Study On The Expression And Resistance To Powdery Mildew Of Novel Stilbene Synthase Gene VqNSTS4 From Chinese Wild Vitis Quinquangularis

Resveratrol is not only a kind of disease-resistant phytoalexin in grapevines,but also has a significant antioxidant benefit in human health.Stilbene synthase(STS)catalyze the bioprocess of resveratrol biosynthesis in plants,therefore,they were recognized as rate-limiting enzymes for stilbenes accumulation.‘Danfeng-2’,a wild Chinese grapevine accession belongs to V.quinquangularis,has been confirmed in a number of studies to contain high levels of resveratrol and is resistant to powdery mildew.Based on the results of transcriptomic analysis,VqNSTS4 was cloned from ‘Danfeng-2’.In this study,we created transgenic plants of V.vinifera Thompson Seedless overexpressing VqNSTS4-GFP,and examined the resistance of transgenic grapevine lines against powdery mildew.In addition,we also identified a PHD transcription factor gene VqPHD via Y1 H screening assays,and examined its ability to regulate the transcription of VqNSTS4.Following are the main results:1.Sequence analysis and expression pattern of VqNSTS4: Based on the results of transcriptomic analysis,VqNSTS4 was cloned from ‘Danfeng-2’.The length of VqNSTS4 coding sequences is 1035 bp,which encodes protein of 344 amino acids.Tissue specific expression analysis demonstrated that VqNSTS4 had the highest expression levels in mature berries of ‘Danfeng-2’,then mainly expressed in young and mature leaves,and had the lowest expression levels in the stem.VqNSTS4 can also respond to powdery mildew inoculation and phytohormones treatments.The relative expression levels of VqNSTS4 gene changed significantly after SA and Me JA treatments.2.Generation and confirmation of transgenic grapevine lines with VqNSTS4-GFP overexpressing: Transgenic V.vinifera cv.Thompson Seedless plants with the overexpression construct 35S::VqNSTS4-GFP(p CAMBIA2300)were obtained via Agrobacterium-mediated transformation.Through 50 mg/L Kanamycin selections and regeneration of somatic embryos,we finally obtained 157 kanamycin-resistant plantlets,and successfully obtained 7 transgenic plants,which were confirmed by PCR and Western blot assays.These results showed that the transformation efficiency was 4.46%.3.Expression analysis of STS and the related genes in transgenic grapevines: q RT-PCR was used to analyze the relative expression levels of VqNSTS4 and several genes involved in phenylalanine metabolic pathway in the 7 transgenic plants.Expectedly,the expression levels of VqNSTS4 were elevated in transgenic lines.In addition,PALs(upstream of STS),RSGT(downstream of STS)and MYB15 that involved in transcriptional regulation of STSs were all significantly up-regulated.However,CHS gene,whose products share common substrate with STSs,was down-regulated in transgenic lines compared to wild type grapevines.HPLC analysis was used to compare the accumulation of stilbenes in transgenic plants(OEVqNSTS4-L1 and OEVqNSTS4-L2)and wild type plants.The results showed that the content of trans-piceid was significantly increased in transgenic lines,and other derivatives were also highly accumulated in these transgenic lines.4.Powdery mildew inoculation assays on transgenic grapevines: Two transgenic lines,OEVqNSTS4-L1 and OEVqNSTS4-L2 that have the highest expression levels of VqNSTS4 were selected to perform powdery mildew inoculations.The results from trypan blue staining showed that the hyphea growth and sporulation of U.necator were obviously restricted in transgenic grapevines leaves,since it showed reduced hyphae length and generated less spores compared to that grown on the wild type plants.The results from qRT-PCR analysis showed that VqNSTS4 kept high transcript levels during the whole stages of pathogens’ s colonization.Analysis of stilbenes content with HPLC showed that the contents of trans-piceid,trans-resveratrol,and ε-viniferin in the transgenic lines were significantly elevated,while the contents of pterostilbene and trans-piceatannol were different in varying plants.Aniline blue staining showed that plenty of callose accumulated in the epidermal cells of VqNSTS4 transgenic grapevine leaves but little callose were observed in the leaves of wild type at 3 dpi.q RT-PCR analysis was performed to examine the expression of several defense-associated genes in different grapevines responses to U.necator inoculations,the results showed that NPR1,PR1,PR4 and LOX2 could be significantly up-regulated at the early stage of inoculation(24 hpi)in the transgenic plants.5.Identification of a PHD transcription factor that regulates the expression of VqNSTS4:Based on the results of Yeast one-hybrid(Y1H)screening assays,a PHD transcription factor VqPHD was cloned from Danfeng-2.It was predicted to be located on chromosome 11,and consist of a 759 bp ORF.Tissue specific expression analysis demonstrated that VqPHD showd the highest expression levels in mature leaves of ‘Danfeng-2’,and had the lowest expression level in the root.In addition,the relative expression levels of VqPHD were obviously changed post SA and ABA treatments.VqPHD proteins localized in the nuclei of the tobacco epidermal cells and exhibited trans-activation ability in yeast cells.Yeast one-hybrid,quantitation of GUS activity and LUC assays were carried out to test if VqPHD can directly bind to the promoter sequences of VqNSTS4,and the results showed that VqPHD could regulate the expression of VqNSTS4 by directly binding to the ’CACCTC’cis-acting element in the promoter of VqNSTS4.When VqPHD transcription factor was transiently overexpressed in the leaves of ‘Danfeng-2’,the expression levels of VqNSTS4 gene was up-regulated by 6-fold.In summary,powdery mildew inoculations could induce the transcription of VqNSTS4 via VqPHD transcription factor in ‘Danfeng-2’.Moerover,overexpression of VqNSTS4 could promote the accumulation of stilbenes and improve the resistance to powdery mildew in Thompson Seedless.Our results reconfirm the significance of ‘Danfeng-2’ for disease resistance breeding in grapevines.