ITMI20112038A1 - "NEW CHEMICAL GPCR RECEPTORS, THEIR USES FOR SCREENING OF PHYTOPHARMANS, AND RELATIVE SCREENING METHODS" - Google Patents

Description

â € œNew chimeric GPCR receptors, their uses for pesticide screening, and related screening methodsâ €

The present invention relates to the construction of novel chimeric G protein coupled receptors (GPCRs) and their uses for the screening of pesticides. In particular, the present invention also refers to the screening methods of pesticides which use such chimeric GPCR receptors. The invention further refers to methods for the construction of chimeric GPCR receptors capable of localizing in the membrane, starting from GPCR receptors lacking this capacity.

Plant parasitic nematodes cause enormous damage to the agricultural economy around the world every year. Among the most common parasites are the endoparasites which include Heterodera and Globodera (cyst nematodes) and Meloidogyne (root knotworms). The species belonging to the genus Meloidogyne are of great importance as they infect plants of considerable economic interest, such as wheat, corn, potatoes, tomatoes and legumes.

In the past, traditional methods such as crop rotation or the search for naturally resistant plants were used to counter the spread of these nematodes, together with the massive use of nematocides.

Most of the nematocides on the market are extremely toxic and have been restricted or banned by the Environmental Protection Agency: among these we find methylene bromide which reduces ozone in the atmosphere, aldicarb (Temik) responsible toxin of the contamination of the aquifers, and the 1,3-dichloropropene substance that has carcinogenic effects on man.

It is necessary to develop a new generation of agropharmaceuticals, much more selective, which allows the control of harmful nematodes, but which at the same time has no effect on the beneficial organisms of the soil and on the environment.

Of fundamental importance for the development of a new generation of crop protection products is the choice of the molecular target.

G-protein-coupled receptors (GPCRs) comprise a large group of structurally and functionally similar proteins that perform vital functions in all eukaryotic organisms.

They consist of a single polypeptide chain that crosses the membrane seven times with the extracellular amino terminal and the intracellular carboxy terminal. Following the binding with extracellular ligands (peptides, small molecules, ions, light and aromatic compounds) the receptor is activated by triggering a response in the cell that results in the production of second messengers (cAMP, Ca <2+>, cGMP, IP3) .

About 800 receptors have been identified in humans and their involvement in multiple functions has been demonstrated: from visual and olfactory perception to the immune response (1).

Given their large number and the importance of the functions they perform, GPCRs represent one of the main interests for the pharmaceutical industry: they are potential targets for new drugs and currently more than 50% of the drugs on the market act on GPCR (2).

The number of GPCR receptors is also very high in the nematode genome: about 1300 genes coding for GPCR have been identified in the model nematode Caenorhabditis elegans and it has been shown that they are involved in vital functions such as reproduction, nutrition and movement ( 3).

Similarly to human receptors, plant parasitic nematode receptors can be used as targets for new crop protection products.

The conventional technologies used by the pharmaceutical industries for the identification of molecules capable of modulating the activity of GPCRs involve the expression of human receptors in heterologous cell systems and the development of biological activity assays.

When the receptor is expressed in a cellular system, following activation by agonists or inhibition by inverse agonists, it is able to modulate a measurable intracellular signal. This receptor expression system makes it possible to analyze collections of thousands of molecules, natural or synthetic, and find those that are able to modulate the activity of the receptor. Once identified, these molecules represent potential drugs which, following optimization, can be tested directly on animals and humans in clinical studies.

The same technology can therefore be applied to research potential pesticides which, by inhibiting or hyperactivating the GPCR function of parasitic nematodes, alter their vital functions. However, the use of GPCR receptors as biological targets has a limit. Many GPCR receptors, when expressed in heterologous systems, present difficulties of localization in the plasma membrane, due to the structure and characteristics of the receptor itself. These difficulties make it impossible to use the receptor for the screening of molecules with pharmacological activity, since the localization of GPCRs in the plasma membrane is a fundamental requirement for the correct functioning of the receptor. If the receptor is not exposed in the membrane, but occurs in an internalized form in the cytoplasm, it is unable to interact with the molecules, agonists or inverse agonists, with which the cells are treated in order to find among these potential modulators of the € ™ receptor activity.

This problem can be solved with techniques that involve modifications to the sequence of the receptor itself, expression of other proteins or chemical treatments of the cells: this is to ensure the correct transport and localization of the receptors in the membrane. Some authors have succeeded in increasing the membrane expression of olfactory GPCR receptors by replacing the aminoterminal region with that of the human rhodopsin receptor (5), co-expressing them with the β-adrenergic receptor (6), or by treating the cells with drugs which block proteolysis (7). However, these systems are not very applicable for the screening of potential modulators of the activity of GPCR receptors of parasitic nematodes. Both the co-expression of a GPCR with the β-adrenergic receptor, and the modification of the aminoterminal portion can easily alter the interaction between the receptor under examination and potential modulators, which usually interact with the transmembrane or extracellular tracts of the protein. The drug treatment is then too invasive, and could easily compromise the response of the cells to the compounds being analyzed.

On the basis of the foregoing, the need for new screening methods and related biological targets is evident that allow the selective selection of specific plant protection products against nematodes and which, consequently, have a lower environmental impact.

The Applicant has now found that GPCR receptors can represent convenient specific targets for the control of plant parasitic nematodes. One of the main advantages associated with the choice of nematocides is in fact the specificity and selectivity of the potential agropharmaceuticals. Many of these GPCRs have a very low degree of identity with receptors belonging to other organisms: between human and nematode receptors there is an identity rate of only 30%. Even among the various species of nematodes, the degree of identity, albeit high, is never complete: between the receptors of the non-parasitic nematode C.elegans and those of the parasitic nematode Meloidogyne incognita the degree of identity is about 50-60 %. Furthermore, the conserved amino acids reside almost all in the transmembrane domains of the receptor, and this guarantees the possibility of finding molecules which, acting on the less conserved extracellular domains, are able to act specifically on the receptors of harmful organisms.

All this would therefore ensure that a molecule acting specifically on a GPCR of M.incognita does not interfere with the viability of other beneficial soil organisms, and is not at all harmful to humans and other mammals.

Besides the choice of GPCRs and the construction of chimeric GPCR receptors that allow the localization of GPCRs in the plasma membrane, a fundamental requirement for the correct functioning of the receptor (given that many GPCR receptors when expressed in heterologous systems present difficulties in localization in the membrane), another innovative aspect of the present invention. The modification of the receptors adopted does not alter the functionality of the receptors. Furthermore, the modification does not alter the interaction with potential modulators since it concerns a portion of the receptor not normally involved in the interaction with the ligands.

The Applicant has now identified a specific pesticide screening method for plant parasitic nematodes comprising the following steps: a) transformation of a heterologous cell line with one or more chimeric GPCR receptors of plant parasitic nematodes: said chimeric GPCR receptors being characterized by having the carboxyterminal domain replaced with that of the GPCR receptors of non-parasitic nematodes capable of localizing in the membrane;

b) putting the compound to be tested in contact with said cell line;

c) measurement of the intracellular signal activated by expression or stimulation with a known agonist of the chimeric GPCR receptor.

Wild type (wt) GPCR receptors are defined as GPCR receptors that have a sequence that is not genetically modified, that is, they possess the sequence of the native protein, as it is found in the organism of origin. It is first identified in a specific organism, then cloned into an appropriate expression vector and transfected into a heterologous cell system. The receptor thus expressed will have a sequence identical to that of the endogenous protein which is produced by the organism of origin. A chimeric GPCR receptor is instead a protein born from the fusion of two or more proteins, and more often also of portions of proteins. These portions can belong to different proteins coming from the thick organism, or from proteins of different organisms. To produce a chimeric protein, the gene sequences corresponding to the portions of proteins that are intended to fuse are joined together, into a single gene sequence. This sequence is first cloned into an expression vector and then transfected into a heterologous cell system. The protein thus expressed will be completely new, resulting from the fusion of several different proteins or parts of them. The protein sequence of the new chimeric receptor will be so different from all other receptor sequences that are natively expressed by living organisms.

According to the present invention, chimeric GPCR receptors of plant parasitic nematodes are meant those GPCRs that derive from the fusion of a GPCR receptor that does not have the ability to localize on the plasma membrane (i.e. the SerR1-like and DopR1-like receptors of M. incognita), or which has a poor localization in the membrane (i.e. the NprR1-like receptor of M. incognita), with the carboxyterminal domain of a GPCR receptor of non-parasitic plant nematodes.

Non-parasitic plant nematodes are defined as free-living nematode species such as, for example, Caenorhabditis elegans, C. briggsae, C. japonica, C. brenneri, C. remanei, Zeldia puntata.

The heterologous cell lines used for the expression of GPCR receptors can be mammalian cell lines chosen from: CHO-K1 (Chinese Hamster Ovary K-1) cells, HEK 293 (Human Embryonic Kidney 293) cells, COS-7 cells, NIH-T3 fibrobasts. The insect lines are: Sf-9 and Sf-21 (Spodoptera frugiperda 9 and 21), S2 (Schneider 2 of Drosophila). Other cellular systems to express receptors are represented by: bacterial cells of E. coli, yeast cells and oocytes of the amphibian Xenopus laevis.

According to a preferred embodiment of the method according to the invention, said GPCR receptors of plant parasitic nematodes (i.e. Meloidogyne, Heterodera and Globodera) are selected from the group consisting of serotonin receptors, dopamine receptors, neuropeptide receptors, galanin, metabotropic glutamate receptors and putative biogenic amine receptors (i.e.

tyramine, melatonin). In particular, when said

Parasitic nematode of plants is Meloidogyne

unknown, said GPCR receptor can be chosen by the

group consisting of amino acid sequences

shown in the following Table 1:

Table 1

TYPE AMINO ACID SEQUENCE

RECEPTOR

M. unknown

SerR1-like<MLENDLENLDDSSLILHHLGGSFVHAQIVEIEENDYINSNTSVSNT>NLNLFSSSSSTSSSSLFSSSSQRSPLLEPNFVDEIWPATSSNSLFY SHPIIAILISILIFLLILITIIGNLGVCAAILLVRKLKAQPANLLL ISLALADFCVGLFVMPLAAVYVLEDRWIFGDILCRFWVTADLTLCT ASILNLCAISVDRHLAVTRALRYSALRTRRRICLYICIVWLGALLV SAAPLALLPFPKIEQYCQVSQNRVYQIYATIIAFWGPSLIMVIVYV KLWSAAKRMHRQDQLVLRWQGVHLPSDGDLEDGLPPTTTTSNATKS LFRNDSIARANARFLFALRMSAQKFGYYDHQNNKTNGGEIINDDEN LRIEVRTSASSSGGGGGGSSTLGTLRVPIVLIIMSAFIICWVPFFI LALAKSQHWVNYVPRWLDSLTLWLGYSNSMLNPIIYCKYNREFRVP FGEMLCCRFRTIQDVMRNESYYAKFGSPRISETKIGGSQQRSSGNN YYTKRKRNNTERRAPQNGFLDSSKTKKNYGIKRNSSPSILMESSK

(SEQ ID NO: 2)

DopR1-like<MLPWWLPLLIFVPIFSVLISLAFSGNLLVIAAIFYDRKLRRQPENL>FLVSLALSDLFVAGMVMVLAAANDLIGFWPFGAAFCQFWICLDIAC STASILNLCAIALDRFIHISRPMRYVRFVGRRVICCSVCAIWIIST AVGTAQIAFGATNGLEIYSVIFNGDYFGEINETDKFRFIEQQHTLV QCQLQLSPFYAVFSSLLSFFLPATLMLFLYYRLYLYARHHARSIQS QLKQATSLLILQLASDRVRQVVVRPSSGFLSPSSIALHYDSRRFSG KSSFDGESLQKAEFSQINVAVNKSPENHSIDATIQQGTPVLRATLR QLRRTESNNSSSFSFVSRNGLGVNSRRVGCGGGNGGERRKSCSPLP SPTTKRGFKSCGWSNNNNGNGGHRSSDKKARVTLGVIMGTFLICWL PFFIVNVLRPFWPEIFPPLLFQTVSWLGYANSSVNPVIYGIFNRDF RRAFSRIMNKLIHCIDEERRGENSRGGTFRTRTESESFNK FNKSLNNASELQQQQKLIFKQKKTNKKLRFLCF

(SEQ ID NO: 4)

Rho1-like MNITVPTTTTSLIINSISPFPPTFSPSISTQQNNFTTSSLTKTTFN FQQKRDVSFHFSLIFGSLIGGVTIVGLTANILVVIAILSDRKMRKS PMNLLLLNLAIADLLYLLAFTPFWLSMSVYGDGGWHFGDMFCPIAR FFGNIFLVISILTYLAICIERYVAIVHPIAMHTSVWCTRSRVLVIA FGIWVFAMTYQFPYLVVFQVFDIPEKNLRVCRNPLASKSKIWKIYK WSEFLLTYALPIIISVLLYSRICRVLWFKNKNGENCGKGQNENENK TELKPNINKRPSGRTIKKVLETRFREAISDVQNKRKVVPKEEEIFN FNNSSEKRQKPMNVSSSANLRARRSVVKMLMLCVGLFFLCYTPMVA YFVWSALFGRPLPLPFEFVLITSALVQFQSAFNPFLYTLFATQFRE KLSKLFLICNKKQKNKIINQHKISAKSTSTFSV

(SEQ ID NO: 6)

Rho2-like MTTANNQGEVDCNQGLVEPLLDRPIFKIPFTFAYVAVFLLCLTGNL FTIVVICAHRSMRTATNFFLANLALADLLVAIFCIMQNMLHLVHLD AQWPLGETLCRMYALILHLVPCTGIGILVCVSVEKYIAVLHPLLAL KLLTPKFRSLMMAAVWICSLLANLPYYTTSKYREWEGGNSACFRGL LTDGFVSTRNMLIISFLVWYCIPLCIIAFLYTRIGFVLWHSAPLKK LTQMRTNSNETTTMTGIQKRSVASCRLSAITNNNGSATIVSEVTIK TPSKRSNSQPNNYSKITNNNLPPLILVNKSSPLPNTASESEGEEED FGREEEGREEEEDKDEEEEEEESNGSHLEEDLDDDEEEEDAEHENG TKCRLALGNGSVGPAHLGQRNSAPSSSTVFVSTVESRKRIIRLLIA IVCSFAVLTLPNHIRLLYSAVYSEERICLGGFEVFTQPVTYLLLFL SSSCNPFLYAFMSQRFRSAIRDIFKCRRGHIRRKSTKTRTQNSDVL CAVNSGDGVALSDCSSLNRLNNNGNNNLLDQSKGEEINSIHGEKNF PWANQKGEEINWLKVKKLKNTERNASGGSGNSTTTASSVLYTTQHS

NGSLQMAVVYRKCAC (SEQ ID NO: 8)

Rho3-like MNDSETIENYEDFLSQLSSTMIPTTTTTSSSSSAIFTSTLATPLIS ETAQIPSPTPKLNINIVAYCYIMPTICVLGIIGNSMNVVTLASPRL KAVSYMYLRALAVSDLLCMLFVLAFACCEVLKESGVPIERHPLYGF YQAHVMLSFINWALATGVYIVVALSLERYVSVVFPLHFRMWNSPKR AMKAIIIAYTVPALFYIPYGIGRYSVSEKINSRGEISYGAIDSEIS KTFGWQVYKWTREAFLRFLPIVILFVLNFQIMIAFRRRQKMFDRLR NRETAARDDTLLYILGGIAVMFFVCNIPAAINLLFINEVVKKRPDY QIFRAAANLLEITNHAAQFYIFCVCSSDYRVTFMQKFPCLRAYYVN KSKFCSFLRNASQLPKRSVARRTISTTTNSKIGNNVNNVCGTMRRS VSASNTPLNAKHGDKNDRRNGKSKKGGGGGGSAIAEMTFVRSTTIT GELLSTCGGHNTEQETIDAQIASLESLSDESETLLKQQKIREESWG SPITTKLCLIGLDGDEEEGIICRRSSNGEGIIAEDEQTDRTDGTSY

L (SEQ ID NO: 10)

Rho4-like MDYAPMPISSNITTWFLLAVFIFVIHRNPSFHSQTNYFLASLAISD LLLILVGVPFDVLFLWKSRFITSPFNGFCEITSTFISWFTFNSILT IVSLTWERLVAICYPFSLKPFFHRDAVIWLIIIIWFISFFPSLFIG LQFKLVIQDFCGHTHVNSNGLGSKCDFVGWSGNYGSGENYTFEVML FFTFVLPVLFVIYCYIRILRTLNEATFNSFHLNAGSSIVRKHTSSS HTNSTLLNAITEENLPSTSNNSNSNTAGNNSDLNARDSFTRSSLRS QRAHKTVMKMLIMIAALFFVCYLPYHLERLIVKYSAKGCTEPQMCL WLYHGTGLLQYISAALNPIIYNVMSRRFRREFKLLCYRIVKKENVT KTRSDNNNQLKMTPMNRFL (SEQ. ID NO:12)

Alternatively, when called parasitic nematode

of plants is Meloidogyne hapla, called the GPCR receptor

has, in a preferred embodiment, the

following amino acid sequence:

MDYAPMPISSNITTWFLLAVFIFVIHRNPSFHSQTNYFLASLAISDLLLILVGVPFDVL FLWKSRFITSPFNGFCEITSTFISWFTFNSILTIVSLTWERLVAICYPFSLKPFFHRDA VIWLIIIIWFISFFPSLFIGLQFKLVIQDFCGHTHVNSNGLGSKCDFVGWSGNYGSGEN YTFEVMLFFTFVLPVLFVIYCYIRILRTLNEATFNSFHLNAGSSIVRKHTSSSHTNSTL LNAITEENLPSTSNNSNSNTAGNNSDLNARDSFTRSSLRSQRAHKTVMKMLIMIAALFF VCYLPYHLERLIVKYSAKGCTEPQMCLWLYHGTGLLQYISAALNPIIYNVMSRRFRREF KLLCYRIVKKENVTKTRSDNNNQLKMTPMNRFL (SEQ. ID NO:14).

Still according to an alternative embodiment of the invention, when said plant parasitic nematode is Globodera rostochiensis, said GPCR receptor has the following amino acid sequence:

MLENDLENLDDSSLILHHLGGSFVHAQIVEIEENDYINSNTSVSNTNLNLFSSSSSTSS SSLFSSSSQRSPLLEPNFVDEIWPATSSNSLFYSHPIIAILISILIFLLILITIIGNLG VCAAILLVRKLKAQPANLLLISLALADFCVGLFVMPLAAVYVLEDRWIFGDILCRFWVT ADLTLCTASILNLCAISVDRHLAVTRALRYSALRTRRRICLYICIVWLGALLVSAAPLA LLPFPKIEQYCQVSQNRVYQIYATIIAFWGPSLIMVIVYVKLWSAAKRMHRQDQLVLRW QGVHLPSDGDLEDGLPPTTTTSNATKSLFRNDSIARANARFLFALRMSAQKFGYYDHQN NKTNGGEIINDDENLRIEVRTSASSSGGGGGGSSTLGTLRVPIVLIIMSAFIICWVPFF ILALAKSQHWVNYVPRWLDSLTLWLGYSNSMLNPIIYCKYNREFRVPFGEMLCCRFRTI QDVMRNESYYAKFGSPRISETKIGGSQQRSSGNNYYTKRKRNNTERRAPQNGFLDSSKT KKNYGIKRNSSPSILMESSK (SEQ. ID NO:16).

According to a particularly preferred embodiment of the invention, said GPCR receptors of non-parasitic nematodes capable of localizing in the membrane are selected from the group consisting of C.elegans, C.briggsae, C. japonica, C. brenneri, C. remanei , Bet Zeldia.

