Field of the invention

The invention relates to ccllulases ot .1 novel class, to their am o acid sequences, and to nucleotides encoding them. The invention also concerns the use of these enzymes in agriculture and in industry and the use ot peptides 01 other compounds inhibiting these enzymes in the protection of crops against plagues Background

Plant parasitic ncmatodcs are an important threat to agriculture because they feed on roots of host plants and reduce the possibilities of the plant to take up nutrients from the soil. Depending on how thev teed t ev can be divided in feeding types: 1. sedentary parasites (have swollen females), 2. migratory cndoparasitcs ( craw l in the root); 3 se i- endoparasites. 4. ectoparasites. Λi\ύ 5. epidermal and root hair feeders

All plant parasitic ncmatodcs tccd hv penetrating one or more root cells with their stylet. Endoparasitic ncmatodcs either disrupt cells or mov e m betw een cells. For these actions more than muscle force is likely to be required

Plant parasitic ncmatodcs comprise

1. Sedentary ncmatodcs; these include a. Cyst ncmatodcs (Hcterodeπdae tamih ) b. Root knot ncmatodcs (Meloidogynidac tamil v) c. genus Natobbus

2. Migratory endoparasitcs d. Pratylenchidae family (genera Pi tin l nduis and Radopholus including the burrowing nematode R smiths which attacks citrus and other (sub)tropιcal crops) c. Anguinidae family (several members) f. Aphelenchoididae family ( including the genera Apli endiυides (leaf ncmatodcs).

Bursap elenduis and Rhadintip elendnts ).

3. Semi-endoparasites g. Hoploiaimidae family (spiral ncmatodcs including the genera Rotylendius and Helicoiy enchus) h. genera Tylencluilus (including the citrus nematodc T seintpenctrans ) and

Rotylen ntlus;

4. Ectoparasites

1. Hemicycliophoπdac family j. Paratylenchidac family (pin ncmatodcs. including the genus P rciivlendws)

k. Tπchodoπdae family (stubbv-root ncmatodcs including the genus Ti idiodorus) 1. Longidoπdac family, 5. Epidermal and root hair feeders m Tylcnchidae family (bulb and stem ncm itodcs including Din lcnduis dtpscict) n. Atylenchidae family

The cyst and root knot ncmatodcs are ot most economic importance

Cyst ncmatodcs are ncmatodcs of the Hcterodeπdae tamih Thev represent an important group of pest organisms in agriculture Subfamilies are the Hcterodeπnae, Meloidodertnae (one genus one species) and Atalodcrinae (4 genera 12 species) The Heterodeπnac are divided into 85 species in 7 genera three of w hich comprise eco¬ nomically important species Hcicrodcta s species including H sdiaduu (sugar beet), H avenae (oat) H btfencsuci (grass) H ui ift'i m (cabbage ) // (soybean) H goettingwna (pea) H oι (ncc) H SUL CIUIΠ (sugarcane) H t/ ifoln (clover sugar beet) and H ze c (maize), Globodei a 1 species including G . ostoihien 'iis and G palhda (potato), G sol nac cii um and G uibac it (tobacco) and Pimttodeia 3 species including P punctata (grass)

Root-knot ncmatodcs (Mcloidogvnulac) include the ceonomicalh important genus Meloidogvnc Meloidogvnc species are pol phagous Thc\ produce c onspicuous knots or gall-like sw ellings on roots Thc\ attack most igπculturalh important plants such as vegetables, cotton straw erries orchard trees garden and ornamental plants Losses can be severe and there can be several generations (the number depending on conditions) per year Species in temperate zones comprise M ap/a (northern root-knot nematode). M chitwoodi and M naasi In (sub)tropιeal zones M nu M jcivcinn. ii and M ω enaria occur Sedentary plant parasitic ncmatodcs feed from their host b\ transforming root cells into multinuclcate feeding cells In the case of cvst ncmatodcs this feeding site consists of a large svncytium w ich results from a fusion of adμcent root cells The successful formation and exploitation of these feeding cells involves a complex interaction between nematodc and host plant, in w hich nematodc secretions ot both polypcptide and polysacchaπde nature arc considered to play an important role

Feeding cells of root-knot ncmatodcs arc so-called giant cells Juveniles of Meloidogvnc migrate interccllularly in the root to the region of c ell differentiation and locate cells in the vascular c\ Under of the stele The luvcnilcs inicct st \ l t secretions into five to seven cells to transform these cells into specialised feeding sites called giant-cells The multinucleate giant cells become the permanent feeding site toi the parasite throughout

its life-cycle Giant cell formation is one of the most complex responses elicited in plant tissue by any parasite Cells parasitised by juveniles undergo repeated division of their nuclei without cell division. Each host cell fed upon increases dramatically in size, the central vacuole diminishes in size, the c\ toplasm increases in volume and density, and cell wall ingrowths form, giving the cells a phenotype similar to that of transfer cells Giant cells are induced and maintained in susceptible hosts only by the feeding activities of Meloidogvnc species Stvlet secretions of root-knot ncmatodcs regulate specific host genes affecting protein synthesis nuclear division cell grow th and differentiation and cell w all synthesis. Most plant parasitic ncmatodcs have three large csophageal gland ce ls one dorsal and tw o subventral Thev inject secretions from these csophageal (also referred to herein as salivary) glands via their sn lct into the cells of their host plant In the case of sedentary nematodes, these saliva proteins are presumabK inv olv ed in feeding site induction and they are also necessary for feeding itself .is follo from the foi ation of mtraccllular feeding tubes

Summary of the invention

The invention is based on the iscov cπ of ccilulolvtic enzymes produced by nematodes in their salivary glands These enzvmes plav a kev role in disrupting ce l walls when produced bv micro-organisms Thev probably play a similar role in root parasitism by endoparasitic nematodes In addition these enzvmes mav be multifunctional and may therefore play a role in the induction of feeding cells w hich are formed b\ species belonging to the plant parasitic nematodes Thus the inv ention pertains to cellulotvtic peptides and parts thereof and their use as w ell as to nucleotides encoding them

Previously, cellulasc activm w as detected in the migrators endoparasitc Bursaphelenchus wlophilu s (sec Odani et al 1985 oj nna et al 1994 and Dcubcrt et al

1971. No sequence information of plant nematodal cellulasc-t\ pe enzvmes has been available thus far Detailed description of the invention

The invention pertains to nov el peptides having cellulasc (cndo-fi- 1 4-glucanasc) activity and exhibiting at least 40 ^r f amino acid identity in the primary structure w ith the amino acid sequence of the celluloh tie subventral csophageal proteins (u>//s) of Heleroderci ghetnes and/or Globoderci lostoducnsis w hich are show n in SEO ID NO 's 1-4 or with the majority sequence show n in Fig 1 By amino acid homology is meant here amino acid identity in the pπmarv structure Ammo acid similarity is usually higher than the figures given for identity Said amino acid identity in the primary structure is

higher for partial sequences In particular the amino acid identity ith the sequence 19- 318 of SEQ ID No 1 (29-330 of SEQ ID No 2 20-324 of SEQ ID No s 3 and 4) is at least 50%, especially at least 60

In another aspect the peptides arc characterised bv comprising a scries of at least 8 contiguous amino acids of the amino acid sequence sho n in one of SEQ ID NO 's 1-4 or of the ammo acid sequence encoded b\ the nucleotide sequence sho n in one of SEQ ID NO 's 1 -7 In particular said series comprises at least 10 especially at least 12 or even at least 15 contiguous amino acids of the amino acid sequence show n in one of SEQ ID NO 's 1 -4 The peptide according to the inv ention preferably corresponds to a nematodal peptide or part thereof especially to a peptide of a sedentary nematodc The c\ st and root- knot nematodes ΔTC most important and thus their c luloh tic enzvmes arc a preferred group according to the present invention Parts of the peptides especially refer to immunogenic parts capable of inducing antibodies ig unst the peptides

The invention furthermore rel ites to a nucleotide sequence encoding a peptide having cellulasc activity and exhibiting at least 40' < ammo aci homolog) w ith the amino acid sequence show n in one of SEQ ID NO s 1 -4 or hy bridising under stringent conditions with a nucleotide sequence sho n in SEQ I D NO s 1 — 1 or 5-7 or a part thereof having at least 15. preferably at least 21 more preferabl y it least 24 or even at least 30 or at least 36 nucleotides of the nucleotide sequence show n in SLO ID NO s 1 -4 and 5-7 or encoding an ammo acid sequence show n m SEQ I D NO s 1 -4

In this context stringent hy bridisation conditions aic as follow s hy bridisation in 6 x SSC (2ϋ\SSC per 1000 ml 175 3 g NaCl 107 1 g sodium c itrate 5H ₂ 0 pH 7 0), 0 1% SDS. () i)5'< sodium pwophosphatc ^s Dcnhardt s solution ( 100 x Dcnhardt's solution per 500 ml 10 g Fιeoll-400 10 g pols v ιn\ lp\ rrolιdone 10 g Bovine Serum Albumin (Pentax Fraction V)) and 20 ug/ml denatured herring sperm DNA at 65°C for

18-24 hrs follow ed bv one 2xSSC 0 5% SDS ( 5 mm ) w ash Λm\ one 2xSSC 0 1 % SDS wash at room temperature one O l xSSC 0 ^r r SDS at 37°C and a final w ash w ith O.lλSSC 0 5% SDS at 65°C

The coding sequences may contain mutations (insertions deletions or both) which serve to modify the structure and/or the activity of the expression product For an active expression product, the minimum identity and/or the hybridisation characteristic as defined above should preferably be maintained The nucleotide sequence may also correspond to regulating or signal sequences of nematodal ccllulases The nucleotide sequence may comprise substantially the encoding and/or regulating sequences of the cellulasc On the other hand, the nucleotide sequence mav be used as a primer or probe in detecting

nematodal cellulasc encoding sequences.

