. Crude extracts were treated at 100°C in a water bath for 60 minutes. When some precipitations occurred, the samples were stirred before the toxicological test. Effect of heating treatment to insecticidal toxicity. After incubation at 100°C for 60 minutes, all extracts were inactivated except for Amanita phalloides (see Table 4). In the same way, protein activities used as controls, were diminished if not completely lost. This denaturation is an important characteristic of macromolecules, especially of proteins. For Amanita phalloides, 57 % of toxic activity remained after heating. This result is in accordance with the presence of , amanitin and phallotoxins. On the other hand, 43% of toxic activity was thermolabile, suggesting that a part of toxicity against the insect was due to one or more proteins. Table 4: Percent of activities left after heating (100°C)

Name of species Toxicity Lectin Hemolysin Serpin

% % % %

Amanita phalloides 57 - 0 -

Xerocomus chrysenteron 3 0 0 83

Xerocomus subtomentosus <1 1 - -

Lepista nuda 3 1 0 -

Xerocomus badius <17 12 25 -

Boletus speciocus <3 50 50 -

Hygrophoropsis aurantiaca <17 - 0 -

Clitocybe nebularis <15 2 0 83

Polyporus squamosus 1 1 - 0 -

Boletus aereus <10 0 25 0

Clitopilus prunulus <10 - 25 0

Albatrellus cristatus <29 0 0 -

Gyrophana lacrymans <20 - 0 -

Hygrophorus chrysodon <30 0 0 -

Example 8

Proteolysis test. Crude extracts were treated by pronase (protease Type XIV : Bacterial, from Streptomyces griseus, Sigma) at the final concentration of 4mg/ml at 37°C for 2 hours, then tested on the larvae of D. melanogaster. The protease presented in the rearing medium was not toxic for D. melanogaster.

Effect of proteases on insecticidal toxicity. As some protease inhibitors have been evidenced in some species of mushroom (Otto and Lipperheide, 1986; Pilgrim et al., 1992), pronase was used because it is composed of a mixture of several proteases. To verify the efficiency of the proteases, crude extracts treated and untreated by proteases were run side by side on a SDS gel. The result (see Figure 1) shows that the majority of the proteins were digested by proteases, but some proteins remained unaffected. Among them, some lectins and hemolysins were resistant to protease treatment (see Table 5). Therefore, the toxicity' s are generally not affected by protease treatment. In some species, the toxicity's were diminished to a small degree, such as in Clitocybe nebularis whereas the toxicity's were augmented such as in the case of Xerocomus subtomentosus.

Table 5: Percent of activities left after proteolysis treatment

Name of species Toxicity Lectin Hemolysin Serpin

% % % %

Amanita phalloides 89 - 50 -

Xerocomus chrysenteron 96 100 100 100

Xerocomus subtomentosus 197 100 100 -

Lepista nuda 65 400 25 -

Xerocomus badius 61 100 50 -

Boletus speciocus 68 100 100 -

Hygrophoropsis aurantiaca 113 - 100 -

Clitocybe nebularis 46 100 100 0

Polyporus squamosus 110 - 50 -

Boletus aereus 165 100 100 0

Clitopilus prunulus 105 - 100 0

Albatrellus cristatus 72 100 100 -

Gyrophana lacrymans 114 - 100 -

Hygrophorus chrysodon 94 50 100 -

These may be explained by two hypotheses : the toxic principles were degraded to sub-units which are toxic or even more toxic, or the toxic principles were not attacked by protease because they are themselves resistant to the pronase.

To test the first hypothesis, the crude extracts treated by proteases were dialysed against lOmM phosphate buffer pH 7 (lv/lv) for overnight at 4°C. The results (see Table 6) showed that the toxic principles were still stayed in the interior of the dialysis tubing and were not degraded into sub-units which were smaller than 10 kDa by proteases. Thus, it seems that the active compounds are resistant to proteases or are protected.

