Mechanism Analysis Of Transcription Factor VqMYB154 Regulating The Resistance To Powdery Mildew In Chinese Wild Vitis Quinquangularis

Grapevine(Vitis spp.)is one of the cultivated fruit trees with the longest cultivation history and the highest economic benefit.Vitis vinifera cultivars have important application value in wine making and table consumption.However,they possess poor disease resistance and are susceptible to grape diseases.Powdery mildew is one of the most serious grape diseases and often causes severe loss of grape yield.Studies have shown that resveratrol in grapes not only has anti-disease effects on plants,but also has anti-cancer and anti-aging effects on human body.Therefore,this study took resveratrol,which is the product of stilbene synthase(STS),and its role as the starting point,and adopted Vitis quinquangularis accession ‘Danfeng-2’,which has strong anti-disease ability and is abundant in resveratrol as the material.By inoculation of Uncinula necator,a disease-resistant transcription factor Vq MYB154,which is highly expressed in response to U.necator,was screened from 106 transcription factor genes of MYB family in this study.Besides,the role of Vq MYB154 in regulating the expression of STS genes,accumulation of stilbenoid and enhancing anti-disease ability was studied.This study provides theoretical guidance and scientific basis for the long-term goal of disease-resistant grapevine breeding by utilizing the germplasm resources of Chinese wild grapevine.The main results were listed as follows:1.The disease-resistance related transcription factor VqMYB154 was screened out by expression analysis after Danfeng-2 leaves were inoculated with U.necator.The leaves of‘Danfeng-2’ grapevine were inoculated with U.necator,and 106 R2R3-MYB genes were analyzed by q RT-PCR.The expressions of 27 MYB members were up-regulated to more than 4 times the normal expression level in response to the induction of U.necator.Among them,Vq MYB154 had the highest induced expression peak after inoculation,which was 21.2times the expression level at 0 h after inoculation;Vq MYB154 gene was a specific disease-resistant transcription factor gene in ‘Danfeng-2’ grapevine and different from Vv MYB154 in V.vinifera,the expression of which was enhanced after the induction of two pathogens,U.necator and Pseudomonas syringae.The Vq MYB154 gene was cloned from‘Danfeng-2’ grapevine utilizing homologous cloning technology.It is located on grapevine chromosome 11,encodes 285 amino acids and is a member from subfamily 14 of MYB family;the expression of Vq MYB154 was not only up-regulated in response to the induction of flg22,hydrogen peroxide and calcium chloride,but also exhibit different expression characteristics under the induction of SA,Me JA,ABA and Eth;Vq MYB154 is located in nucleus and has the transcriptional activation function.2.The promoter sequence of VqMYB154 was cloned,and the promoter activity was analyzed by U.necator and flg22 induction.The promoter sequences of Vq MYB154 and Vv MYB154 in Cabernet Sauvignon were obtained using homologous cloning technology.There were 6 deletion mutations,3 insertion mutations and an extra ERE element was found in Vq MYB154 promoter compared with Vv MYB154 by sequence alignment and element difference analysis;the promoter sequences of MYB154 gene in two grapevines were fused to GUS protein expression vector,and transient transformation mediated by Agrobacterium was adopted to introduce them into ‘Danfeng-2’ leaves.It was found that both U.necator and P.syringae could activate Vq MYB154 promoter,but not Vv MYB154 promoter by GUS protein activity analysis.Besides,it was found that salicylic acid,hydrogen peroxide,and flg22 could activate Vq MYB154 promoter.The activation of Vq MYB154 promoter by flg22 requires the involvement of reactive oxygen species and MAPK cascade pathways.By construction of deletion mutants and treatment with flg22 after transient transformation of grape leaves,it was found that the ERE element is the core of Vq MYB154 promoter in response to flg22 induction.3.The VqSTS genes regulated by VqMYB154 was screened and identified,and their expression was studied under U.necator and various stress conditions.The co-expression analysis of Vq MYB154 and Vq STSs was performed on the basis of the transcriptome data of‘Danfeng-2’ and Cabernet Sauvignon grapevines.Preliminary screening results showed that Vq MYB154 might interact with 18 Vq STSs.The Y1 H verification