Also part of the invention are expression vec tors and plasmids containing the nucleotide sequences described above under the control of a homologous or hctcrologous promoter. Furthermore, the invention is concerned w ith the use of these sequences for the production of ccllulases by a different host under the control of its own. or hctcrologous regulatory sequences. The expression vectors and host cells may contain multiple copies of the cellulase-encoding sequences (altered or not w ith respect to SEO ID NO's 1 -4 or 5-7) and also of other genes. Host organisms may be homologous production strains or alternatively hetero- logous hosts. Suitable host organisms include fungi y easts bacteria and plants Examples are A pergdlus species. Ti id xk'i inci species Dae din s spec ies klnw romvc cs species and Sacchcironivc cs species For food applic ations of the ce llulasc the host organism is preferably food-grade Examples of ow n control regions and hctcrologous regulatory regions include fungal constitutive and/or inducible promoters such as the p ruvatc kinase promoter (pkiA) and the glyceraldehyde-3-phosphatc dchvdrogenasc (gpd) promoters Examples of strong yeast promoters are alcohol dehydrogenasc 3-phosρhogl vcerate kinase and tπose phosphate lsomcrase promoters. Examples of bac terial promoters arc α-amyiase. spo2 and promoters of extracellular protease genes

The host cell may advantageously express or ov erexprcss other relevant proteins, including enzvmes. in particular other glycanolvtic enzy mes such as xvlanascs. amvlases. glucanascs, -glucosidascs and/or other enzvmes such as oxidoreductascs such as hexose oxidase, u-glucuronidase. lipases. cstcrascs and/or proteases The corresponding genes may be under the control of homologous control regions or under the control region of the cellulasc gene.

The invention is also concerned w nh antibodies directed at the ccllulolvtic enzymes described above or to immunogenie parts thereof These antibodies or one or more of the variable parts thereof can be used in protecting crops against the action of parasitic plant nematodes, especially sedentary ncmatodcs cither as such or as an antibody-encoding insert in a transgenic plant

SEQ ID NO's 1 and 2 depict the genes HG-c/nA (about 1 kb) and UG-engl (about 1.5 kb) respectively encoding fi-1 .4-endoglucan<ιscs of H icrodera glvcincs with deduced amino acid sequence; these listings show the complete protein including the signal sequences. SEQ ID NO's 3 and 4 depict the genes GR-enι;2 and GR-eng I respectively

encoding β-1.4-endoglucanascs of Globoderci lostocluensis w ith deduced amino acid sequence. SEQ ID NO's 5, 6 and 7 depict the partial nucleotide sequences of the corresponding genes respectively encoding — 1,4— endoglucanases of Meloidogvnc incognita

The mutual degrees of homology on amino acid level bet een the endoglucanases of Globoderci rostoduensis and Heleroder ci iζ ancs < ύ w ith the Eni imci dirvscinthemi (Erwch) endoglucanase are shown below

The organisation of the proteins encoded by these genes is as follow s GR-ENG1. amino acids 1 -19 signal sequence ammo acids 20-324 catalytic domain, amino acids 325-375 linker sequence amino acids 376-472 cellulose binding domain,

GR-ENG2. amino acids 1 - 19 signal sequence amino acids 20-324 catalytic domain, amino acids 325-386 linker sequence This protein has no cellulose binding domain, HG-ENG1 ammo acids 1 -28 signal sequence amino acids 29-330 catalytic domain, am o acids 331 -380 linker sequence 381 — 176 cellulose binding domain HG-ENG2 1-18 signal sequence amino acids 19-318 catalytic domain This protein has no linker sequence and no cellulose binding domain

The catalytic domain of HG-ENG 1 having a homology of 80' ^" ? w ith the catalytic domain of HG-ENG2 shows homologies w ith the follo ing proteins Endoglucanase Z precursor (cndo- 1 4- -glucanasc cellulasc) from Ft wima du wanihemi, 35.6% in 385 amino acids overlap (AC P07103)

Endoglucanase precursor (endo-1 4-|3-glucanasc cellulasc) fro Closiπduini aceto- butyltcii ; 37 4% in 390 amino acids overlap (AC PI 5704) Endoglucanase precursor (endo-1 4-fi-glucanase alkaline cellulasc) from Bacillus sp (Strain 1139), 34.5% in 171 ammo acids overlap (AC P065 4) Endoglucanase precursor (cndo-1.4-β-glucaπasc cellulasc) tram Bacillus suhnli s 34.7% in 383 amino acids overlap (AC P07983) Endoglucanase E-5 precursor (cndo-l ,4-(i-glucanasc E-4 cellulasc E-5) from Tlicrmo- monosjwrci fuscci, 35 4% in 280 am o acids ov erlap (AC 001786)

Endoglucanase precursor (cπdo-l .4-[Aglucanasc; carboxymethyl-ccllulase: CMCasc; cellulasc) from Bacillus siώiilis; 34.4% in 392 amino acids overlap (AC PI 0475)

Endoglucanase IV (cndo-1.4-| -glucanase: cellulasc: EG-IV) from Ruininococcus albus;

33.0% in 297 amino acids overlap (ACQ07940) Endoglucanase precursor (cndo- 1.4- -glucanase: carboxymethyl-cellulasc: CMCase; cellulasc) from Bacillus subtilis 35.0% in 383 amino acids overlap (AC P23549)

Endoglucanase CELA precursor (endo- 1.4-(Aglucanase; cellulasc) from Sircptonivces lividans; 35.4% in 297 amino acids overlap (AC P27035) Endoglucanase A precursor (endo-1.4- i-glucaπasc; cellulasc; EGA) from Butyrivtbrio fibrisolvcns; 36.5% in 277 amino acids overlap (AC P22541 )

Endoglucanase A (endo- 1.4-|Aglucanasc; cellulasc; clone PNK 1 ) from Bacillus sp.

(Strain N-4) 35.4% in 362 amino acids overlap (AC P06566) Endoglucanase B (cndo- 1 .4- - lucaπasc: cellulasc: clone PN K.2) from Bacillus sp. (Strain N-4) 34.9% in 358 amino acids overlap (AC P06565) Endoglucanase C precursor (cndo- 1.4- - lucanasc; cellulasc) from Bacillus sp. (Strain

N-4) 31.6% in 187 amino acids overlap (AC P19570) Endoglucanase precursor (endo- 1 .4-|Aglucanasc: cellulasc: alkaline cellulasc) from Bacillus sp. (Strain KSM-635) 28.7% in 188 amino acids overlap (AC PI 9424) The first region of the G.ros. 1.2 kb and 1 .5kb genes shows a 45.6 and 44.1 homology in 270 amino acids of the endoglucanase Z precursor (endo- 1 .4-β-glucaπase; cellulasc) from Erwinia chrysanihe i (AC P07103).

From the described homoiogies it is concluded that the present genes encode a endo-l ,4-β-glucanase (EC 3.2.1.4) or cellulasc.

Figure 1 shows an alignment of the four new nematodal cellulases of SEO ID No's 4 to 1 (GRENG1 = CellPCN = G.ros 1 .5 kb. GRENG2 = CeI2PCN = G.ros 1.2 kb,

HGENG1 = CcllSCN = H.gly 1.5 kb. HGENG2 = Cel2SCN = H.gly 1 .0 kb) with the known cellulasc of Erwinia chrysanihemi (GunZ-Frwch). which was found to have the highest homology wth the new cellulases. Identical matches arc shaded. Also the majority sequence is mentioned. Figure 2 shows an alignment of the partial nucleotide sequences of the

Meloidogvnc inocgniui genes Ml-engl . (SEO ID NO's 5-7) together with the majority sequence.

Figures 3-5 show the putative partial amino acid sequences of M. incognita endoglucanases as derived from SEQ ID NO's 5-7. respectively, in the boxed areas.