Table 6: Percent of activities in the interior of dialysis tubing after treated by pronase

Name of species Toxicity

%

Amanita phalloϊdes 49

Xerocomus chrysenteron 98

Xerocomus subtomentosus 98

Lepista nuda 80

Xerocomus badius 78

Boletus speciocus 85

Hygrophoropsis aurantiaca 77

Clitocybe nebularis 83

Polyporus squamosus 86

Boletus aereus 88

Clitopilus prunulus 89

Albatrellus cristatus 90

Gyrophana lacrymans 65

Hygrophorus chrysodon 81

All above results show that the insecticidal principles follow a change of protein activities with the exception of protease resistance. This last property is shared with other mushroom proteins such as lectins therefore suggesting that some proteins greater in size to 10 kDa are the active compounds responsible for the insecticidal activity in mushroom fruitbodies.

Example 9 Purification of lectins.

X. chrysenteron lectin: was purified to homogeneity by affinity chromatography and presented one band on a gel (see Figure 2). Mushrooms were homogenised in PBS. The supernatant, collected after centrifugation (10000 rpm, 4°C, 10 min), was filtered and applied to a column of lactosyl sepharose prepared according to Levi and Teichber (1981). The elution was made with PBS added with 0.14 M lactose and followed at 280 nm. The lectin was dialysed against 10 mM phosphate buffer pH7 and lyophilised.

Lectin of Lathyrus ochrus: Purification was performed according to Rouge and Sousa-Cavada (1984) using an affinity chromatography on Sephadex G100.

Lectin of Galanthus nivalis: Purification was performed according to Van Damme et al (1987) using an affinity chromatography on immobilised D-mannose. The lectin activity was determined by agglutination. The erythrocytes were washed three times in PBS by centrifugation (3500 rpm, 20 min.). The pellet obtained was used to prepare a suspension of 4% (v/v) erythrocytes in PBS. Determination of the agglutination activity was carried out in a final volume of 300 μl. The sample was serially diluted in PBS, with two fold increments in order to obtain a volume of 200 μl. Fifty microlitres of PBS or PBS with sugar (50mM) were added. Fifty microlitres of the suspension of erythrocytes were finally added. The tubes had been left for two hours at room temperature and the agglutination was monitored visually. The control contained 250 μl of PBS and 50 μl of erythrocytes suspension. Protein concentration was determined by the method of Lowry et al. (1951) using bovin serum albumin as standard.

Example 10

Cloning of the lectin. Internal peptidic sequences of X. chrysenteron lectin were determined.

6 μg of purified lectin were deposed in a SDS-PAGE. The band corresponding to the lectin was cut and digested by trypsin. The peptides were isolated and five of them sequenced (Institut Pasteur, Paris). The five peptidic sequences of the fungal lectin obtained after trypsin digestion were ITVAVG (SEQ ID No. 4), QLAEYSV (SEQ ID No. 5), GYFSIVEK (SEQ ID No. 6), TVWHFANG (SEQ ID No. 7) and GYFSIVESTV (SEQ ID No. 8).

The poly(A) RNA from X. chrysenteron was prepared according to Chomczyski and Sacchi (1987). cDNA was obtained with a cDNA synthesis kit (Stratagene). The cDNA library was constructed with a λ-ZAP cloning kit (Stratagene) and screened by PCR with synthesised probes from sequenced peptides of X chrysenteron lectin.

Example 11 Toxicological tests

Test on D. melanogaster. The flies were allowed to lay eggs overnight on a rearing medium (2.25% agar, 2.5% glucose, 25% fruit juice and 0.5% -ethylhydroxybenzoate). Three groups of ten eggs were harvested and put on the rearing medium (2.5% agar, 10% yeast extract and 0.5% p-methylhydroxybenzoate) containing the lectin. After 14 days at 25°C, the development was completed and the adults were counted. Normal development of eggs was followed with rearing medium devoid of lectin. The corrected mortality was determined with the Abbot method (Abbot, 1925). Test on Acyrthosiplion pisum: The standard test 0- 10-50-250 μg/ml performed according to Rahbe and Febvay ( 1993) was used. 20 neonate larvae (aged 0-24h) were deposited at day 0 on artificial diets of three protein concentrations. At day 7, mortality was determined in each cage and the aphids were individually weighed (0.01 mg precision) in order to determine the growth inhibition.

Test on Meloidogyne incognita: 100 second instar larvae were immersed in water containing different concentrations of lectin (from 0.25 μg/ml to 2 mg/ml). The percent of immobilised larvae was estimated after 24 hours.