According to a preferred embodiment of the invention said non-parasitic nematode is C.elegans and the GPCR receptors of C.elegans can be selected from the group consisting of SER-1 serotonin receptors (Gene Bank: F59C12.2), SER -2 (C02D4.2), SER-3 (K02F2.6), SER-4 (Y22D7AR.13), SER-5 (F16D3.7), SER-6 (Y54G2A.35), SER-7 (C09B7. 1); dopamine receptors DOP-1 (F15A8.5), DOP-2 (K09G1.4), DOP-3 (T14E8.3), DOP-4 (C52B11.3); NPR-1 (C39E6.6), NPR-2 (T05A1.1), NPR-3 (C10C6.2) neuropeptide receptors; putative NPR1-like neuropeptide receptors (C16D6.2, C25G6.5, F41E7.3); GAR-1 (C15B12.5), GAR-2 (F47D12.1), GAR-3 (Y40H4A.1) galanin receptors; metabotropic glutamate receptors MGL-1 (ZC506.4), MGL-2 (F45H11.4); or putative receptors of biogenic amines (clones F01E11.5, C24A8.1, T02E9.3).

Therefore, according to preferred embodiments of the method according to the invention, the carboxyterminal domain of the GPCR receptors of C.elegans, which are able to localize in the membrane, has an amino acid sequence chosen from the group consisting of the sequences SEQ ID NO: 17 - SEQ ID NO: 41 shown in the following Table 2, which also indicates the name of the relative C.elegans receptor and the access number in Gene Bank:

Table 2

NAME NUMBER AMINO ACID SEQUENCE

CARBOXY TERMINAL ACCESS RECEPTOR

C.elegans GENE

BANK SER-1 F59C12.2 TVFNKRFRQAFVRILRCQCFHPLRDSHQMYSRNFT TTIVPDTYTCSRSNQERTTSVITRDETRSARSSER PEPSRARSEISEEPVARTNGKLTSEKKKISLPSFP RVSSSRDSRATTEASTTDEETKPLIPKSTVPATVI NIPEQLINPIKKSLTTIINMPLLDETIPEKAQVHH KSQTLLTSSTLNFATFSTCPQQPTRSYSCVDCKKA EKMLSSDVSDMMTTSTASTASTVNGAPRKHLTLFN

HFDSAIKETFL

(SEQ ID NO: 17)

SER-2 C02D4.2 GILNLEFRRAFKKILCPKAVLEQRRRRMSAQP

(SEQ ID NO: 18)

SER-3 K02F2.6 TVFNREFRICFKRLLTCHHLNHPTQKYTNNNSYNS TAIRSTNAVNRVPQTSLGNYTQTQNSEKSSAAVTF NTPTN (SEQ ID NO: 19)

SER-4 Y22D7AR. TVFSQDFRAAFKRIIKRMCLIHDY

13 (SEQ ID NO: 20)

SER-5 F16D3.7 SFTVKEFKRSAFRLVVPIWQFVNRCLPFVPAPPDN ILQRIARHVHRHKEKEMQTRHRSFEMSSNKNGMLT TKVRSKRRQTEPNVVGLITPDHKLQTVAS

(SEQ ID NO: 21)

SER-6 Y54G2A.3 AAFSRDFRIALKRLFFQKPKF

5 (SEQ ID NO: 22)

SER-7 C09B7.1 YCKYNKEFRIPFREMLACRCATLQTVMRQQSFTSR YGPPVRYRTQSSSYRPLLSRRNDSHEASDV

(SEQ ID NO: 23)

DOP-1 F15A8.5 NRDFRRAFKKIIVRVFGCCWEEPDLNKSISSRYAA PDNIERRRSCTRSSESAHDNNNDANATRLNLLSNN NEETIPE (SEQ ID NO: 24)

DOP-2 K09G1.4 TIFNTEFRRAFKSIIFGRNSTRHHFSNKQAHV

(SEQ ID NO: 25)

DOP-3 T14E8.3 TVFDQRFRNAFRNILSCGIFKKR

(SEQ ID NO: 26)

DOP-4 C52B11.3 SRFSRDFRRAFKQILTCQRQQKVKTAFKTPLSLVF TQLISVTQMWEQPPNTSIE

(SEQ ID NO: 27)

GAR-1 C15B12.5 ALANRQFRSAFMRMFRGNFNKVA

(SEQ ID NO: 28)

GAR-2 F47D12.1 AMANQQFKKTLTRIFKGDFRRV

(SEQ ID NO: 29)

GAR-3 Y40H4A.1 ALCNARFRHTYMRILRCKFKAERPTMNQGYVRRN

(SEQ ID NO: 30)

NPR-1 C39E6.6 AWLNPSFRQLVIKTYFGDRRKSDRIINQTSVYKTK IVHDTKHLNGRAKIGGGGSHEALKERELNSCSENL SYHVNGHTRTPTPEVQLNEVSSPEISKLVAEPEEL IEFSVNDTLV (SEQ ID NO: 31)

NPR-2 T05A1.1 AWLNPMFKEMLIKTLRGGSKSPKPADIKQTSFIRM PNSGAPSQSSYL (SEQ ID NO: 32)

NPR-3 C10C6.2 SWFNPQFRQSITTLFKGTDEARLIKKKPQSTSKMV SYPTNFSEIRKETEIASTKTKITIAENDYRAGDQL

L (SEQ ID NO: 33)

NPR-like C16D6.2 LNLQLRAAFIDLMPHWLRRHLNLEGDNSSPLLNHP TMTITNKYGSTATKTVKATYINTSNGQPYVSTSLV GKVQPEAPSFKFNGSGRKKSAMMRILVQKRNAEEE EQLQITKESPSPPEISAPM

TNEKVVLPRKASF

(SEQ ID NO: 34)

C25G6.5 receptor AFFNHNFRIEFMHLFDRVGLRSLRVVIFGEQESLK putative KSMRTEFRSRGGCKTVTTAEPATFQRMNESMILSA neuropeptide MEQDEQLSSGGKLFVLKKYILKMFQKGGHKQSTPA SPRLGNSVFS

(SEQ ID NO: 35)

F41E7.3 receptor AFMNETFREEFAKVVPCLFARRPGTGPIRVITERT putative AMITNPFRRANRKKKVEEQPVTVISESPLQTAVEP neuropeptide QRSIVYLDEPENGSSCQTLLL

(SEQ ID NO: 36)

MGL-1 ZC506.4 RKQEGESMLNKSSRSLGNCSSRLCANSIDEPNQYT ALLTDSTRRRSSRKTSQPTSTSSAHDTFL

(SEQ ID NO: 37)

MGL-2 F45H11.4 IRASYTTTKLIRCHFGNSQAAYDSTSKQQHLGSKT TARTSVQSGSASKSSSMGGGVTRTASVHVPVSRGS THSTDVSTQTEAASKFSRSFSIVGRKKQGLDDDVQ QLVDACRRYQDEKINSSAANLLLEESEDEVGALLA DSIENSMRTVLSTVAGKAVVPLVPMVPMIPVVTLP TAPSQEDNFEQLLRSRGVQPLALSQATPL

(SEQ ID NO: 38)

Putative F01E11.5 TVFNRDYQIALKRLFTSEKKPSSTSRV receptor (SEQ ID NO: 39)

tyramine

Receptor STINPVSIFISNLISKKIIFLIFQVQFALYKTLIH homolog C24A8.1 GIETRNSVIHSAAYLASVDLLDVREKNRGCYRHEI dopamine AAEVLQNVDAVSIKS (SEQ ID NO: 40) Receptor T02E9Q NO homologue C24A8.1

melatonin

Still preferably, the chimeric GPCR receptor of step a) of the method of the invention has an amino acid sequence chosen from among all the sequences SEQ ID NO: 42-SEQ ID NO: 49, shown in Fig.7. The following are the protein sequences of chimeric receptors in which the carboxyterminal portion has been replaced: the underlined amino acids indicate the carboxyterminal sequence of the C.elegans GPCR receptor used for the construction of the chimera (i.e. C-terminals having SEQ ID NO: 23, SEQ ID NO: 24; SEQ ID NO: 34 shown in Table 2):

i)MLENDLENLDDSSLILHHLGGSFVHAQIVEIEENDYINSNTSVSNTNLNLFSSSSST SSSSLFSSSSQRSPLLEPNFVDEIWPATSSNSLFYSHPIIAILISILIFLLILITIIGN LGVCAAILLVRKLKAQPANLLLISLALADFCVGLFVMPLAAVYVLEDRWIFGDILCRFW VTADLTLCTASILNLCAISVDRHLAVTRALRYSALRTRRRICLYICIVWLGALLVSAAP LALLPFPKIEQYCQVSQNRVYQIYATIIAFWGPSLIMVIVYVKLWSAAKRMHRQDQLVL RWQGVHLPSDGDLEDGLPPTTTTSNATKSLFRNDSIARANARFLFALRMSAQKFGYYDH QNNKTNGGEIINDDENLRIEVRTSASSSGGGGGGSSTLGTLRVPIVLIIMSAFIICWVP FFILALAKSQHWVNYVPRWLDSLTLWLGYSNSMLNPIIYYCKYNKEFRIPFREMLACRC ATLQTVMRQQSFTSRYGPPVSRRNDSHEASDV

(MiSerR1-Chimera B; SEQ ID NO: 43); ii)MEASTMELLPPPTILFNVSSAPSVICEDMNAYLWNTRRDLTTCPFVMAVFASLYSS IILLGIIGNSCVIMAIARIKSLQTVPNMFIFSLSCSDILVCFISATITPIAAFKKDWIF GQFLCSFAPFVAGGSLCFSTFTLAAISVDRFLLILFPTRKAFSHTQALFIILVTFLLAS GFSLPMLFMQKLKPVTHYCGRFCFEDWGEMIGIRRIYGTLLLTVQFIIPLALITFCYTA ISFRLGKSVNLRTRKKCEWQMPISAQRNGATKRRQRTNRMFIAMVIAFSVSWIWSVLYN LLRDYDGLPKFVHDQEFFFGILTHCIAMSSTVWNPILYALLYCKYNKEFRIPFREMLAC RCATLQTVMRQQSFTSRYGPPVSRRNDSHEASDV

(MiNpR1 :: CtermCeSer-7; SEQ ID NO: 46); iii)MLPWWLPLLIFVPIFSVLISLAFSGNLLVIAAIFYDRKLRRQPENLFLVSLALSD LFVAGMVMVLAAANDLIGFWPFGAAFCQFWICLDIACSTASILNLCAIALDRFIHISRP MRYVRFVGRRVICCSVCAIWIISTAVGTAQIAFGATNGLEIYSVIFNGDYFGEINETDK FRFIEQQHTLVQCQLQLSPFYAVFSSLLSFFLPATLMLFLYYRLYLYARHHARSIQSQL KQATSLLILQLASDRVRQVVVRPSSGFLSPSSIALHYDSRRFSGKSSFDGESLQKAEFS QINVAVNKSPENHSIDATIQQGTPVLRATLRQLRRTESNNSSSFSFVSRNGLGVNSRRV GCGGGNGGERRKSCSPLPSPTTKRGFKSCGWSNNNNGNGGHRSSDKKARVTLGVIMGTF LICWLPFFIVNVLRPFWPEIFPPLLFQTVSWLGYANSSVNPVIYGIFYCKYNKEFRIPF REMLACRCATLQTVMRQQSFTSRYGPPVSRRNDSHEASDV

(MiDop1 :: CtermCeSer-7; SEQ ID NO: 47); iv)MEASTMELLPPPTILFNVSSAPSVICEDMNAYLWNTRRDLTTCPFVMAVFASLYSS IILLGIIGNSCVIMAIARIKSLQTVPNMFIFSLSCSDILVCFISATITPIAAFKKDWIF GQFLCSFAPFVAGGSLCFSTFTLAAISVDRFLLILFPTRKAFSHTQALFIILVTFLLAS GFSLPMLFMQKLKPVTHYCGRFCFEDWGEMIGIRRIYGTLLLTVQFIIPLALITFCYTA ISFRLGKSVNLRTRKKCEWQMPISAQRNGATKRRQRTNRMFIAMVIAFSVSWIWSVLYN LLRDYDGLPKFVHDQEFFFGILTHCIAMSSTVWNPILYALLLNLQLRAAFIDLMPHWLR RHLNLEGDNSSPLLNHPTMTITNKYGSTATKTVKATYINTSNGQPYVSTSLVGKVQPEA PSFKFNGSGRKKSAMMRILVQKRNAEEEEQLITKESPSPPEIQMDTLCAASIIPRRKSA QPRSTNEKVVLPRKASF

(MiNpR1 :: Cterm2 (C16D6.2); SEQ ID NO: 48); v)MLPWWLPLLIFVPIFSVLISLAFSGNLLVIAAIFYDRKLRRQPENLFLVSLALSDLF VAGMVMVLAAANDLIGFWPFGAAFCQFWICLDIACSTASILNLCAIALDRFIHISRPMR YVRFVGRRVICCSVCAIWIISTAVGTAQIAFGATNGLEIYSVIFNGDYFGEINETDKFR FIEQQHTLVQCQLQLSPFYAVFSSLLSFFLPATLMLFLYYRLYLYARHHARSIQSQLKQ ATSLLILQLASDRVRQVVVRPSSGFLSPSSIALHYDSRRFSGKSSFDGESLQKAEFSQI NVAVNKSPENHSIDATIQQGTPVLRATLRQLRRTESNNSSSFSFVSRNGLGVNSRRVGC GGGNGGERRKSCSPLPSPTTKRGFKSCGWSNNNNGNGGHRSSDKKARVTLGVIMGTFLI CWLPFFIVNVLRPFWPEIFPPLLFQTVSWLGYANSSVNPVIYGIFNRDFRRAFKKIIV RVFGCCWEEPDLNKSISSRYAAPDNIERRRSCTRSSESAHDNNNDANATRLNLL SNNNEETIPE (MiDop1::Cterm3(F15A8.5); SEQ ID NO:49).

According to a preferred embodiment of the method according to the invention, said intracellular signal of phase c) is selected from the variation of the levels of cAMP, Ca <2+>, IP3, cGMP, kinase activity.

A further object of the present invention is a method for assisting the expression in a heterologous cellular system of GPCR receptors which have difficulty in localizing correctly on the cellular plasma membrane comprising the following steps:

a) creation of the chimeric GPCR receptor gene by replacing the sequence corresponding to the carboxyterminal domain of said GPCR receptor with that of the non-parasitic plant nematode GPCR receptor capable of localizing in the membrane;

b) transfection of the plasmid containing the chimeric gene into heterologous cell systems, preferably through the use of lipofectamine; c) expression of the chimeric GPCR receptor in cells. The level of this expression can be verified by means of an ELISA assay.

In a preferred embodiment, said GPCR receptors which have difficulty in localizing correctly on the cell plasma membrane are GPCRs of plant parasitic nematodes (i.e. Meloidogyne, Heterodera and Globodera). The term `` difficulty in localizing correctly on the cellular plasma membrane '' can be understood as both a GPCR receptor that does not localize at all on the plasma membrane, and a receptor that is partially localized but not in such a way as to allow the detection of a response. Preferably, said GPCR receptors are selected from the group consisting of serotonin receptors, dopamine receptors, neuropeptide receptors, galanin receptors, metabotropic glutamate receptors and biogenic amine receptors.

In particular, when said parasitic nematode of plants is Meloidogyne incognita, said GPCR receptor can be chosen from the group consisting of the amino acid sequences SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; SEQ ID NO: 10, SEQ ID NO: 12, shown in the previous Table 1 (and in Figure 1); when said plant parasitic nematode is Meloidogyne hapla, said GPCR receptor preferably has the amino acid sequence SEQ. ID NO: 14 shown in Figure 1; when said plant parasitic nematode is Globodera rostochiensis it preferably has the amino acid sequence SEQ. ID NO: 16 shown in Figure 1.

According to a particularly preferred embodiment of the invention, said GPCR receptors of non-parasitic nematodes capable of localizing in the membrane are selected from the group consisting of C.elegans, C.briggsae, C. japonica, C. brenneri, C. remanei , Bet Zeldia.

In a particularly preferred embodiment, said GPCR receptors of non-parasitic nematodes capable of localizing in the membrane are of C.elegans. Preferably, said C.elegans GPCR receptors are selected from the group consisting of serotonin receptors SER-1 (No. Gene Bank F59C12.2), SER-2 (C02D4.2), SER-3 (K02F2.6), SER-4 (Y22D7AR.13), SER-5 (F16D3.7), SER-6 (Y54G2A.35), SER-7 (C09B7.1); dopamine receptors DOP-1 (F15A8.5), DOP-2 (K09G1.4), DOP-3 (T14E8.3), DOP-4 (C52B11.3); NPR-1 (C39E6.6), NPR-2 (T05A1.1), NPR-3 (C10C6.2) neuropeptide receptors; putative NPR1-like neuropeptide receptors (C16D6.2, C25G6.5, F41E7.3); GAR-1 (C15B12.5), GAR-2 (F47D12.1), GAR-3 (Y40H4A.1) galanin receptors; metabotropic glutamate receptors MGL-1 (ZC506.4), MGL-2 (F45H11.4); or putative receptors of biogenic amines (clones F01E11.5, C24A8.1, T02E9.3). Still preferably, the carboxyterminal domain of the membrane-localizing GPCR receptors of C.elegans has an amino acid sequence selected from the group consisting of the sequences SEQ ID NO: 17-SEQ ID NO: 41 shown in the above mentioned Table 2.

A further object of the present invention is a chimeric GPCR receptor obtainable according to the method defined above.

A further object of the invention is a chimeric GPCR receptor characterized by the fact that it comprises the amino acid sequence of a plant parasitic nematode GPCR receptor (i.e. Meloidogyne, Heterodera and Globodera) which presents difficulties in locating correctly on the cell plasma membrane, in which the carboxyterminal domain of said sequence is replaced with that of a GPCR receptor of non-parasitic nematodes (i.e. C.elegans, C.briggsae, C. japonica, C. brenneri, C. remanei, Zeldia puntata) capable of localizing in the membrane.