Figure 6 shows an alignment of the seven nematodal cellulases of SEQ ID No's 4-1 (GR-ENG1. GR-ENG2, HG-ENGl . HG-ENG2) and of figures 3-5 (MI-ENG1 , MI-ENG2. MI-ENG3) with the Erwinia chrysanihemi cellulasc (GunZ-Erwch). Identical matches are shaded. Also the majority sequence is mentioned. The criteria for peptides of the invention (minimum homology and minimum identical sequence length) as defined above, can also be used with regard to the sequences of Figure 6 including the majority sequence.

The enzymes may show multifunctionality including e.g. ccllobiohydrolase,

Iichcnase, xyloglucanase or .xylanasc activity. The cellulases according to the invention can be used in enzyme preparations. The invention is also concerned with such enzyme preparations. Such preparation may further contain other enzvmes, such as other glucanases. xylanascs. amvlascs. pascs. proteinases. oxulorcductascs etc

Cellulasc containing enzv e preparations can be applied:

- in treating ccllulosic fibres to impiove colour and feeling and various other properties. while preventing a lowering in strength of the ccllulosic fibres ( 'loss in w eight treatment" or "stone-free stone-washed process"), as described m WO 94/19528):

- as additive in detergents tor clothing, acting only on the non-crvstallinc region to remove soils and not having an effect of improving colour or feeling of the ccllulosic fibre (see also WO 94/19528); - as additive to feed for domestic animals to more effectively utilise the nutritive components, thus improving feed growth and feed conversion, as described in EP-A- 674843;

- for increasing the filtration rate of beer by degrading ccllulosic fibres;

- for clarification of fruit luiccs and in vegetable processing; - as a bread improver and in bakery applications;

- as a modifier for cellulose for use in dietary food, clinical foods and medicaments;

- for waste water treatment in the pulp and paper industry and in the starch industry; the cellulase assists in removing cellulose residues and also facilitates drainage in paper making and deinking of paper (see WO 94/00578 and WO 96/195 9).

Examples

General: Sequencing of N. IAtermini u[ vps Sample preparation

1-3.1 ' J-> of G rostochtensis or H. glyanes were homogenised in Potter Elvejhem homogeniser and teflon pestle (750 rpm: 40 times up-and-down) in 10 mM

Tris-Cl pH 7.4, Pefabloc (AEBSF, Bochπnger Mannheim) was added to a final concentration of 1 mM. The homogenatc was ccntπtuged for 5 mm. at 14.000 rpm (16.000 x g), and the supernatant and the pellet underneath the fat layer were collected. Shortly before use 1/3 sample volume of 4x SDS-sample buffer (240 mM Tris-Cl (pH 6.8), 40% glycerol, 8% SDS, 0.1 % bromophenol blue. 20 % 2-mcrcaptoethanol) was added, the sample was heated for 3 min at 100 °C and centrituged tor 5 mm. at 14.000 rpm ( 16.000 x g). The overlaying supernatant was used in the preparativ e purification. Purification of proteins

A 10% SDS-polyacrylamide gel was poured in a preparative elcctrophoresis system (Prep Cell, Bio-Rad, Richmond. Ca, USA) and overlaid w ith a 4% stacking gel according to the manufacturer. The 28 mm diameter gel tube was used. Standard SDS- PAGE buffers were used (Lacmmli. 1971 ). The follow mg running conditions were applied: 40 mA constant current (about 2X0 V). After 2.00 h ciution ys s started at 1 ml/min and collected in a 50 ml tube. After approximately 2:35 h the bromophenol blue migrated into the ciution chamber, w hereafter the traction collector as started and 2 ml per fraction was collected. After about 6:00 h and 100 tractions collected, elcctrophoresis was stopped. 25 μ\ of each third fractions was kept separate for a SDS- PAGE and immunoblot, the remaining part stored in the -80 °C freezer. After analysis on a SDS-PAGE slab gel and immunoblot (sec de Boer et al. 1996) the tractions containing the s\ p were combined to three pools of 31/32 kDa. 39 kDa and 49 kDa.

An immunoaffinity column w as prepared of monoclonal antibody MGR 48 (de Boer et al., 1996) by cross-linking it to piotcm A ScphaiosA 4 hist Flow ( Pharmacia. Uppsala, Sweden) using dimethyl pimclimidatc (Sigma. St Louis. MO. USA). The pooled svp samples were dialysed against 20 mM Tπs-HCl pH 8 overnight (two 2 litre buffer changes) and. one sample at a time, loaded onto a. w ith the same buffer, equilibrated

MGR48 protein A affinity column, at 1 ml/min. The sample s 'reloaded ' until the UV (280 ran) signal remained constant. The column w as then equilibrated with 20 mM Tris- HCI pH 8 to the base line whereafter 3 column volumes of 0.2 M glycine pH 3 were applied for elution. The UV 280 nm peak observed was collected and the pH immediately adjusted to pH = 7 with 1.0 M Tris- HCI pH 8. The column w as regenerated with 20 mM

Tris-HCI pH 8 whereafter the two other samples of svp's were subsequently purified. Sample preparation for NH-,-termιnal sequencing

To the protein samples Pefabloc y as added to a concentration of 1 mM where¬ after they were concentrated by lyophilisation The concentrated samples were subjected to SDS-polyacrylamide gel elcctrophoresis using a Tπs/tπciπc buftci system (Schaggcr,

H. and von Jagovv, G 1987) whereafter the proteins were transferred to an PVDF- membrane (Immobiion-Millφorc. Bedford MA) by semi-drv blotting The blot was stained with Coomassic Brilliant Blue R250. stained bands ere cut out and destained with

90% n-propanol. Sequencing of NH- _> -terιnιnι of svp

NH -terminal sequencing. Amino terminal sequencing w as carried out by ARIAD

Pharmaceuticals, Cambridge, MA. USA The follo mg N-tcrmini w ere obtained

SVP49 SCN

AVAPPFGQLSVSGSNLVGANKQPVQLISNSLFEH SVP39 PCN.

LTATPPPYGQLSVSGTKLVGSNGQPVOLIGNSLf-WPOFQGQYWNA

SVP39 PCN (internal sequence obtained after C N Bi digestion of the protein)

YNLAVAVIEAAISQGMYVIVD

SVP32 PCN VTAPPYGPLAVNGKFLVQKSTKQTVD

Oligonucleotidcs were designed based on the N-tcrmini and used for the cloning of the genes encoding these proteins as described in examples 1 and 2

Example 1 : Cloning of svps from // glycines

From 1 10 ' w ater hatched second stage juv eniles of // ι>l\ c ιnes RNA w as isolated using the RNA Extraction Kit (Pharmacia Biotech Uppsala Sw eden) Subsequently. cDNA was synthesised and the target DNA amplified using the 3' RACE System for Rapid amplification of cDNA Ends (Gibco-BRL Life Technologies Gaithersburg MD, USA). For amplification of the putative cellulasc gene the follow ing degenerate primer w as used: 5'-GTIGGIGCIAA(C/T)AA(A/G)CA(A/G)CCTGTICAA '

PCR resulted in tw o bands of approximately 1000 and 1 51)0 base pairs respectively Both bands were purified from the agarose gel using Easv prep PCR product prep kit (Pharmacia, SE) and ligated in the TA cloning vector ( Invitrogc , San Diego. CA. USA). DNA sequencing (ALF automated sequencing Pharmacia Biotech. SE) using Thermo Sequenase fluorescent labelled primer cycle sequencing kit w ith 7-deaza-dGTP

(Amersham. Buckinghamshire. UK) resulted in the sequences according to SEQ ID No's 1 and 2. The sequences show a homology with cellulasc from other organisms as described above.

Example 2: Cloning of svp's from G. rostochiensts

From 1 * 10 ⁷ juveniles of G rosioduensis hatched in potato root chffusate RNA was isolated using RNA Extraction Kit (Pharmacia Biotech. SE). PolyA RNA was purified using mRNA Purification Kit (Pharmacia Biotech. SE). A cDNA library containing 2.5 * 10 ⁶ primary rccombinants w ith an average insert size of 1500 bascpairs was constructed by Invitrogen (San Diego CA USA) This library in cloning vector pcDNAII was used as target DNA for amplification of svp encoding sequences using primers: 5'- CTTCCGTGTCTTCCTCCTCCATG -3' v s as 5' primer 5'- GGGTCG ACGCGCAACCAC 1 Ti Tl TATCATCATC - 3'. and

5'- GGGTCGACCGCAACAATTTTATCGTCAA1 AAATT - 3' as 3' primers

PCR resulted in t bands appr x l ^{ς =;} 0 bp M ά 1280 bp respectiv ely Both bands were purified from agarosc using Easy Prep PCR Pioduet Prep kit ( Pharmacia SE), and ligated in a TA Cloning vector (pCR2.1 ) of the One Shot Cloning kit (Invitπgen. San Diego. USA). DNA sequencing resulted in sequences according to SEO I D No's 3 and

4.