The lectin toxicity determined on D. melanogaster is shown in Figure 4. The . ochrus lectin presented a LC50 of 8.51 mg/ml and the G. nivalis lectin a LC₅₀ of 0.72 mg/ml. The fungal lectin which showed a LC50 of 0.38 mg/ml, was the more toxic. The fungal lectin has also been tested on Acyrthosiphon pisum (see Figure 4) and presented at 0.25 mg/ml, a growth inhibition of 43% and a larvae mortality of 18.5%. The X chrysenteron lectin, when tested on Meloidogyne incognita, induced a 50% immobilised larvae at 0.014 mg/ml (see Figure 5).

Example 12

Sugar inhibition of the X. chrysenteron lectin: The presence of some sugars with the lectin involved an inhibition of erythocyte agglutination of 91% with lactose and 94% with galactose. No inhibition was observed with glucose, fructose, sorbitol, mannose and sucrose.

Example 13

Cloning and sequencing lectin: cDNA library hybridisation is performed with oligonucleotides designed from peptide sequences and with a partial probe obtained from PCR amplification using the same oligonucleotides. A full length cDNA is obtained with RACE (Rapid Amplification of cDNA Ends). Nucleotide sequences are obtained according to the method by Sanger and the open reading frame is deduced.

Example 14 Construction of plasmid: the full length cDNA is inserted into suitable plasmids for DNA work (sequencing, mutagenesis), for in vitro production and for plant transformation, following methods well known in the art. - 9? .

Example 15

Transformation of plasmid: the plasmids are transformed into plant cells using standard procedures. Any transformation method suitable for the target plant or plant cells may be employed, including infection by Agrobacterium tumefaciens containing recombinant Ti plasmids, electroporation, microinjection of plant cells and protoplasts, microprojectile bombardment, bacterial bombardment, particularly the "fibre" or "whisker" method, and pollen tube transformation. The transformed cells may then in suitable cases be regenerated into whole plants in which the new nuclear material is stably incorporated into the genome. Both transformed monocot and dicot plants may be obtained in this way. Full details of the methods of transformation are known to the person skilled in the art.

Example 16

Nematicidal effect on Heterodera schachtii in Arabidopsis thaliana 16.1 General Plant-parasitic nematodes are known to cause a severe reduction in crop yield. The sedentary endoparasites have the most complex interaction with their host plant. Two groups, cyst- and root-knot nematodes, induce specialised feeding structures in the vascular cylinder of the plant root. The method used enables substances to be injected into the nematode feeding structure (syncytium) established by Heterodera schachtii (beet cyst nematode, BCN) in roots of Arabidopsis thaliana. This method is described in detail in A. Bδckendorff and F. M. W. Grundler (1994): Studies on the nutrient uptake by the beet cyst nematode Heterodera schachtii by in situ microinjection of fluorescent probes into the feeding structures in Arabidopsis thaliana. Parasitology 109: 249-254. The injections were performed with InjectMan™ and CellTram Oil™ (Eppendorf, Hamburg, Germany) and we used a Nikon Eclipse™ TE200 microscope. Injections were made into syncytia of J3 juveniles, female J4 juveniles and adult females. Together with the tested substance we inject Lucifer Yellow CH (Molecular Probes, Leiden, Holland). This enables the injection process and the uptake of the substances by the nematode under UV-light to be followed. It has been shown that Lucifer Yellow has no impact on the development of H. schachtii on Arabidopsis. Development of the nematodes over at least seven days after injection was followed. 16.2 Results Xerocomus chrysenteron purified protein sequence (SEQ ID No. 1) The purified protein depicted as SEQ ID No. 1 and Lucifer Yellow were dissolved in distilled water and the final concentration of the lectin was 1000 ppm. After injecting the lectin into the syncytia 93% of the nematodes stopped development and died. Mortality in the control was only 0-10%. Later the protein was heat treated (20 min at 100°C) and the microinjection experiment was repeated at the same concentration (1000 pm). Mortality was 0% and all nematodes developed to adult males or females.