Preferably the amino acid sequence of said plant parasitic nematode GPCR receptor is chosen from the group consisting of the amino acid sequences reported in Table 3 below, in relation to the parasitic nematode species (Meloidogyne incognita, Meloidogyne hapla, Globodera rostochiensis):

Table 3

Meloidogyne AMINO ACID SEQUENCE unknown

SerR1-like MLENDLENLDDSSLILHHLGGSFVHAQIVEIEENDYINSNTS VSNTNLNLFSSSSSTSSSSLFSSSSQRSPLLEPNFVDEIWPA TSSNSLFYSHPIIAILISILIFLLILITIIGNLGVCAAILLV RKLKAQPANLLLISLALADFCVGLFVMPLAAVYVLEDRWIFG DILCRFWVTADLTLCTASILNLCAISVDRHLAVTRALRYSAL RTRRRICLYICIVWLGALLVSAAPLALLPFPKIEQYCQVSQN RVYQIYATIIAFWGPSLIMVIVYVKLWSAAKRMHRQDQLVLR WQGVHLPSDGDLEDGLPPTTTTSNATKSLFRNDSIARANARF LFALRMSAQKFGYYDHQNNKTNGGEIINDDENLRIEVRTSAS SSGGGGGGSSTLGTLRVPIVLIIMSAFIICWVPFFILALAKS QHWVNYVPRWLDSLTLWLGYSNSMLNPIIYCKYNREFRVPFG EMLCCRFRTIQDVMRNESYYAKFGSPRISETKIGGSQQRSSG NNYYTKRKRNNTERRAPQNGFLDSSKTKKNYGIKRNSSPSIL

MESSK (SEQ ID NO: 2)

DopR1-like MLPWWLPLLIFVPIFSVLISLAFSGNLLVIAAIFYDRKLRRQ PENLFLVSLALSDLFVAGMVMVLAAANDLIGFWPFGAAFCQF WICLDIACSTASILNLCAIALDRFIHISRPMRYVRFVGRRVI CCSVCAIWIISTAVGTAQIAFGATNGLEIYSVIFNGDYFGEI NETDKFRFIEQQHTLVQCQLQLSPFYAVFSSLLSFFLPATLM LFLYYRLYLYARHHARSIQSQLKQATSLLILQLASDRVRQVV VRPSSGFLSPSSIALHYDSRRFSGKSSFDGESLQKAEFSQIN VAVNKSPENHSIDATIQQGTPVLRATLRQLRRTESNNSSSFS FVSRNGLGVNSRRVGCGGGNGGERRKSCSPLPSPTTKRGFKS CGWSNNNNGNGGHRSSDKKARVTLGVIMGTFLICWLPFFIVN VLRPFWPEIFPPLLFQTVSWLGYANSSVNPVIYGIFNRDFRR AFSRIMNKLIHCIDEERRGENSRGGTFRTRTESESFNK FNKSLNNASELQQQQKLIFKQKKTNKKLRFLCF

(SEQ ID NO: 4)

Rho1-like MNITVPTTTTSLIINSISPFPPTFSPSISTQQNNFTTSSLTK TTFNFQQKRDVSFHFSLIFGSLIGGVTIVGLTANILVVIAIL SDRKMRKSPMNLLLLNLAIADLLYLLAFTPFWLSMSVYGDGG WHFGDMFCPIARFFGNIFLVISILTYLAICIERYVAIVHPIA MHTSVWCTRSRVLVIAFGIWVFAMTYQFPYLVVFQVFDIPEK NLRVCRNPLASKSKIWKIYKWSEFLLTYALPIIISVLLYSRI CRVLWFKNKNGENCGKGQNENENKTELKPNINKRPSGRTIKK VLETRFREAISDVQNKRKVVPKEEEIFNFNNSSEKRQKPMNV SSSANLRARRSVVKMLMLCVGLFFLCYTPMVAYFVWSALFGR PLPLPFEFVLITSALVQFQSAFNPFLYTLFATQFREKLSKLF

LICNKKQKNKIINQHKISAKSTSTFSV

(SEQ ID NO: 6)

Rho2-like MTTANNQGEVDCNQGLVEPLLDRPIFKIPFTFAYVAVFLLCL TGNLFTIVVICAHRSMRTATNFFLANLALADLLVAIFCIMQN MLHLVHLDAQWPLGETLCRMYALILHLVPCTGIGILVCVSVE KYIAVLHPLLALKLLTPKFRSLMMAAVWICSLLANLPYYTTS KYREWEGGNSACFRGLLTDGFVSTRNMLIISFLVWYCIPLCI IAFLYTRIGFVLWHSAPLKKLTQMRTNSNETTTMTGIQKRSV ASCRLSAITNNNGSATIVSEVTIKTPSKRSNSQPNNYSKITN NNLPPLILVNKSSPLPNTASESEGEEEDFGREEEGREEEEDK DEEEEEEESNGSHLEEDLDDDEEEEDAEHENGTKCRLALGNG SVGPAHLGQRNSAPSSSTVFVSTVESRKRIIRLLIAIVCSFA VLTLPNHIRLLYSAVYSEERICLGGFEVFTQPVTYLLLFLSS SCNPFLYAFMSQRFRSAIRDIFKCRRGHIRRKSTKTRTQNSD VLCAVNSGDGVALSDCSSLNRLNNNGNNNLLDQSKGEEINSI HGEKNFPWANQKGEEINWLKVKKLKNTERNASGGSGNSTTTA SSVLYTTQHSNGSLQMAVVYRKCAC(SEQ ID NO:8) Rho3-like MNDSETIENYEDFLSQLSSTMIPTTTTTSSSSSAIFTSTLAT PLISETAQIPSPTPKLNINIVAYCYIMPTICVLGIIGNSMNV VTLASPRLKAVSYMYLRALAVSDLLCMLFVLAFACCEVLKES GVPIERHPLYGFYQAHVMLSFINWALATGVYIVVALSLERYV SVVFPLHFRMWNSPKRAMKAIIIAYTVPALFYIPYGIGRYSV SEKINSRGEISYGAIDSEISKTFGWQVYKWTREAFLRFLPIV ILFVLNFQIMIAFRRRQKMFDRLRNRETAARDDTLLYILGGI AVMFFVCNIPAAINLLFINEVVKKRPDYQIFRAAANLL EITN HAAQFYIFCVCSSDYRVTFMQKFPCLRAYYVNKSKFCSFLRN ASQLPKRSVARRTISTTTNSKIGNNVNNVCGTMRRSVSASNT PLNAKHGDKNDRRNGKSKKGGGGGGSAIAEMTFVRSTTITGE LLSTCGGHNTEQETIDAQIASLESLSDESETLLKQQKIREES WGSPITTKLCLIGLDGDEEEGIICRRSSNGEGIIAEDEQTDR

TDGTSYL (SEQ ID NO: 10)

Rho4-like MDYAPMPISSNITTWFLLAVFIFVIHRNPSFHSQTNYFLASL AISDLLLILVGVPFDVLFLWKSRFITSPFNGFCEITSTFISW FTFNSILTIVSLTWERLVAICYPFSLKPFFHRDAVIWLIIII WFISFFPSLFIGLQFKLVIQDFCGHTHVNSNGLGSKCDFVGW SGNYGSGENYTFEVMLFFTFVLPVLFVIYCYIRILRTLNEAT FNSFHLNAGSSIVRKHTSSSHTNSTLLNAITEENLPSTSNNS NSNTAGNNSDLNARDSFTRSSLRSQRAHKTVMKMLIMIAALF FVCYLPYHLERLIVKYSAKGCTEPQMCLWLYHGTGLLQYISA ALNPIIYNVMSRRFRREFKLLCYRIVKKENVTKTRSDNNNQL

KMTPMNRFL (SEQ. ID NO: 12)

Meloidogyne MDYAPMPISSNITTWFLLAVFIFVIHRNPSFHSQTNYFLASL hapla AISDLLLILVGVPFDVLFLWKSRFITSPFNGFCEITSTFISW FTFNSILTIVSLTWERLVAICYPFSLKPFFHRDAVIWLIIII WFISFFPSLFIGLQFKLVIQDFCGHTHVNSNGLGSKCDFVGW SGNYGSGENYTFEVMLFFTFVLPVLFVIYCYIRILRTLNEAT FNSFHLNAGSSIVRKHTSSSHTNSTLLNAITEENLPSTSNNS NSNTAGNNSDLNARDSFTRSSLRSQRAHKTVMKMLIMIAALF FVCYLPYHLERLIVKYSAKGCTEPQMCLWLYHGTGLLQYISA ALNPIIYNVMSRRFRREFKLLCYRIVKKENVTKTRSDNNNQL

KMTPMNRFL (SEQ. ID NO: 14)

Globodera MLENDLENLDDSSLILHHLGGSFVHAQIVEIEENDYINSNTS rostochiensis VSNTNLNLFSSSSSTSSSSLFSSSSQRSPLLEPNFVDEIWPA TSSNSLFYSHPIIAILISILIFLLILITIIGNLGVCAAILLV RKLKAQPANLLLISLALADFCVGLFVMPLAAVYVLEDRWIFG DILCRFWVTADLTLCTASILNLCAISVDRHLAVTRALRYSAL RTRRRICLYICIVWLGALLVSAAPLALLPFPKIEQYCQVSQN RVYQIYATIIAFWGPSLIMVIVYVKLWSAAKRMHRQDQLVLR WQGVHLPSDGDLEDGLPPTTTTSNATKSLFRNDSIARANARF LFALRMSAQKFGYYDHQNNKTNGGEIINDDENLRIEVRTSAS SSGGGGGGSSTLGTLRVPIVLIIMSAFIICWVPFFILALAKS QHWVNYVPRWLDSLTLWLGYSNSMLNPIIYCKYNREFRVPFG EMLCCRFRTIQDVMRNESYYAKFGSPRISETKIGGSQQRSSG NNYYTKRKRNNTERRAPQNGFLDSSKTKKNYGIKRNSSPSIL

MESSK (SEQ. ID NO: 16)

In a particularly preferred embodiment, said GPCR receptors of non-parasitic nematodes capable of localizing in the membrane are of C.elegans. According to a preferred embodiment, the carboxyterminal domain of the GPCR receptors of C.elegans capable of localizing in the membrane has an amino acid sequence chosen from the group consisting of the sequences SEQ ID NO: 17-SEQ ID NO: 41 shown in the previous Table 2.

In una forma particolarmente preferita di realizzazione dell’invenzione detto recettore GPCR chimerico può avere una sequenza amminoacidica scelta dal gruppo che consiste in: i)MLENDLENLDDSSLILHHLGGSFVHAQIVEIEENDYINSNTSVSNTNLNLFSSSSST SSSSLFSSSSQRSPLLEPNFVDEIWPATSSNSLFYSHPIIAILISILIFLLILITIIGN LGVCAAILLVRKLKAQPANLLLISLALADFCVGLFVMPLAAVYVLEDRWIFGDILCRFW VTADLTLCTASILNLCAISVDRHLAVTRALRYSALRTRRRICLYICIVWLGALLVSAAP LALLPFPKIEQYCQVSQNRVYQIYATIIAFWGPSLIMVIVYVKLWSAAKRMHRQDQLVL RWQGVHLPSDGDLEDGLPPTTTTSNATKSLFRNDSIARANARFLFALRMSAQKFGYYDH QNNKTNGGEIINDDENLRIEVRTSASSSGGGGGGSSTLGTLRVPIVLIIMSAFIICWVP FFILALAKSQHWVNYVPRWLDSLTLWLGYSNSMLNPIIYYCKYNKEFRIPFREMLACRC ATLQTVMRQQSFTSRYGPPVSRRNDSHEASDV (SEQ ID NO:43); ii)MEASTMELLPPPTILFNVSSAPSVICEDMNAYLWNTRRDLTTCPFVMAVFASLYSS IILLGIIGNSCVIMAIARIKSLQTVPNMFIFSLSCSDILVCFISATITPIAAFKKDWIF GQFLCSFAPFVAGGSLCFSTFTLAAISVDRFLLILFPTRKAFSHTQALFIILVTFLLAS GFSLPMLFMQKLKPVTHYCGRFCFEDWGEMIGIRRIYGTLLLTVQFIIPLALITFCYTA ISFRLGKSVNLRTRKKCEWQMPISAQRNGATKRRQRTNRMFIAMVIAFSVSWIWSVLYN LLRDYDGLPKFVHDQEFFFGILTHCIAMSSTVWNPILYALLYCKYNKEFRIPFREMLAC RCATLQTVMRQQSFTSRYGPPVSRRNDSHEASDV (SEQ ID NO: 46); iii)MLPWWLPLLIFVPIFSVLISLAFSGNLLVIAAIFYDRKLRRQPENLFLVSLALSD LFVAGMVMVLAAANDLIGFWPFGAAFCQFWICLDIACSTASILNLCAIALDRFIHISRP MRYVRFVGRRVICCSVCAIWIISTAVGTAQIAFGATNGLEIYSVIFNGDYFGEINETDK FRFIEQQHTLVQCQLQLSPFYAVFSSLLSFFLPATLMLFLYYRLYLYARHHARSIQSQL KQATSLLILQLASDRVRQVVVRPSSGFLSPSSIALHYDSRRFSGKSSFDGESLQKAEFS QINVAVNKSPENHSIDATIQQGTPVLRATLRQLRRTESNNSSSFSFVSRNGLGVNSRRV GCGGGNGGERRKSCSPLPSPTTKRGFKSCGWSNNNNGNGGHRSSDKKARVTLGVIMGTF LICWLPFFIVNVLRPFWPEIFPPLLFQTVSWLGYANSSVNPVIYGIFYCKYNKEFRIPF REMLACRCATLQTVMRQQSFTSRYGPPVSRRNDSHEASDV

(SEQ ID NO: 47); iv)MEASTMELLPPPTILFNVSSAPSVICEDMNAYLWNTRRDLTTCPFVMAVFASLYSS IILLGIIGNSCVIMAIARIKSLQTVPNMFIFSLSCSDILVCFISATITPIAAFKKDWIF GQFLCSFAPFVAGGSLCFSTFTLAAISVDRFLLILFPTRKAFSHTQALFIILVTFLLAS GFSLPMLFMQKLKPVTHYCGRFCFEDWGEMIGIRRIYGTLLLTVQFIIPLALITFCYTA ISFRLGKSVNLRTRKKCEWQMPISAQRNGATKRRQRTNRMFIAMVIAFSVSWIWSVLYN LLRDYDGLPKFVHDQEFFFGILTHCIAMSSTVWNPILYALLLNLQLRAAFIDLMPHWLR RHLNLEGDNSSPLLNHPTMTITNKYGSTATKTVKATYINTSNGQPYVSTSLVGKVQPEA PSFKFNGSGRKKSAMMRILVQKRNAEEEEQLITKESPSPPEIQMDTLCAASIIPRRKSA QPRSTNEKVVLPRKASF (SEQ ID NO:48); v)MLPWWLPLLIFVPIFSVLISLAFSGNLLVIAAIFYDRKLRRQPENLFLVSLALSDLF VAGMVMVLAAANDLIGFWPFGAAFCQFWICLDIACSTASILNLCAIALDRFIHISRPMR YVRFVGRRVICCSVCAIWIISTAVGTAQIAFGATNGLEIYSVIFNGDYFGEINETDKFR FIEQQHTLVQCQLQLSPFYAVFSSLLSFFLPATLMLFLYYRLYLYARHHARSIQSQLKQ ATSLLILQLASDRVRQVVVRPSSGFLSPSSIALHYDSRRFSGKSSFDGESLQKAEFSQI NVAVNKSPENHSIDATIQQGTPVLRATLRQLRRTESNNSSSFSFVSRNGLGVNSRRVGC GGGNGGERRKSCSPLPSPTTKRGFKSCGWSNNNNGNGGHRSSDKKARVTLGVIMGTFLI CWLPFFIVNVLRPFWPEIFPPLLFQTVSWLGYANSSVNPVIYGIFNRDFRRAFKKIIV RVFGCCWEEPDLNKSISSRYAAPDNIERRRSCTRSSESAHDNNNDANATRLNLL SNNNEETIPE (SEQ ID NO:49);

in which the underlined amino acids indicate the carboxy terminal portion of the GPCR receptor of C.elegans used for the construction of the chimera (i.e. C-terminals having SEQ ID NO: 23, SEQ ID NO: 24; SEQ ID NO: 34 reported in Table 2) .

The invention further refers to the use of chimeric GPCR receptors of plant parasitic nematodes (i.e. Meloidogyne, Heterodera and Globodera) obtainable by the method according to the invention, as targets of potential pesticides specific for said nematodes.

A further object of the present invention is an expression vector comprising a nucleotide sequence encoding a chimeric GPCR receptor obtainable by means of the method according to the invention. Preferably, the nucleotide sequences encode a chimeric GPCR receptor having one of the sequences SEQ ID NO: 43, SEQ ID NO: 46-SEQ ID NO: 49 mentioned above. Still preferably, said nucleotide sequences are the sequences SEQ ID NO: 51, SEQ ID NO: 54-SEQ ID NO: 57, shown in Figure 7.

Finally, the invention relates to a heterologous cell line (i.e. CHO cells, HEK 293 cells, COS-7 cells, NIH-T3, Sf-9 and Sf-21 fibrobasts (Spodoptera frugiperda 9 and 21), S2 (Schneider 2 of Drosophila; bacterial cells of E. coli, yeast cells and oocytes of the amphibian Xenopus laevis) transformed with an expression vector like the one described above. This cell line can be advantageously used in a screening method of specific pesticides for plant parasitic nematodes (i.e. Meloidogyne, Heterodera and Globodera).

The present invention will now be described by way of illustration, but not of limitation, according to its preferred embodiments with particular reference to the figures of the attached drawings, in which: Figure 1 shows the nucleotide and protein sequences of the cloned GPCR receptors: MiSerR1-like ( SEQ ID NO: 1-2); MiDopR1-like (SEQ ID NO: 3-4); MiRho1-like (SEQ ID NO: 5-6); MiRho2-like (SEQ ID NO: 7-8), MiRho3-like (SEQ ID NO: 9-10); MiRho4-like of M. incognita (SEQ ID NO: 11-12); MhRho1 of Meloidogyne hapla (SEQ ID NO: 13-14) and GrSer1 of Globodera rostochiensis (SEQ ID NO: 15-16);

Figure 2 shows the analysis of the number of adult C.elegans worms produced by non-transgenic â € œwild typeâ € (WT) lines and those hyper-expressing the MiSerR1-like receptor of M. incognita and CeSer-4 of C .elegans: in transgenic worms, MiSerR1-like and CeSer-4 receptors were expressed under the control of the egl-15 (Pegl-15) promoter, activated specifically in the vulva muscles of the worm; unlike the WT control, both receptor-expressing lines exhibit similar reproductive impairment behavior, measured as the number of individuals in the progeny;

Figure 3 shows the GPCR receptor expression analysis 24 hours after transfection; CHO cells (15,000 / well in 96-well plates) were transfected with 100, 50 and 25 ng of plasmid DNA each containing one gene of the indicated GPCRs; the expression was analyzed with an ELISA assay using a specific antibody against the HA epitope fused to the aminoterminal of the expressed receptor: unlike the CeSer-7 receptor of C. elegans, the other 3 GPCR of M.incognita show a scarce (MiNpR1-like) or no (MiSerR1-like and MiDopR1-like) membrane expression;

Figure 4 shows the schematic representation of the chimeric proteins constructed by replacing portions of the MiSerR1-like receptor of Meloidogyne incognita with homologous portions of the CeSer-7 receptor of C. elegans; the unmodified traits of M. incognita are represented in dark gray, while those modified with the receptor of C. elegans are shown in light gray. ChA = chimera A, amino-terminal substitution; ChB = chimera B, substitution of the carboxy-terminal tract; ChC = chimera C, replacement of the intermediate portion, including all seven transmembrane tracts, the three â € œextracellular loopsâ € and the three â € œ intracellular loopsâ €; ChD = Chimera D, replacement of the carboxyterminal tract and of the third â € œ intracellular loopâ €;

Figure 5 shows the GPCR receptor expression analysis 24 hours after transfection; CHO cells (15,000 / well in 96 plates) were transfected with 50 ng of plasmid DNA each containing a GPCR gene: CeSer-7 of C.elegans, MiSerR1-like of M.incognita, and those of the chimeric ChA constructs, ChB, ChC and ChD; the expression of the receptors was analyzed with an ELISA assay, using a specific antibody against the HA epitope fused to the aminoterminal of the expressed receptors; unlike the MiSerR1-like receptor, the four chimeric proteins all show significant membrane expression;

Figure 6 shows the analysis of the expression and cellular localization of the CeSer-7, MiSerR1-like and MiSerR1-ChB receptors. These receptors were fused to the fluorescent protein GFP and transiently expressed in mammalian CHO cells to visualize their localization in the cell. Unlike the C.elegans CeSer-7 receptor which is totally localized in the membrane (panel A), MiSerR1-like of M.incognita is predominantly localized in the cytoplasm (panel B). The chimeric protein MiSerR1-ChimeraB, consisting of the MiSerR1-like receptor of M.incognita in which the carboxy-terminal tract has been replaced with that of the CeSer-7 receptor of C.elegans, instead shows a prevalent localization in the membrane (panel C);

Figure 7 shows the nucleotide and protein sequences of all the chimeras of the MiSer1-like receptor [Chimera A (SEQ ID NO: 50; SEQ ID NO: 42), Chimera B (SEQ ID NO: 51; SEQ ID NO: 43) , Chimera C (SEQ ID NO: 52; SEQ ID NO: 44) and Chimera D (SEQ ID NO: 53; SEQ ID NO: 45)], those of chimeric receptors MiNpR1 :: CtermCeSer-7 (SEQ ID NO: 54 ; SEQ ID NO: 46) and MiDop1 :: CtermCeSer-7 (SEQ ID NO: 55; SEQ ID NO: 47), and those of MiNpR1-ChB (Cterm2) (SEQ ID NO: 56; SEQ ID NO: 48) and MiDopR1-ChB (Cterm3) (SEQ ID NO: 57; SEQ ID NO: 49);

Figure 8 shows the GPCR receptor expression analysis 24 hours after transfection; CHO cells (15,000 / well in 96-well plates) were transfected with 100 and 50 ng of plasmid DNA each containing one gene of the indicated GPCRs; the expression of the receptors was analyzed with an ELISA assay, using a specific antibody against the HA epitope fused to the aminoterminal of the expressed receptors; for both chimeric receptors (MiNpR1-ChB and MiDopR1-ChB) the level of expression is significantly higher than in the unmodified form of receptor (MiNpR1-like and MiDopR1-like);

Figure 9 shows the results of the CRE (cAMP Response Element) assay conducted on the MiSerR1-like and MiSerR1-ChimeraB receptors; 25 ng of plasmid DNA containing the indicated GPCR genes was transfected into CHO cells together with 125 ng of plasmid containing the luciferase gene upstream of the CRE promoter induced by intracellular cAMP increases; following the expression of the receptors or stimulation by the activator forskolin (forsk), the level of cAMP varies and these variations are measurable as an expression of the reporter luciferase protein; unlike MiSerR1-like which does not produce any effect on cAMP (because the receptor is not expressed in the membrane), the chimeric protein MiSerR1-ChB behaves similarly to the CeSer-4 receptor of C.elegans, resulting in reductions in the level of intracellular cAMP, and in the opposite way to CeSer-7;

Figure 10 shows the results of the CRE (cAMP Response Element) assay conducted on the MiNpR1-ChB and MiDopR1-ChB receptors; 25 ng of plasmid DNA containing the indicated GPCR genes were transfected into CHO cells together with 125 ng of plasmid containing the luciferase gene upstream of the CRE promoter induced by intracellular cAMP increases; following the expression of the receptors, induction by specific agonists (dopamine) or stimulation by the activator forskolin (forsk), the level of cAMP varies and these variations are measurable as expression of the luciferase reporter protein; unlike MiDopR1-like and MiNpR1-like, which do not produce any effect on cAMP, the chimeric proteins MiDopR1-ChB and MiNpR1-ChB determine, respectively, increases in cAMP following stimulation with the specific dopamine agonist or its reduction;

Figure 11 shows the results of the ELISA and CRE (cAMP Response Element) assay, conducted on the chimeric receptors MiNpR1-ChB (Cterm2) and MiDopR1-ChB (Cterm3): in the ELISA assay CHO cells (15,000 / well in 96-well plates ) were transfected with 100 and 50 ng of plasmid DNA each containing a gene of the indicated GPCRs and the expression level of the receptors was measured using a specific antibody against the HA epitope fused to the aminoterminal of the expressed receptors; in the CRE assay 25 ng of plasmid DNA containing the indicated GPCR genes were transfected into CHO cells together with 125 ng of plasmid containing the luciferase gene upstream of the CRE promoter induced by intracellular cAMP increases; following the expression of the receptors, induction by specific agonists (dopamine) or stimulation by the activator forskolin (forsk), the level of cAMP varies and these defects are measurable as expression of the luciferase reporter protein; both chimeric receptors MiNpR1-ChB (Cterm2) and MiDopR1-ChB (Cterm3) show levels of expression in ELISA and activation higher than those of the corresponding unmodified receptors (MiNpR1-like and MiDopR1-like).

