Expression And Regulation Of Chinese Wild Vitis Stilbene Synthase Genes And Their Resistance To Powdery Mildew

Grapevines（Vitis L.）are an important global perennial fruit crop.Vitis vinifera is the main cultivar widely cultivated due to its quality of berries.However,most varieties lack disease resistance to a range of pathogenic microorganisms,especially the ascomycete fungus Uncinula necator.Resveratrol and its derivatives in grape berry and wine are not only phytoalexins against fungal diseases,but also the benefit for human health,including cancer chemo-preventive activity,anti-aging and antioxidant.Thus,the further investigation of the roles of stilbene synthase genes and their metabolites is one of the core issues in our team to improve the susceptible wine and table grape with high content of resveratrol and disease resistance.Based on the previous reseaches,this study was designed to investigate the expression profiles of VpSTS29 in tissue and subcellular level under natural and stress conditions by cell biology and molecular biology,explore the signal pathway and key node gene function of regulating the expression of stilbene synthase genes,clear the synthesis,transport,degradation and action of STSs and its derivatives,and study the relationship of stilbenes and grape powdery mildew interaction.This study provides scientific basis for disease-resistant breeding by hybridization between resistant Chinese wild grape germplasms,especially using the STS genes to improve the disease resistance of V.vinifera.The main results were obtained as follows:1.The expresstion pattern of V.pseudoreticulata stilbene synthase gene VpSTS29and its metabolites showed tissue-specific.STS29 was highly expressed in the aging leaves of V.pseudoreticulata and V.vinifera and mature berries of V.quinquangularis Danfeng-2.Stilbene synthase VpSTS29 was accumulated in root,senescing leaves of the transgenic grapevines.HPLC analysis indicated that resveratrol and piceid were mainly distributed in root,stem and senescing leaves.In the transgenic Arabidopsis,Stilbene synthase VpSTS29 and trans-piceid were accumulated in matured leaves.Intriguingly,in comparison to the controls,over-expression of VpSTS29 in Arabidopsis resulted in relatively high levels of chlorophyll content and photochemical efficiency accompanied by delayed leaf senescence.2.Stilbene synthase gene VpSTS29 was responsive to a wide range of biotic and abiotic stresses.VpSTS29 was induced both from 48h to 72h and at 144h after Uncinula necator inoculation.RT-PCR analysis revealed that VpSTS29 was also induced by both the biotic stresses such as the SA,ABA,MeJA and Ethylene treatments and abiotic stresses including NaCl,drought,wounding,low temperature and UV-C treatments.What’s more,UV-C treatment showed the highest induction of VpSTS29.The further investigation indicated that VpSTS29 protein displayed UV-induced feature coupled with the accumulation of stilbene compounds and translocated from cytoplasm into chloroplast upon UV-irradiation.Leaves from the two VpSTS29 transgenic grapevines displayed more serious UV damage,showing the wizened and marginal scorching phenotype,and decreased content of H₂O_2,compared to the untransformed plant.Also,overexpression of VpSTS29 altered the expression of genes related to redox regulation,stilbene biosynthesis,and light stimulus.Co-expression of VpSTS29-GFP with Glycolate oxidase 1（myc-VpGLO1）confirmed the ability of stilbenes to decrease the content of H₂O₂ in Arabidopsis mesophyll protoplasts.These results provide evidence that the change of translocation pattern of stilbene synthase could adapt to the environmental stimulus.Together VpSTS29 was induced by the biotic and abiotic stresses and functioned in the disease resistance and enhanced the adapt to adversity stress during the growth and development in grape.3.The cross-grafting experiment confirmed that VpSTS29 does not exist the transportation among tissues.Over-expression of VpSTS29 in rootstock could significantly increase the content of stilbenes in scion.In situ fluorescence observation showed that VpSTS29 was mainly distributed in the meristematic