Example 3: Heterologous expression of GR-engl and GR-eng2

Heterologous expression of GR-rai;/ and GR-eng2 w as done using the maltose binding protein, protein fusion and purification system (Nevs England Biolabs). O gonuclcotide primer 5'-TGGGATCCACTGCCACGCCTCCCCCA-3 ' w as used in combination w ith primer 5'- GGAAGCTGCGAACCACTTTTTTATCATCATC-3 ' and 5'- GGAAGCTTCGCAACAATTΓΓA7 CGTCAATAAATT-3 ' to amplify PCR resp GR-engl and GR-eng2 The amplified PCR products y\ cre digested ith restrictiv e endo- nucleases Hindlll and B.imHI and ligated into the pMALc2 m.ilE-cxprcssion vector E coli strain TB1 w as transformed w ith pMAL-c 2 containing fusion proteins w ith cither

GR-engl or GR-eng2

Single colony transtormants w ere selected tor insert, and used for overexpression of fusion proteins and malE GR-ENG2 Overexpression w as done in LB culture medium including 0.2% glucose, and 100 μg/ml ampillicin Cultures were grown to A600 of 0.5 at 30°C, and 250 rpm rotation, w hen IPTG was added to a final concentration of 0.3 mM. After IPTG induction w as maintained for 3 h at 30 °C. while rotating at 250 rpm. the cells w ere harvested by eentπfuging at 4000 x g for 20 mm The supernatant w as discarded, and the cells w ere resuspended in 6 M urea. 20 mM Tris-Cl pH 7.4, 200 mM NaCl. 1 mM EDTA for 2 h at room temperature The resuspended cells

were subsequently ccntπfuged for 30 m at 10.000 x g. The supernatant was collected, and dialysed against 20 mM Tris-Cl, 200 mM NaCl, 1 mM EDTA to remove the urea.

The rccombinant fusion proteins w ere purified from the E. co lysate ith affinity chromatography on amylose resin (New England Biolabs). and tested on Western blot for detection with a cocktail of svp31 , svp39, and svp49 specific monoclonal antibodies (MGR

46,47,48,49,53,54.55.56.57.59.60; WO 96/22372. De Boer et al, 1996), and a malE specific polyclonal antiserum. The rccombinant proteins w ere recognized as fusion proteins, and migrated in the SDS-PAGE betw een 80 and 95 kDa. w ith approx. 10 kDa difference in molecular mass. The fusion proteins w ere incubated 17 h at 30 °C in a cup- plate assay including 0.2% carbo.xy methyl cellulose in 0.5% agarosc (see example 4).

Cleared halos were measured having a size of around 65 mm"

Example 4: Functionality assay I

Carboxymcthylccllulose ( CMC). 0 4% in 50 mM K-phosphaie/citπc acid buffer pH 5.2 (PCA buffer) w as mixed with an equal volume of 1 % agarosc (Seakem) in PCA buffer and a layer of 4 mm w as poured in a 10 cm pctπ dish ( Matcos et al. 1992). Holes were punched out able to contain 4 μ[. From a homogenate of second stage larvae of G. rosioduensis in 50 mM Na-phosphate buffer pH 7.2 containing \ b μvjμ\ protein. 3 μl was added to a hole. After 18 h incubation at 30 °C the plates were flooded w ith 0.1 % Congo Red in water and incubated for 30 min. at room temperature and then rinsed with 1 M NaCl. A translucent halo w ith a diameter of 16 mm vs as observed proving the presence of cellulolytic activity in nematode homogenates This sample could be 256-fold diluted before the activity had become invisible.

When the same experiment w as carried out with 20-fold concentrated salivary gland secretions (potato root diffusate wherein second stage juveniles w ere hatched), also cellulolytic activity was observed. A translucent halo w ith a diameter of 8 mm was seen.

Example 5: Functionality assay II

To establish which proteins have cellulasc activity in second stage juveniles (J2s) of G. rosioduensis and H glyctnes and in collected salivary secretions of J2s of G. rostochiensi.s and thus with the gene cloned as described in examples 1 and 2, to test the cellulasc activity of these cloned and hcterologously expressed proteins malE::GR-ENGl and malE::GR-ENG2 (example 3) and to assess the presence of cellulases in J2s of Meloidogvnc incognua and Globoderci labacum. homogenates were prepared in 50 mM Na-phosphate buffer pH 7.2 and subjected to elcctrophoresis on a 10% polvacrylamidc

gel containing 20 μg per ml bovine serum albumin. Samples were prepared with a sample buffer having a final SDS concentration of 0.4% while reducing agent was omitted. Of the J2 homogenates 1 to 5 μg of protein and 15 j of 20-fold concentrated salivary gland secretions (standard pore water (Schouten & VandcrBrugge, 1989) w herein the juveniles were incubated overnight after hatching in potato root diffusatc) were loaded on two gels without having been boiled. One gel was blotted onto PVDF membrane and stained with MGR 48 or, for the hetcrologously expressed fusion proteins, with a cocktail of MGRs 46-60 (see example 3). The other gel was shed three times in 100 ml PCA buffer for 40 minutes and then placed on top of a 1 mm layer of 0,5% Seakem agarosc containing 0.2% carboxymcthylccllulo.se in PCA buffer. After incubation for at least 3 hrs at room temperature, the gel w as removed and (he agarosc lay er stained w ith Congo Red for 30 minutes. After dcstaining by rinsing ith 1 NaCl clcai hands became visible representing cellulasc activity. The position ot these bands tor the protein homogenate of G rosioduensis J2s corresponded to that of the 49 and 32/31 kD bands detected on Western blot with MGR 48. In salivary gland secretions tvy o bands became visible and their positions were the same as the 32/31 kD and 39 kD bands on the immunoblot.

For homogenates of H glycines J2s hands representing cellulasc activity were found corresponding to 66 kD. 49 kD and 32 kD The latter tw o bands w ere also recognised by MGR48. For (7 uibacum homogcn.iles a cellulasc activity w as found corresponding to an apparent molecular w eight of 70 kD. On a Western blot probed with

MGR48 eight bands are visible in the molecular w eight range of 30 to 50 kD. For M incognita cellulasc activities w ere found corresponding to apparent molecular weights of 32. 49 and 60 kD. These proteins w ere not recognised bv MGR48 The hetcrologously expressed proteins MalE:./GR-ENGl and MalE /GR-ENG2 sho ed cellulasc activity and could be detected on a Western blot w ith the cocktail ot MGRs 46-60.

Example 6: Testing inhibiting capacity of anti-cellulase monoclonal antibodies.

The assay described in example 4 (Functionality assay I) was used. Instead of holes able to contain 4 μl. holes able to contain 8 id were punched out. To test the inhibiting capacity of MGRs 48. 49. 56, 59 and 60 they were mixed with the hetero- logously expressed proteins MalE::/GR-ENG l and Mali: 'GR-ENG2 Thereto. 4 μl of a 1 mg/ l solution in PCA buffer of each of the antibodies w as mixed with 4 μl of a 1 mg ml (ENG1) or 0.4 mg/ml (ENG2) solution of each of the fusion proteins. As controls PCA buffer, the aspecific anti-cmyc monoclonal antibody 9E10 (in a 1 mg ml concentration) and cellulasc tτom Λspergtllus mgei in a 25 ng/ml concentration were used.

The results are shown in figure 7 and in the table below. It was shown that MGR60 inhibited MalE::/GR-ENGl in this set up to about 50% of its activity.

Table: Inhibition of ccllulase activity by specific monoclonal antibodies

References

De Boer, J.M., G. Smant. A. Goversc. E.L. Davis. H.A. Overmars. H. Pomp. M. Van Gent-Pclzcr, J.F. Zilverentant. J.P.W.G. Stokkermans. R.S. Hussey. F.J. Gommers. J. Bakker. and A. Schots. Secretory granule proteins from the subventral csophageal glands ot the potato cyst-nematodc identified by monoclonal antibodies to a protein traction from the second-stage juveniles (1996) Molecular Plant -Microbe Interactions 9. 39-46. Deubert, K.H.. & R.A. Rohde. 1971. Nematodc enzymes (Chapter 16). In: Plant Parasitic Nematodes. Volume II. Eds. Zuckcrman. B.M.. W.F. Mai. k. R.A. Rohde.

Academic Press, pp. 73-90. Koji a K., Kamijyo A., Masumori M.. λ: Sasaki S 1994. Cellulasc activities of pine-wood nematodc isolates with different v irulences Journal t>f the Japanese Forestry Society 76 (3): 258-262. Mateos. P.F.. Jimenez-Zurdo. J.I.. Chen. J., Squartini. A.S.. KjHaack. S K.. Martinez-

Molina, E., Hubbcil. D.H. and Dazzo. F.B. 1992. Cell-associated pectinolytic and cellulolytic enzymes in RJuzobiu/n leguniinosarum bivar infolu . -ippl. Environ. Microbiol. 58, 1816-1822. Odani, K., Sasaki, S., Nishiyama Y. & Yamamoto N. 1985; Early symptom development of the pine wilt disease by hydrolytic enzymes produced by the pine wood nematodes - cellulasc as a possible candidate of the pathogen. Journal of the Japanese Forestry Society 67 (9): 366-372. Ray, C, Abbott. A.G., and Hussey. R.S. 1994 Mol. Biochem. Parasitol. 68, 93-101. Schagger, H. and von Jagow. G. 1987. Tricine-sodium doclccyl sulfatc-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa.