EXAMPLE 1: Expression in mammalian CHO cells of three GPCR receptors of the parasitic nematode M.incognita.

The validity of the invention proposed here is exemplified below, with illustrative and non-limiting purposes, through the expression in a heterologous cell system (mammalian CHO cells) of three GPCR receptors of the parasitic nematode M.incognita.

Two examples of application of the technology object of this patent are provided: 1) in the first the modification made to the two receptors allows their expression in the membrane, 2) in the other the modification introduced increases the expression of the third receptor which , in the unmodified form (wild-type), is expressed at very low levels in the plasma membrane.

In the experimental part reported below, the modifications made to the three receptors, with the relative new nucleotide and protein sequences, are described, by way of non-limiting example. ELISA assays are also reported for the verification of membrane expression of modified receptors, and observation of the localization of these receptors under a fluorescence microscope, thanks to the fusion with the fluorescent protein GFP. MATERIALS AND METHODS

Cloning of M.incognita receptors

To clone the receptors from M.incognita, M.hapla and G.rostochiensis the database of the expressed sequences of these species (available at: http://www.nematode.net/BLAST/Cluster.BLAST/index.php) She was interrogated with various peptide sequences of the receptor homologues of C. elegans.

The identified clones were:

AW 735607 for MiSerR1-like and GrSerR1-like;

AW 571066 for MiDopR1-like;

BQ 548106 for MiRho1-like;

AW 735557 for MiRho2-like;

AW 829676 for MiRho3-like;

CD749350 for MiRho4-like;

CN 572214 for MhRho1-like.

Starting from the sequences of the identified clones, specific oligonucleotides were drawn which were used to amplify the entire receptors using the 5â € ™ and 3â € ™ Race method. The oligonucleotides used for amplification are the following:

For MiSerR1-like:

MiserR F1: GATTTGGAAAATTTGGACGAT (SEQ ID NO: 58) MiserR F2: GGAGGGTCTTTTGTCCATGCA (SEQ ID NO: 59) MiserR F3: TCTCATCCAATAATTGCAATT (SEQ ID NO: 60) MiserR R1: ACGTAATC CAAGTGAAT: R2 ID NO: 62) MiserR R3: AGCCAAAGCAAGTGATATAAG (SEQ ID NO: 63)

For MiDopR1-like:

MidopR F1: ACTCTTGGTGTTATTATGGGC (SEQ ID NO: 64) MidopR F2: TGGCTAGGTTATGCCAATTCT (SEQ ID NO: 65) MidopR F3: CGAGACTTTCGACGTGCCTTT (SEQ ID NO: 66) MidopR R1: AGQAGTCGC ID NO: 68) MidopR R3: GCCCATAACACCAAGAGT (SEQ ID NO: 69) For MiRho1-like:

MILYMN F1: CCAACAACAACAACTCGTTAATT (SEQ ID NO: 70) MILYMN F2: CCCCCAACATTTTCTCCTTCT (SEQ ID NO: 71) MILYMN F3: GATAGAAAAATGAGAAAATCTCCA (SEQ ID NO: 72) MILYMN R1 SEQACACGA (TTGATMAT) ID No. MiRho2-like:

MIOPIOD F1: GGAATTCTCGTGTGTGTTTCA (SEQ ID NO: 79) MIOPIODF2: GCTCTAAAATTGCTAACGCCT (SEQ ID NO: 80) MIOPIODF3: GTCTGGTATTGTATTCCTTTG (SEQ ID NO: 81) ) MIOPIODR3: TGAAACACACACGAGAATTCC (SEQ ID NO: 84) MIOPIODF0: ATGTATGCACTCATTCTACAC (SEQ ID NO: 85) For MiRho3-like:

MIRHO F1: GTTGCCTATTGTTATATAATG (SEQ ID NO: 86) MIRHO F2: GCTGTTTCTTATATATGTATTTG (SEQ ID NO: 87) MIRHO R1: AATATAAAACAATGCTGGAAC (SEQ ID NO: 88) MIRHO R2: CAQACACGTCTAG ID NO: 90) For MiRho4-like:

MIFRZ F1: TCTGGATTTGGATATCTGGAT (SEQ ID NO: 91) MIFRZ F2: AATATACCTCATTTGACAAGA (SEQ ID NO: 92) MIFRZ F3: AATTGTTGGTGGTTAATACTTGGT (SEQ ID NO: 94) MIFRZ AGTCAGT: 94

MIFRZ R2: ACCAAGTATTAACCACCAACAATT (SEQ ID NO: 95)

MIFRZ R3: ATCCAGATATCCAAATCCAGA (SEQ ID NO: 96)

MIFRZ F4: GTAGACGCTTCTGAATTAACT (SEQ ID NO: 97)

For MhRho1-like:

MHXF87 R0: TTACAAAAATCTATTCATTGG (SEQ ID NO: 98)

MHXF87 R1: GTAAATTATTGGATTTAAAGC (SEQ ID NO: 99)

MHXF87 R2: ACTGTATTTAACAATTAAACG (SEQ ID NO: 100)

MHXF87 R3: CTTGTGAGCTCTTTGACTGCG (SEQ ID NO: 101)

For GrSer1-like:

GRSER F1: ATGTTAGAAAATGATTTGGAA (SEQ ID NO: 102)

GRSER R1: TTATTTTGATGATTCCATCAA (SEQ ID NO: 103)

The amplified receptors were cloned into the PCR2.1 vector (Invitrogen) and sequenced with the appropriate oligonucleotides.

Generation of expression vectors

The cDNA coding for the different GPCRs were amplified starting from suitable templates using specific oligonucleotides, which contain at 5â € ™ a sequence for suitable restriction enzymes (shown in bold) used for the introduction of the amplified product in the vector of expression.

The sequences of the oligonucleotides used are the following:

MiserR Forward: ACCAAGCTTTTAGAAAATGATTTGGAAAA (SEQ ID NO: 104)

MiserR Reverse: GACGAATTCTTATTTTGATGATTCCATCA (SEQ ID NO: 105)

MiDopR Forward: ACCAAGCTTTTGCCCTGGTGGCTACCTCT (SEQ ID NO: 106)

MiDopR Reverse: CGGGGTACCTTAAAAACATAAAAATCTCA (SEQ ID NO: 107)

MiNprR Forward: CGGACTAGTGAAGCATCTACAATGGAATT (SEQ ID NO: 108)

MiNprR Reverse: AGTTTAGCGGCCGCTCATATCCTCTCATCTGTAT (SEQ ID NO: 109)

CeSer-7 Forward: ACTGGTACCGCCCGTGCAGTCAACATATC (SEQ ID NO: 110)

CeSer-7 Reverse: CTAGAATTCCTAGACGTCACTTGCTTCGTGAC (SEQ ID NO: 111)

CeSer-4 Forward: CGGGGTACCATCGACGAGACGCTTCTCAATC (SEQ ID NO: 112)

CeSer-4 Reverse: CCGGAATTCTCAATAATCGTGAATAAGGCAC (SEQ ID NO: 113)

For the construction of the chimeric receptors a protocol of â € œoverlapping PCRâ € was followed which consists of two consecutive amplification reactions: in the first the fragments to be fused are amplified using oligonucleotides which at 5â € ™ contain an extra (underlined) complementary sequence to the other fragment; in the second reaction the fragments obtained in the first amplification are mixed and fused thanks to the amplification with the two outer 5â € ™ and 3â € ™ oligos corresponding to the entire sequence to be obtained.

The sequences of the oligonucleotides used for the amplifications are the following:

First MiSerR1-ChimeraA amplifications:

CeSer-7 Forward: ACTGGTACCGCCCGTGCAGTCAACATATC (SEQ ID NO: 110)

CeSer-7 Nterm fus Reverse: GATTAAAATTGCAATAGCTTTT CCAGTCGT (SEQ ID NO: 114)

MiSer 1TR fus Forward: ACGACTGGAAAAGCTATTGCAATTTTAATC (SEQ ID NO: 115)

MiSerR Reverse: GACGAATTCTTATTTTGATGATTCCATCA (SEQ ID NO: 105).

The most external oligonucleotides were used for the second amplification and fusion reaction: CeSer-7 Forward and MiSerR Reverse.

First MiSerR1-ChimeraB amplifications:

MiSerR Forward 2: ACCGGTACCTTAGAAAATGATTTGGAAAA (SEQ ID NO: 116)

MiSer TR fus Reverse: GTTGTACTTGCAGTAATAAATTATTGGGTT (SEQ ID NO: 117)

CeSer-7 C-term fus Forward: AACCCAATAATTTATTACTGCAA GTACAAC (SEQ ID NO: 118)

CeSer-7 Reverse: CTAGAATTCCTAGACGTCACTTGCTTCGTGAC (SEQ ID NO: 111).

The most external oligonucleotides were used for the second amplification and fusion reaction: MiSerR Forward 2 and CeSer-7 Reverse.

First MiSerR1-ChimeraC amplifications:

MiSerR Forward 2: ACCGGTACCTTAGAAAATGATTTGGAAAA (SEQ ID NO: 116)

MiSer-7 N-term fus Reverse: GGCAATGGCTAGCAGTATTGGATG AGAATA (SEQ ID NO: 119)

CeSer-7 1TR fus Forward: TATTCTCATCCAATACTGCTAGCCA TTGCC (SEQ ID NO: 120)

CeSer-7TR fus Reverse: TCGATTATATTTGCAGATCAATGGATTTAA (SEQ ID NO: 121)

MiSer-7 C-term fus Forward: TTAAATCCATTGATCTGCAAA TATAATCGA (SEQ ID NO: 122)

MiSerR Reverse: GACGAATTCTTATTTTGATGATTCCATCA (SEQ ID NO: 105).

The most external oligonucleotides were used for the second amplification and fusion reaction: MiSerR Forward 2 and MiSerR Reverse.

First MiSerR1-Chimera D amplifications:

MiSerR Forward 2: ACCGGTACCTTAGAAAATGATTTGGAAAA (SEQ ID NO: 116)

Mi-CeSer-7 3 int loop Reverse: CCAAATTTTGATGTT ACTCCATAATTTAAC (SEQ ID NO: 123)

Mi-CeSer-7 3 int loop Forward: GTTAAATTATGGAGTAACAT CAAAATTTGG (SEQ ID NO: 124)

Ce-MiSer-7 3 int loop Reverse: AATTAACACTATTGGCAATG TTTTCCGGGC (SEQ ID NO: 125)

Ce-MiSer-7 3 int loop Forward: GCCCGGAAAACATTGCCAATAGT GTTAATT (SEQ ID NO: 126)

CeSer-7 Reverse: CTAGAATTCCTAGACGTCACTTGCTTCGTGAC (SEQ ID NO: 111).

The most external oligonucleotides were used for the second amplification and fusion reaction: MiSerR Forward 2 and CeSer-7 Reverse.

First amplifications MiNpR1 :: CtermCeSer-7 MiNpr F Kpn I: ACTGGTACCGAAGCATCTACAATGGAATT (SEQ ID NO: 127).

MiCeNpr ser-7 Cterm fus R: GTTGTACTTGCAGTATAATAAGG CATATAA (SEQ ID NO: 128)

MICE NPRSER FUS F: TTATATGCCTTATTATACTGCAAGTACAAC (SEQ ID NO: 129)

CE SER-7 R NOT I: ATAAGAATGCGGCCGCCTAGACGTCACTTGCTTCGT (SEQ ID NO: 130).

For the second amplification and fusion reaction the outermost oligonucleotides were used: MiNprF-KpnI and CE SER-7 R NOT I.

First amplifications MiDopR1 like :: CtermCeSer-7: MI DOP-1 F KPN I: CGGGGTACCTTGCCCTGGTGGCTACCTCT (SEQ ID NO: 131)

MI CE DOPSER FUS R: GTTGTACTTGCAGTAAAATATTCCATAAAT (SEQ ID NO: 132)

MI CE DOPSER FUS F: ATTTATGGAATATTTTACTGCAAGTACAAC (SEQ ID NO: 133)

CE SER-7 R ECORI: CTAGAATTCCTAGACGTCACTTGCTTCGTGAC (SEQ ID NO: 134).

For the second amplification and fusion reaction the outermost oligonucleotides were used: MI DOP-1 F KPNI and CESER-7 R ECORI.

First amplifications MiDopR1like :: Cterm2 (F15A8.5): MI DOP-1 F Kpn I: CGGGGTACCTTGCCCTGGTGGCTACCTCT (SEQ ID NO: 131)

MiCedop1CtermfusReverse:

TCTAAAGTCACGATTAAATATTCCATAAAT (SEQ ID NO: 135)

Mi Ce dop1CtermfusF: ATTTATGGAATATTTAATCGTGACTTTAGA (SEQ ID NO: 136)

Ce dop-1 R ECO RI: CCGGAATTCCTATTCCGGAATGGTTTCCT (SEQ ID NO: 137)

For the second amplification and fusion reaction the outermost oligonucleotides were used: MI dop-1 F Kpn I and ce dop-1 R ECO RI.

First MiNPR-Cterm3 amplifications (C16D6.2):

Mi npr F Kpn I: ACTGGTACCGAAGCATCTACAATGGAATT (SEQ ID NO: 138)

Mi Ce npr Cterm fusR: CAGTTGGAGGTTGAGTAATAAGGCATATAA (SEQ ID NO: 139)

Mi Ce npr Cterm fusF: TTATATGCCTTATTACTCAACCTCCAACTG (SEQ ID NO: 140)

CE npr R Note: ATAAGAATGCGGCCGCTTAGAAAGAAGCCTTCCTTG (SEQ ID NO: 141)

For the second amplification and fusion reaction the outermost oligonucleotides were used: Mi npr F Kpn I and CE npr R Not I.

The typical amplification scheme was:

- denaturation: 98 ° C for 10 seconds;

- annealing: 50 ° C for 30 seconds;

- extension: 72 ° C for 1 minute and 30 seconds.

The cycles were repeated 35 times, then a final extension of 7 minutes was performed at 72 ° C, followed by cooling the samples to 4 ° C. The DNA Polymerase used was Phusion High-Fidelity (Finzymes).

The fragments obtained, checked by agarose gel electrophoresis for concordance with the expected size, were digested with the appropriate restriction enzymes (New England Biolabs), purified from agarose gel and ligated in the appropriate vectors.

The cDNAs coding for the MiSerR1-like (SEQ ID NO: 1), MiDopR1-like (SEQ ID NO: 3), MiNpR1-like (Q2TGX5_MELIC), CeSer-7 (Gene Bank C09B7.1), CeSer-4 (Gene Bank Y22D7AR.13), MiSerR1-ChimeraA (SEQ ID NO: 50), MiSerR1-ChimeraB (SEQ ID NO : 51), MiSerR1-ChimeraC (SEQ ID NO: 52), MiSerR1-ChimeraD (SEQ ID NO: 53), MiNpR1 :: CtermCeSer-7 (SEQ ID NO: 54) and MiDopR1like: CtermCeSer-7 (SEQ ID NO: 55), MiNpR1 :: Cterm2 (C16D6.2) (SEQIDNO: 56), MiDop1 :: Cterm3 (F15A.

5) (SEQ ID NO: 57) (see Figures 1 and 7). The cDNAs encoding the MiSerR1-like (SEQ ID NO: 1), CeSer-7, MiSerR1-ChimeraB (SEQ ID NO: 1), CeSer-7, MiSerR1-ChimeraB (SEQ ID NO: 51) (see Figures 1 and 7), through appropriate subcloning.

The correct insertion of the fragments and the respect of the correct reading frames were checked by sequence analysis.

Transfection

Mammalian CHO cells were grown at 37 ° C in the presence of 5% CO2, in a DMEM culture medium (DMEM F-12, Lonza) supplemented with 10% bovine fetal serum (FBS).

All receptors were transiently transfected into cells using the Lipofectamine 2000 (Invitrogen) method.

For transfection the cells were seeded in 96-well plates, 1.5 x 10 <4> cells per well, allowed to grow for 24 hours and then incubated with the transfection mixture containing the lipofectamine and DNA (in a 5: 1 ratio) to 5 hours.

The amount of DNA used for all receptors was 25 to 100 ng. The cells were analyzed 24-48 hours after transfection.

ELISA assay

This assay is based on the principle of specific immunorecognition: the N-terminal portion of the receptor is recognized by a primary anti-HA antibody recognized, in turn, by a secondary antibody conjugated with an enzyme whose activity can be easily detected by a colorimetric reaction. The signal can be detected only when the receptors, locating correctly in the membrane, expose their N-terminal portion on the cell surface.

The protocol followed was as follows: the cells transiently transfected with the receptors and still attached to the well surface of the 96-well plates were washed with buffer (1x PBS, 0.5 mM CaCl2, 1 mM MgCl2) and fixed to 20 minutes in 4% formaldehyde.

After two washes with the same buffer, the fixed cells were incubated with a 1: 500 dilution of the primary anti-HA (rabbit polyclonal Santa Cruz Biotechnology) antibody in 1% BSA for two hours.

At the end of the incubation period, the cells were washed 3 times to remove the excess of unbound primary antibody, and incubated with a secondary antibody conjugated with β-galactosidase (goat anti-rabbit IgG β-galactosidase) for about an hour.

After this second incubation they were subjected to a new series of washes and incubated with the β-galactosidase substrate, CPRG (Chloro phenol red-β-D-galactopyranoside, Roche Diagnostics GmbH).

