Analysis Of Differentially Expressed Genes In The Infecton Of Different Hosts By Rhizoctonia Solani AG1-IA And The Function Analysis Of A Secreted Protein Inducing Plant Cell Death

Rhizoctonia solani AG1-IA is the pathogen causing rice sheath blight which occurs frequently in rice growing areas around the world with a trend of increasing year by year.The occurrence of this disease can lead to the reduction of rice yield and rice quality,thus causing serious economic losses.Its genome,which was the first to be sequenced from the Rhizoctonia genus,may serve as a model for studying pathogenic mechanisms in rice.In this study,in order to explore the different mechanisms of R.solani AG1-IA during the infection of different hosts at the transcriptional level and lays a foundation for the screening of key pathogenic genes,the differentially expressed genes produced by the transcriptome sequencing data of R.solani AG1-IA from different hosts during the infection of rice,maize and soybean was further analyzed and excavated.First of all,the accuracy of transcriptome data was verified by real-time fluorescence quantification.In addition,a total of 2,319 differentially expressed genes were identified by Edge R software,and 64 out of104 predicted differentially expressed effectors were found up-regulated during the infection of different hosts.At the same time,the different expression patterns of pathogenicity-related genes（mainly including candidate effectors,carbohydrases,etc.）of R.solani AG1-IA from different hosts during the infection of different hosts or the same host were revealed,and these diferentially expressed genes could also refect the adaptation of R.solani AG1-IA to diferent host plants.Also,it was found that there is a high proportion of alternatively spliced genes in R.solani AG1-IA during the infection of different hosts,especially some genes related to pathogenicity.On the other hand,the expression of CAZymes in R.solani AG1-IA existed preference during the infection of different hosts.CAZymes that mediate cellulose,hemicellulose and starch could be more necessary during infection of rice.Therefore,CAZymes that mediate the degradation of celluloses,hemicellulose and chitin could be likely more required during the infection of maize,and those that mediate pectin degradation could be more important during the infection of soybean.An agrobacterium-mediated transient expression system was used to screen and 10predicted cysteine-rich secreted proteins wheather they could cause programmed cell death of N.benthamiana were identifid and a preliminary study on the function of a secreted protein（temporarily named Rs IA＿CEP32）which could cause plant cell death was also conducted.The results show that the Rs IA＿CEP32 was firstly found to induce the cell death of N.benthamiana in small cysteine-rich proteins of R.solani AG1-IA.However,after removing its signal peptide,it could not induce the cell death of N.benthamiana.Functional verification of signal peptide showed that the predicted signal peptide of Rs IA＿CEP32 was able to function normally.Sequence homology analysis showed that Rs IA＿CEP32 was conserved and specific in R.solani with a strong positive selection and the 101st amino acid in Rs IA＿CEP32 have an important influence on its function.It was strongly induced during the infection of Teqing and Lement and showed persistent up-regulated expression pattern in the early stage of infection.And the transient expression of Rs IA＿CEP32 in N.benthamiana could stimulate the expression of resistance-defense-related genes and lead to the accumulation of H₂O₂.The subcellular localization of Rs IA＿CEP32 in N.benthamiana showed that there was no positional preference.Rs IA＿CEP32 is non-toxic to the yeast strains but existed self-activating.The GST Pull-down method was used to screen the host target protein,and 10 candidate objective protein has been initially obtained.