Anal. Biochem. 166, 368-379. Schouten A.J. and VanderBrugge, I.R. 1989. Acute to icitcit van aluminium en H ⁺ ionen concentratie voor bodemnematoden uit ecn zuur en kalknjk denncbos. Rep. 718603001. National Institute of Publ ic Health and Environmental Protection. Bilthoven, The Netherlands.

SEQUENCE LISTING

( 1 ) GENERAL INFORMATION :

(i) APPLICANT:

(A) NAME: Rijkslandbou universitei Wageningen

(B) STREET: P.O. Box 9101

(C) CITY: Wageningen

(E) COUNTRY: The Netherlands

(F) POSTAL CODE (ZIP): 6700 HB

(G) TELEPHONE: 0317-489111

(ii) TITLE OF INVENTION: Novel cellulases (iii) NUMBER OF SEQUENCES: 7

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0. Version #1.25 (EPO)

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: EP 96201890.9

(B) FILING DATE: 08-JUL-1996

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: EP 97200136.6

(B) FILING DATE: 17-JAN-1997

12) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1191 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Heterodera glycines

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 6 ..1023

(D) OTHER INFORMATION: /product= "Endoglucanase2"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

TCTTAATAAA AAAATACTCT TCAAATAAAT AATCATTCCT AGCTGCTTTT GGTTTGATCA 60

CAA ATG TTC GTC CAA CTC GTC CTC CTT GCC ATC GTT GGC ATT ACT TTT 108 Met Phe Val Gin Leu Val Leu Leu Ala He Val Gly He Thr Phe

GTC GAT GCT GCC GCA CCG CCG TTC GGC CAA TTG TCC GTC AAA GGC ACC 156 Val Asp Ala Ala Ala Pro Pro Phe Gly Gin Leu Ser Val Lys Gly Thr 20 25 30

AAT TTG GTC GGC TCA AAC GGC AAA CCG GTG CAG CTG ATC GGC AAT TCG 204 Asn Leu Val Gly Ser Asn Gly Lys Pro Val Gin Leu He Gly Asn Ser 35 40 4

TTG TTC TGG CAC CAG TGG TAC CCA CAA TTT TGG AAT GCC CAA ACA GTG 252 Leu Phe Trp His Gin Trp Tyr Pro Gin Phe Trp Asn Ala Gin Thr Val 50 55 60

AAG GCA CTC AAA TGC AAT TGG AAT TCC AAT GTC GTG CGC ACC GCA ATG 300 Lys Ala Leu Lys Cys Asn Trp Asn Ser Asn Val Val Arg Thr Ala Met 65 70 75

GGC GTG GAA CAG GGC GGC TAT CTG ACT GAC GCG AAC ACC GCC TAC CGA 348 Gly Val Glu Gin Gly Gly Tyr Leu Ser Asp Ala Asn Thr Ala Tyr Arg 80 85 90 95

CTG ACG GCA GCT GTG ATT GAG GCG GCC ATT GCA CAG CGC ATT TAC GTG 396 Leu Thr Ala Ala Val He Glu Ala Ala He Ala Gin Gly He Tyr Val 100 105 110

ATC GTC GAT TGG CAC GCG CAT GAG CCG AAC GCG GAC AAG GCG ATT GAA 444 He Val Asp Trp His Ala His Glu Pro Asn Ala Asp Lys Ala He Glu 115 120 125

TTC TTC ACC AAA ATT GCG AAA GCG TAC GGC TCC AAC CCT CAC TTG CTT 92 Phe Phe Thr Lys He Ala Lys Ala Tyr Gly Ser Asn Pro His Leu Leu 130 135 140

TAC GAA ACG TTT AAC GAG CCA TTG GAT GTG TCT TGG AAC CAT GTG CTT 50 Tyr Glu Thr Phe Asn Glu Pro Leu Asp Val Ser Trp Asn Asp Val Leu 145 150 155

GTC CCT TAC CAT AAA AAG GTT ATT TCT GCA ATT CGT GCC ATC GAC AAA 588 Val Pro Tyr His Lys Lys Val He Ser Ala He Arg Ala He Asp Lys 160 165 170 175

AAG AAT GTG ATC ATT CTC CGC ACT CCC AAA TGG TCT CAA GAT GTT GAC 636 Lys Asn Val He He Leu Gly Thr Pro Lys Trp Ser Gin Asp Val Asp 180 185 190

GTG GCG GCC CAA AAT CCG ATC AAA GGA TTC AGT AAT TTG ATG TAC ACT 684 Val Ala Ala Gin Asn Pro He Lys Gly Phe Ser Asn Leu Met Tyr Thr 195 200 205

CTC CAC TTC TAT GCG TCC AGT CAC TTT GTT GAT GGA CTT GGC AAT AAG 732 Leu His Phe Tyr Ala Ser Ser His Phe Val Asp Gly Leu Gly Asn Lys 210 215 220

CTT AAG ACC GCC GTA AAT AAG GGT CTT CCG GTG TTC GTC ACT GAG TAC 78O Leu Lys Thr Ala Val Asn Lys Gly Leu Pro Val Phe Val Thr Glu Tyr 225 230 235

GGT ACA TGC GAA GCG TCT AGC AAC GGT AAT CTG AAC TCC GGC TCA ATG 828 Gly Thr Cys Glu Ala Ser Ser Asn Gly Asn Lou Asn Ser Gly Ser Met 240 245 250 255

TCA AGC TGG TGG AGC CTG TTG GAC CAA CTG AAA ATT TCG TAC GTC AAT 876 Ser Ser Trp Trp Ser Leu Leu Asp Gin Leu Lys He Ser Tyr Val Asn 260 265 270

TGG TCA ATC ACT GAC AAA AGC GAA GCT TGT GCA GCG CTC ACT GGC GGA 924 Trp Ser He Thr Asp Lys Ser Glu Ala Cys Ala Ala Leu Thr Gly Gly 275 280 285

ACA TCG GCT GCC AAT GTT GGC ACT TCC TCC CGC TGG ACG CAG TCT GGC 972 Thr Ser Ala Ala Asn Val Gly Thr Ser Ser Arg Trp Thr Gin Ser Gly 290 295 300

AAT ATG GTA GCT TCG CAA CAC AAG AAA AAA TCC ACC GGT GTG AAA TGC 1020 Asn Met Val Ala Ser Gin His Lys Lys Lys Ser Thr Gly Val Lys Cys 305 310 315

TAAATAAAGT CTGAAGTCCG GATGATCAAC TGAAACTGAA GAAAATGGAA AATGTGAAGA lθ8θ

TTGCGAAAAA TAAATATATA TAAAATTTGC ATGCG Yl'GT TIYTCGA T TCCGATTAAA ll4θ

TTGTTTTTTG AGCGCAGATA AATCGCAAAG CΛTAAAAΛAΛ A-\ \AAΛΛA A 1191

(2) INFORMATION FOR SEQ ID NO: 2:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1597 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: He erodera glycmes

(IX) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 4..1 34

(D) OTHER INFORMATION: /product^ "Endoglucanasel"

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

CAA ATG TGC CGA CTC CAA GCA ACT CAT CTG CTC GCT CGA CTC TTT CTG 48 Met Cys Arg Leu Gin Ala Thr His Leu Leu Ala Arg Leu Phe Leu

1 10 15

CTT CTT GCG CTT TGC ACT GCT CTC GTT AGC TCT CTC ACT GCT GTT GCC 96 Leu Leu Ala Leu Cys Thr Ala Leu Val Ser Ser Leu Thr Ala Val Ala 20 25 30

CCG CCA TTC GGC CAA TTG TCC GTT TCC GGC ACC AAT TTG GTC GGC GCC 144 Pro Pro Phe Gly Gin Leu Ser Val Ser Gly Thr Asn Leu Val Gly Ala 35 40 45

AAC GGA CAA CCC GTA CAG CTG ATC GGC AAC TCA CTG TTC TGG CAC CAA 192 Asn Gly Gin Pro Val Gin Leu He Gly Asn Ser Leu Phe Trp His Gin 50 55 60