The colorimetric reaction that developed following degradation of the chromogenic substrate was detected by reading the absorbance of the samples at 550 nm after about 5 hours with the Victor3 instrument (PerkinElmer): the intensity of the color is correlated to the amount of receptor expressed and correctly localized in the plasma membrane.

CRE essay

Cells transiently transfected with the reporter vector (125 ng per well) and the different receptors (25 ng per well) were grown for 24 hours post transfection in culture medium containing 10% serum.

The next day they were treated with only buffer (control samples) and with 10 microM of forskolin, known activator of the cAMP pathway, for 3 hours.

Subsequently the culture medium was removed, the cells washed with buffer (1x PBS, 0.5 mM CaCl2, 1 mM MgCl2) and incubated for 5 minutes with 10 mg / ml of the luciferase substrate (Steady-Glo luciferase substrate, Promega) dissolved in a lysis buffer supplied by the manufacturer together with the substrate.

The photometric reaction that developed following degradation of the substrate by luciferase was detected by reading the luminescence with the Victor3 instrument (PerkinElmer). RESULTS

Receptors cloning

6 different receptors were cloned from the species Meloidogyne incognita (MiSerR1-like, MiDopR1-like, MiRho1-like, MiRho2-like, MiRho3-like, MiRho4-like), one from the species Meloidogyne hapla (MhRho1-like) and one from the species Globodera rostochiensis (GrSerR1-like). A study of these receptors was therefore carried out, both at the sequence level and at the functional level, in order to demonstrate the validity of the GPCR of parasitic nematodes as nematocide targets.

The GPCRs of M.incognita that we cloned and chosen as the target of new nematocides are:

1) a homolog of the serotonin receptors of C. elegans, which we have called MiSerR1-like;

2) a homolog of the dopamine receptor CeDop-1 of C. elegans, which we have called MiDopR1-like;

3) a homolog of a putative GPCR of C.elegans (T27D1.3) (belonging to the Rhodopsinlike GPCR family), which we have called MiRho1-like;

4) a homolog of a putative GPCR of C.elegans (T07D4.1) (belonging to the Rhodopsinlike GPCR family), which we have called MiRho2-like;

5) a homolog of a putative GPCR of C.elegans (B0563.6) (belonging to the Rhodopsinlike GPCR family), which we have called MiRho3-like;

6) a homolog of a putative GPCR of C.elegans (F02E8.2) (belonging to the Rhodopsinlike GPCR family), which we have called MiRho4-like.

The GPCRs of M.hapla and G.rostochiensis cloned and chosen as targets of new nematocides are:

1) a homolog of a putative GPCR of C.elegans (F02E8.2) (belonging to the Rhodopsinlike GPCR family), which we have called MhRho1-like.

2) a homolog of the serotonin receptors of C. elegans, which we have called GrSerR1-like.

The sequences encoding the GPCRs were isolated starting from the reverse transcribed cDNA from the total RNA extracted from M.incognita, M.hapla or G.rostochiensis.

For the cloning of the complete sequences, specific oligonucleotides, drawn on the basis of the expressed sequences (EST) of the nematode M.incognita, M.hapla and G.rostochiensis, were used in 5â € ™ and 3â € ™ RACE experiments. The nucleotide and protein sequences (SEQ ID NO: 1-16) of the cloned receptors are shown in Figure 1.

A particular aspect of the present invention concerns the possibility of validating the importance of the chosen targets thanks to studies in the C. elegans model system. Thanks to the functional and sequence homology between the model system and the parasitic nematodes, it is possible to understand which GPCRs of the parasitic nematode are of fundamental importance for its viability. For example, the MiSerR1-like receptors of M.incognita and GrSerR1-like of G.rostochiensis we identified and cloned are homologous to CeSer-7 and CeSer-4, two serotonin receptors of C.elegans, both involved in important functions biological, such as the reproduction and contraction of the pharynx (4). The overexpression of the MiSerR1-like receptor in the C.elegans system produced alterations in reproduction similar to the same CeSer-4 receptor of C.elegans: this, in addition to indicating a functional homology between the receptors of these two organisms, confirms the importance of this receptor as a target of nematocides (Figure 2).

Membrane expression of cloned receptors

Good expression and localization in the membrane of parasitic nematode receptors was obtained thanks to modifications based on the substitution of the carboxy-terminal region. All parasitic nematode receptors tested presented difficulties in localization in the membrane.

By replacing portions of these receptors with homologous domains of the CeSer-7 receptor (see SEQ ID NO: 23 - Table 2), belonging to the C.elegans species that does not present problems of localization in the membrane, it was possible to express in membrane these receptors. The best results have been obtained by replacing the carboxyterminal portion of these receptors with that of CeSer-7. Following this modification, the receptor is correctly localized in the membrane, exposing its extracellular domains to the interaction with potential modulators, without the risk that the modifications can alter the structure of domains important for the interaction with these modulators.

In particular, the study focused on two of these MiSerR1-like and MiDopR1-like receptors and on a receptor for a neuropeptide (MiNpR1-like), whose sequence was already present in the database (Q2TGX5_MELIC) and appears to be homologous to that of a C. elegans neuropeptide receptor. The receptor chosen for the localization studies and for the construction of chimeras is that of the serotonin of C.elegans (CeSer-7), which localizes in the membrane and functions correctly in mammalian cells.

The cDNAs coding for these receptors have been cloned in the eukaryotic expression vector pHM6, downstream of a sequence of an HA epitope consisting of 9 amino acids and recognized by specific commercial antibodies: in this way all the receptors contain N-terminal the sequence of the epitope HA. If the cells contain a receptor in the membrane that carries the HA epitope, they respond positively to the ELISA assay which consists in an immunorecognition of the HA epitope by a specific antibody.

MiSerR1-like, MiDopR1-like and MiNprR1-like receptors were transiently transfected into Chinese Hamster Ovary cells (CHO) mammalian cells and analyzed with an ELISA assay to verify their correct expression and localization in the membrane. As shown in Figure 3, the MiSerR1-like and MiDopR1-like receptors do not show membrane expression, while the MiNprR1-like receptor shows very weak expression; the CeSer-7 receptor shows a strong signal.

Using MiSerR1-like, a study was conducted to understand which portion of the receptor was to be modified in order to have a correct expression and localization in the membrane. For this purpose, several chimeric proteins have been constructed by modifying the MiSerR1-like receptor with portions of the CeSer-7 receptor of C.elegans, and their expression and localization have been verified.

The modifications made to the MiSerR1-like receptor are schematized in Figure 4: in chimera A (SEQ ID NO: 42) the aminoterminal portion has been replaced, in chimera B (SEQ ID NO: 43) the carboxyterminal portion, in chimera C ( SEQ ID NO: 44) the seven transmembrane tracts and in chimera D (SEQ ID NO: 45) the third intracellular tract and the carboxyterminal portion.

The nucleotide (SEQ ID NO: 50-53) and protein (SEQ ID NO: 42-45) sequences of the chimeric proteins are shown in Figure 7.

As can be seen from the result of the ELISA assay reported in Figure 5, all the chimeric proteins show a significant expression in the membrane compared to the unmodified protein MiSerR1-like, and it is also clear that for the chimera B and D this increase turns out to be more evident. Since the chimeraD is nothing more than a chimeraB with a further substitution of the third intracellular domain, it is evident that the substitution of the carboxy-terminal tract alone is sufficient to guarantee the maximum expression in the membrane for this protein. Chimera B was then used for subsequent localization studies with the fluorescent protein GFP.

As confirmation of the data obtained with the ELISA assays, the cDNAs coding for MiSerR1-like, CeSer-7 and for MiSerR1-Chimera B were cloned into a eukaryotic expression vector upstream of the GFP gene, transiently transfected into cells CHO and the expression of these proteins analyzed under a fluorescence microscope.

The observation under the fluorescence microscope, shown in Figure 6, showed that in the cells transfected with the MiSerR1-like receptor (panel B) the fluorescence is all localized in the cytoplasm, while in the cells transfected with CeSer-7 and with MiSerR1 -ChimeraB (panels A and C respectively) the fluorescent signal is also localized in the membrane.

These results allow us to conclude that the carboxyterminal tract plays a decisive role in the membrane localization of these receptors and for the receptor under examination to be correctly localized it is sufficient to replace its C-terminal portion with another receptor that does not show problems of expression and localization in the membrane. This other receptor was chosen from among those belonging to the C.elegans species, as long as it does not present problems of expression and localization in the membrane.

As confirmation of the validity of this invention, the carboxy terminal portion of MiDop1-like was also replaced with that of CeSer-7 and the expression in the membrane was measured.

It has also been verified whether the replacement of the carboxy terminal portion of the MiNprR1-like receptor with the corresponding portion of the CeSer-7 receptor is able to improve its expression. In particular, we wanted to verify whether even for a receptor showing low levels of expression it is possible to use portions of another receptor to improve its expression in the membrane.

The nucleotide and protein sequences of the two chimeric receptors that have been constructed and named MiNpR1 :: CtermCeSer-7 (SEQ ID NO: 54, SEQ ID NO: 46) and MiDop1 :: CtermCeSer-7 (SEQ ID NO: 55, SEQ ID NO: 47) is always shown in Figure 7.

As can be seen from the ELISA assay reported in Figure 8, the chimeric protein MiNpR1 :: CtermCeSer-7 is expressed at higher levels than the receptor in its unmodified form, and the chimeric protein MiDop1 :: CtermCeSer-7 shows appreciable expression in membrane. These results suggest that this type of modification can be applied to all GPCR receptors of M.incognita, M.hapla and G.rostochiensis, thus allowing their expression and correct localization in the membrane.

This method therefore allows to express in the membrane receptors that usually do not express themselves in their unmodified form and to increase the expression for those showing a weak signal.

Check functionality of chimeric receptors

To demonstrate that the receptors thus modified were functionally active, that is, capable of transducing a measurable intracellular signal with a variation of one or more cytoplasmic second messengers, we conducted a series of biochemical assays on the cells expressing the chimeras in question. One of the assays reported in the experimental part for illustrative purposes is that of the CRE (cAMP response element) which measures intracellular increases of the second cyclic AMP messenger (cAMP) following the simple expression and / or activation of the receptors with agonists.

To verify if MiSerR1-ChimeraB, located in the membrane, is also functionally active, capable of transducing a signal from the outside to the inside of the cell, changes in the level of cAMP induced by the activation of the GPCR were measured in analysis. Literature data show, in fact, the involvement of this second messenger in the pathway activated by the serotonin receptors in C. elegans (8).

In this regard, a reporter assay (CRE assay) was adopted which measures the expression of a reporter gene, luciferase, induced by variations in the level of cAMP: the pCRE vector used in the assay contains, in fact, the luciferase under the transcriptional control of a promoter with elements that are activated by cAMP.

The measurement of luciferase activity in cells transfected with pCRE and with vectors coding for CeSer-7, MiSerR1-like and MiSerR1-ChimeraB showed that both ChimeraB and CeSer-7, thanks to their correct localization in the membrane, show a feature detectable as a variation of the intracellular cAMP level.

As can be seen in Figure 9, in fact, the cells transfected with the CeSer-7 receptor show an increase in the activity of luciferase due to increases in the cAMP level, the cells transfected with the MiSerR1-like receptor show no variation, while the cells transfected with MiSerR1-ChimeraB show a decrease in activity due to a reduction in cAMP levels. This reduction is similar to that observed in cells transfected with another member of the C.elegans serotonin receptor family, CeSer-4, of which MiSerR1-like appears to be the functional homologue. This reduction is more evident if the cells are previously treated with forskolin, a well-known activator of the enzyme adenylate cyclase which synthesizes cAMP. A similar result was obtained by testing variations in the activity of luciferase induced by the expression and activation of the chimeras MiNpR1 :: CtermCeSer-7 and MiDop1 :: CtermCeSer-7.

The expression of the chimera MiNpR1 :: CtermCeSer-7 (in the figure indicated MiNpR1-ChB) also determines a reduction in cAMP levels, while the chimera MiDop1 :: CtermCeSer-7 (indicated as MiDop1-ChB) produces a increased cAMP, only following stimulation with its specific dopamine agonist (Figure 10).

To verify that even the use of a carboxy terminal portion different from Ser-7, but belonging to another GPCR of C.elegans, produced a similar response of membrane expression and activation, we built other chimeric receptors using the receptors of M.incognita MiNpR1-like and MiDopR1-like and replacing the carboxyterminal tract with that of the NPR1-like (C16D6.2) and DOP-1 (F15A8.5) receptors of C.elegans. The resulting chimeras were named MiNpR1-ChB (Cterm2) (SEQ ID NO: 56; SEQ ID NO: 48), in which the carboxy terminal was replaced with that of C16D6.2 at the M.incognita MiNpR1-like receptor, and MiDopR1-ChB (Cterm3) (SEQ ID NO: 57; SEQ ID NO: 49), in which the carboxy terminal has been replaced with that of F15A8.5 at the M.incognita MiDopR1-like receptor.

The resulting chimeric constructs were transfected into CHO cells to measure their expression level with the ELISA assay and their activation with the cAMP Response Element assay (CRE). As shown in Figure 11, both chimeric receptors show a level of expression in the membrane higher than their respective unmodified receptors: in the case of MiNpR1-ChB (Cterm2) the expression increases compared to that of MiNpR1-like, while for MiDopR1 -ChB (Cterm3) is now appreciable since the unmodified form MiDopR1-like is not expressed in the membrane. The functionality of the two chimeric receptors was also confirmed by the subsequent CRE assay (Figure 11). The MiNpR1-ChB (Cterm2) construct, once expressed, causes a reduction in intracellular cAMP levels, while the MiDopR1-ChB (Cterm3) construct produces increases in cAMP only following stimulation with its specific dopamine agonist. The results of both assays reported in Figure 11 suggest that even these two chimeras, unlike the unmodified receptor forms, express themselves, localize in the membrane and are functionally active.

We can therefore conclude that similarly to what previously observed with the chimeras MiNpR1 :: CtermCeSer-7 and MiDop1 :: CtermCeSer-7, also the expression of the chimeras MiNpR1-ChB (Cterm2) and MiDopR1-ChB (Cterm3) produces receptors that they correctly localize in the cell membrane and are functionally active, therefore usable for analysis and screening systems, unlike non-engineered receptors.

All these results therefore suggest that for all the tested receptors the modifications in the carboxy-terminal region allow their correct localization in the membrane and their correct functioning, thus confirming the validity of the developed technology.

Furthermore, we think that this technology we intend to protect could potentially be extended to all those GPCR receptors that exhibit membrane localization problems, such as many human GPCRs with taste and smell perception functions.

BIBLIOGRAPHY

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(3) Keating CD, Kriek N, Daniels M, Ashcroft NR, Hopper NA, Siney EJ, Holden-Dye L, Burke JF (2003). Curr Biol., 13 (19): 1715-20.

(4) Hobson RJ, Hapiak VM, Xiao H, Buehrer KL, Komuniecki PR, Komuniecki RW (2006). Genetics. 172 (1): 159-69.

(5) Krautwurst D, Yau KW, Reed RR. (1998). Cell, 95: 917-926.

(6) Hague C, Uberti MA, Chen Z, Bush CF, Jones SV, Ressler KJ, Hall RA, Minnerman KP. (2004). PNAS, 101 (37): 13672-13676.

(7) Lu M, Staszewski L, Echeverri F, Xu H, Moyer BD. (2004). BMC Cell Biol, 5: 34-44.

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Claims (3)

