Pathogenic Function Of A Key Effector BC1G＿14481 Of Botrytis Cinerea And Identification Of Effecter-targeted Proteins In Grape

Grape(Vitis vinifera)is a deciduous vine fruit tree,which has the characteristics of short childhood,high yield and high economic benefit,but it is easy to be affected by a variety of pathogens leading to yield reduction.Grape gray mold is a fungal disease caused by Botrytis cinerea.In severe cases,the whole ear will rot and be covered with mildew.When Botrytis cinerea infects host,it secretes a series of Effector proteins(Effector)that interact with the resistance and susceptible genes in plants,thus affecting the plants’ resistance and susceptibility to disease.Therefore,it is of great significance for grape disease resistance breeding to screen the key effectors of Botrytis cinerea and understand their interaction mechanism with grape disease resistance and susceptibility genes.In this study,28 effectors were screened based on amino acid number less than 300 and cysteine number more than 3%.Among them,the amino acid number of BC1G＿14481 was less than 300 and contained Y/F/WxC motif,which was homologous to Effector 5 reported in Eutypa lata.BC1G＿03591 amino acid number less than 300 and cysteine number greater than 5%.Therefore,taking BC1G＿14481 and BC1G＿03591 as entry points,BC1G＿14481 was first verified as the key effector,and then the interaction target proteins of the key effector in‘Thompson Seedless’ were screened.The main results are as follows:1.Identification of the key effector BC1G＿14481 from Botrytis cinerea.Bioinformatics revealed that the N-terminal of BC1G＿14481 contained signal peptide and Y/F/WxC motif,which was homologous to Effector 5 reported in Eutypa lata.The N terminal of BC1G＿03591 contains signal peptide and the cysteine content is as high as 7.4%.The mutant strains △BC1G＿14481 and △BC1G＿03591 were obtained by gene knockout technique.Compared with NN,the mycelium growth of BC1G＿14481 grew slowly and the pathogenicity decreased significantly.The mycelia of △BC1G＿03591 grew slowly,and the pathogenicity did not change significantly.Transversal analysis showed that BC1G＿14481was the key effector in Botrytis cinerea.Expression analysis showed that the expression of BC1G＿14481 was significantly up-regulated in the early stage of the infection of Botrytis cinerea.Subcellular localization shows its distribution in the endoplasmic reticulum.2.Pathogenic function analysis of the key effector BC1G＿14481.The transient overexpression of BC1G＿14481 in the leaves of ‘Thompson Seedless’ enhanced the infection of Botrytis cinerea,while the transient interference of BC1G＿14481 inhibited the infection of Botrytis cinerea.These results indicated that BC1G＿14481 is the key pathogenicity protein.3.Study on Function of BC1G＿14481 Interacting protein VvPP2A from Botrytis cinerea.The decoy vectors of BC1G＿14481 showed no self-activation activity.The decoy vectors of BC1G＿14481 were used in the yeast screening library of Chinese wild grapevine‘Shuang you’ yeast cDNA library inoculated with Botrytis cinerea to search for the target proteins interacting with BC1G＿14481.VvPP2A was cloned in ‘Thompson Seedless’,and the interaction between BC1G＿14481 and VvPP2A was confirmed by yeast two-hybrid recovery verification and BiFC experiment.Bioinformatics revealed that VvPP2A contains serine/threonine PP2A domain and calc-like regulatory phosphatase domain.The regulation of protein activity is related to phosphorylation.Subcellular mapping results showed that VvPP2A was distributed in cell membrane,cytoplasm and nucleus.After transient overexpression of VvPP2A in the leaves of ‘Thompson Seedless’,leaf disease index,mycelium number and biomass increased compared with wild type and empty carrier leaves.These results indicated that VvPP2A is associated with Botrytis cinerea susceptibility.