TGG TAC CCG CAG TTT TGG AAC GCT GAC ACA GTG AAG GCA CTC AAA TGC 240

Trp Tyr Pro Gin Phe Trp Asn Ala Asp Thr Val Lys Ala Leu Lys Cys

65 70 75

AAT TGG AAT GCC AAT GTC ATC CGG GGG GCC ATG GGC GTG GAC GAG GGC 288

Asn Trp Asn Ala Asn Val He Arg Gly Ala Met Gly Val Asp Glu Gly

80 85 90 95

GGC TAT CTG AGT GAC GCG AAC ACG GCT TAC AAT CTG ATG GTG GCA GTG 336

Gly Tyr Leu Ser Asp Ala Asn Thr Ala Tyr Asn Leu Met Val Ala Val

100 105 110

ATC GAA GCG GCC ATT TCC AAT GGC ATT TAC GTG ATC GTC GAT TGG CAT 384

He Glu Ala Ala He Ser Asn Gly He Tyr Val He Val Asp Trp His

115 120 125

GCC CAC AAT TCA CAT CCG GAC GAA GCG GTC AAA TTC TTC ACC CGA ATT 32

Ala His Asn Ser His Pro Asp Glu Ala Val Lys Phe Phe Thr Arg He

130 135 140

GCT CAA GCG TAC GGC TCC TAC CCT CAC ATT TTG TAC CAG GAT TTC AAC 480

Ala Gin Ala Tyr Gly Ser Tyr Pro His He Leu Tyr Glu Asp Phe Asn

145 150 155

GAG CCG CTG AGC GTT TCG TGG ACC GAT GTG CTG GTG CCA TAC CAC AAA 528

Glu Pro Leu Ser Val Ser Trp Thr Asp Val Leu Val Pro Tyr His Lys

160 165 170 175

AAG GTC ATT GCT GCC ATC CGA GCT ATT GAC AAG AAA AAT GTG ATC ATT 576

Lys Val He Ala Ala He Arg Ala He Asp Lys Lys Asn Val He He

180 185 190

CTC GGC ACT CCA ACA TGG TCC CAA GAC GTG GAT GTG GCA TCA CAG AAC 624

Leu Gly Thr Pro Thr Trp Ser Gin Asp Val Asp Val Ala Ser Gin Asn

195 200 205

CCA ATC AAA GAC TAC CAA AAT CTG ATG TAC ACT CTC CAC TTT TAC CCG 672

Pro He Lys Asp Tyr Gin Asn Leu Met Tyr Thr Leu His Phe Tyr Ala

210 215 220

TCC AGT CAC TTC ACG AAT GAT CTT GGT GCC AAG CTC AAA ACA GCC GTG 720

Ser Ser His Phe Thr Asn Asp Leu Gly Ala Lys Leu Lys Thr Ala Val

225 230 235

AAC AAC GGT TTG CCT GTG TTC GTC ACT GAG TAC GGC ACA TGC GAA GCG 768

Asn Asn Gly Leu Pro Val Phe Val Thr Glu Tyr Gly Thr Cys Glu Ala

240 245 250 25

TCG GGC AAC GGC AAC TTG AAC ACT GAC TCG ATG TCC AGC TGG TGG ACT 8l6

Ser Gly Asn Gly Asn Leu Asn Thr Asp Ser Met Ser Ser Trp Trp Thr

260 265 270

CTG CTG GAC AGC TTG AAG ATT TCA TAC GCC AAC TGG GCA ATC TCC GAC 864

Leu Leu Asp Ser Leu Lys He Ser Tyr Ala Asn Trp Ala He Ser Asp

275 280 285

AAA AGT GAG GCC TGC TCA GCA CTG AGC CCC CGT ACA ACT GCT GCC AAT 912

Lys Ser Glu Ala Cys Ser Ala Leu Ser Pro Gly Thr Thr Ala Ala Asn

290 295 300

GTC GGT GTT TCG TCC CGT TGG ACA TCC TCC GGA AAT ATG GTT GCT TCG 960 Val Gly Val Ser Ser Arg Trp Thr Ser Ser Gly Asn Met Val Ala Ser 305 310 315

TAC TAC AAG AAA AAA TCC ACC GGC GTA AGC TGT AGC GGC TCA AGT TCC 1008 Tyr Tyr Lys Lys Lys Ser Thr Gly Val Ser Cys Ser Gly Ser Ser Ser 320 325 330 335

GGC AGC TCT TCG GGC TCG AGT TCT GGC TCT TCC GGT TCA AGT TCC GGC 1056 Gly Ser Ser Ser Gly Ser Ser Ser Gly Ser Ser Gly Ser Ser Ser Gly 340 34 350

AGC TCT TCC GGC TCA AGT TCC GGC AGC TCT TCC GGC TCA AGT TCG GGC 1104 Ser Ser Ser Gly Ser Ser Ser Gly Ser Ser Ser Gly Ser Ser Ser Gly 355 360 36

TCC TCT GGT TCG AGC TCA GGG TCC AGC TCG GGT TCG GGA TCC GCC AGC 1152 Ser Ser Gly Ser Ser Ser Gly Ser Ser Ser Gly Ser Gly Ser Ala Ser 370 375 380

ATC TCT GTA GTC CCA TCC AAC ACG TGG AAT GGC GGT GGT CAG GTC AAC 1200 He Ser Val Val Pro Ser Asn Thr Trp Asn Gly Gly Gly Gin Val Asn 385 390 395

TTC GAA ATC AAA AAC ATC GGG TCC GTG CCA TTG TGT GGC GTT GTG TTC 1248 Phe Glu He Lys Asn He Gly Ser Val Pro Leu Cys Gly Val Val Phe 400 405 410 415

AGC GTC TCT CTT CCC TCA GGG ACC ACG CTT GGT GGA TCG TGG AAC ATG 1296 Ser Val Ser Leu Pro Ser Gly Thr Thr Leu Gly Gly Ser Trp Asn Met 420 425 430

GAA TCC GCA GGC TCC GGC CAA TAC AGC TTG CCA AGT TGG GTC AGA ATT 1344 Glu Ser Ala Gly Ser Gly Gin Tyr Ser Leu Pro Ser Trp Val Arg He 43 440 445

GAG GCC GGA AAA TCG AGC AAA GAC GCG GGG CTG ACA TTC AAC GGA AAA 1392 Glu Ala Gly Lys Ser Ser Lys Asp Ala Clγ Leu Thr Phe Asn Gly Lys 450 460

GAT AAG CCA ACG GCG AAA ATT GTG ACG ACG AAG AAA TGT TAGGGAAAAG l44l Asp Lys Pro Thr Ala Lys He Val Thr Thr Lys Lys Cys 465 470 475

ACAAAGCGCA AAATGGGAGG AAAAGAATGG GAAAATGAAA GAGTGGAGAA AAAGAACAAA 1501

GGGAAAGAAA GACAAAGAAT TAATGATTAT GATAAGAGAT AATAACTGAT AAAGTTTAGC I56I

ATAAAATGTT GACCAAAGTA AAAAAAAAAA AAAAAA 1597

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 128l base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Globodera rostochiensis

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..1161

(D) OTHER INFORMATION: /ρroduct= "Endoglucanase2"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: :

ATG TCC TTC CGT GTC TTC CTC CTC CAT GCC CTG CAC ATT GTG TTC TGT 48 Met Ser Phe Arg Val Phe Leu Leu His Ala Leu His He Val Phe Cys 1 10 15

AAC GCT TTG ACT GCC ACG CCT CCC CCA TAC GGG CAA TTG TCC GTC TCC 96 Asn Ala Leu Thr Ala Thr Pro Pro Pro Tyr Gly Gin Leu Ser Val Ser 20 25 30

GGC ACC AAA TTG GTC GGC TCC AAC GGA CAA CCC GTT CAG CTG ATC GGA 144 Gly Thr Lys Leu Val Gly Ser Asn Gly Gin Pro Val Gin Leu He Gly 35 40 45

AAT TCG CTG TTC TGG CAC CAA TTT CAA GGA CAG TAC TGG AAC GCC GAT 192 Asn Ser Leu Phe Trp His Gin Phe Gin Gly Gin Tyr Trp Asn Ala Asp 50 55 60

ACA GTG AAG GCG CTC AAA TGC AAT TGG AAC GCC AAC GTC GTG AGG GCG 240 Thr Val Lys Ala Leu Lys Cys Asn Trp Asn Ala Asn Val Val Arg Ala 65 70 75 80

GCC GTC GGC GTG GAC TTG GAG CGC GGC TAT ATG AGC GAC CCG ACC AGC 288 Ala Val Gly Val Asp Leu Glu Arg Gly Tyr Met Ser Asp Pro Thr Ser 85 90 95

GCT TAC AAT CTG GCG GTT GCC GTG ATT GAG GCC GCC ATC TCC CAG GGC 336 Ala Tyr Asn Leu AJa Val Ala Val He Glu Ala Ala He Ser Gin Gly 100 105 110

ATG TAC GTG ATC GTT GAT TGG CAT TCG CAC GAG GCA CAC GCG GAC AAA 38 Met Tyr Val He Val Asp Trp His Ser His Glu Ala His Ala Asp Lys 115 120 125

GCG GTT GAA TTC TTC ACC AAA ATT GCC AAA GCC TAC GGC TCA TAC CCA 432 Ala Val Glu Phe Phe Thr Lys He Ala Lys Ala Tyr Gly Ser Tyr Pro 130 135 1 0