CLAIMS 1. Screening method for specific pesticides for plant parasitic nematodes comprising the following steps: a) transformation of a heterologous cell line with one or more chimeric GPCR receptors of plant parasitic nematodes, called chimeric GPCR receptors being characterized by have the carboxyterminal domain replaced with that of the GPCR receptors of non-parasitic nematodes capable of localizing in the membrane; b) putting the compound to be tested in contact with said cell line; c) measurement of the intracellular signal activated by the expression or stimulation of the chimeric GPCR receptor with a known agonist, and measurement of the possible variations of the activity of said receptor following treatment with the compound to be tested. Method according to claim 1, wherein said plant parasitic nematode GPCR receptors are selected from the group consisting of serotonin receptors, dopamine receptors, neuropeptide receptors, galanin receptors, metabotropic glutamate receptors and putative amine receptors biogenic. Method according to any one of claims 1-2, wherein said plant parasitic nematodes are selected from the group consisting of Meloidogyne, Heterodera and Globodera. 4. Method according to claim 3 wherein when said plant parasitic nematode is Meloidogyne incognita, said GPCR receptor is selected from the group consisting of the amino acid sequences shown in the following Table: AMINO ACID SEQUENCE TYPE M.incognita SerR1-like >NLNLFSSSSSTSSSSLFSSSSQRSPLLEPNFVDEIWPATSSNSLFY SHPIIAILISILIFLLILITIIGNLGVCAAILLVRKLKAQPANLLL ISLALADFCVGLFVMPLAAVYVLEDRWIFGDILCRFWVTADLTLCT ASILNLCAISVDRHLAVTRALRYSALRTRRRICLYICIVWLGALLV SAAPLALLPFPKIEQYCQVSQNRVYQIYATIIAFWGPSLIMVIVYV KLWSAAKRMHRQDQLVLRWQGVHLPSDGDLEDGLPPTTTTSNATKS LFRNDSIARANARFLFALRMSAQKFGYYDHQNNKTNGGEIINDDEN LRIEVRTSASSSGGGGGGSSTLGTLRVPIVLIIMSAFIICWVPFFI LALAKSQHWVNYVPRWLDSLTLWLGYSNSMLNPIIYCKYNREFRVP FGEMLCCRFRTIQDVMRNESYYAKFGSPRISETKIGGSQQRSSGNN YYTKRKRNNTERRAPQNGFLDSSKTKKNYGIKRNSSPSILMESSK (SEQ ID NO:2) DopR1-like<MLPWWLPLLIFVPIFSVLISLAFSGNLLVIAAIFYDRKLRRQPENL>FLVSLALSDLFVAGMVMVLAAANDLIGFWPFGAAFCQFWICLDIAC STASILNLCAIALDRFIHISR PMRYVRFVGRRVICCSVCAIWIIST AVGTAQIAFGATNGLEIYSVIFNGDYFGEINETDKFRFIEQQHTLV QCQLQLSPFYAVFSSLLSFFLPATLMLFLYYRLYLYARHHARSIQS QLKQATSLLILQLASDRVRQVVVRPSSGFLSPSSIALHYDSRRFSG KSSFDGESLQKAEFSQINVAVNKSPENHSIDATIQQGTPVLRATLR QLRRTESNNSSSFSFVSRNGLGVNSRRVGCGGGNGGERRKSCSPLP SPTTKRGFKSCGWSNNNNGNGGHRSSDKKARVTLGVIMGTFLICWL PFFIVNVLRPFWPEIFPPLLFQTVSWLGYANSSVNPVIYGIFNRDF RRAFSRIMNKLIHCIDEERRGENSRGGTFRTRTESESFNK FNKSLNNASELQQQQKLIFKQKKTNKKLRFLCF (SEQ ID NO:4) Rho1-like MNITVPTTTTSLIINSISPFPPTFSPSISTQQNNFTTSSLTKTTFN FQQKRDVSFHFSLIFGSLIGGVTIVGLTANILVVIAILSDRKMRKS PMNLLLLNLAIADLLYLLAFTPFWLSMSVYGDGGWHFGDMFCPIAR FFGNIFLVISILTYLAICIERYVAIVHPIAMHTSVWCTRSRVLVIA FGIWVFAMTYQFPYLVVFQVFDIPEKNLRVCRNPLASKSKIWKIYK WSEFLLTYALPIIISVLLYSRICRVLWFKNKNGENCGKGQNENENK TELKPNINKRPSGRTIKKVLETRFREAISDVQNKRKVVPKEEEIFN FNNSSEKRQKPMNVSSSANLRARRSVVKMLMLCVGLFFLCYTPMVA YFVWSALFGRPLPLPFEFVLITSALVQFQSAFNPFLYTLFATQFRE KLSKLFLICNKKQKNKIINQHKISAKSTSTFSV (SEQ ID NO:6) Rho2-like MTTANNQGEVDCNQGLVEPLLDRPIFKIPFTFAYVAVFLLCLTGNL FTIVVICAHRSMRTATNF FLANLALADLLVAIFCIMQNMLHLVHLD AQWPLGETLCRMYALILHLVPCTGIGILVCVSVEKYIAVLHPLLAL KLLTPKFRSLMMAAVWICSLLANLPYYTTSKYREWEGGNSACFRGL LTDGFVSTRNMLIISFLVWYCIPLCIIAFLYTRIGFVLWHSAPLKK LTQMRTNSNETTTMTGIQKRSVASCRLSAITNNNGSATIVSEVTIK TPSKRSNSQPNNYSKITNNNLPPLILVNKSSPLPNTASESEGEEED FGREEEGREEEEDKDEEEEEEESNGSHLEEDLDDDEEEEDAEHENG TKCRLALGNGSVGPAHLGQRNSAPSSSTVFVSTVESRKRIIRLLIA IVCSFAVLTLPNHIRLLYSAVYSEERICLGGFEVFTQPVTYLLLFL SSSCNPFLYAFMSQRFRSAIRDIFKCRRGHIRRKSTKTRTQNSDVL CAVNSGDGVALSDCSSLNRLNNNGNNNLLDQSKGEEINSIHGEKNF PWANQKGEEINWLKVKKLKNTERNASGGSGNSTTTASSVLYTTQHS NGSLQMAVVYRKCAC(SEQ ID NO:8) Rho3-like MNDSETIENYEDFLSQLSSTMIPTTTTTSSSSSAIFTSTLATPLIS ETAQIPSPTPKLNINIVAYCYIMPTICVLGIIGNSMNVVTLASPRL KAVSYMYLRALAVSDLLCMLFVLAFACCEVLKESGVPIERHPLYGF YQAHVMLSFINWALATGVYIVVALSLERYVSVVFPLHFRMWNSPKR AMKAIIIAYTVPALFYIPYGIGRYSVSEKINSRGEISYGAIDSEIS KTFGWQVYKWTREAFLRFLPIVILFVLNFQIMIAFRRRQKMFDRLR NRETAARDDTLLYILGGIAVMFFVCNIPAAINLLFINEVVKKRPDY QIFRAAANLLEITNHAAQFYIFCVCSSDYRVTFMQKFPCLRAYYVN KSKFCSFLRNASQLPKRSVARRTISTTTNSKIGNNVNNV CGTMRRS VSASNTPLNAKHGDKNDRRNGKSKKGGGGGGSAIAEMTFVRSTTIT GELLSTCGGHNTEQETIDAQIASLESLSDESETLLKQQKIREESWG SPITTKLCLIGLDGDEEEGIICRRSSNGEGIIAEDEQTDRTDGTSY L (SEQ ID NO:10) Rho4-like MDYAPMPISSNITTWFLLAVFIFVIHRNPSFHSQTNYFLASLAISD LLLILVGVPFDVLFLWKSRFITSPFNGFCEITSTFISWFTFNSILT IVSLTWERLVAICYPFSLKPFFHRDAVIWLIIIIWFISFFPSLFIG LQFKLVIQDFCGHTHVNSNGLGSKCDFVGWSGNYGSGENYTFEVML FFTFVLPVLFVIYCYIRILRTLNEATFNSFHLNAGSSIVRKHTSSS HTNSTLLNAITEENLPSTSNNSNSNTAGNNSDLNARDSFTRSSLRS QRAHKTVMKMLIMIAALFFVCYLPYHLERLIVKYSAKGCTEPQMCL WLYHGTGLLQYISAALNPIIYNVMSRRFRREFKLLCYRIVKKENVT KTRSDNNNQLKMTPMNRFL (SEQ. ID NO:12) 5. Metodo secondo la rivendicazione 3, in cui quando detto nematode parassita delle piante à ̈ Meloidogyne hapla, detto recettore GPCR ha la seguente sequenza amminoacidica: MDYAPMPISSNITTWFLLAVFIFVIHRNPSFHSQTNYFLASLAISDLLLILVGVPFDVL FLWKSRFITSPFNGFCEITSTFISWFTFNSILTIVSLTWERLVAICYPFSLKPFFHRDA VIWLIIIIWFISFFPSLFIGLQFKLVIQDFCGHTHVNSNGLGSKCDFVGWSGNYGSGEN YTFEVMLFFTFVLPVLFVIYCYIRILRTLNEATFNSFHLNAGSSIVRKHTSSSHTNSTL LNAITEENLPSTSNNSNSNTAGNNSDLNARDSFTRSSLRSQRAHKTVMKMLIMIAALFF VCYLPYHLERLIVKYSAKGCTEPQMCLWLYHGTGLLQYISAALNPIIYNVMSRRFRREF KLLCYRIVKKENVTKTRSDNNNQLKMTPMNRFL (SEQ. ID NO:14) . 6. Metodo secondo la rivendicazione 3, in cui quando detto nematode parassita delle piante à ̈ Globodera rostochiensis, detto recettore GPCR ha la seguente sequenza amminoacidica: MLENDLENLDDSSLILHHLGGSFVHAQIVEIEENDYINSNTSVSNTNLNLFSSSSSTSS SSLFSSSSQRSPLLEPNFVDEIWPATSSNSLFYSHPIIAILISILIFLLILITIIGNLG VCAAILLVRKLKAQPANLLLISLALADFCVGLFVMPLAAVYVLEDRWIFGDILCRFWVT ADLTLCTASILNLCAISVDRHLAVTRALRYSALRTRRRICLYICIVWLGALLVSAAPLA LLPFPKIEQYCQVSQNRVYQIYATIIAFWGPSLIMVIVYVKLWSAAKRMHRQDQLVLRW QGVHLPSDGDLEDGLPPTTTTSNATKSLFRNDSIARANARFLFALRMSAQKFGYYDHQN NKTNGGEIINDDENLRIEVRTSASSSGGGGGGSSTLGTLRVPIVLIIMSAFIICWVPFF ILALAKSQHWVNYVPRWLDSLTLWLGYSNSMLNPIIYCKYNREFRVPFGEMLCCRFRTI QDVMRNESYYAKFGSPRISETKIGGSQQRSSGNNYYTKRKRNNTERRAPQNGFLDSSKT KKNYGIKRNSSPSILMESSK (SEQ. ID NO:16). Method according to any one of claims 1-6, wherein said GPCR receptors of non-parasitic nematodes capable of localizing in the membrane are selected from the group consisting of C.elegans, C. briggsae, C. japonica, C. brenneri, C . remanei, bet Zeldia. 8. Method according to claim 7, wherein said C. elegans GPCR receptors are selected from the group consisting of SER-1 (Gene Bank: F59C12.2), SER-2 (C02D4.2), SER- 3 (K02F2.6), SER-4 (Y22D7AR.13), SER-5 (F16D3.7), SER-6 (Y54G2A.35), SER-7 (C09B7.1); dopamine receptors DOP-1 (F15A8.5), DOP-2 (K09G1.4), DOP-3 (T14E8.3), DOP-4 (C52B11.3); NPR-1 (C39E6.6), NPR-2 (T05A1.1), NPR-3 (C10C6.2) neuropeptide receptors; putative NPR1-like neuropeptide receptors (C16D6.2, C25G6.5, F41E7.3); GAR-1 (C15B12.5), GAR-2 (F47D12.1), GAR-3 (Y40H4A.1) galanin receptors; metabotropic glutamate receptors MGL-1 (ZC506.4), MGL-2 (F45H11.4); or putative receptors of biogenic amines (F01E11.5, C24A8.1, T02E9.3). Method according to claim 8, wherein the carboxyterminal domain of the GPCR receptors of C.elegans capable of localizing in the membrane has an amino acid sequence selected from the group consisting of the sequences SEQ ID NO: 17-SEQ ID NO: 41 shown in following Table: NAME NUMBER AMINO ACID SEQUENCE CARBOXY TERMINAL ACCESS RECEPTOR C.elegans GENE BANK SER-1 F59C12. TVFNKRFRQAFVRILRCQCFHPLRDSHQMYSR 2 NFTTTIVPDTYTCSRSNQERTTSVITRDETRS ARSSERPEPSRARSEISEEPVARTNGKLTSEK KKISLPSFPRVSSSRDSRATTEASTTDEETKP LIPKSTVPATVINIPEQLINPIKKSLTTIINM PLLDETIPEKAQVHHKSQTLLTSSTLNFATFS TCPQQPTRSYSCVDCKKAEKMLSSDVSDMMTT STASTASTVNGAPRKHLTLFNHFDSAIKETFL (SEQ ID NO:17) SER-2 C02D4.2 GILNLEFRRAFKKILCPKAVLEQRRRRMSAQP (SEQ ID NO:18) SER-3 K02F2.6 TVFNREFRICFKRLLTCHHLNHPTQKYTNNNS YNSTAIRSTNAVNRVPQTSLGNYTQTQNSEKS SAAVTFNTPTN (SEQ ID NO:19) SER-4 Y22D7AR TVFSQDFRAAFKRIIKRMCLIHDY .13 (SEQ ID NO: 20) SER-5 F16D3.7 SFTVKEFKRSAFRLVVPIWQFVNRCLPFVPAP PDNILQRIARHVHRHKEKEMQTRHRSFEMSSN KNGMLTTKVRSKRRQTE54 AAFSRDFRIALKRLFFQKPKF 35 (SEQ ID NO:22) SER-7 C09B7.1 YCKYNKEFRIPFREMLACRCATLQTVMRQQSF TSRYGPPVRYRTQSSSYRPLLSRRNDSHEASD V (SEQ ID NO:23) DOP-1 F15A8.5 SIFNRDFRRAFKKIIVRVFGCCWEEPDLNKSI SSRYAAPDNIERRRSCTRSSESAHDNNNDANA TRLNLLSNNNEETIPE (SEQ ID NO:24) DOP-2 K09G1.4 TIFNTEFRRAFKSIIFGRNSTRHHFSNKQAHV (SEQ ID NO: 25) DOP-3 T14E8.3 TVFDQRFRNAFRNILSCGIFKKR (SEQ ID NO: 26) DOP-4 C52B11. SRFSRDFRRAFKQILTCQRQQKVKTAFKTPLS 3 LVFTQLISVTQMWEQPPNTSIE (SEQ ID NO: 27) GAR-1 C15B12. ALANRQFRSAFMRMFRGNFNKVA 5 (SEQ ID NO: 28) GAR-2 F47D12. AMANQQFKKTLTRIFKGDFRRV 1 (SEQ ID NO: 29) GAR-3 Y40H4A. ALCNARFRHTYMRILRCKFKAERPTMNQGYVR 1 RN (SEQ ID NO:30) NPR-1 C39E6.6 AWLNPSFRQLVIKTYFGDRRKSDRIINQTSVY KTKIVHDTKHLNGRAKIGGGGSHEALKERELN SCSENLSYHVNGHTRTPTPEVQLNEVSSPEIS KLVAEPEELIEFSVNDTLV (SEQ ID NO:31) NPR-2 T05A1.1 AWLNPMFKEMLIKTLRGGSKSPKPADIKQTSF IRMPNSGAPSQSSYL (SEQ ID NO:32) NPR-3 C10C6.2 SWFNPQFRQSITTLFKGTDEARLIKKKPQSTS KMVSYPTNFSEIRKETEIASTKTKITIAENDY RAGDQLL (SEQ ID NO:33) NPR-like C16D6.2 AVLNLQLRAAFIDLMPHWLRRHLNLEGDNSSP LLNHPTMTITNKYGSTATKTVKATYINTSNGQ PYVSTSLVGKVQPEAPSFKFNGSGRKKSAMMR ILVQKRNAEEEEQLITKESPSPPEIQMDTLCA ASIIPRRKSAQPRSTNEKVVLPRKASF (SEQ ID NO:34) Recettore C25G6.5 AFFNHNFRIEFMHLFDRVGLRSLRVVIFGEQE putativo SLKKSMRTEFRSRGGCKTVTTAEPATFQRMNE neuropeptide SMILSAMEQDEQLSSGGKLFVLKKYILKMFQK GGHKQSTPASPRLGFGYNSIMTSELFSIVEGV LS (SEQ ID NO:35) Recettore F41E7.3 AFMNETFREEFAKVVPCLFARRPGTGPIRVIT putativo ERTAMITNPFRRANRKKKVEEQPVTVISESPL neuropeptide QTAVEPQRSIVYLDEPENGSSCQTLLL (SEQ ID NO: 36) MGL-1 ZC506.4 RKQEGESMLNKSSRSLGNCSSRLCANSIDEPN QYTALLTDSTRRRSSRKTSQHDSA NO: 37) MGL-2 F45H11. IRASYTTTKLIRCHFGNSQAAYDSTSKQQHLG 4 SKTTARTSVQSGSASKSSSMGGGVTRTASVHV PVSRGSTHSTDVSTQTEAASKFSRSFSIVGRK KQGLDDDVQQLVDACRRYQDEKINSSAANLLL EESEDEVGALLADSIENSMRTVLSTVAGKAVV PLVPMVPMIPVVTLPTAPSQEDNFEQLLRSRG VQPLALSQATPL (SEQ ID NO:38) Recettore F01E11. TVFNRDYQIALKRLFTSEKKPSSTSRV putativo 5 (SEQ ID NO:39) tiramina Recettore C24A8.1 STINPVSIFISNLISKKIIFLIFQVQFALYKT omologo LIHGIETRNSVIHSAAYLASVDLLDVREKNRG dopamina CYRHEIAAEVLQNVDAVSIKS (SEQ ID NO:40) Recettore T02E9.3 MCLNKNFRNTMRKMMQKTNRAKMAEQEG omologo (SEQ ID NO:41) melatonina 10. Metodo secondo ognuna delle rivendicazioni 4- 9, in cui detto recettore GPCR chimerico della fase a) ha una sequenza amminoacidica scelta tra: i)MLENDLENLDDSSLILHHLGGSFVHAQIVEIEENDYINSNTSVSNTNLNLFSSSSSTS SSSLFSSSSQRSPLLEPNFVDEIWPATSSNSLFYSHPIIAILISILIFLLILITIIGNLG VCAAILLVRKLKAQPANLLLISLALADFCVGLFVMPLAAVYVLEDRWIFGDILCRFWVTA DLTLCTASILNLCAISVDRHLAVTRALRYSALRTRRRICLYICIVWLGALLVSAAPLALL PFPKIEQYCQVSQNRVYQIYATIIAFWGPSLIMVIVYVKLWSAAKRMHRQDQLVLRWQGV HLPSDGDLEDGLPPTTTTSNATKSLFRNDSIARANARFLFALRMSAQKFGYYDHQNNKTN GGEIINDDENLRIEVRTSASSSGGGGGGSSTLGTLRVPIVLIIMSAFIICWVPFFILALA KSQHWVNYVPRWLDSLTLWLGYSNSMLNPIIYCKYNKEFRIPFREMLACRCATLQTVMRQ QSFTSRYGPPVSRRNDSHEASDV (SEQ ID NO:43); ii)MEASTMELLPPPTILFNVSSAPSVICEDMNAYLWNTRRDLTTCPFVMAVFASLYSSI ILLGIIGNSCVIMAIARIKSLQTVPNMFIFSLSCSDILVCFISATITPIAAFKKDWIFGQ FLCSFAPFVAGGSLCFSTFTLAAISVDRFLLILFPTRKAFSHTQALFIILVTFLLASGFS LPMLFMQKLKPVTHYCGRFCFEDWGEMIGIRRIYGTLLLTVQFIIPLALITFCYTAISFR LGKSVNLRTRKKCEWQMPISAQRNGATKRRQRTNRMFIAMVIAFSVSWIWSVLYNLLRDY DGLPKFVHDQEFFFGILTHCIAMSSTVWNPILYALLYCKYNKEFRIPFREMLACRCATLQ TVMRQQSFTSRYGPPVSRRNDSHEASDV (SEQ ID NO:46); iii)MLPWWLPLLIFVPIFSVLISLAFSGNLLVIAAIFYDRKLRRQPENLFLVSLALSDL FVAGMVMVLAAANDLIGFWPFGAAFCQFWICLDIACSTASILNLCAIALDRFIHISRPMR YVRFVGRRVICCSVCAIWIISTAVGTAQIAFGATNGLEIYSVIFNGDYFGEINETDKFRF IEQQHTLVQCQLQLSPFYAVFSSLLSFFLPATLMLFLYYRLYLYARHHARSIQSQLKQAT SLLILQLASDRVRQVVVRPSSGFLSPSSIALHYDSRRFSGKSSFDGESLQKAEFSQINVA VNKSPENHSIDATIQQGTPVLRATLRQLRRTESNNSSSFSFVSRNGLGVNSRRVGCGGGN GGERRKSCSPLPSPTTKRGFKSCGWSNNNNGNGGHRSSDKKARVTLGVIMGTFLICWLPF FIVNVLRPFWPEIFPPLLFQTVSWLGYANSSVNPVIYGIFYCKYNKEFRIPFREMLACRC ATLQTVMRQQSFTSRYGPPVSRRNDSHEASDV (SEQ ID NO:47); iv)MEASTMELLPPPTILFNVSSAPSVICEDMNAYLWNTRRDLTTCPFVMAVFASLYSSI ILLGIIGNSCVIMAIARIKSLQTVPNMFIFSLSCSDILVCFISATITPIAAFKKDWIFGQ FLCSFAPFVAGGSLCFSTFTLAAISVDRFLLILFPTRKAFSHTQALFIILVTFLLASGFS LPMLFMQKLKPVTHYCGRFCFEDWGEMIGIRRIYGTLLLTVQFIIPLALITFCYTAISFR LGKSVNLRTRKKCEWQMPISAQRNGATKRRQRTNRMFIAMVIAFSVSWIWSVLYNLLRDY DGLPKFVHDQEFFFGILTHCIAMSSTVWNPILYALLAVLNLQLRAAFIDLMPHWLRRHLN LEGDNSSPLLNHPTMTITNKYGSTATKTVKATYINTSNGQPYVSTSLVGKVQPEAPSFKF NGSGRKKSAMMRILVQKRNAEEEEQLITKESPSPPEIQMDTLCAASIIPRRKSAQPRSTN EKVVLPRKASF (SEQ ID NO:48); v)MLPWWLPLLIFVPIFSVLISLAFSGNLLVIAAIFYDRKLRRQPENLFLVSLALSDLFV AGMVMVLAAANDLIGFWPFGAAFCQFWICLDIACSTASILNLCAIALDRFIHISRPMRYV RFVGRRVICCSVCAIWIISTAVGTAQIAFGATNGLEIYSVIFNGDYFGEINETDKFRFIE QQHTLVQCQLQLSPFYAVFSSLLSFFLPATLMLFLYYRLYLYARHHARSIQSQLKQATSL LILQLASDRVRQVVVRPSSGFLSPSSIALHYDSRRFSGKSSFDGESLQKAEFSQINVAVN KSPENHSIDATIQQGTPVLRATLRQLRRTESNNSSSFSFVSRNGLGVNSRRVGCGGGNGG ERRKSCSPLPSPTTKRGFKSCGWSNNNNGNGGHRSSDKKARVTLGVIMGTFLICWLPFFI VNVLRPFWPEIFPPLLFQTVSWLGYANSSVNPVIYGIFSIFNRDFRRAFKKIIVRVFG CCWEEPDLNKSISSRYAAPDNIERRRSCTRSSESAHDNNNDANATRLNLLSNNNE ETIPE (SEQ ID NO:49). 