Accumulation And Resistant Mechanism Of Resveratrol And Derivatives During Grape Defense To Downy Mildew

Downy mildew is one of pathogen and insect diseases mainly affecting grape yield and quality,caused by Plasmopara viticola (Pv). In the previous studies, the PV-resistant abilities of different grape cultivars are various. What's more, the accumulations of stilbene (resveratrol, piceid,pterostilbene, viniferin, and so on) are completely different in the different resistant grape cultivars defense to Plasmopara viticola. Based on this phenomenon, this study focused on the accumulation and resistant mechanism of stilbene in the different resistant grape cultivars.Pv-immune Muscadine rotundifolia 'Noble' and 'Carlos', Pv-moderate resistant Vitis amurensis'Zuoshanyi' and 'Shuanghong', Pv-susceptive Vitis vinifera 'Thompson Seedless' and 'Chardonnay'were inoculated with Plasmopara viticola artificially. The results of stilbene contents and the qRT-PCR of related genes showed that the expressions of MrSTS gene and MrROMT gene were up-regulated and a large number of resveratrol, pterostilbene and ?-viniferin were accumulated during 2 hpi - 1 dpi in the Pv-immune Muscadine rotundifolia grapes. However, the expressions of VvSTS gene and VvGT gene were up-regulated and a mass of resveratrol and piceid were accumulated at 3 dpi in the Pv-susceptive Vitis vinifera grapes.The promoters of STS gene, ROMT gene and GT gene of Muscadine rotundifolia 'Noble' and Vitis vinifera 'Thompson Seedless' were gained through genome walking method. The alignment results revealed that the similarity between MrSTS promoter and VvSTS promoter was only 81.12 %, the similarity between MrROMT promoter and VvROMT promoter was 99.83 %, and the similarity between MrGT promoter and VvGT promoter was 98.3 %. These promoters were studies in vitro through the transient transformation of grape leaves. The results of GUS staining discovered that the VvSTS and MrSTS promoter was induced by SA, ABA, MeJA, GA3 and PV. Besides, MrROMT promoter and VvROMT promoter were both induced by SA, ABA and PV, and MrGT promoter and VvGT promoter were both induced by SA, ABA, MeJA and PV.The phytohormone contents were detected with UPLC-ESI-Ms/MS in the Pv-inoculated leaves. The contents of ABA, SA and MeJA in the Muscadine rotundifolia and Vitis amurensis grapes were much more than those in the Vitis vinifera grapes. To verify the phytohormone role in stilbene regulation,'Noble' and 'Thompson Seedless' were inoculated with ABA, SA and MeJA artificially. The results of phytohormone contents showed that ABA and SA inhibited each other. ABA, SA and MeJA induced the stilbene accumulations and the up-regulations of related genes at some different level, which was lower than Pv induction. 'Noble' and 'Thompson Seedless' were inoculated with ABA or SA firstly and then with Pv artificially, the qRT-PCR results indicated that hormone pre-treatment invigorate the accumulation of stilbene and improve the host to defense Pv.The MrSTS and MrROMT co-expression vectors were constructed by Gateway recombination and double-enzyme digestion methods and then verified through the transient transformation of tobacoo leaves. The results of fluorescence observation of GFP protein, resveratrol and pterostilbene contents and the qRT-PCR of related genes showed that the vectors of pH7FWG2/P35s::MrSTS/P35s::MrROMT and pH7FWG2/PMrSTS::MrSTS/PMrROMT::MrROMT were constructed successfully and had transcriptional activity to compound resveratrol and pterostilbene.