Example 17

Cloning of plant expression construct for nematicidal protein

The coding sequence of the X chrysenteron protein was made synthetically to optimise codon usage for plants (e.g. dicotyledonous plants). The synthetic sequence fragment has a 5' overlap with the 3' end of the Arabidopsis thaliana actin 7 promoter, which confers strong expression in roots, and a Pstl site 3' to the stop codon of the open reading frame to allow linking the coding region to the proteinase inhibitor II gene terminator from potato. The sequence of this fragment is shown in SEQ ID No. 9. In order to link the gene coding sequence to the actin 7 promoter a 3' fragment of this promoter up to an Sphl site is PCR amplified from a genomic actin 7 clone using oligonucleotide primers SEQ ID No. 10 and SEQ ID No 11. Primer SEQ ID No 11 is designed to create an overlapping homology with the coding sequence. The resulting 474 bp PCR product and the synthetic coding sequence are mixed as template in a second PCR amplification reaction with primers SEQ ID No 10 and primer SEQ ID No 12 which is homologous to the 3' end of the coding sequence spanning the Pstl site. The resulting 907 bp PCR fragment is then cloned as Sphl/Pstl fragment into the vector pMOG1467. This multicopy cloning vector (pBKS,Stratagene) contains a 1889 bp Actin 7 promoter fragment and a 273 bp proteinase inhibitor II terminator fragment flanked by BamHI sites. The entire expression cassette is then cloned as BamHI fragment into the BamHI site of the binary vector pMOG800, which is described in WO 98/22599.

Example 18 Mobilisation of binary vectors into Agrobacterium

The binary vectors described in Example 17 are mobilised in a triparental mating with E. coli K-12 strain HB101 (containing plasmid RK2013) (Ditta et al., 1980, Proc. Nat. Acad. Sci. USA 77, 7347-7351 ), into an Agrobacterium tumefaciens strain for example LBA4404 (Hoekema et al. 1983, Nature 303, 179-180) that contains a plasmid with the virulence genes necessary for T-DNA transfer to plants.

Example 19

Plant Transformation a) Transformation of Arabidopsis thaliana

Arabidopsis is transformed by co-cultivation of plant tissue with Agrobacterium tumefaciens strain containing one of the binary vectors as described in Example 17. Transformation is carried out using co-cultivation of Arabidopsis thaliana (ecotype C24) root segments as described by Valvekens et al. (1988, Proc. Nat. Acad. Sci. USA 85, 5536-5540). Transgenic plants are regenerated from shoots that grow on selection medium (containing either kanamycin or hygromycin, depending on the originating binary plasmid pMOG23 or pMOG22 respectively - both of these vectors are described in International Patent application publication number WO93/10251 and pMOG 23 is deposited at the Centraal Bureau voor schimmelcultures in Baarn under CBS 102.90), rooted and transferred to germination medium or soil. Young plants are grown to maturity and allowed to self-pollinate and set seed. b) Transformation of potato (Solanum tuberosum ssp.) Potato is transformed by co-cultivation of plant tissue with Agrobacterium tumefaciens strain LBA4404 containing one of the binary vectors described in Example III. Transformation is carried out using co-cultivation of potato (Solanum tuberosum var. Desiree) tuber disks as described by Hoekema et al. 1989, Bio/Techn. 7, 273-278). Transgenic plants are regenerated from shoots that grow on selection medium (containing either kanamycin or hygromycin, depending on the originating binary plasmid pMOG22 or pMOG23), rooted, multiplied axenically by meristem cuttings and transferred to soil. Young plants are grown to maturity and allowed to develop tubers. c) Transformation of tobacco (Nicotiana tabacum SRI)

Tobacco is transformed by co-cultivation of plant tissue with Agrobacterium tumefaciens strain LBA4404 (Hoekema et al. 1983, Nature 303, 179-180) containing one of the binary vectors described in Example III. Transformation is carried out using co-cultivation of tobacco (Nicotiana tabacum SRI) leaf disks as described by Horsch et al. 1985, Science 227, 1229- 1231 ). Transgenic plants are regenerated from shoots that grow on selection medium (containing either kanamycin or hygromycin, depending on the originating binary plasmid pMOG22 or pMOG23), rooted and transferred to soil. Young plants are grown to maturity and allowed to self-pollinate and set seed.

