The Mechanism Of The Enhanced Basal Resistance To Magnaporthe Oryzae By Silencing The OsDCL1Gene In Rice

Rice is one of the most important food crops in the world. Rice is consumed asthe staple food by nearly half the population in the world. Rice blast disease causesserious damage to the rice growth and great economic losses in rice productionyearly. To adapt to environmental changes and resist the unfavorable factors bydiverse pathogens, plants have evolved two-layered immune systems:PTI and ETI,which can protect against the invasion of the pathogen. Small RNAs are20-to24-nucleotide (nt) noncoding RNAs, widely existing ineukaryotic. miRNAs in plants are processed from gene spacer or introns, which canbe divided into miRNAs (microRNAs) and siRNAs (short interfering RNAs)according to the synthetic pathway. miRNAs and siRNAs are produced by differentRNase Ⅲ-like Dicer enzymes and manifested as key characteristic of RNAsilencing. The DCL1gene is known to engage mainly in the biogenesis of miRNAsand plays a determinant role in plant growth and development, it’s reported thatDCL1genes play an important role in plant immune responses against pathogens inArabidopsis. The function of OsDCL1in the immunity to rice blast pathogen wasexplored in the current studythe enhanced basal resistance to Magnaporthe oryzaeby silencing the OsDCL1gene in rice was found, the main results are as follows:1. The phenotype and resistance of OsDCL1-RNAi mutant. Expressionprofiling demonstrated that different OsDCLs responded dynamically and OsDCL1reduced its expression upon the challenge of rice blast pathogen. In contrast,miR162a predicted to target OsDCL1increased its expression, implying a negativefeedback loop between OsDCL1and miR162a in rice. To further verify the OsDCL1gene indeed participated the rice immunity, we creat the OsDCL1-silencingmutants.In addition to developmental defects, e.g., the dwarf phenotype, narrowleaves, less tillers, chloroplast ectopic expression, the OsDCL1-silencing mutantsshowed enhanced resistance to three virulent rice blast strains in a non-race specificmanner. Accumulation of hydrogen peroxide and cell death were observed in the contact cells with infectious hyphae, revealing that silencing of OsDCLl activated cellular defense responses. Furthermore, the expression of11pathogenesis related (PR) genes and2PTI related genes OsKS4and OsNAC4were activated in OsDCL1-RNAi mutant. The dynamics of these13genes in the interaction between rice and rice blast showed two expression patterns. In the first group, the genes culminated their expression at36hpi or48hpi, then gradually decreased their expression until72hpi. In the other group, the abundance of genes were increased overall upon the challenge of pathogen.2. The analysis of the changed transcriptome and differentially expressed genes induced by silencing OsDCL1gene. To investigate the mechanism, we analysed and compared the transcriptome profiling in between wild type (Nipponbare, NPB) and OsDCL1-RNAi mutant plants upon the challenge of rice blast pathogen. Out of7129differentially expressed genes (DEGs) responsive to the infection of rice blast,5382and5180were identified in NPB and OsDCL1-RNAi mutant, respectively. The genes involved in4pathway groups including biotic stress response, signaling, protein metabolism and RNA regulation were significantly identified in DEGs. Moreover, we identified1318DEGs in OsDCL1-RNAi mutant compared to wild type plants without pathogen infection. Of those DEGs in OsDCL1-RNAi mutant plants,70%were up-regulated and most of them were involved in two pathway groups, i.e., biotic stress response and signaling. Interestingly, the genes encoding various families of pathogenesis related (PR) and cell wall related proteins were frequently identified in biotic stress response genes. We also identified10jasmonic acid and11salicylic acid related genes down-and up-regulated in OsDCLl-RNAi mutant plants, respectively. The differentially expressed genes (up-regulated more than2folds in OsDCL1-RNAi mutant plants compared to wild type plants and up-regulated more than2folds in wild type plants after infection of rice blast) were selected for qRT-PCR validation, the result is consistent with DEGs. Five differentially expressed genes were selected for constructing overexpression vector, which were used for obtaining transgenic plants. The altered transcriptome profiling of OsDCL1-RNAi mutant plants documented in this study could provide new insights into the enhanced resistance mediated by the silencing of OsDCL1in rice.3. The analysis of the differentially expressed miRNAs induced by silencing OsDCL1gene. Small-RNA sequencing was adopted to identify miRNAs related to immune response and12miRNAs’ expression pattern upon the challenge of pathogen were verified using stem-loop RT-PCR, of which5and7down-and up-regulated their expressions, respectively. Finally, three differentially expressed miRNAs were selected to create transgenic plants, through excessive expressing the mimic of target genes in plants. After obtained the mutant plants, we focus on the phenotypic analysis and validation of miRNA159a.1/b mutant.In a word, transcriptome rearrangement and miRNAs biosynthesis that were regulated by OsDCL1gene, activated the basal immune response against rice blast fungus in rice.