Functional Study Of Os-miR169a In Rice Blast Resistance And Small RNAs In AR156-Mediated Systemic Disease Resistance Against Rice Blast

Rice blast,which is caused by fungal pathogen Magnaporthe oryzae,is one of the most serious diseases of rice.In epidemic years it can lead to rice production reduction and cause huge economic losses.MicroRNAs(miRNAs)are endogenous no-coding RNAs that are typically encoded by endogenous genes and regulate gene expression post-transactionally by mRNA cleavage and translation inhibition.miRNAs are important factors at response to biotic and abiotic stresses that control development,differentiation,metabolism and signaling pathways.However,rice miRNAs involved in the rice blast resistacne are rarely reported.The screening of rice miRNAs differentially expressed by rice blast and understanding the function of the downstream target genes will help to reveal the molecular mechanisms of the interaction between rice blast fungus and plants.In this study,rice blast strain(Guy11)was used to infect susceptible accession Oryza sativa L.japonica cv.Nipponbare.Four small RNA libraries were constructed by using samples from 0,24,48 and 120 hours post inoculation of the rice blast fungus.Differential expressed miRNAs were screened during the infection of the blast fungus by using high-throughput sequence and bioinformatics analysis.The main results are as follows:372 kinds of conserved miRNAs and 133 novel miRNAs were identified.Three hundred and seventy-two kinds of differential expression of miRNAs were identified,with 113 conserved and 42 novel miRNAs have more than doubled induction or reduction.Through Northern blot verification experiment,we detected the abundance of miR169a increased in samples of infection,which is most obvious in the sample of 24 h.Using qRT-PCR and agrobacterium-mediated transient gene expression system in tobacco,we identified 25 target genes of miR169a,from which OsNFYA had a negative correlation with miR1 69a.Furthermore,miR169a could suppress the expression of OsNFYA both mRNA and protein level.In order to study the function of OsNFYA in rice immune system,we established a rice seedling protoplasts overexpression and RNAi.The overexpression of OsNFYA stimulates accumulation of callose and the expression of OsPR1a and OsPBZl,whereas silencing of OsNFYA showed opposite trends.Therefore,we speculate that OsNFYA participates in the plant immume response through SA-dependent signaling pathways.The plant growth-promoting rhizobacteria(RGPR)can optimize the rhizosphere environment,improve crop growth,antagonize pathogens and induce induced systemic resistance(ISR).With Pst(EV),Arabidopsis leaves form plants with AR156 pre-treatment show higher PR1 and reactive oxygen species(ROS)accumulation compared to leaves from control treated plants.AR156-treated plants also activate MPK3/6 signaling and FRK1 gene expression rapidly.Our results indicate that AR156 can induce SAR and trigger PAMP-triggered immunity(PTI).We further studied AR156 in the rice-M.grisea interaction system.The AR156 pre-treatment could improve the resistance against M.grisea and reduce hypha accumulation in the rice leaf.By qRT-PCR analysis,the miR1862e,miR5794,miR396d and miR160a exhibited differential expression profiles in the plants with or without AR156 pre-treatment,indicating a potential role they play in AR156-mediated systemic resistance against rice blast.