Example 20

Analysis of transgenic Arabidopsis plants for susceptibility to Plant parasitic nematodes

(PPN)

Transgenic Arabidopsis plants are assayed both in vitro or in soil for resistance against M. incognita or the cyst nematode H. schachtii. For in vitro analysis, seeds are surface sterilised, grown and inoculated as described by Sijmons et al. (1991, Plant J. 1; 245-254). Two weeks after infection, the root systems can be scored visually for the number of successful infections and compared to wild type Arabidopsis plants. Plant lines are considered resistant when they show a significantly decreased susceptibility to PPN (i.e. a significant decrease in the number of females found on control roots). For soil-grown plants, seedlings are germinated on selective medium (10 mg/1 hygromycin or 50 mg/1 kanmaycin). Resistant seedlings are transferred to soil/sand mixtures (1 :3 v/v) in 1 x 1 x 6cm transparent plastic tubes. Once the rozettes are well developed (ca. 14 days) the containers are inoculated with ca. 300 hatched J2 of H. schachtii each. Hatching of H. schachtii is stimulated by submerging cysts for several days in a 3 mM ZnC12 solution at a temperature of ca. 20°C. Eighteen days after inoculation, the roots are carefully removed from the soil/sand mixture and stained with acid fuchsin (Dropkin, 1989 in: Introduction to plant nematology, 2nd edition, Wiley & Sons, New York). In this assay, susceptible plants score a mean of 17 cysts per root system (range 4-40 cyst per root system). A genotype is considered resistant when the mean number of cysts is reduced to 2 per root system. Similarly, plants can be inoculated with hatched J2 of M. incognita or with egg-masses that are mixed through the soil/sand mixture. The plants are then be scored for the presence of galls which are clearly visible after the soil/sand mixture is removed from the roots.

Example 21

Analysis of transgenic tobacco plants for susceptibility to PPN For analysis of nematode resistance, the soil is pre-infected with M. incognita egg masses. This inoculum can be produced by maintaining a stock culture of M. incognita on soil grown celery plants (Apium graveolens) under standard greenhouse conditions, below 25°C. Mature celery root systems, containing a high number of root knots and mature females of M. incognita, are carefully dusted off to remove the soil, homogenised briefly in a Waring blendor (2 seconds) and weighed in portions of 60 gram. These root samples are mixed with 1 kg sand:potting soil (1:1) mixtures and used for growth of transgenic tobacco plants for 6 weeks. For each genotype, at least 100 individual plants are used for each test. The soil/sand mixture is then carefully washed away and the number of galls / root system is counted with a binocular. In this assay, control plants have a mean of 25 plus or minus 15 galls. A genotype is considered resistant when the mean number of galls is reduced to 2 per root system.

Example 22 Analysis of transgenic potato plants for susceptibility to PPN

Transgenic potato plants are assayed for resistance against M. incognita using soil that is pre- infected with Mi. incognita egg masses mixed with sand (1:3 w/w), growing the potato plants in that soil mixture for 6 weeks and, after removing the soil, counting the developed number of galls on a root system. Alternatively, to assay for resistance against Globodera ssp. a closed container is used. For this assay, three replicate 2-4 cm tubers are transferred to soil which is pre-inoculated with cysts from G. rostochiensis or G pallida in transparent containers. The peripheral root systems are analysed visually 7-8 weeks after germination for the presence of cysts. A genotype is scored as resistant if none of the three replicates had cysts and susceptible if at least one of the three replicates shows cysts.

Example 23

Comparison of protein sequences to SEQ ID No. 1 using the FASTA Algorithm

A FASTA comparative search of SEQ ID No. 1 to a database of protein sequences was carried out.

SEQ ID No. 1 was compared to all publicly available protein sequences using the FASTA method and algorithm as described above. The results were given in the form of an opt score which is also described above.

Other modifications of the present invention will be apparent to those skilled in the art without departing from the scope of the invention. References

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Claims

1. A protein comprising the amino acid sequence depicted as SEQ ID No. 1 or a variant having at least about 60% identity therewith wherein said protein or variant is insecticidal and/or nematicidal.

