Research On Disease Resistance Function And Regulation Mechanism Of VqWRKY52 From Chinese Wild Vitis Quinquangularis

Grape is one of the most important fruit trees in the world and ha s important economic value.Wine,jam,juice,jelly,grape seed extract,raisins,and vinegar are all inseparable from grapes.At present,the European grape(Vitis vinifera L.)has a wide cultivation area and good fruit quality,but its disease resistance is poor,especially powdery mildew.Every year,it causes huge losses to production.In recent years,the molecular mechanism of plant disease resistance has been extensively studied in Arabidopsis,tobacco,rice and other model plants.Among them,WRKY transcription factor has an important function in the process of plant disease resistance.However,the research on the gene function and molecular mechanism of WRKY transcription factor on grape is slow,and the longer genetic transformation cycle and lack of mutant materials limit the development of grape functional genomics and affect the genetic modification breeding process of grape.In this study,a mature CRISPR/Cas9 genome editing system was established on the grape,and the apex mutation of VvWRKY52 gene was successfully achieved,and the homozygous functional deletion lines was obtained.It proves that CRISPR/Cas9 technology can be successfully applied to the precise editing of grape genome,which provides an effective means for grape functional genomics and genetic modification breeding.At the same time,we also identified the function of VqWRKY52 gene in grape disease resistance,and conducted a preliminary analysis of the regulation mechanism of VqWRKY52 involved in powdery mildew resistance.Candidate genes are provided for grape genetic modification breeding.The study has achieved the following results:1.Previous studies have shown that VqWRKY52 gene is strongly induced 24 hours after inoculation with powdery mildew.In this study,we treated grape leaves with salicylic acid(SA).The results showed that VqWRKY52 could be induced by SA,and then we performed a transient transformation experiment of tobacco.The results also proved that VqWRKY52 can be affected by SA.To further identify the function of VqWRKY52,we over-expressed VqWRKY52 in Arabidopsis.Its overexpressing lines enhance resistance to powdery mildew and P.syringae in Arabidopsis.However,the resistance to gray mold is reduced.At the same time,trypan blue staining found that overexpressing lines showed more cell death after inoculation of pathogenic bacteria.It is suggested that the VqWRKY52 gene may enhance the resistance to pathogenic bacteria by regulating the strength of cell death.In addition,after infecting different pathogens,we compared the relative expression levels of various defense-related genes between transgenic lines and wild-type plants,and the results showed that the SA signaling pathway was enhanced in overexpressing lines.All of these results indicate that VqWRKY52 plays an important role in the SA-dependent signal transduction pathway and that it can enhance pathogen-induced cell death to regulate plant resistance to pathogens.2.Based on the CRISPR/Cas9 multi-target editing system,we designed four targets for the first exon region of VvWRKY52 gene,constructed a plant expression vector,and transformed the grape PEM by agrobacterium-mediated transformation methods,72 transgenic plants were successfully obtained.DNA level identification of the first generation of transgenic plants confirmed that 72 lines were positive plants,and 22 mutant lines were identified in 72 positive lines.Among them,15 lines were biallelic mutations,and 7 liness were heterozygous mutations.We found a series of editing events in mutant lines,including deletions of large fragments,deletions and insertions of small bases,and the deletion of a small number of bases is the most common type of mutation.At the same time,we also found that different targets can work together to achieve accurate knockout of the targe t area.It is proved that CRISPR/Cas9 technology can achieve simultaneous loss of function of one gene or multiple genes on grapes,and can also accurately knock out specific domains of certain genes.In addition,we performed mutation detection on potential off-target sites of all four targets,and no off-target events were found.In addition,the functionally deficient lines of VvWRKY52 in the grape increased resistance to gray mold.At the same time,we constructed the VqWRKY52 overexpression vector and obtained the overexpressed lines of VqWRKY52 gene by genetic transformation of grape.The positive overexpression lines were obtained by DNA and RNA level identification.Afterwards,all overexpressing lines and functionally deficient lines were transplanted to the solar greenhouse.3.The VqWRKY52 overexpressing lines,functionally deficient lines and WT plant were inoculated with powdery mildew.The results showed that the overexpressing lines significantly enhanced the resistance to powdery mildew,while the functionally deficient lines decreased.We then performed a transcriptome analysis of the leaves after inoculation with powdery mildew.Through the functional enrichment of differential expression genes,the lignin synthesis pathway-related genes in the overexpressing lines were enhanced at the background level and inhibited in the functionally deficient strains,suggesting that the VqWRKY52 gene can regulate the resistance to pathogens by regulating plant lignin synthesis.In function-deficient lines,a large number of differential expression genes beyond the normal level were found.We speculate that this may be a signal that the immune response of the functionally-deficient lines is broken by the deletion of the VqWRKY52 gene,and the plant needs to call more genes to response to the invasion of pathogens.Subsequently,the binding regions of bHLH,NAC,and ERF were enriched in the promoter region of each differential expression gene affected by the VqWRKY52 gene,and bHLH,NAC,and ERF transcription factors were also enriched in these differential expression sets.After the analysis of WGCNA,we found a grey60 module similar to the VqWRKY52 gene expression pattern,and then we also enriched bHLH,NAC,ERF and WRKY in the gene set that was differentially expressed after VqWRKY52 overexpression and loss of function in the grey60 module.In addition,a large number of W-boxes in the promoter regions of these genes were found.Taken together,these results demonstrate that the VqWRKY52 gene can regulate transcription factors such as bHLH,NAC,and ERF,and these transcription factors then regulate downstream disease-resistant signaling pathways.