Bertsch, C.

Joly, D.

Walter, B.

English

JKI Open Journal Systems (Julius Kühn-Institut)

103Vitis 42 (2), 103–104 (2003)Research NoteSequence of a putative Vitis viniferaPR-1C. BERTSCH1), D. JOLY2) and B. WALTER1)1) Laboratoire de Biologie, Université de Haute-Alsace, Colmar,France2) INRA, UMR 1131, Vigne et Vin d’Alsace, Colmar, FranceK e y   w o r d s :  pathogenesis-related (PR) protein, gene,defense.Introduction: Incompatible host-pathogen interactionsinclude localized hypersensitive reaction (HR) and systemicresistances, e.g. systemic acquired resistance (SAR) andinduced systemic resistance (ISR). Amongst various meta-bolic alterations associated with HR and SAR, various pro-teins are induced which are collectively referred to as‘pathogenesis-related proteins’ (PRs). These proteins aredefined as coded for by the host plant but induced specifi-cally in pathological or related situations (VAN LOON andVAN STRIEN 1999). The association of PRs with SAR - but notwith ISR - suggests that accumulation of PRs contributes tothe induction of resistance but is not a prerequisite for it(VAN LOON 1997; VAN WEES et al. 1999). Fourteen families ofPRs have been proposed (VAN LOON and VAN STRIEN 1999).The only PR family for which no function is known consistsof the PR-1 proteins. Tobacco and tomato PR-1 proteinshave antifungal activity, but their mechanism of action isnot known.PR genes have been identified in Vitis species codingfor chitinases, glucanases, osmotins, a ribonuclease-likeprotein and a lipid-transfer protein (BUSAM et al. 1997;SALZMAN et al. 1998; JACOBS et al. 1999; ROBERT et al. 2001).In addition, peroxydases, glucanases, chitinases and PR-1like proteins have been serologically detected in grapevines(DELOIRE et al. 1997; REPKA et al. 2000, 2001). In the presentnote, we report cloning by RT-PCR of the open reading frameof a putative Vitis vinifera cv. Gewürztraminer PR-1.Material and Methods: Adult grapevines (Vitis viniferacv. Gewürztraminer) were maintained in a growth chamber at25 ± 2 °C, at 80 % RH and 16 h light. Leaves were sprayedwith salicylic acid 300 µM, pH 5.1 (Sigma Chemical) in DMSO(0.5 % final concentration). E x t r a c t i o n   a n d   a m p l i f i c a t i o n : Total RNAswere extracted 96 h after spraying from 50 mg of leaves ac-cording to a modified method from the RNeasy plant proto-col (Qiagen). Based on comparison of various plant PR-1protein sequences,  sense primer and antisense primer cor-responding to the consensus peptide motifs SPQDY andHYTQVVW were synthesized. cDNA synthesis and polymer-ase chain reaction (PCR) were performed using the Super-script One-Step RT-PCR kit with Platinium Taq polymerase(Invitrogene). cDNA synthesis was performed at 55 °C for30 min and PCR amplification was 35 cycles for 30 s at 94 °C,60 s at 57 °C and 90 s at 72 °C. 5’ and 3’ cDNA ends wereobtained by elongation with the 5’/3’ Race kit (Roche Mo-lecular Biochemicals). The different PCR products wereseparated by electrophoresis on a 1.2 % agarose gel. Theexpected band was excised and the amplification productsextracted and purified by using the nucleospin extract kit(Macherey-Nagel). The product was cloned in the TA clon-ing vector (Invitrogene) and double strand sequenced (Ge-nome express).S o u t h e r n   b l o t  a n a l y s i s :  Genomic DNA wasisolated from leaves of V. rupestris, V. vinifera cvs Chardon-nay, Gewürztraminer, Merlot, Muscat, Sauvignon, V. Ber-landieri x V. rupestris 110 R