2. A polynucleotide encoding a protein or variant according to claim 1.

3. A polynucleotide sequence which is the complement of one which hybridises to the polynucleotide according to claim 2 at a temperature of about 65°C in a solution containing 6xSSC, 0.01% SDS and 0.25% skimmed milk powder, followed by rinsing at the same temperature in a solution containing 0.2xSSC and 0.1% SDS wherein the said polynucleotide sequence still encodes a protein having insecticidal and/or nematicidal properties.

4. A polynucleotide according to claim 2 or claim 3 comprising the sequence depicted as SEQ ID No. 2 or SEQ ID No. 3.

5. A method of evolving a polynucleotide which encodes a protein having insecticidal and or nematicidal properties comprising:

(b) shuffling said variants and further polynucleotides to form recombinant polynucleotides; and

(c) selecting or screening for recombinant polynucleotides which have evolved towards the said insecticidal and/or nematicidal properties; and

(d) repeating steps (b) and (c) with the recombinant polynucleotides according to step (c) until an evolved polynucleotide which encodes a protein having insecticidal and/or nematicidal properties has been acquired wherein said population of variants in part (a) contains at least one polynucleotide according to any one of claims 2 to 4.

6. A polynucleotide obtainable by a method of claim 5.

7. A polynucleotide obtained by the method of claim 5.

8. An insecticidal and/or nematicidal protein encoded by a polynucleotide according to claim 6 or claim 7.

9. A method of isolating a polynucleotide encoding an insecticidal and/or nematicidal protein from a Xerocomus sp. DNA library comprising: (a) constructing a DNA library of Xerocomus sp. or a particular strain thereof preferably Xerocomus chrysenteron; and

(b) probing said library with at least one oligonucleotide probe which is capable of detecting said polynucleotide present in said library wherein said probe is derived from at least one peptide sequence selected from the group consisting of SEQ ID Nos. 4-10; and

(c) identifying and isolating the thus detected polynucleotide.

10. An isolated polynucleotide obtainable by a method of claim 9.

11. An insecticidal and/or nematicidal protein encoded by the polynucleotide of claim 10.

12. A protein or variant according to any one of claims 1, 8 or 11 which has a molecular weight of greater than or equal to 10 kDa.

13. A protein or variant according to any one of claims 1, 8, 11 or 12 which is a lectin.

14. A DNA construct comprising in sequence a plant operable promoter operably linked to a polynucleotide according to any one of claims 2, 3, 4, 6, 7 and 10 or a polynucleotide encoding a protein according to any one of claims 1, 8, 11, 12 or 13 operably linked to a transcription termination region.

15. A DNA construct according to claim 14 which further comprises a region which provides for the targeting of the protein product to a particular location.

16. A DNA construct according to claim 14 or claim 15 which further comprises a region which provides for the production of a selectable marker.

17. A DNA construct according to any one of claims 14 to 16 wherein the plant operable promoter is selected from the group consisting of: CaMV35S, FMV35S, NOS, OCS, Patatin, E9, alcA/alcR switch, GST switch, RMS switch, oleosin, ribulose bisphosphate carboxylase-oxygenase small sub-unit or root specific promoters including MR7 promoter (maize), Gos 9 (rice), GOS2 promoters, MasOcs (or super promoter) and the Agrobacterium rhizogenes RolD promoter.

18. A method of providing a plant or plant part with an insecticidal and/or nematicidal protein comprising:

(a) inserting into the genome of plant material a polynucleotide according to any one of claims 2, 3, 4, 6, 7 and 10 or a polynucleotide encoding a protein according to any one of claims 1, 8, 11, 12 or 13 or a DNA construct according to any one of claims 14 to 17; and (b) regenerating plants or plant parts therefrom; and

(c) selecting those plants or plant parts having said protein.

19. Plants or plant parts obtained according to the method of claim 18.

20. Plants or plant parts according to claim 19 wherein said plants or plant parts are selected from the group consisting of: melons, mangoes, soybean, cotton, tobacco, sugarbeet, oilseed rape, canola, flax, sunflower, potato, tomato, alfalfa, lettuce, maize, wheat, sorghum, rye, bananas, barley, oat, turf grass, forage grass, sugar cane, pea, field bean, rice, pine, poplar, apple, peaches, grape, strawberries, carrot, lettuce, cabbage, onion, citrus, cereal or nut plants or any other horticultural crops.

