Identification Of Effectors Of Podosphaera Xanthii And Preliminary Analysis Of Their Interaction Mechanism With Cucumber Proteins

Cucumber powdery mildew is one of the most serious cucumber diseases worldwide,which is an obligate living parasitic fungus mainly caused by Podosphaera xanthii.Cucumber powdery mildew is an air borne disease.Under the condition of high-temperature and highhumidity,the disease develops and spreads rapidly.After infecting cucumber leaves,Podosphaera xanthii releases effector proteins that enter host cells,interfering with the immune system and facilitating pathogen infection and proliferation.The screening and functional analysis of effector proteins of Podosphaera xanthii can reveal the interaction mechanism between cucumber and Podosphaera xanthii,which is of great importance to elucidate the resistance mechanism against powdery mildew and to breed disease-resistant varieties.In this study,candidate effector proteins were screened by genome sequencing of Podosphaera xanthii.The expression changes of candidate effectors in cucumber with high powdery mildew sensitivity were analyzed by qRT-PCR,and the HIGS(host-induced gene silencing)was used to elucidate the interaction mechanism between key candidate effector proteins and cucumber proteins.The main research results are as follows:1.In this study,PacBio and Illumina sequencing technology was used to conduct deep sequencing for the genome of Podosphaera xanthii.After assembly of sequencing data with MECAT software,the reference genome of Podosphaera xanthii with a length of 65046586bp was obtained,encoding 32397 proteins.Using SignalP 5.0,TMHMM 2.0 and TargetP 2.0 to predict secreted proteins and the general characteristics of effector proteins,20 effector proteins were finally predicted.2.qRT-PCR was used to analysis the expression levels of candidate effector protein genes in the leaves of cucumber susceptible variety D8 inoculated with Podosphaera xanthii at 0h,6h,12h,24h,48h,72h,96h,192h and in the haustoria at 48h.Five down-regulated genes(Cspm＿G22590,Cspm＿G19598,Cspm＿G09091,Cspm＿G03455 and Cspm＿G00698)were found during the infection of Podosphaera xanthii,while other candidate genes showed no obvious regularity during the infection.Cspm＿G04704 was highly expressed at the early stage of infection of Podosphaera xanthii as well as in the haustoria.And it has a highly conserved Cerato-platanin protein structure.3.Subcellular localization of Arabidopsis thaliana protoplasts showed that Cspm＿G04704-GFP which carries signal peptide is widely localized in the nucleus and cytoplasm,while Cspm＿G04704-m-GFP,lacking the signal peptide,is also widely localized in the nucleus and cytoplasm.Comparing with the yeast strain YTK12 only carrying the pSUC2Avr1b,yeast strain YTK12 carrying pSUC2-Cspm＿G04704 and pSUC2-Avr1b could secrete invertase which hydrolyze sucrose into monosaccharides,and turn red after reacting with TTC.The secretory system of yeast confirmed that the protein encoded by Cspm＿G04704 effector gene was a typical secreted protein.4.Using host-induced gene silencing to analyze the function of Cspm＿G04704 gene.The results showed that comparing with injecting with an empty plasmid,plants injected with Cspm＿G04704 silencing plasmid significantly improved their resistance to powdery mildew,indicating Cspm＿G04704 has a significant pathogenicity.Yeast two-hybridization,GST pull down and rotation verification experiment proved that CsaV3＿2G015280(Fructosebisphosphate aldolase)is the target protein of Cspm＿G04704 protein during the infection of Podosphaera xanthii.The above results provide a basis for revealing the pathogenic mechanism of Podosphaera xanthii and its interaction mechanism with cucumber.It also provides a genetic target by controlling the cucumber fructose-bisphosphate aldolase(CsaV3＿2G015280)target protein precisely,for regulating cucumber powdery mildew resistance with molecular biological techniques in the future.