and V. riparia x V. rupestris3309 C. Samples of 10 µg were digested with the endonucle-ase Hind III. The restriction fragments were separated byelectrophoresis on a 1% agarose gel and blotted by down-ward capillary transfer to a nylon membrane. The blots werehybridized at 42 °C overnight with a digoxigenin-labeled PCRPR-1 fragment, washed subsequently under stringent con-ditions and developed with a chemiluminescence substrateaccording to the manufacturer’s recommendation (Roche).Results and Discussion: Like other PR-1 proteins, theprimary translation product (161 amino acids) deduced fromour nucleotide sequence (EMBL: AJ 536326) contains a hy-drophobic signal sequence (Table), which is cleaved offupon entry in the endoplasmic reticulum (VAN LOON andVAN STRIEN 1999). The mature protein is 137 amino acidslong  with a theoretical pI of 6.97 and a calculated molecularweight of 14 999.40 Da (Peptide Mass-Expasy) close to the15.5 kDa of gPR-1a. GPR-1a is one of the three grapevine PR-1 proteins detected by immunoblotting after fungal infec-tion of V. vinifera cv. Limberger (REPKA et al. 2000).Southern hybridization analysis shows several bandsin the different restriction digests, suggesting a variablegenomic organization for the PR-1 gene in the different spe-cies and cultivars that were analysed (Figure).Analysis of the amino acid sequence of our V. viniferaPR-1 protein reveals the presence of the structural elementshighly conserved in the PR-1 family: 4 α-helices andCorrespondence to: Prof. Dr. B. WALTER, Laboratoire de Biologie, Université de Haute-Alsace, 29 rue de Herrlisheim,  F-68008 Colmar,France. Fax: +33-3-8920-2357. E-mail: b.walter@uha.frFigure: Leaf DNA of cvs Sauvignon (1), Merlot (2), Chardonnay (3),Muscat (4), V. rupesris (5), 3309 C (6), 110 R (7) and Gewürz-traminer (8) was used for the restriction with the endonucleaseHindIII and the Southern hybridization. MW: DNA Molecularweight DIG-labeled (Roche).104 C. BERTSCH, D. JOLY and B. WALTER4 β-strands, as well as 6 conserved Cys residues were in-volved in the formation of disulphide bridges (Table). Theidentity to the type member, tobacco PR-1a, is 64 % andranges from 53 % to 68 % to different PR-1 proteins (Table).Identity to a partial sequence (99 aa) of a putative Vitisvinifera cv. Pinot noir PR-1 (CAA05868) is 81 %.Homologies and common structural motifs have shownto be between plant PR-1 proteins and proteins from fungi,invertebrate and vertebrate animals and humans suggest-ing a possible functional link between the human immunesystem and the defense-related activity in plants (VAN LOONand VAN STRIEN 1999). Our V. vinifera PR-1 shows respectiveidentities with proteins from yeasts (29 to 42 % - M81722,M81723, Z28238), insects (17 to 24 % - C44583, L49036,J03601, B37330), and vertebrates (24 to 29 % - X95240,U13619, U16307, S80310, X94323, D45027).BUSAM, G.; KASSEMEYER, H.H.; MATERN, U.; 1997: Differential expres-sion of chitinases in Vitis vinifera L. responding to systemicacquired resistance activators or fungal challenge. Plant Physiol.115, 1029-1038.DELOIRE, A.; KRAEVA, E.; MAURO, M.C.; BONNET, E.; BIERNE, J.; 1997:Immunodetection of chitinase-like and β-1,3-glucanase-like pro-teins secreted in vitro by embryogenic and non-embryogeniccells of grapevines. Vitis 36, 51-52.T a b l eA) Amino acid sequence of V. vinifera putative PR-1 including the signal sequence. Amino acid residues identical to the consensussequences (VAN LOON and VAN STRIEN 