CAC GTT CTC TAC GAA ACT TTC AAC GAG CCG TTG CAG GGC GTG TCG TGG 480 His Val Leu Tyr Glu Thr Phe Asn Glu Pro Leu Gin Gly Val Ser Trp 145 150 155 160

ACC AAC ATT CTG GTG CCA TAC CAC AAG AAG GTA ATT GCT GCC ATC CGC 528 Thr Asn He Leu Val Pro Tyr His Lys Lys Val He Ala Ala He Arg 165 170 175

GCG CTT GAT CCC AAA AAT GTG ATA ATT CTC GGC ACG CCC ACA TGG TGC 576 Ala Leu Asp Ala Lys Asn Val He He Leu Gly Thr Pro Thr Trp Cys l8θ 18 190

CAA GAT GTG GAT CTG GCC TCG CAG AAT CCG ATC AAA GAG TAC AAA AAT 624 Gin Asp Val Asp Leu Ala Ser Gin Asn Pro He Lys Glu Tyr Lys Asn 195 200 205

CTG ATG TAC ACC TTC CAC TTC TAC GCC TCC ACC CAC TTC GTC AAC GGT 672 Leu Met Tyr Thr Phe His Phe Tyr Ala Ser Thr His Phe Val Asn Gly 210 215 220

CTT GGT GCC AAA CTT CAG ACG GCC ATA AAC AAC GGT CTG CCC ATC TTC 720 Leu Gly Ala Lys Leu Gin Thr Ala He Asn Asn Gly Leu Pro He Phe 225 230 235 240

GTG ACT GAG TAC GGC ACA TGC GCT GCG GAC GGC AAC GGC AAC ATC GAC 768 Val Thr Glu Tyr Gly Thr Cys Ala Ala Asp Gly Asn Gly Asn He Asp 245 250 255

ACC AAC TCC ATC TCG AGC TGG TGG ACT CTG TTG GAC AAC CTG AAG ATT 8l6 Thr Asn Ser He Ser Ser Trp Trp Thr Leu Leu Asp Asn Leu Lys He 260 265 270

TCG TAC CTA AAT TGG GCA ATC AGC GAC AAA AGC GAA TCT TGC TCA GCA 864 Ser Tyr Leu Asn Trp Ala He Ser Asp Lys Ser Glu Ser Cys Ser Ala 275 280 285

CTC AAA CCG GGC ACA CCA GCA GCC AAT GTT GGC CTT TCG TCC GCT TGG 912 Leu Lys Pro Gly Thr Pro Ala Ala Asn Val Gly Val Ser Ser Ala Trp 290 295 300

ACA ACC TCC GGA AAT TTG GTA GCC GCC CAT GAT AAG AAA AAG TCC ACC 96O Thr Thr Ser Gly Asn Leu Val Ala Ala His Asp Lys Lys Lys Ser Thr 305 310 315 320

GGC GTG AGC TGC AAC GGC GCC ACT CCC GCC CGT GGT TCG TCG TCG GCT 1008 Gly Val Ser Cys Asn Gly Ala Thr Pro Ala Arg Gly Ser Ser Ser Ala 325 330 335

AAT TCG GGC AAA CCT GCC GCT GCC AAA AAG CCT CCG GCC AAA CCT GCC 1056 Asn Ser Gly Lys Pro Ala Ala Ala Lys Lys Pro Pro Ala Lys Pro Ala 340 3 350

GCT GCC AAA AAA TCG CCT CCG GCC AAA CCT GCC GCT GCC AAA AAG CCC 1104 Ala Ala Lys Lys Ser Pro Pro Ala Lys Pro Ala Ala Ala Lys Lys Pro 355 360 365

CCG GCC AAA TCG GCC AAA GCT GCC CCT GCC AAA AAG CCC AAG TCC AAA 1152 Pro Ala Lys Ser Ala Lys Ala Ala Pro Ala Lys Lys Pro Lys Ser Lys 370 375 ^" 380

TCA GGC TAAGAAATGA CGGAGAAGCG AAACATTGGA GCAAAATGGG GGATTATGAT 1208

Ser Gly

385

AAATAACGGA CGATGTTTCT TTAAATTTAT TGACGATAAA ATTGTTGCGA ATAAAAAAAA 1268 AAAAAAAAAA AAA 128l

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1551 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Globodera rostochiensis

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..1 19

(D) OTHER INFORMATION: /product= "Endoglucanasel"

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

ATG TCC TTC CGT GTC TTC CTC CTC CAT GGC CTG CAC ATT GTG TTC TGT 48 Met Ser Phe Arg Val Phe Leu Leu His Gly Leu His He Val Phe Cys 1 5 10 15

AAC GCT CTG ACT GCC ACG CCT CCC CCA TAC GGG CAA TTG TCC CTT TCC 96 Asn Ala Leu Thr Ala Thr Pro Pro Pro Tyr Gly Gin Leu Ser Val Ser 20 25 30

GGC ACC AAA TTG GTC GGC TCC AGT GGA CAA CCC GTT CAG CTG ATC GGA 144 Gly Thr Lys Leu Val Gly Ser Ser Gly Gin Pro Val Gin Leu He Gly 35 40 5

AAT TCG CTG TTC TGG CAT CAA TTT CAA GCA CAG TAC TGG AAT GCC GAA 192 Asn Ser Leu Phe Trp His Gin Phe Gin Ala Gin Tyr Trp Asn Ala Glu

50 55 60

ACA GTA AAG GCG CTC AAA TGC AAT TGG AAC GCA AAC GTC GTG AGG GCG 240 Thr Val Lys Ala Leu Lys Cys Asn Trp Asn Ala Asn Val Val Arg Ala 65 70 75 80

GCC GTG GGC GTG GAC TTG GAG CGC GGC TAT ATG AGC GAC CCG ACC ACC 288 Ala Val Gly Val Asp Leu Glu Arg Gly Tyr Met Ser Asp Pro Thr Thr 85 90 95

GCT TAC AAT CAG GCG GTT GCC GTC ATC GAG GCA GCC ATC TCC CAG GGC 336 Ala Tyr Asn Gin Ala Val Ala Val He Glu Ala Ala He Ser Gin Gly

100 105 110

TTG TAC GTA ATC GTT GAT TGG CAT TCG CAC GAA TCT CAC GTG GAC AAA 384 Leu Tyr Val He Val Asp Trp His Ser His Glu Ser His Val Asp Lys 115 120 125

GCG ATT GAA TTC TTC ACC AAA ATT GCC AAA GCC TAC GGC TCA TAC CCA 432 Ala He Glu Phe Phe Thr Lys He Ala Lys Ala Tyr Gly Ser Tyr Pro 130 135 140

CAC GTT CTC TAC GAA ACT TTC AAC GAG CCG TTG CAA GGC GTG TCG TGG 480 His Val Leu Tyr Glu Thr Phe Asn Glu Pro Leu Gin Gly Val Ser Trp 145 150 155 160

ACC GAC ATT CTG GTG CCA TAC CAC AAG AAG GTA ATT GCT GCC ATC CGC 528 Thr Asp He Leu Val Pro Tyr His Lys Lys Val He Ala Ala He Arg 165 170 175

GCG CTT GAT TCC AAA AAT GTG ATA ATT CTC GGC ACG CCC ACA TCG TGC 76 Ala Leu Asp Ser Lys Asn Val He He Leu Gly Thr Pro Thr Trp Cys 180 185 190

CAA GAT GTG GAT ATT GCT TCG CAG AAT CCG ATC AAA GAG TAC AAA AAT 624 Gin Asp Val Asp He Ala Ser Gin Asn Pro He Lys Glu Tyr Lys Asn 195 200 205

CTG ATG TAC ACC TTT CAC TTC TAC GCC GCC ACT CAC TTC GTC AAC GGT 672 Leu Met Tyr Thr Phe His Phe Tyr Ala Ala Thr His Phe Val Asn Gly 210 215 220

CTT GGT GCC AAA CTT CAG ACG GCC ATA AAC AAC GGC CTG CCA ATC TTT 720 Leu Gly Ala Lys Leu Gin Thr Ala He Asn Asn Gly Leu Pro He Phe 225 230 235 240

GTG ACT GAG TAC GGC ACA TGC TCT GCG GAC GGC AAC GGC AAC ATC GAC 768 Val Thr Glu Tyr Gly Thr Cys Ser Ala Asp Gly Asn Gly Asn He Asp 245 250 2

ACA AAC TCC ATC TCG AGC TGG TGG AGT CTG ATG GAC AAC CTG AAG ATT 8l6 Thr Asn Ser He Ser Ser Trp Trp Ser Leu Met Asp Asn Leu Lys He 260 265 270

TCG TAC CTA AAT TGG GCA ATC AGC GAC AAA AGC GAA ACT TGT TCA GCA 864 Ser Tyr Leu Asn Trp Ala He Ser Asp Lys Ser Glu Thr Cys Ser Ala 275 280 285

