Cloning And Functional Analysis Of Stilbene Synthase And Pathogenesis-related10Protein Genes From Chinese Wild Grapevines

The grapevine is one of the most important fruit crops in the world. However, most ofcultivated Vitis vinifera varieties worldwide are generally susceptible to Erysiphe necatorand Plasmopara viticola. Grape resveratrol is a phytoalexin, plays an important role in plantdefense against pathogens infection, and also shows significant health-promoting effectsincluding anti-cancer, prevention of cardiovascular disease and neuroprotective properties.Stilbene synthase (STS) is the key enzyme for producing the resveratrol. Plantpathogenesis-related (PR) protein PR10plays an important role in the grape resistance tofungal disease. In this study, stilbene synthase genes were cloned from Chinese wild V.quinquangularis accession 'Danfeng-2' which showed high resistance to powdery mildewand high content of resveratrol in mature berries. A PR10gene, designated VpPR10.2, wasclone from Chinese wild V. pseudoreticulata accession 'Baihe-35-1' which showed highresistance to powdery mildew and downy mildew. Expression and functional analyses ofstilbene synthase genes and VpPR10.2were performed in this study. The main results aredescribed as followings.1. BLAT and Blastn searches were performed in genoscope grape genomic database tofind homologous sequences of stilbene synthase gene using the nucleotide of stilbenesynthase gene from V. pseudoreticulata as query sequence. A total of53STS genehomologous sequences were found, and9of them have no STS feature motif'IPNSAGAIAGN' in the deduced amino acid sequences, thus, they should be chalconesynthase-like sequences. STS multi-gene families of V. vinifera contained44members, and11of them were pseudogenes which do not encode stilbene synthase correctly. All STSswere clustered into two groups,6STSs on chromosome10and38on chromosome16. STSsequences covered all coding region and3' UTR were cloned from E. necatorinoculated-leaves and berries of Chinese wild V. quinquangularis accession 'Danfeng-2' byRACE amplification. RT-PCR products were analyzed by gel electrophoresis, cloned andmore than800individual clones were sequenced. Sequence analysis identifiedthat'Danfeng-2' have41single STS genes, one of which were pseudogene which do notencode stilbene synthase correctly. GenBank Accession numbers of STS from V. quinquangularis were from JQ868658to JQ868698. STS sequences from V. vinifera and V.quinquangularis sequences were homologies, but most of them cannot be grouped in asingle cluster.2. Expression profiles of STS in different organ tissues or in developing of berries of V.quinquangularis and V. vinifera were analyzed by real-time quantitative PCR. Thegrapevine STS genes were expressed in root with the highest level and in leaves with thelowest, compared to the expression levels of STS in stems, tendrils, flowers and berries.During berries development, there is an expression level peak of STS at the veraison stageof grapevine development. At the ripening stage, the expression levels of STS genes wereincreased in berries of V. quinquangularis but decreased in berries of V. vinifera. Therewere significant differences among the genes involved in pathway for phenolicsbiosynthesis in V. quinquangularis and V. vinifera.. The higher expression levels of STS andPAL genes might result the higher accumulation level of resveratrol in berries of V.quinquangularis than that of V. vinifera. HPLC was used to analyze the resveratrol contentin grapevine berries. The highest accumulation levels of resveratrol in two grapevine berrieswere at the veraison stage,16.897μg·FW-1for berries of V. quinquangularis and8.908μg·FW-1for berries of V. vinifera. In mature berries, accumulation level of resveratrol wasincreased in V. quinquangularis but decreased in V. vinifera. The resveratrol content inripen berries of V. quinquangularis is higher than that of V. vinifera,7.297μg·gFW-1forberries of V. quinquangularis and4.791μg·gFW-1for berries of V. vinifera.3. Expression profiles of grapevine STS genes in response to biotic or abiotic stresseswere analysis by real-time quantitative PCR. STS genes from three grapevine genotypes, V.pseudoreticulata, V. quinquangularis and V. vinifera, were induced by E. necator and P.viticola, and their expression levels were consistent their resistances to powdery mildew anddowny mildew. Members of STS multi-gene families from V. quinquangularis or V. viniferainduced by E. necator showed different expression profiles and expression levels. STSgenes from V. quinquangularis were induced by compatible pathogen E. necator, P. viticolaand incompatible pathogen E. cichoracearum. In addition, STS genes were induced by ABA,MeJA, SA, coldness, hotness and drought treatment. The highest expression level wasinduced by SA treatment among the other abiotic stresses. Thus, the expression of STSmight be involved in the SA pathway in plant. However, the expression of STS wasdown-regulated by hotness treatment. Interestingly, the expression of STS in grapevineleaves was consistent with the expression of pathogenesis-related genes, such as PR1, PR2,PR10, and PR14. However, the STSs showed the highest normalized fold expression levelamong that of other PR genes. 