1999) are highlighted in gray. Positions of the cysteine residues, α helices and β strands are indicatedB) Homology of PR-1 like protein from V. vinifera and other organisms in percentage amino acid identity (upper) and similarity (lower),including the signal sequence: PR-1a Nicotiana tabacum (Nt PR1a) (CAA30017), PR-1b Nicotiana tabacum (Nt PR-1b) (CAA47374),basic form of PR-1 Nicotiana tabacum (Nt basic) (CAA32228),  PR-1c Nicotiana tabacum (Nt PR-1c) (CAA29023), PR-1 Capsicumannuum (C. annu) (AAK30143), PR-1b Solanum phrija (Sp PR-1b) (E08876), PR-1b2 Solanum phrija (Sp PR-1b2) (CAD38277), PR-1Solanum tuberosum (St PR-1) (CAB58263), PR-1b Solanum tuberosum (St PR-1b) (AAL01594), PR-1a Glycine max (Gm PR-1a)(AAD33696), PR-1a Lycopersicon esculentum (Le PR-1a) (CAA09671), PR-1a Brassica napus (Bn PR-1a) (AAB01666), PR-1-1aCucumis sativus (Cs PR-1-1a) (AAL84767), PR-1 Arabidopsis thaliana (At PR-1) (AAD24401), PR-1 Hordeum vulgare (Hv PR-1)(CAA88618), PR-1b Hordeum vulgare (Hv PR-1b) (AQ294250), PR-1 Oryza sativa (Oz PR-1) (AAM93438)Nt PR-1a Nt PR-1b Nt basic Nt PR-1c C. annuV. vinifera PR-1 64 % 63 % 68 % 64 % 66 %78 % 74 % 78 % 77 % 79 %Sp PR-1b Sp PR-1b2 St PR-1 St PR-1b Gm PR-1aV. vinifera PR-1 66 % 64 % 65 % 64 % 61 %75 % 74 % 75 % 75 % 77 %Le PR1a Bn PR1a Cs PR-1-1a At PR-1 Hv PR-1V. vinifera PR-1 63 % 58 % 63 % 58 % 53 %75 % 72 % 78 % 75 % 67 %Hv PR-1b Oz PR-1V. vinifera PR-1 57 % 55 %72 % 69 %JACOBS, A.K.; DRY, I.B.; ROBINSON, S.P.; 1999: Induction of differentpathogenesis-related cDNAs in grapevine infected with powderymildew and treated with ethephon. Plant Pathol. 48, 325-336.REPKA, V.; FISCHEROVA, I.; SILHAROVA, K.; 2000: Immunodetection of PR-1 like proteins in grapevine leaves infected with Oidium tuckeriand in elicited suspension cell cultures. Vitis 39, 49-52.REPKA, V.; FISCHEROVA, I.; SILHAROVA, K.; 2001: Methyl jasmonate in-duces a hypersensitive-like response of grapevine in the absenceof avirulent pathogens. Vitis 40, 5-10.ROBERT, N.; FERRAN, J.; BREDA, C.; COUTOS-THÉVENOT, P.; BOULAY, M.;BUFFARD, D.; ESNAULT, R.; 2001: Molecular characterization of theincompatible interaction of Vitis vinifera leaves with Pseu-domonas syringae pv pisi: Expression of genes coding for thestilbene synthase and class 10 PR protein. European Journal ofPlant Pathology 107, 249-261.SALZMAN, R.A.; TIKHONOVA, I.; BORDELON, B.P.; HASEGAWA, P.M.; BRESSAN,R.A.; 1998: Coordinate accumulation of antifungal proteins andhexoses constitutes a developmentally controlled defense re-sponse during fruit ripening in grape. Plant Physiol. 117, 465-472.VAN LOON, L.C.; 1997: Induced resistance in plants and the role ofpathogenesis-related proteins. European Journal of Plant Pa-thology 103, 753-765.VAN LOON, L.C.; VAN STRIEN, E.A.; 1999: The families of pathogenesis-related proteins, their activities, and comparative analysis ofPR-1 type proteins. Physiol. Mol. Plant Pathol. 55, 85-97.VAN WEES, S.C.M.; LUIJENDIJK, M.; SMOORENBURG, I.; VAN LOON, L.C.;PIETERSE, C.M.J., 1999: Rhizobacteria-mediated induced systemicresistance (ISR) in Arabidopsis is not associated with a directeffect on expression of known defense-related genes but stimu-lates the expression of the jasmonate-inducible gene Atvsp upon chal-lenge. Plant Mol. Biol. 41, 537-549.

Sequence of a putative Vitis vinifera PR-1

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Sequence of a putative Vitis vinifera PR-1

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