小西葫芦黄花叶病毒弱毒疫苗的构建及其交叉保护防治作用OA

[Objective]By investigating the effects of the mutation at the 364th conserved lysine(K364)in helper component-proteinase(HC-Pro)on the virulence of zucchini yellow mosaic virus(ZYMV),an attenuated ZYMV mutant were developed,and its genetic stability and cross-protection efficacy were assessed.Our study provided the theoretical basis for developing sustainable control strategies against ZYMV using attenuated strains.[Method]Using site-directed mutagenesis,the K364 residue in HC-Pro was substituted with aspartic acid(D)to investigate the effect of the K364D mutation on the virulence of ZYMV and the RNA silencing suppression activity of HC-Pro,thereby constructing the attenuated ZYMV-HC-ProK364D mutant.Furthermore,the genetic stability of ZYMV-HC-ProK364D mutant was assessed through serial passage of progeny viruses.The cross-protection efficacy of ZYMV-HC-ProK364D mutant was evaluated through cross-protection assays.[Result]The K364D mutation in HC-Pro attenuated ZYMV virulence,alleviated typical yellowing symptoms and reduced ZYMV accumulation in Cucumis melo plants.Simultaneously,K364D mutation suppressed the RNA silencing suppression activity of ZYMV HC-Pro.The attenuated mutant ZYMV-HC-ProK364D-GFP maintained genetic stability without reversion mutation after three successive passages in C.melo plants.In cross-protection assays,ZYMV-HC-ProK364D mutant induced significant resistance in C.melo plants against the wild-type ZYMV infection when the protection interval was 11 d.[Conclusion]The conserved K364 residue in HC-Pro was crucial in determining the RNA silencing suppression activity and the virulence of ZYMV.The ZYMV-HC-ProK436D mutant present excellent cross-protection efficacy and is a potential mild vaccine against the wild-type ZYMV infection in C.melo plants.