analysis was further used to screen out 3 Vq STS genes directly bound and regulated by Vq MYB154,namely Vq STS9,Vq STS32 and Vq STS42.Vq MYB154 played a transcriptional regulatory role by binding to 2kinds of MYB binding elements L5-box(GAGTTGGTGAGA)and AC-box(ACCAACT)in3 Vq STS promoter sequences.The results of GUS activity analysis showed that Vq MYB154 is the positive regulatory factor for 3 Vq STSs and up-regulates the expression of Vq STS9,Vq STS32 and Vq STS42 by binding and activating promoter activity.Vq MYB154 was then fused with overexpression vector,and the transient transformation assay on ‘Danfeng-2’grape leaves showed that overexpression of Vq MYB154 not only significantly enhanced the expression of 3 Vq STSs under natural conditions,but also enhanced the transcription levels of phenylalanine ammonia lyase PAL gene upstream of the STS pathway,thus activating the STS pathway and increasing the stilbenoid content including piceid and resveratrol.q RT-PCR analysis showed that U.necator,P.syringae,chitin,wound,ultraviolet,high salt,low temperature(-2°C and 4°C)and high temperature(37°C)could up-regulate the co-expression of Vq MYB154,Vq STS9,Vq STS32 and Vq STS42.Besides,q RT-PCR,GUS histochemical staining and protein activity analysis after transient transformation of tobacco leaves verified that Vq MYB154 interacted with the micro RNA genes Vqmi R171 c and Vqmi R171 i in grapevines.Vqmi R171 c and Vqmi R171 i could target Vq MYB154 and degrade its m RNA.Co-expression of Vq MYB154 with Vqmi R171 c,Vq MYB154 and Vqmi R171 i could inhibit stilbenoid synthesis regulated by Vq MYB154 compared with overexpression of Vq MYB154 in ‘Danfeng-2’ leaves alone.The analysis of the transcription levels of MYB154 and 2 mi R171 genes in ‘Danfeng-2’ and Cabernet Sauvignon leaves after inoculation with U.necator and P.syringae showed that Vq MYB154 and the two Vqmi R171 genes in ‘Danfeng-2’were negatively correlated in pathogen-induced expression,that is,two pathogens inhibited the transcriptional activity of the two Vqmi R171 genes while increasing the expression level of Vq MYB154.There was no correlation between the expressions of Vv MYB154 with two Vvmi R171 genes in Cabernet Sauvignon.4.The transcription factor VqMYB154 was transferred into grapevine and Arabidopsis to study the regulation role of Vq MYB154 in plant disease resistance.To explore the molecular mechanism of Vq MYB154 regulating plant disease resistance,the Vq MYB154 fusion overexpression vector was constructed,and Vq MYB154 was overexpressed in Danfeng-2,Cabernet Sauvignon and Arabidopsis and then the resistance identification of powdery mildew was carried out.Overexpression of Vq MYB154 could significantly inhibit hyphae development of U.necator in grapevines and Arabidopsis and enhance plant resistance to powdery mildew by inducing allergic necrosis,stimulating production of reactive oxygen species,accumulation of callose and expression of resistance-related genes.On the one hand,overexpression of Vq MYB154 in two grapevines could enhance the expression of genes related to stilbenoid synthesis induced by U.necator,and increase the accumulation of piceid,resveratrol,viniferin and piceatannol in grape leaves after U.necator inoculation.At the same time,it could enhance the expression levels of resistance-related genes in grapevine SA pathway and JA/ET pathway under U.necator induction.On the other hand,the transgenic mutants of Arabidopsis overexpressing Vq MYB154 were obtained.After inoculation with U.necator,overexpression of Vq MYB154 could activate the expression of resistance-related genes in SA and JA/ET pathway in Arabidopsis.It could enhance the resistance of Arabidopsis plants to powdery mildew by multigene synergy in different disease resistance pathways.In addition,by evaluation of disease resistance for the Arabidopsis mutants inoculated with Pst DC3000,it was found that overexpression of Vq MYB154 could induce cell allergic necrosis,stimulate ROS production,callose accumulation and activate the expression of resistance-related genes At ICS1 and At PR5 in SA pathway,thus enhance the resistance of Arabidopsis to Pst DC3000.In summary,the transcription factor VqMYB154 in Chinese wild grapevine regulates the expression of 3 Vq STSs target genes and enhances plant disease resistance.Therefore,Vitis quinquangularis accession ‘Danfeng-2’ is a valuable disease-resistant germplasm resource for improving disease resistance of V.vinifera cultivars.