21. Plants or plant parts according to claim 19 or claim 20 which comprise a further agronomic trait selected from the group consisting of: herbicide resistance; insect resistance; nematode resistance; altered stress tolerance; altered yield and altered nutritional content or any desired agronomic trait.

22. Use of a polynucleotide according to any one of claims 2, 3, 4, 6, 7 and 10 or a polynucleotide encoding a protein according to any one of claims 1, 8, 11, 12 or 13 or a DNA construct according to any one of claims 14 to 17 in a method of producing plants which are resistant and/or tolerant to insects and/or nematodes.

23. Use of a Xerocomus sp. protein extract as an active ingredient in the production of an insecticide and/or nematicide.

24. Use according to claim 23 wherein the Xerocomus sp. is Xerocomus chrysenteron.

25. Use according to either of claims 23 or 24 wherein the protein extract comprises a protein or variant according to any one of claims 1, 8, 11, 12 or 13.

26. Use according to any one of claims 23 to 25 wherein said extract further comprises an agriculturally acceptable carrier and/or a diluent.

27. Use according to any one of claims 23 to 26 wherein said extract is formulated for use as a spray.

28. Use of a protein or variant according to any one of claims 1 , 8, 11 , 12 or 13 as an insecticide and/or nematicide.

29. A method of controlling insects and/or nematodes comprising providing at a locus where the said insects and/or nematodes feed a protein or variant according to any one of claims 1, 8, 11, 12 or 13 or an extract according to any one of claims 23 to 27.

30. An insecticidal and/or nematicidal protein obtained from the mushroom Xerocomus sp.

31. A composition comprising an insecticidally and/or nematicidally effective amount of a protein or variant according to any one of claims 1, 8, 1 1, 12 or 13 or an extract according to any one of claims 23 to 27 and optionally an agriculturally acceptable carrier and/or a diluent and or an insect/nematode attractant.

32. A plant cell comprising a protein or variant according to any one of claims 1, 8, 11, 12 or 13.

33. An insecticidal and/or nematicidal synergistic combination comprising a protein or variant according to any one of claims 1, 8, 11, 12 or 14 and a further protein.

34. A synergistic combination according to claim 33 wherein said protein comprises the sequence depicted as SEQ ID No 1 and said further protein is insecticidal and/or nematicidal.

35. A synergistic combination according to claim 33 or claim 34 wherein said further protein comprises an insecticidal CRY protein.

36. A polynucleotide having a first region encoding a protein or variant according to any one of claims 1, 8, 11, 12 or 13 and a second region encoding a further protein.

37. An insecticidal and/or nematicidal protein having a FASTA opt score greater than 489 when compared with SEQ ID No. 1 calculated using FASTA V.3.

38. An insecticidal and or nematicidal protein or variant according to any one of claims 1, 8, 11 to 13, 30 or 37 having a specific galactose and/or lactose binding ability.

39. An insecticidal and/or nematicidal protein obtainable from mushroom and having a specific galactose and/or lactose binding ability

40. A recombinant micro-organism which provides for production of a protein or variant according to any one of claims 1, 8, 11, 12, 13, 30, 38 or 39.

41. A recombinant micro-organism according to claim 40 which is an endophyte or a Pseudomonas sp.

42. A recombinant baculovirus which provides for production of a protein or variant according to any one of claims 1, 8, 11, 12, 13, 30, 38 or 39.

43. Use of a recombinant micro-organism according to claim 40 or 41 or a baculovirus according to claim 42 in a method of controlling insects.

44. An insecticidal and/or nematicidal protein which is capable of reacting with a monoclonal antibody raised to the protein depicted as SEQ ID No. 1.

45. Use of a protein or variant according to any one of claims 1, 8, 11, 12, 13, 30, 38 or 39 in a method of detection, isolation and characterisation of specific glycans.

46. An insecticidal and/or nematicidal protein obtainable from mushroom which protein has a molecular weight greater than or equal to 10 kDa and comprises at least one amino acid selected from the group consisting of: Asparagine; Cysteine; Glutamine; Histidine and Tryptophan.

47. A protein according to claim 46 obtainable from the mushroom Xerocomus sp.