zone,vascular bundle of stem and mesophyll tissue.Subcellular localisation analyses indicated VpSTS29 was present in the cytoplasm and in stilbene synthase-containing bodies.Nile red staining,co-localisation and immunohistochemistry analyses of leaves confirmed that the stilbene synthase-containing bodies were oil bodies and that these moved randomly in the cytoplasm and vacuole.Detection of protein profiles revealed that no free GFP was detected in the pVpSTS29::VpSTS29-GFP-expressing protoplasts or in Arabidopsis during the dark-light cycle,demonstrating that GFP fluorescence distributed in the stilbene synthase-containing bodies and vacuole was the VpSTS29-GFP fusion protein.These results provide exciting new insights into the subcellular localisation of stilbene synthase in plant cells and information about stilbene synthesis and storage,especially the trafficking of VpSTS29 from ER to vacuole by autophagic pathway in grapevine.Stilbenes were dynamically redistributed after their synthesis in order to meet the needs of plant growth and development.4.The expression of STSs in the transgenic grapevines was significantly higher than that of in the UT.Over-expression of VpSTS29 led to the rapid induction of the endogenous STS genes as well as the expression intensity in response to U.inoculation.WheatGermAgglutininstrain,biolisticbombardment,immunofluorescence in situ imaging and Western blot analysis confirmed that VpSTS29 showed a pathogen-induced expression pattern uniquely in the leaf mesophyll cells andε-viniferin were specially accumulated in the infection sites to strength the defense in the transgenic grapevines.Transformation of VpSTS29 into the STS-deficient Arabidopsis thaliana showed that VpSTS29 and trans-piceid were induced by G.cichoracearum inoculation,leading to the inhibition of mycelial growth,local hypersensitive response and reduction of penetration frequency.Furthermore,exogenous SA and resveratrol treatments also enhanced the accumulation of VpSTS29 and trans-piceid,indicating their potential function in the defense signaling molecule to mitigate pathogen stress.These results showed that VpSTS29 transgenic plants displayed the resistance to U.in grapevine and Golovinomyces cichoracearum in Arabidopsis.Stilbene synthase and its metabolic products could enhance the powdery mildew-resistance.5.The R2R3-MYB transcription factors MYB14 and MYB15 showed a high level of conservation between Chinese wild Vitis species and V.vinifera.And MYB14and MYB15 binded to the promoter of VpSTS29 from V.pseudoreticulata.Furthermore,the bHLH-type transcription factor gene VqICE1 was identified to negatively regulated the expression of STS genes through the transcriptome data of V.quinquangularis Danfeng-2.Co-expression analysis showed that VqICE1 was correlated with 26 STS genes.The yeast one-hybrid assay showed that VqICE1 binded to the promoter regions of group-B-type STS gene VqSTS15,VqSTS27 and VqSTS48and reduced their transcriptional activity.While VqMYB14 and VqMYB15 activated the expression of the selected STS genes.VqICE1 directly binded to the promoter of VqMYB14 but not VqMYB15 and over-expression of VqICE1 decreased the mRNA level of VqMYB14 and the accumulation of trans-piceid.VqICE1 interacted with VqMYB14 and VqMYB15 at the nucleus,respectively.The expression of STS genes and MYB14/15 were induced by U.inoculation in the wild type,while over-expression of VqICE1 released pathogen induced STS accumulation at the late stage of infection.These results indicated that VqICE1 acted as a repressor to negatively regulate the expression of stilbene synthase genes to maintain the balance level of stilbenes to the normal after their actions.In summary,STS genes and their products are tissue-specific and induced by biotic and abiotic stresses;VpSTS29 and stilbens travel to vacuole by autophagic pathway at the particular stage of plant growth and development.Under the U.necator inoculation,stilbenes are rapidly accumulated in the infection sites,and then being accurately regulated by transcription factors.Thus,gene VpSTS29 functions in the accumulation of stilbenes and enhancing the disease resistance.