Functional Study Of MiR319 In Modulating Rice Defense Responses To The Blast Disease And Identification Of Sheath Blight Resistance-Related Long SiRNA

Rice is one of the major food crops in the world.Rice blast and rice sheath blight are considered as two of the major diseases that lead to significant yield losses and quality deterioration each year.The system has become a model for studying plant-fungus interactions.Non-coding RNAs,especially small RNAs,play crucial roles in regulating plant responses to diverse stresses.In this paper,we sequenced and compared small RNA libraries to discover miRNA species for rice immunity to Magnaportheoryzae infection.We hope to understand the molecular mechanism of small RNA in the rice-M.oryzae interaction system;we also performed and ayalized deep sequencing results to identify long small interfering RNAs(Osa-lsiRNAs)that may participate in response against Rhizoctonia solani infection.We also studied the biogenesis and functional mechanism of Osa-lsiRNAs,especially in a context of defense responses against pathogen infection.We hope that this study will provide build the fundation for prevention and control of rice blast and sheath blight diseases.The main research results are summarized as follows:1.M.oryzae defeats rice defense by inducing miR319b and suppressing Jasmonic acid signaling.By comparing small RNAs from mock-treated and Guy11-infected rice plant,we identified candidate miRNAs that may be involved in rice immunity because of their differential expression to the infection of the Guyll.We found that Guy11 induced miR319b accumulation,while the expression of miR319-regulated TCP(TEOSINTE BRANCHED/CYCLOIDEA/PCF)gene OsTCP21 was negatively correlated.The expression of miR319b was not affected by an avirulent M.oryzae strain(strain 2539).Moreover,overexpressing miR319b in rice exhibited enhanced susceptibility to Guy11.In contrast,overexpressing OsTCP21 in rice demonstrated lower susceptibility to Guy11.Mutation of miR319 or Os TCP21affects the conversion of linoleic acid(LnA)to hydroperoxy-octadecadienoic acid(HPODE),as well asmodulates the expression of JA biosynthesis and signaling-related genes(OsLOX2and OsLOX5).Taken together,the miR319/OsTCP21 module regulates rice defense responses to the blast disease through affecting the JA signaling pathway.2.Rice ls:iRNAs may participate in response against R.solani infectionby silencing defense-related genes.By comparing deep sequencing data,we identified a group of rice lsiRNAs(Osa-lsiRNAs)that are within the range of 25-40 nt in length.Our results show that some rice IsiRNAs are differentially expressed upon infection of R.solani,the causal agent of the rice sheath blight disease.Bioinformatic analysis and experimental validation indicate that some rice lsiRNAs can target defense-related genes.lsiR51031 was suppressed by R,solani infection.In contrast,lsiR118183,194568,and 73750 showed a sustained increase with R.solani infection.lsiR51031 targets a gene encoding the putative cytochrome b559 subunit alpha(Os08g15322).Osa-lsiR118183 targets a gene encoding a putative transferase Osa-lsiR194568 targets a gene encoding a putative receptor-like protein kinase 2 precursor(Os06938990).lsiR73750 targets a gene encoding a TIR-NBS type disease resistance protein(Os09g14490).We further demonstrate that rice lsiRNAs are neither derived from RNA degradation nor originated as secondary small interfering RNAs(siRNAs).Moreover,lsiRNAs require OsDCL4 for biogenesis and OsAGO18 for function.Therefore,our study indicates that rice lsiRNAs are a unique class of endogenous sRNAs produced in rice,which may participate in response against pathogens.