11. Method according to any one of the preceding claims, in which said intracellular signal of step c) is selected from variation in the levels of cAMP, Ca <2+>, IP3, cGMP, kinase activity. 12. Method for assisting the expression in a heterologous cellular system of GPCR receptors that have difficulty in localizing correctly on the cell plasma membrane, comprising the following steps: a) creation of the chimeric GPCR receptor gene by replacing the sequence corresponding to the carboxy terminal domain of said GPCR receptor with that of non-parasitic nematode GPCR receptor of plants capable of localizing in the membrane; b) transfection of the plasmid containing the chimeric gene into heterologous cell systems; c) expression of the chimeric GPCR receptor in cells. The method according to claim 12, wherein said GPCR receptors are selected from the group consisting of serotonin receptors, dopamine receptors, neuropeptide receptors, galanin receptors, glutamate receptors and biogenic amine receptors. The method according to claim 13 wherein said GPCR receptors are plant parasitic nematode GPCRs. Method according to claim 13, wherein said plant parasitic nematodes are selected from the group consisting of Meloidogyne, Heterodera and Globodera. 16. Method according to claim 15 wherein when said plant parasitic nematode is Meloidogyne incognita, said GPCR receptor is selected from the group consisting of the amino acid sequences shown in the following Table: AMINO ACID SEQUENCE TYPE M.incognita RECEPTOR SerR1-like <MLENDLENLDRSSLSSLHHLGSSLSSFSSLSSLSSL FYSHPIIAILISILIFLLILITIIGNLGVCAAILLVRKLKAQPAN LLLISLALADFCVGLFVMPLAAVYVLEDRWIFGDILCRFWVTADL TLCTASILNLCAISVDRHLAVTRALRYSALRTRRRICLYICIVWL GALLVSAAPLALLPFPKIEQYCQVSQNRVYQIYATIIAFWGPSLI MVIVYVKLWSAAKRMHRQDQLVLRWQGVHLPSDGDLEDGLPPTTT TSNATKSLFRNDSIARANARFLFALRMSAQKFGYYDHQNNKTNGG EIINDDENLRIEVRTSASSSGGGGGGSSTLGTLRVPIVLIIMSAF IICWVPFFILALAKSQHWVNYVPRWLDSLTLWLGYSNSMLNPIIY CKYNREFRVPFGEMLCCRFRTIQDVMRNESYYAKFGSPRISETKI GGSQQRSSGNNYYTKRKRNNTERRAPQNGFLDSSKTKKNYGIKRN SSPSILMESSK (SEQ ID NO:2) DopR1-like<MLPWWLPLLIFVPIFSVLISLAFSGNLLVIAAIFYDRKLRRQPEN>LFLVSLALSDLFVAGMVMVLAAANDLIGFWPFGAAFCQFWICLDI ACSTASILNLCAIALDRFIHISRP MRYVRFVGRRVICCSVCAIWI ISTAVGTAQIAFGATNGLEIYSVIFNGDYFGEINETDKFRFIEQQ HTLVQCQLQLSPFYAVFSSLLSFFLPATLMLFLYYRLYLYARHHA RSIQSQLKQATSLLILQLASDRVRQVVVRPSSGFLSPSSIALHYD SRRFSGKSSFDGESLQKAEFSQINVAVNKSPENHSIDATIQQGTP VLRATLRQLRRTESNNSSSFSFVSRNGLGVNSRRVGCGGGNGGER RKSCSPLPSPTTKRGFKSCGWSNNNNGNGGHRSSDKKARVTLGVI MGTFLICWLPFFIVNVLRPFWPEIFPPLLFQTVSWLGYANSSVNP VIYGIFNRDFRRAFSRIMNKLIHCIDEERRGENSRGGTFRTRTES ESFNK FNKSLNNASELQQQQKLIFKQKKTNKKLRFLCF (SEQ ID NO:4) Rho1-like MNITVPTTTTSLIINSISPFPPTFSPSISTQQNNFTTSSLTKTTF NFQQKRDVSFHFSLIFGSLIGGVTIVGLTANILVVIAILSDRKMR KSPMNLLLLNLAIADLLYLLAFTPFWLSMSVYGDGGWHFGDMFCP IARFFGNIFLVISIL TYLAICIERYVAIVHPIAMHTSVWCTRSRVLVIAFGIWVFAMTYQ FPYLV VFQVFDIPEKNLRVCRNPLASKSKIWKIYKWSEFLLTYALPIIIS VLLYSRICRVLWFKNKNGENCGKGQNENENKTELKPNINKRPSGR TIKKVLETRFREAISDVQNKRKVVPKEEEIFNFNNSSEKRQKPMN VSSSANLRARRSVVKMLMLCVGLFFLCYTPMVAYFVWSALFGRPL PLPFEFVLITSALVQFQSAFNPFLYTLFATQFREKLSKLFLICNK KQKNKIINQHKISAKSTSTFSV (SEQ ID NO:6) Rho2-like MTTANNQGEVDCNQGLVEPLLDRPIFKIPFTFAYVAVFLLCLTGN LFTIVVICAHRSMRTAT NFFLANLALADLLVAIFCIMQNMLHLVH LDAQWPLGETLCRMYALILHLVPCTGIGILVCVSVEKYIAVLHPL LALKLLTPKFRSLMMAAVWICSLLANLPYYTTSKYREWEGGNSAC FRGLLTDGFVSTRNMLIISFLVWYCIPLCIIAFLYTRIGFVLWHS APLKKLTQMRTNSNETTTMTGIQKRSVASCRLSAITNNNGSATIV SEVTIKTPSKRSNSQPNNYSKITNNNLPPLILVNKSSPLPNTASE SEGEEEDFGREEEGREEEEDKDEEEEEEESNGSHLEEDLDDDEEE EDAEHENGTKCRLALGNGSVGPAHLGQRNSAPSSSTVFVSTVESR KRIIRLLIAIVCSFAVLTLPNHIRLLYSAVYSEERICLGGFEVFT QPVTYLLLFLSSSCNPFLYAFMSQRFRSAIRDIFKCRRGHIRRKS TKTRTQNSDVLCAVNSGDGVALSDCSSLNRLNNNGNNNLLDQSKG EEINSIHGEKNFPWANQKGEEINWLKVKKLKNTERNASGGSGNST TTASSVLYTTQHSNGSLQMAVVYRKCAC (SEQ ID NO:8) Rho3-like MNDSETIENYEDFLSQLSSTMIPTTTTTSSSSSAIFTSTLATPLI SETAQIPSPTPKLNINIVAYCYIMPTICVLGIIGNSMNVVTLASP RLKAVSYMYLRALAVSDLLCMLFVLAFACCEVLKESGVPIERHPL YGFYQAHVMLSFINWALATGVYIVVALSLERYVSVVFPLHFRMWN SPKRAMKAIIIAYTVPALFYIPYGIGRYSVSEKINSRGEISYGAI DSEISKTFGWQVYKWTREAFLRFLPIVILFVLNFQIMIAFRRRQK MFDRLRNRETAARDDTLLYILGGIAVMFFVCNIPAAINLLFINEV VKKRPDYQIFRAAANLLEITNHAAQFYIFCVCSSDYRVTFMQKFP CLRAYYVNKSKFCSFLRNASQLPKRSVARRTISTTTNSKIGNNV N NVCGTMRRSVSASNTPLNAKHGDKNDRRNGKSKKGGGGGGSAIAE MTFVRSTTITGELLSTCGGHNTEQETIDAQIASLESLSDESETLL KQQKIREESWGSPITTKLCLIGLDGDEEEGIICRRSSNGEGIIAE DEQTDRTDGTSYL (SEQ ID NO:10) Rho4-like MDYAPMPISSNITTWFLLAVFIFVIHRNPSFHSQTNYFLASLAIS DLLLILVGVPFDVLFLWKSRFITSPFNGFCEITSTFISWFTFNSI LTIVSLTWERLVAICYPFSLKPFFHRDAVIWLIIIIWFISFFPSL FIGLQFKLVIQDFCGHTHVNSNGLGSKCDFVGWSGNYGSGENYTF EVMLFFTFVLPVLFVIYCYIRILRTLNEATFNSFHLNAGSSIVRK HTSSSHTNSTLLNAITEENLPSTSNNSNSNTAGNNSDLNARDSFT RSSLRSQRAHKTVMKMLIMIAALFFVCYLPYHLERLIVKYSAKGC TEPQMCLWLYHGTGLLQYISAALNPIIYNVMSRRFRREFKLLCYR IVKKENVTKTRSDNNNQLKMTPMNRFL (SEQ. ID NO:12) 17. Metodo secondo la rivendicazione 15, in cui quando detto nematode parassita delle piante à ̈ Meloidogyne hapla, detto recettore GPCR ha la seguente sequenza amminoacidica: MDYAPMPISSNITTWFLLAVFIFVIHRNPSFHSQTNYFLASLAISDLLLILVGVPFDVL FLWKSRFITSPFNGFCEITSTFISWFTFNSILTIVSLTWERLVAICYPFSLKPFFHRDA VIWLIIIIWFISFFPSLFIGLQFKLVIQDFCGHTHVNSNGLGSKCDFVGWSGNYGSGEN YTFEVMLFFTFVLPVLFVIYCYIRILRTLNEATFNSFHLNAGSSIVRKHTSSSHTNSTL LNAITEENLPSTSNNSNSNTAGNNSDLNARDSFTRSSLRSQRAHKTVMKMLIMIAALFF VCYLPYHLERLIVKYSAKGCTEPQMCLWLYHGTGLLQYISAALNPIIYNVMSRRFRREF KLLCYRIVKKENVTKTRSDNNNQLKMTPMNRFL (SEQ. ID NO:14) . 18. Metodo secondo la rivendicazione 15, in cui quando detto nematode parassita delle piante à ̈ Globodera rostochiensis, detto recettore GPCR ha la seguente sequenza amminoacidica: MLENDLENLDDSSLILHHLGGSFVHAQIVEIEENDYINSNTSVSNTNLNLFSSSSSTSS SSLFSSSSQRSPLLEPNFVDEIWPATSSNSLFYSHPIIAILISILIFLLILITIIGNLG VCAAILLVRKLKAQPANLLLISLALADFCVGLFVMPLAAVYVLEDRWIFGDILCRFWVT ADLTLCTASILNLCAISVDRHLAVTRALRYSALRTRRRICLYICIVWLGALLVSAAPLA LLPFPKIEQYCQVSQNRVYQIYATIIAFWGPSLIMVIVYVKLWSAAKRMHRQDQLVLRW QGVHLPSDGDLEDGLPPTTTTSNATKSLFRNDSIARANARFLFALRMSAQKFGYYDHQN NKTNGGEIINDDENLRIEVRTSASSSGGGGGGSSTLGTLRVPIVLIIMSAFIICWVPFF ILALAKSQHWVNYVPRWLDSLTLWLGYSNSMLNPIIYCKYNREFRVPFGEMLCCRFRTI QDVMRNESYYAKFGSPRISETKIGGSQQRSSGNNYYTKRKRNNTERRAPQNGFLDSSKT KKNYGIKRNSSPSILMESSK (SEQ. ID NO:16). 19. Method according to any one of claims 12-18, wherein said GPCR receptors of non-parasitic nematodes capable of localizing in the membrane are selected from the group consisting of C.elegans, C. briggsae, C. japonica, C. brenneri, C . remanei, bet Zeldia. Method according to claim 19, wherein said C.elegans GPCR receptors are selected from the group consisting of SER-1 (Gene Bank F59C12.2), SER-2 (C02D4.2), SER-3 serotonin receptors (K02F2.6), SER-4 (Y22D7AR.13), SER-5 (F16D3.7), SER-6 (Y54G2A.35), SER-7 (C09B7.1); dopamine receptors DOP-1 (F15A8.5), DOP-2 (K09G1.4), DOP-3 (T14E8.3), DOP-4 (C52B11.3); NPR-1 (C39E6.6), NPR-2 (T05A1.1), NPR-3 (C10C6.2) neuropeptide receptors; putative NPR1-like neuropeptide receptors (C16D6.2, C25G6.5, F41E7.3); GAR-1 (C15B12.5), GAR-2 (F47D12.1), GAR-3 (Y40H4A.1) galanin receptors; metabotropic glutamate receptors MGL-1 (ZC506.4), MGL-2 (F45H11.4); or putative receptors of biogenic amines (F01E11.5, C24A8.1, T02E9.3). 21. A method according to claim 20, wherein the carboxyterminal domain of the GPCR receptors of C.elegans capable of localizing in the membrane has an amino acid sequence selected from the group consisting of the sequences SEQ ID NO: 17-SEQ ID NO: 41 shown in seguente Tabella: NOME NUMERO SEQUENZA AMMINOACIDICA RECETTORE ACCESSO CARBOSSITERMINALE C.elegans GENE BANK SER-1 F59C12.2 TVFNKRFRQAFVRILRCQCFHPLRDSHQMYSRNF TTTIVPDTYTCSRSNQERTTSVITRDETRSARSS ERPEPSRARSEISEEPVARTNGKLTSEKKKISLP SFPRVSSSRDSRATTEASTTDEETKPLIPKSTVP ATVINIPEQLINPIKKSLTTIINMPLLDETIPEK AQVHHKSQTLLTSSTLNFATFSTCPQQPTRSYSC VDCKKAEKMLSSDVSDMMTTSTASTASTVNGAPR KHLTLFNHFDSAIKETFL (SEQ ID NO:17) SER-2 C02D4.2 GILNLEFRRAFKKILCPKAVLEQRRRRMSAQP (SEQ ID NO:18) SER-3 K02F2.6 TVFNREFRICFKRLLTCHHLNHPTQKYTNNNSYN STAIRSTNAVNRVPQTSLGNYTQTQNSEKSSAAV TFNTPTN (SEQ ID NO: 19) SER-4 Y22D7AR. TVFSQDFRAAFKRIIKRMCLIHDY 13 (SEQ ID NO:20) SER-5 F16D3.7 SFTVKEFKRSAFRLVVPIWQFVNRCLPFVPAPPD NILQRIARHVHRHKEKEMQTRHRSFEMSSNKNGM LTTKVRSKRRQTEPNVVGLITPDHKLQTVAS (SEQ ID NO:21) SER-6 Y54G2A.3 AAFSRDFRIALKRLFFQKPKF 5 (SEQ ID NO:22) SER-7 C09B7.1 YCKYNKEFRIPFREMLACRCATLQTVMRQQSFTS RYGPPVRYRTQSSSYRPLLSRRNDSHEASDV (SEQ ID No. -4 C52B11.3 SRFSRDFRRAFKQILTCQRQQKVKTAFKTPLSLV FTQLISVTQMWEQPPNTSIE (SEQ ID NO:27) GAR-1 C15B12.5 ALANRQFRSAFMRMFRGNFNKVA (SEQ ID NO:28) GAR-2 F47D12.1 AMANQQFKKTLTRIFKGDFRRV (SEQ ID NO:29) GAR-3 Y40H4A.1 ALCNARFRHTYMRILRCKFKAERPTMNQGYVRRN ( SEQ ID NO: 30) NPR-1 C39E6.6 AWLNPSFRQLVIKTYFGDRRKSDRIINQTSVYKT KIVHDTKHLNGRAKIGGGGSHEALKERELNSCSE NLSYHVNGHTRTPIKTPEVQLNEVSSPEISKLVAEP EELIEFSVQLNDTLV NPR-1 TSFIR MPNSGAPSQSSYL (SEQ ID NO:32) NPR-3 C10C6.2 SWFNPQFRQSITTLFKGTDEARLIKKKPQSTSKM VSYPTNFSEIRKETEIASTKTKITIAENDYRAGD QLL (SEQ ID NO:33) NPR-like C16D6.2 AVLNLQLRAAFIDLMPHWLRRHLNLEGDNSSPLL NHPTMTITNKYGSTATKTVKATYINTSNGQPYVS TSLVGKVQPEAPSFKFNGSGRKKSAMMRILVQKR NAEEEEQLITKESPSPPEIQMDTLCAASIIPRRK SAQPRSTNEKVVLPRKASF (SEQ ID NO:34) Recettore C25G6.5 AFFNHNFRIEFMHLFDRVGLRSLRVVIFGEQESL putativo KKSMRTEFRSRGGCKTVTTAEPATFQRMNESMIL neuropeptide SAMEQDEQLSSGGKLFVLKKYILKMFQKGGHKQS TPASPRLGFGYNSIMTSELFSIVEGVLS (SEQ ID NO:35) Recettore F41E7.3 AFMNETFREEFAKVVPCLFARRPGTGPIRVITER putativo TAMITNPFRRANRKKKVEEQPVTVISESPLQTAV neuropeptide EPQRSIVYLDEPENGSSCQTLLL (SEQ ID NO:36) MGL-1 ZC506.4 RKQEGESMLNKSSRSLGNCSSRLCANSIDEPNQY TALLTDSTRRRSSRKTSQPTSTSSAHDTFL (SEQ ID NO:37) MGL-2 F45H11.4 IRASYTTTKLIRCHFGNSQAAYDSTSKQQHLGSK TTARTSVQSGSASKSSSMGGGVTRTASVHVPVSR GSTHSTDVSTQTEAASKFSRSFSIVGRKKQGLDD DVQQLVDACRRYQDEKINSSAANLLLEESEDEVG ALLADSIENSMRTVLSTVAGKAVVPLVPMVPMIP VVTLPTAPSQEDNFEQLLRSRGVQPLALSQATPL (SEQ ID NO: 3 8) Recettore F01E11.5 TVFNRDYQIALKRLFTSEKKPSSTSRV putativo (SEQ ID NO:39) tiramina Recettore C24A8.1 STINPVSIFISNLISKKIIFLIFQVQFALYKTLI omologo HGIETRNSVIHSAAYLASVDLLDVREKNRGCYRH dopamina EIAAEVLQNVDAVSIKS(SEQ ID NO:40) Recettore T02E9.3 MCLNKNFRNTMRKMMQKTNRAKMAEQEG omologo (SEQ ID NO:41) melatonina 22. Recettore GPCR chimeric obtainable with the method defined according to each of the claims 12-21. 