Identification And Pathogenic Function Analysis Of Effector Gene Of Valsa Mali

Apple Valsa canker,caused by the weakly parasitic ascomycete Valsa mali（Vm）Mayabe et Yamada,anamorph: Cytospora sacculus [Schwein.] Gvrit.,has always been key problem in apple production.However,lacking of deep understanding of the mechanism of pathogenesis of canker on tree bark,no efficient control measures developed so far.Therefore,analysis of the interaction between Vm and apple would be the critical problem.More and more research suggests that effectors secreted by pathogens promoted its infection and colonization,which would provide an important material resources for the target loci design of new fungicides as well as establish disease-resistant breeding theory.Thus,it is very urgent to discover and analysis the pathogenicity of effector gene during infecting apple.In this study,we confirmed that Vm contained effector genes as well as some of them has always been virulence factor during infection.The main results are as follows.1.Prediction and identification of effector gene in Vm.The Vm genome contains 11261protein-coding genes.Using bioinformatics analysis,193 secreted proteins with signal peptide,no transmembrane structure,cysteine-rich and no known function were predicted from genomic data in total.Candidate effector proteins（CEPs）have shorter sequence length（about233 aa）and markov clustering suggests that there is no obvious expansion of Vm CEP families.Based on Agrobacterium-mediated transient over-expression in Nicotiana benthamiana performed for 74 randomly selected CEPs,67 V.mali Effector Proteins（VmEPs）could not induce cell death on N.benthamiana and not suppress BAX-induced PCD,7VmEPs were shown to significantly suppress BAX-induced PCD.Analysis of RT-PCR revealed that 5 of the 7 VmEP genes were up-regulated（fold change >2）within 48 hours after inoculation.Particularly,VM1G₀5336 was remarkably induced up to 30 fold.BLAST analysis showed that VM1G₀2400（VmEP1）contained a HeLo domain and VM1G₀5336（VmEp2）was Vm-specific gene.Polymorphism analyses of the two effector genes in different intraspecific（Vm）and interspecific（V.pyri）isolates revealed that VmEP1 and VmEP2 have conserved sequences,indicating the two genes might play an important role during early infection.2.Pathogenicity analysis of effector genes VmEP1 and VmEP2.Firstly,the deletion cassette was generated by double-joint PCR.For generating deletion mutants,the VmEP1gene-replacement construct was transformed into protoplasts of Vm.Among the resulting 125hygromycin-resistant transformants of VmEP1,four were identified by PCR analysis.These two VmEp1 deletion mutants ? VmEp1-74 and ? VmEp1-85 were further confirmed by Southern hybridization.And then the ?VmEp1/VmEp1 complementary mutants?VmEp1-74#-1 and ?VmEp1-85#-1 were obtained.Simultaneously,VmEP2 over-expressed mutants ΔVmEp2-6# andΔVm Ep2-15# were achieved as well.Results of vegetative growth and conidiation certified that VmEp1 deletion and VmEp2 over-expression had no effect on mycelial growth and conidiation on PDA medium.Intriguingly,pathogenicity assays showed that both VmEp1 deletion mutants ? VmEp1-74 and ? VmEp1-85 had significantly reduced virulence（about 45%）（P < 0.05）on apple twigs and leaves.Complemented mutants?VmEp1/VmEp1 exhibit similar virulence as the wild type isolate 03-8.However,VmEp2over-expression unexpectedly had no effect on pathogenicity.These results indicate that VmEp1 is undoubtedly involved in virulence of Vm.Further functional analysis of piecewise deletion VmEp1 revealed that the function motif which contribute to suppress BAX-induced PCD exist in the 340-354 aa for secreted protein.Above all,the pathogenicity returned 30%when this domain was complemented to the deletion mutant ?VmEp1-85.Also subcellular localization of VmEp1 in tobacco cells showed that VmEp1-eGFP fusion proteins were located nucleus.3.Construction of cDNA library.A normalized cDNA library of V.m-apple shoots was constructed by SMART technique.Quality evaluation indicate that the normalized cDNA library contained about 3.6×107independent clones and the insertion size of cDNA was between 750 to 2000 bp with 96% recombination rate.Sequencing analysis for randomly selected 96 clones,91% of the fragments were corrected and completed.What’s more,the proportion of host genes and pathogen genes included in the cDNA library was nearly to 8:1.GO functional annotation exhibited that gene functions involved 14 classes with nearly 200 biological processes,including basal metabolism,cell development and growth process,plant resistance,biological regulation,immune and stress reaction.Which indicating the cDNA library contained all the information of Vm-apple interaction.4.Preliminary identify and prediction of the target.Based on the cDNA library and yeast two hybrid（Y2H）,30 blue clones were obtained on the SD/-Ade/-His/-Leu/-Trp/X-α-Gal（B）plate,after two generation successive culturing,4 stable clones were gained at last.After sequencing and bioinformatics analysis,a gene（tentatively named Mwrky33）with high similarity to the WRKY33 transcription factor in Arabidopsis thaliana was obtained.And then constructed the prey vector pGADT7-Mwrky33,and verified VmEp1 indeed interacted with Mwrky33 again by Y2 H.Analysis of RT-PCR demonstrated that the target gene Mwrky33 were significantly down – regulation at 12-36 h,particularly at 24 h after inoculating deletion mutant ?VmEp1-85.Subsequently,and the autophagy genes were significantly down– regulation as well at 36 and 48 h.Numerous studies showed that transcription factor WRKY33 regulates the expression of host defense related autophagy gene during necrotrophic fungus infection.As a result,we speculated VmEp1 interacted with Mwrky33,which might affect the Vm infection by regulating the expression of host autophagy related gene.Of course,this possibility still needed to be further verification.