4. Subcellular localization of STS in grapevine leaves inoculated with E. necator and P.viticola was analyzed. Grapevine STSs were located in the cytoplasm and on cell wall ofepidermal cells on leaves. Moreover, the STSs were also located on cell wall of hyphae andhaustorium of P. viticola at the later infection stage. The ORF of VqSTS1, a STS geneselected from V. quinquangularis, was amplified and sub-cloned into vector pGEX-4T-1tocreate an in-frame fusion of the VqSTS1with the GST tag protein. The VqSTS1recombinant protein was then expressed in Escherichia coli and purified. The purifiedVqSTS1recombinant protein showed grown inhibitation against E. necator, P. viticola,Botrytis cinerea and tobacco Ralstonia solanacearum. In addition, the coding region ofVqSTS1was sub-cloned into expression vector pCAMBIA1302and driven by an enhanced35S promoter, and then, were transformed into Nicotiana tabacum cv. NC89byAgrobacterium-mediated method. Over-expression of VqSTS1in tobacco could enhance thehost resistance to E. cichoracearum. The expression levels of PR1, PR2and PR4intransgenic tobacco plants were much higher than that in control plants which weretransformed with Agrobacterium harboring vector pCAMBIA1302. In addition, theexpression levels of PR5and PR10in transgenic plants were slight higher than that incontrol plants, and the expression levels of CHS were down-regulated by over-expression ofVqSTS1. The results above suggested that the anti-fungal activities of grapevine STS and itsproducts resveratrol combined with the induction of host PR genes all contribute to theirhost resistance to pathogens.5. The expression levels of grapevine PR10.2genes in V. pseudoreticulata and V.vinifera were higher than that of other PR10multi-gene family members in the twograpevine genotypes. The PR10.2gene was cloned from Chinese wild V. pseudoreticulata,and was designated as VpPR10.2(GenBank accession no. HQ634186). The genomicVpPR10.2and its promoter sequence (GenBank accession no. HQ634185) were also clonedby PCR amplification. The coding sequence, deduced amino acid sequence and promoterregion sequence of VpPR10.2had99.6%,100%and96.2%identities with thecorresponding sequences of VvPR10.2from V. vinifera, respectively. The low sequencesimilarity of PR10promoters might result their different expression levels and profiles ofgenes in response to pathogen attack.6. The expression levels of VpPR10.2in roots, stems, leaves, flowers, berries wereanalyzed by real-time quantitative PCR. VpPR10.2was expressed in roots with the highestlevel and in leaves with the lowest. The expression of VpPR10.2was induced by theinfection of E. necator and P. viticola. The expression level of VpPR10.2in leavesinoculated with E. necator peaked at12hpi, while VvPR10.2peaked at72hpi. The expression level of VpPR10.2was induced approximately13times by P. viticola in leavesat72hpi. In contrast, the expression level of VvPR10.2in leaves inoculated with P. viticolawas first down-regulated and then slightly increased. Moreover, the expression of VpPR10.2was induced by the ABA, MeJA and SA treatments, and ABA treatment induced theexpression of VpPR10.2with the highest level. The expression of VpPR10.2was alsoinduced by the coldness, hotness and drought treatments. The results above suggested thatthe expression of VpPR10.2was induced by biotic and abiotic stresses, and showeddifferent profiles in resistant-and susceptible-grapevine plants in response to pathogenattack.7. The coding region of VpPR10.2was amplified and sub-cloned into the vectorpGEX-4T-1to create an in-frame fusion of158amino acids of the VpPR10.2protein withthe GST tag protein. The VpPR10.2recombinant protein was then expressed in E. coli andpurified. The purified VpPR10.2recombinant protein showed RNase against yeast totalRNA and DNase against grapevine genomic DNA in vitro, and also showed growthinhibitation against tobacco fungus Alternaria alternata. In addition, the coding region ofVpPR10.2was sub-cloned into expression vector pCAMBIA1302and driven by anenhanced35S promoter, and then, were transiently transformed into V. vinifera cv.Carignane using Agrobacterium-mediated vacuum method. The over-expression ofVpPR10.2gene in V. vinifera could improve the host resistance to P. viticola. The resultsfrom subcellular localization analysis showed that VpPR10.2proteins were distributeddynamically inside or outside of host cell. Moreover, they were found in haustorium of P.viticola and nucleus of host cell which was associated with a nucleus collapse, a hallmark ofprogrammed cell death, at10days post-inoculation.