23. Chimeric GPCR receptor characterized by comprising the amino acid sequence of a plant parasitic nematode GPCR receptor which has difficulty in localizing correctly on the cell plasma membrane, in which the carboxy terminal domain of said sequence is replaced with that of a GPCR receptor of non-parasitic nematodes able to localize in the membrane. 24. Chimeric GPCR receptor according to claim 23, wherein said plant parasitic nematode is selected from the group consisting of Meloidogyne, Heterodera and Globodera. 25. Recettore GPCR chimerico secondo ognuna delle rivendicazioni 23-24, in cui detto recettore GPCR à ̈ scelto dal gruppo che consiste nelle seguenti sequenze amminoacidiche: Meloidogyne SEQUENZA AMMINOACIDICA incognita SerR1-like MLENDLENLDDSSLILHHLGGSFVHAQIVEIEENDYINSNTS VSNTNLNLFSSSSSTSSSSLFSSSSQRSPLLEPNFVDEIWPA TSSNSLFYSHPIIAILISILIFLLILITIIGNLGVCAAILLV RKLKAQPANLLLISLALADFCVGLFVMPLAAVYVLEDRWIFG DILCRFWVTADLTLCTASILNLCAISVDRHLAVTRALRYSAL RTRRRICLYICIVWLGALLVSAAPLALLPFPKIEQYCQVSQN RVYQIYATIIAFWGPSLIMVIVYVKLWSAAKRMHRQDQLVLR WQGVHLPSDGDLEDGLPPTTTTSNATKSLFRNDSIARANARF LFALRMSAQKFGYYDHQNNKTNGGEIINDDENLRIEVRTSAS SSGGGGGGSSTLGTLRVPIVLIIMSAFIICWVPFFILALAKS QHWVNYVPRWLDSLTLWLGYSNSMLNPIIYCKYNREFRVPFG EMLCCRFRTIQDVMRNESYYAKFGSPRISETKIGGSQQRSSG NNYYTKRKRNNTERRAPQNGFLDSSKTKKNYGIKRNSSPSIL MESSK (SEQ ID NO: 2) DopR1-like MLPWWLPLLIFVPIFSVLISLAFSGNLLVIAAIFYDRKLRRQ PENLFLVSLALSDLFVAGMVMVLAAANDLIGFWPFGAAFCQF WICLDIACSTASILNLCAIALDRYRFIHISR FIEQQHTLVQCQLQLSPFYAVFSSLLSFFLPATLM LFLYYRLYLYARHHARSIQSQLKQATSLLILQLASDRVRQVV VRPSSGFLSPSSIALHYDSRRFSGKSSFDGESLQKAEFSQIN VAVNKSPENHSIDATIQQGTPVLRATLRQLRRTESNNSSSFS FVSRNGLGVNSRRVGCGGGNGGERRKSCSPLPSPTTKRGFKS CGWSNNNNGNGGHRSSDKKARVTLGVIMGTFLICWLPFFIVN VLRPFWPEIFPPLLFQTVSWLGYANSSVNPVIYGIFNRDFRR AFSRIMNKLIHCIDEERRGENSRGGTFRTRTESESFNK FNKSLNNASELQQQQKLIFKQKKTNKKLRFLCF (SEQ ID NO:4) Rho1-like MNITVPTTTTSLIINSISPFPPTFSPSISTQQNNFTTSSLTK TTFNFQQKRDVSFHFSLIFGSLIGGVTIVGLTANILVVIAIL SDRKMRKSPMNLLLLNLAIADLLYLLAFTPFWLSMSVYGDGG WHFGDMFCPIARFFGNIFLVISILTYLAICIERYVAIVHPIA MHTSVWCTRSRVLVIAFGIWVFAMTYQFPYLVVFQVFDIPEK NLRVCRNPLASKSKIWKIYKWSEFLLTYALPIIISVLLYSRI CRVLWFKNKNGENCGKGQNENENKTELKPNINKRPSGRTIKK VLETRFREAISDVQNKRKVVPKEEEIFNFNNSSEKRQKPMNV SSSANLRARRSVVKMLMLCVGLFFLCYTPMVAYFVWSALFGR PLPLPFEFVLITSALVQFQSAFNPFLYTLFATQFREKLSKLF LICNKKQKNKIINQHKISAKSTSTFSV (SEQ ID NO:6) Rho2-like MTTANNQGEVDCNQGLVEPLLDRPIFKIPFTFAYVAVFLLCL TGNLFTIVVICAHRSMRTATNFFLANLALADLLVAIFCIMQN MLHLVHLDAQWPLGETLCRMYALILHLVPCTGIGILVCVSV E KYIAVLHPLLALKLLTPKFRSLMMAAVWICSLLANLPYYTTS KYREWEGGNSACFRGLLTDGFVSTRNMLIISFLVWYCIPLCI IAFLYTRIGFVLWHSAPLKKLTQMRTNSNETTTMTGIQKRSV ASCRLSAITNNNGSATIVSEVTIKTPSKRSNSQPNNYSKITN NNLPPLILVNKSSPLPNTASESEGEEEDFGREEEGREEEEDK DEEEEEEESNGSHLEEDLDDDEEEEDAEHENGTKCRLALGNG SVGPAHLGQRNSAPSSSTVFVSTVESRKRIIRLLIAIVCSFA VLTLPNHIRLLYSAVYSEERICLGGFEVFTQPVTYLLLFLSS SCNPFLYAFMSQRFRSAIRDIFKCRRGHIRRKSTKTRTQNSD VLCAVNSGDGVALSDCSSLNRLNNNGNNNLLDQSKGEEINSI HGEKNFPWANQKGEEINWLKVKKLKNTERNASGGSGNSTTTA SSVLYTTQHSNGSLQMAVVYRKCAC(SEQ ID NO:8) Rho3-like MNDSETIENYEDFLSQLSSTMIPTTTTTSSSSSAIFTSTLAT PLISETAQIPSPTPKLNINIVAYCYIMPTICVLGIIGNSMNV VTLASPRLKAVSYMYLRALAVSDLLCMLFVLAFACCEVLKES GVPIERHPLYGFYQAHVMLSFINWALATGVYIVVALSLERYV SVVFPLHFRMWNSPKRAMKAIIIAYTVPALFYIPYGIGRYSV SEKINSRGEISYGAIDSEISKTFGWQVYKWTREAFLRFLPIV ILFVLNFQIMIAFRRRQKMFDRLRNRETAARDDTLLYILGGI AVMFFVCNIPAAINLLFINEVVKKRPDYQIFRAAANLLEITN HAAQFYIFCVCSSDYRVTFMQKFPCLRAYYVNKSKFCSFLRN ASQLPKRSVARRTISTTTNSKIGNNVNNVCGTMRRSVSASNT PLNAKHGDKNDRRNGKSKKGGGGGGSAIAEMTFVRSTTITGE LLS TCGGHNTEQETIDAQIASLESLSDESETLLKQQKIREES WGSPITTKLCLIGLDGDEEEGIICRRSSNGEGIIAEDEQTDR TDGTSYL (SEQ ID NO:10) Rho4-like MDYAPMPISSNITTWFLLAVFIFVIHRNPSFHSQTNYFLASL AISDLLLILVGVPFDVLFLWKSRFITSPFNGFCEITSTFISW FTFNSILTIVSLTWERLVAICYPFSLKPFFHRDAVIWLIIII WFISFFPSLFIGLQFKLVIQDFCGHTHVNSNGLGSKCDFVGW SGNYGSGENYTFEVMLFFTFVLPVLFVIYCYIRILRTLNEAT FNSFHLNAGSSIVRKHTSSSHTNSTLLNAITEENLPSTSNNS NSNTAGNNSDLNARDSFTRSSLRSQRAHKTVMKMLIMIAALF FVCYLPYHLERLIVKYSAKGCTEPQMCLWLYHGTGLLQYISA ALNPIIYNVMSRRFRREFKLLCYRIVKKENVTKTRSDNNNQL KMTPMNRFL (SEQ. ID NO:12) Meloidogyne MDYAPMPISSNITTWFLLAVFIFVIHRNPSFHSQTNYFLASL hapla AISDLLLILVGVPFDVLFLWKSRFITSPFNGFCEITSTFISW FTFNSILTIVSLTWERLVAICYPFSLKPFFHRDAVIWLIIII WFISFFPSLFIGLQFKLVIQDFCGHTHVNSNGLGSKCDFVGW SGNYGSGENYTFEVMLFFTFVLPVLFVIYCYIRILRTLNEAT FNSFHLNAGSSIVRKHTSSSHTNSTLLNAITEENLPSTSNNS NSNTAGNNSDLNARDSFTRSSLRSQRAHKTVMKMLIMIAALF FVCYLPYHLERLIVKYSAKGCTEPQMCLWLYHGTGLLQYISA ALNPIIYNVMSRRFRREFKLLCYRIVKKENVTKTRSDNNNQL KMTPMNRFL (SEQ. ID NO:14) Globodera MLENDLENLDDSSLILHHLGGSFVHAQIVEIEENDYINSNTS rostochiensis VSNTNLNLFSSSSSTSSSSLFSSSSQRSPLLEPNFVDEIWPA TSSNSLFYSHPIIAILISILIFLLILITIIGNLGVCAAILLV RKLKAQPANLLLISLALADFCVGLFVMPLAAVYVLEDRWIFG DILCRFWVTADLTLCTASILNLCAISVDRHLAVTRALRYSAL RTRRRICLYICIVWLGALLVSAAPLALLPFPKIEQYCQVSQN RVYQIYATIIAFWGPSLIMVIVYVKLWSAAKRMHRQDQLVLR WQGVHLPSDGDLEDGLPPTTTTSNATKSLFRNDSIARANARF LFALRMSAQKFGYYDHQNNKTNGGEIINDDENLRIEVRTSAS SSGGGGGGSSTLGTLRVPIVLIIMSAFIICWVPFFILALAKS QHWVNYVPRWLDSLTLWLGYSNSMLNPIIYCKYNREFRVPFG EMLCCRFRTIQDVMRNESYYAKFGSPRISETKIGGSQQRSSG NNYYTKRKRNNTERRAPQN GFLDSSKTKKNYGIKRNSSPSIL MESSK (SEQ. ID NO: 16) 26. Chimeric GPCR receptor according to any one of claims 23-25, wherein said GPCR receptors of non-parasitic nematodes capable of localizing in the membrane are selected from the group consisting of C.elegans, C. briggsae, C. japonica, C. brenneri, C. remanei, Zeldia puntata. 27. Chimeric GPCR receptor according to claim 26, wherein the carboxyterminal domain of the membrane-localizing C.elegans GPCR receptors has an amino acid sequence selected from the group consisting of the sequences SEQ ID NO: 17-SEQ ID NO: 41 shown in the following Table: NAME NUMBER AMINO ACID SEQUENCE CARBOXY TERMINAL ACCESS RECEPTOR C.elegans GENE BANK SER-1 F59C12. TVFNKRFRQAFVRILRCQCFHPLRDSHQMYSR 2 NFTTTIVPDTYTCSRSNQERTTSVITRDETRS ARSSERPEPSRARSEISEEPVARTNGKLTSEK KKISLPSFPRVSSSRDSRATTEASTTDEETKP LIPKSTVPATVINIPEQLINPIKKSLTTIINM PLLDETIPEKAQVHHKSQTLLTSSTLNFATFS TCPQQPTRSYSCVDCKKAEKMLSSDVSDMMTT STASTASTVNGAPRKHLTLFNHFDSAIKETFL (SEQ ID NO:17) SER-2 C02D4.2 GILNLEFRRAFKKILCPKAVLEQRRRRMSAQP (SEQ ID NO:18) SER-3 K02F2.6 TVFNREFRICFKRLLTCHHLNHPTQKYTNNNS YNSTAIRSTNAVNRVPQTSLGNYTQTQNSEKS SAAVTFNTPTN (SEQ ID NO:19) SER-4 Y22D7AR TVFSQDFRAAFKRIIKRMCLIHDY .13 (SEQ ID NO: 20) SER-5 F16D3.7 SFTVKEFKRSAFRLVVPIWQFVNRCLPFVPAP PDNILQRIARHVHRHKEKEMQTRHRSFEMSSN KNGMLTTKVRSKRRQTE54 AAFSRDFRIALKRLFFQKPKF 35 (SEQ ID NO:22) SER-7 C09B7.1 YCKYNKEFRIPFREMLACRCATLQTVMRQQSF TSRYGPPVRYRTQSSSYRPLLSRRNDSHEASD V (SEQ ID NO:23) DOP-1 F15A8.5 SIFNRDFRRAFKKIIVRVFGCCWEEPDLNKSI SSRYAAPDNIERRRSCTRSSESAHDNNNDANA TRLNLLSNNNEETIPE (SEQ ID NO:24) DOP-2 K09G1.4 TIFNTEFRRAFKSIIFGRNSTRHHFSNKQAHV (SEQ ID NO: 25) DOP-3 T14E8.3 TVFDQRFRNAFRNILSCGIFKKR (SEQ ID NO: 26) DOP-4 C52B11. SRFSRDFRRAFKQILTCQRQQKVKTAFKTPLS 3 LVFTQLISVTQMWEQPPNTSIE (SEQ ID NO: 27) GAR-1 C15B12. ALANRQFRSAFMRMFRGNFNKVA 5 (SEQ ID NO: 28) GAR-2 F47D12. AMANQQFKKTLTRIFKGDFRRV 1 (SEQ ID NO: 29) GAR-3 Y40H4A. ALCNARFRHTYMRILRCKFKAERPTMNQGYVR 1 RN (SEQ ID NO:30) NPR-1 C39E6.6 AWLNPSFRQLVIKTYFGDRRKSDRIINQTSVY KTKIVHDTKHLNGRAKIGGGGSHEALKERELN SCSENLSYHVNGHTRTPTPEVQLNEVSSPEIS KLVAEPEELIEFSVNDTLV (SEQ ID NO:31) NPR-2 T05A1.1 AWLNPMFKEMLIKTLRGGSKSPKPADIKQTSF IRMPNSGAPSQSSYL (SEQ ID NO:32) NPR-3 C10C6.2 SWFNPQFRQSITTLFKGTDEARLIKKKPQSTS KMVSYPTNFSEIRKETEIASTKTKITIAENDY RAGDQLL (SEQ ID NO: 33) NPR-like C16D6. 2 AVLNLQLRAAFIDLMPHWLRRHLNLEGDNSSP LLNHPTMTITNKYGSTATKTVKATYINTSNGQ PYVSTSLVGKVQPEAPSFKFNGSGRKKSAMMR ILVQKRNAEEEEVQLITPRESPSPPEIQMDTLCA ASQL (SEQ ID NO: 34) C25G6.5 receptor AFFNHNFRIEFMHLFDRVGLRSLRVVIFGEQE putative SLKKSMRTEFRSRGGCKTVTTAEPATFQRMNE neuropeptide SMILSAMEQDEQLSSGGKLFVLKKYILKMFQK GGHKQSTPEGASPRLGFGNS (LSK) F41E7.3 receptor AFMNETFREEFAKVVPCLFARRPGTGPIRVIT putative ERTAMITNPFRRANRKKKVEEQPVTVISESPL neuropeptide QTAVEPQRSIVYLDEPENGSSCQTLLL (SEQ ID NO: 36) MGL-1 ZC506.4 RKQEGESMLNKSSRSLGNCSSRLCANSIDEPN QYTALLTDSTRRRSSRKTSQPTSTSSAHDTFL (SEQ ID NO: 37) MGL-2 F45H11. IRASYTTTKLIRCHFGNSQAAYDSTSKQQHLG 4 SKTTARTSVQSGSASKSSSMGGGVTRTASVHV PVSRGSTHSTDVSTQTEAASKFSRSFSIVGRK KQGLDDDVQQLVDACRRYQDEKINSSAANLLL EESEDEVGALLADSIENSMRTVLSTVAGKAVV PLVPMVPMIPVVTLPTAPSQEDNFEQLLRSRG VQPLALSQATPL (SEQ ID NO:38) Recettore F01E11. TVFNRDYQIALKRLFTSEKKPSSTSRV putative 5 (SEQ ID NO: 39) tyramine C24A8.1 receptor STINPVSIFISNLISKKIIFLIFQVQFALYKT homolog LIHGIETRNSVIHSAAYLASVDLLDVREKNRG dopamine CYRHEIAAEVLQNVDAVSIKS (SEQ ID NO: 40) T02E9 receptor. 3 MCLNKNFRNTMRKMMQKTNRAKMAEQEG homolog (SEQ ID NO: 41) melatonin 28. Chimeric GPCR receptor according to each of the claims 25-27, characterized by the fact of have an amino acid sequence chosen from the group that consists of: i)MLENDLENLDDSSLILHHLGGSFVHAQIVEIEENDYINSNTSVSNTNLNLFSSSSST SSSSLFSSSSQRSPLLEPNFVDEIWPATSSNSLFYSHPIIAILISILIFLLILITIIGN LGVCAAILLVRKLKAQPANLLLISLALADFCVGLFVMPLAAVYVLEDRWIFGDILCRFW VTADLTLCTASILNLCAISVDRHLAVTRALRYSALRTRRRICLYICIVWLGALLVSAAP LALLPFPKIEQYCQVSQNRVYQIYATIIAFWGPSLIMVIVYVKLWSAAKRMHRQDQLVL RWQGVHLPSDGDLEDGLPPTTTTSNATKSLFRNDSIARANARFLFALRMSAQKFGYYDH QNNKTNGGEIINDDENLRIEVRTSASSSGGGGGGSSTLGTLRVPIVLIIMSAFIICWVP FFILALAKSQHWVNYVPRWLDSLTLWLGYSNSMLNPIIYCKYNKEFRIPFREMLACRCA TLQTVMRQQSFTSRYGPPVSRRNDSHEASDV (SEQ ID NO:43); ii)MEASTMELLPPPTILFNVSSAPSVICEDMNAYLWNTRRDLTTCPFVMAVFASLYSS IILLGIIGNSCVIMAIARIKSLQTVPNMFIFSLSCSDILVCFISATITPIAAFKKDWIF GQFLCSFAPFVAGGSLCFSTFTLAAISVDRFLLILFPTRKAFSHTQALFIILVTFLLAS GFSLPMLFMQKLKPVTHYCGRFCFEDWGEMIGIRRIYGTLLLTVQFIIPLALITFCYTA ISFRLGKSVNLRTRKKCEWQMPISAQRNGATKRRQRTNRMFIAMVIAFSVSWIWSVLYN LLRDYDGLPKFVHDQEFFFGILTHCIAMSSTVWNPILYALLYCKYNKEFRIPFREMLAC RCATLQTVMRQQSFTSRYGPPVSRRNDSHEASDV (SEQ ID NO: 46); iii)MLPWWLPLLIFVPIFSVLISLAFSGNLLVIAAIFYDRKLRRQPENLFLVSLALSD LFVAGMVMVLAAANDLIGFWPFGAAFCQFWICLDIACSTASILNLCAIALDRFIHISRP MRYVRFVGRRVICCSVCAIWIISTAVGTAQIAFGATNGLEIYSVIFNGDYFGEINETDK FRFIEQQHTLVQCQLQLSPFYAVFSSLLSFFLPATLMLFLYYRLYLYARHHARSIQSQL KQATSLLILQLASDRVRQVVVRPSSGFLSPSSIALHYDSRRFSGKSSFDGESLQKAEFS QINVAVNKSPENHSIDATIQQGTPVLRATLRQLRRTESNNSSSFSFVSRNGLGVNSRRV GCGGGNGGERRKSCSPLPSPTTKRGFKSCGWSNNNNGNGGHRSSDKKARVTLGVIMGTF LICWLPFFIVNVLRPFWPEIFPPLLFQTVSWLGYANSSVNPVIYGIFYCKYNKEFRIPF REMLACRCATLQTVMRQQSFTSRYGPPVSRRNDSHEASDV (SEQ ID NO:47); iv)MEASTMELLPPPTILFNVSSAPSVICEDMNAYLWNTRRDLTTCPFVMAVFASLYSS IILLGIIGNSCVIMAIARIKSLQTVPNMFIFSLSCSDILVCFISATITPIAAFKKDWIF GQFLCSFAPFVAGGSLCFSTFTLAAISVDRFLLILFPTRKAFSHTQALFIILVTFLLAS GFSLPMLFMQKLKPVTHYCGRFCFEDWGEMIGIRRIYGTLLLTVQFIIPLALITFCYTA ISFRLGKSVNLRTRKKCEWQMPISAQRNGATKRRQRTNRMFIAMVIAFSVSWIWSVLYN LLRDYDGLPKFVHDQEFFFGILTHCIAMSSTVWNPILYALLAVLNLQLRAAFIDLMPHW LRRHLNLEGDNSSPLLNHPTMTITNKYGSTATKTVKATYINTSNGQPYVSTSLVGKVQP EAPSFKFNGSGRKKSAMMRILVQKRNAEEEEQLITKESPSPPEIQMDTLCAASIIPRRK SAQPRSTNEKVVLPRKASF (SEQ ID NO:48); v)MLPWWLPLLIFVPIFSVLISLAFSGNLLVIAAIFYDRKLRRQPENLFLVSLALSDLF VAGMVMVLAAANDLIGFWPFGAAFCQFWICLDIACSTASILNLCAIALDRFIHISRPMR YVRFVGRRVICCSVCAIWIISTAVGTAQIAFGATNGLEIYSVIFNGDYFGEINETDKFR FIEQQHTLVQCQLQLSPFYAVFSSLLSFFLPATLMLFLYYRLYLYARHHARSIQSQLKQ ATSLLILQLASDRVRQVVVRPSSGFLSPSSIALHYDSRRFSGKSSFDGESLQKAEFSQI NVAVNKSPENHSIDATIQQGTPVLRATLRQLRRTESNNSSSFSFVSRNGLGVNSRRVGC GGGNGGERRKSCSPLPSPTTKRGFKSCGWSNNNNGNGGHRSSDKKARVTLGVIMGTFLI CWLPFFIVNVLRPFWPEIFPPLLFQTVSWLGYANSSVNPVIYGIFSIFNRDFRRAFKK IIVRVFGCCWEEPDLNKSISSRYAAPDNIERRRSCTRSSESAHDNNNDANATRL NLLSNNNEETIPE (SEQ ID NO:49). 29. Use of chimeric GPCR receptors of plant parasitic nematodes as defined according to each of claims 22-28, as targets of potential pesticides specific for said nematodes. 30. Expression vector comprising a nucleotide sequence encoding a chimeric GPCR receptor as defined according to any one of claims 22-28. 31. Heterologous cell line transformed with the expression vector as defined in claim 30. 32. Use of a cell line as defined according to claim 31 in a method of screening potential pesticides specific for plant parasitic nematodes.