Functional Analysis Of PvCRN11 And PvCRN20 Effectors Of Plasmopara Viticola

Grapevine（Vitis L.）is one of the most widely-grown fruits in the world with high economic value.Plasmopara viticola could infect the leaves,inflorescences and tender tissues of grapes,resulting in a decrease in its yield and quality.Many plant pathogens can secrete effector proteins to counteract the immune response of plant.At present,we know little about the molecular basis of the pathogenicity of effectors.The CRN effector produced by oomycete pathogens can manipulate host physiological and biochemical events inside host cells.From the genome information of the grape downy mildew strain collected of in Yangling,Shaanxi preliminary,we found 31 CRN proteins.In this study,the effects of different PvCRNs on plant immune responses were investigated by subcellular localization analysis,PCD and mechanism of PvCRN11 and PvCRN20.By analysing the expression pattern of PvCRN11 at different stages when infecting the susceptible grape‘Pinot Noir’（Vitis vinifera）,it was found that PvCRN11 could be induced by downy mildew.Subcellular localization analysis showed that PvCRN11 was localized in the cell membrane and nucleus.To explore the influence of PvCRN11 on plant immunity,pCAMBIA2300-GFP/PvCRN11 was transformed to N.benthamiana leaves by A.tumefaciens,and after 48 hours,P.capsicia was inoculated.The result showed that could inhibit the infection of Phytophthora capsici.The same result showed in over-expression PvCRN11 transgenic Arabidopsis thaliana,which meant that PvCRN11 could enhance the resistance of Phytophthora capsici in Arabidopsis thaliana and tobacco.In addition,the relative expression levels of both SA pathway-related genes PR1 and PR2 and JA/ET pathway-related genes PDF1.2 and COR1 wrere analysed.The results showed that PvCRN11 could up-regulate the SA pathway-related genes and down-regulate JA/ET pathway-related genes to regulate the pathogen resistance of transgenic plant.PvCRN20 expressed at early during the host infection and significantly up-regulated at72 h.PvCRN20 could supress the cell death induced by elicitor-induced INF1 in N.benthamiana.The expression analysis of NbEDS,NbSGT1,NbRAR1 and NbHSP90 in INF1pathway indicated that PvCRN20 can down-regulate these genes inhibiting the cell death induced by INF1.After transiently expression of PvCRN20 in N.benthamiana leaves and P.capsici inoculation,it was found that overexpression of PvCRN20 could enhance the infection of Phytophthora capsici.To exlore the mechanisms behind it,diami-nobenzidine（DAB）was used to evaluate H₂O₂ accumulation in infected tissues,and the result showed that PvCRN20 impair plants’pathogen resistance by repressing the accumulation of hydrogen peroxide.We found PvCRN20 was localized in the cell membrane and nucleus,and then,we added a nuclear export signal NES（NELALKLAGLDINK）at the C-terminus in order to search weather PvCRN20 employ the nucleus to work.As a result,it was found that PvCRN20^NES could still supress the cell death induced by INF1 and enhance the infection of P.capsici,indicating that PvCRN20 didn’t rely on the nuclear to work.