The Exploration Of The Underlying Functional SRNAs In Phytophthora And The Functional Study Of Dicer And Argonaute Genes In SRNA Pathway

Phytophthora could infect a variety of crops,causing serious economic losses worldwide.Small RNA(sRNA)is widespread in many organisms,and it plays an important role in many biological processes as an epigenetic regulator.In this research,the functional study of sRNAs and their relative pathways in development and infection processes of Phytophthora were conducted,and the application of HIGS in Phytophthora disease control was also studied.The results were summarized below:1.The miRNAs with significantly different expression levels after infected by P.capsici mainly took part in plant-pathogen interaction,catalysis of plant biological processes,etc.The miRNA with increasing expression level after infection was involved in the plant response to biotic and abiotic stress and the regulation of resistant genes expression under the non-stress condition,which could reduce the toxicity of the resistant proteins.2.Among all the endogenous sRNA of P.capsici,25-26 nt sRNA was found to have the highest abundance.All the microRNA candidates have a strong preference of adenine at the 8th nucleotide.The differently expressed sRNAs in different stages of P.capsici.were mainly involved in the processes associated with combination and catalytic activity.This is the first time to identify sRNA in different life stages of P.capsici,which could be an useful database to further explore the function of Phytophthora sRNAs.3.Two Dicer genes were identified in both P.capsici and P.sojae,three and at least five Argonaute genes were identified in P.capsici and P.sojae,respectively.The functional study of DCL1,DCL2,AGO1 and AGO4 in P.capsici,and DCL1,DCL2 in P.sojae show that they were involved in hyphal growth,sporangium production,zoospore production,oospore production and virulence in Phytophthora,and the virulence of ?PcDCL1 and ?PcAGO1 isolates on tobacco were regained after co-expressing PcDCL1 and PcAGO1 in Nicotiana benthamiana.All these data implicated that sRNAs and their related pathways were essential for Phytophthora.There was a big difference of sRNA profiles between Empty vector isolate(EV)and dcl1 silencing isolate,which implies that sRNA could be a key factor in the phenotype change.4.The AGOs were over-expressed in P.capsici,and the RNA Co-IPed with AGOs were analyzed.RNA with different molecular weight could bind to PcAGO1 and PcAGO4.The sRNA sequencing of PcAGO1-binding sRNA shows that many functional sRNAs were loaded into AGO1 of P.capsici,which implicated that they may induce gene silencing through a canonical miRNA pathway.5.Two miRNA candidates were found in Phytophthora,and functional study shows that they were involved in development and virulence of P.capsici.The tight regulation of functional genes with miRNA was also found,which means that miRNA could trigger a change in downstream biological process through regulating their target gene(s).6.The artificial siRNA and trans-kingdom gene silencing of P.capsici sRNA related genes in plant could significantly enhance the resistance of plant against Phytophthora.Therefore,it could be a new alternative approach to control plant disease caused by Phytophthora.