CTC AAA CCG GGC ACA CCA GCA GCC AAT GTT GGC GTT TCG TCC AGT TGG 912 Leu Lys Pro Gly Thr Pro Ala Ala Asn Val Gly Val Ser Ser Ser Trp 290 29 300

ACA ACC TCC GGA AAT ATG GTG GCC GAC CAT GAT AAG AAA AAA TCC ACC 960 Thr Thr Ser Gly Asn Met Val Ala Asp His Asp Lys Lys Lys Ser Thr 305 310 315 320

GGC GTG AGC TGC AGC GGC TCC ACT TCC AGT GGT TCG TCG TCG TCT AAT 1008 Gly Val Ser Cys Ser Gly Ser Thr Ser Ser Gly Ser Ser Ser Ser Asn

325 330 335

TCT GGC AAT TCT GCC GCC ACA ACG ACC ACC AAA AAG CCC CCA TCT AAT 1056 Ser Gly Asn Ser Ala Ala Thr Thr Thr Thr Lys Lys Pro Pro Ser Asn 340 345 350

TCT GGT CAA ACC ACT AAC CAA AAG CCT TCG TCT TCG GCG GGG TCC AGT 1104 Ser Gly Gin Thr Thr Asn Gin Lys Pro Ser Ser Ser Ala Gly Ser Ser 355 360 365

TCC AGC TCG GGG TCG GCC TCT GCC AGC GTC ACT GTT GTC TCA ACC AAC 1152 Ser Ser Ser Gly Ser Ala Ser Ala Ser Val Thr Val Val Ser Thr Asn 370 375 380

ACA TGG AAC GGA GGT GGA CAA GTC AAC TTT GAA GTC AAG AAC ACC GGA 1200 Thr Trp Asn Gly Gly Gly Gin Val Asn Phe Glu Val Lys Asn Thr Gly 385 390 395 400

AGC ACG ACA TTG TGT GGC GTG AAG TTC AGC GTC ACT CTT CCA GCG GGG 1248 Ser Thr Thr Leu Cys Gly Val Lys Phe Ser Val Thr Leu Pro Ala Gly 405 410 415

ACA ACT GTC GCC GGC TCA TGG AAC ATG AAC GCG GCC GGC TCT AAC GAA 1296 Thr Thr Val Ala Gly Ser Trp Asn Met Asn Ala Ala Gly Ser Asn Glu 420 425 430

TAC ACA TTG CCC AGC TAC ATA AAT ATT AAA GCA AAG GAA GCC AAC AAA 134 Tyr Thr Leu Pro Ser Tyr He Asn He Lys Ala Lys Glu Ala Asn Lys 435 440 445

GAC GCG GGA ATG ACG CTG AAC GGA AGT GGA AAG CCA ACG GCC AAA GTT 1 92 Asp Ala Gly Met Thr Leu Asn Gly Ser Gly Lys Pro Thr Ala Lys Val 450 455 460

TTG TCA ACA ACG GCA TGT TCG GGC TAAGAAATAA ATGGAGAAGA AGCGAAACAT 1446 Leu Ser Thr Thr Ala Cys Ser Gly 465 470

TGAAGAAAAA ACTAAGAGAT TTTGTTAAAT AACGGCGAAT TTTCTTTTAΛ TTTAATGATG 1506

ATGATAAAAA AGTGGTTGCG CCAAAAAAAA AAAAAAAAAA AAAAA I55I

(2) INFORMATION FOR SEQ ID NO- 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 836 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE-

(A) ORGANISM: Vjeloidogyne incognita

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: :

TACGTGATTG TGGATTGGCA TGACCATAAT GCTCAGAATC ATGTTGATCA GGCGGTGAGT 60

TAGAAGTTAT TTGAGGGAAT ATCAATCTAA TTAACATCTC TCAGTTTCAA TCTTACAATT 120

AGTATGTTAA AAAATAATTT TGAGTCAACA TCGGTATTTA ATCTGTGAGT TGAGTGTTTG 180

TGGTATATCT CAATAAATCG AGGAGGAATC CATTTATTTT TTGATATTGA TTCTTATAAT 24θ

GTTTCAATAA TTAAAATTGA CAATGATTTA TTCAATTTTT TAAAAATTGA TAAGGAAAAG 300

GAGGGGTAAT TATTAAAGGG GTAATTATTT GAGAGAGGGT AAAATATTTG AGCATTTACG 36O

GTAACGATTA TTGTTTTCAA GAAAAATACT TACCATGTAT AACCTGAGTT TGTCTAGACT 420

AAACAATGTC GAGTATTAAT CTTTATTGTT TTACAGATCA ΛTTTCTTCAC CTACATTGCT 480

AAACATACGG ATCGAATCCA AACATTATTT ACGAGACTTT CAATGAACCT TTACAAATTG 54θ

ATTGGAGTAT TGTAAAAGCT ATCACGAAAA AGTCGTTGCC GCCATACGCA AATATGACAA 600

AAAGAATTTA ATAGTACTCG GACAACAACA TGGTCTCAAG ATGTAGATAT TGCTGCAGCΛ 660

AATCCAGTTA GTGGATCTAA CCTTTGCTAC CTCTCCACTA TTACGCGGCT TCCCACAAAC 720

AATCTCTTCG TGACAAAGCC CAAACAGCCT TAAACAAGGA AAAATCTGTC AAAAAATCTT 7δO

TCCTAACTTT GTTTΛTAATT TAAGGGAGTT GGCACCTTCG TCACCGAAAC GGCACA 836

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 783 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Meloidogyne incognita

(xi) SEQUENCE DESCRIPTION: SEQ ID \ϋ: b:

TACGTGATTG TGGACTGGCA TGACCATAAT GCTCAGAATC ΛTGTTGAICA GGCGGTGAGT 60

TAGAGTTAAT GATGGAATTT TTACCCTCTC GCAAATΛATT ACCACCTCTA TAATTACCCC 120

CTAATTTTTC CGAAAACCTT AAAATAATTA AAAAATTATA AACAΛATTTA ATTATTGGAA l8θ

CATTTTAAGA CTTAATGTCA AAAATAATTA ATTTCGCCTC AATTAGATTλ AGATATATCC 240

CAAATCCAGC ACATCCGCCC TAATAATCAA AATCTGATTT AATCTCTATG TGTTAAGTGT 300

TTAATCCACA TAACGATTAT TGTCTTTAAT AAAAATACTT CCATGATAAC CTGAATTTGT 36O

CTTAGACTAA ATAAATGTCG AGTATTAGCC TTTATTGTTT AACAGATCAA TTTCTCACTT 420

ACATTGCTAA AACATACGGA TCAAATCCAA ACATTATTTA CGAGACTCTC AATGAACCTT 480

TACAATTGAT TGGAGTATTG TTAAAAGCTA TCACGAAAAA GTCGTTGCCG CCATACGCAA 0

ATATGACAAA AAAATTTAAT AGTACTCGGA ACAACAACAT GGTCTCAAGA TGTAGATATT 600

GCTGCAGCAA ATCCAGTTAG TGATCTAACC TTTGCTACAC TCTTCAC'IΛT TACGCGGCTT 660

CCCACAAACA ATCTCTTCGT GACAAAGCCC AACAGCCTTΛ AACAAGGCAA AAACTTTCAA 720

AAAATCTTTC CATACTTTGC TTATAATTTA AGGGAGTTTC ATTTTCGTCA CCGAATACGG 780

CAC 783

(2 ) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 2 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Meloidogyne incognita

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

TACGTGATTG TGGACTGGCA TGACCATAAT GCTCAGAATC ATGTTGATCA GGCGGTTAGT 60

TAGAAGTTAT TTTGAGGGAA TATCAATCTA ATTGACATCT CGΛGΛAATAT CTTAGTAATG 120

TTATAAAATA ATTTGTGTAA CATTTTTATT TAATCTCTGT GTGTTCAGTG TTTAATCCAC l8θ

ATAACGATTA TTGTCTTTCA AAAAAATCTT TTATAAATAA CCTGAGTTTA TCTTAGACTA 240

AACAATTTTC TAGTATTAAT CTTTATTGTT TCACAGTCAA TTTCTTCACC TACATTGCTA 300

AGACATACGG ATCAAATCCA AACATTATTT ACGAGACTTT CAATGACCTT TACAAATTGA 360

TTGGAGTATT GTTAAAAGCT ATCACGAAAA AGTCGTTGCC GCCATACGCA A TAGΛCAAA 420

AAGAATTTAA TAGTACTCGG AACAACAACA TGGTCTCAAG ATGTAGATAT TGCTGCAGCA 480

AATCAGTTAG TGGATCTAAC CTTTGCTACA CTCTTCATTA TT.ACGCGGCT TCCCACAAAC 40

AATCTCTTCG TGCAAAGCCC AAACAGCCTT AAΛCAAGGCA AAGATCTGTC AAAAAATCTT 600

TCCATACTTT GCTTTTTAAG GAGTTTGCAT TTTCGTCACC GΛGTΛCGGCΛ \ 652