Molecular Mechanism Of High Blast Resistance Rice H4 Induced By MicroRNA And CircRNA

In recent years,due to the vigorous development of high-throughput sequencing technology,research on nc RNAs has progressed from the initial stage of identification to the functional research and even the advanced stages of the regulatory network,and made breakthrough progress.Numerous studies have shown that micro RNAs(mi RNAs)play important roles in plant cell growth,tissue differentiation,hormone regulation,and fighting against stress.In addition,a number of micro RNAs that are induced by rice blast are also identified in rice,but the specific mechanisms that they participate in immune responses are unclear.Recent reports indicate that circular RNAs(circ RNAs)have been found to competitively bind to mi RNAs as "sponges," thereby preventing mi RNAs from cleavage of target gene m RNA.Whether the function of mi RNA and its involved regulatory network are regulated or influenced by circ RNA,or how endogenous nc RNAs regulate genes expression,and thus mediating the plants against stress,is still need to be further explored and clarified.In this study,we used a stable high-resistance rice cultivar H4 and susceptible control line Zhongshuangzhan as materials,and further statistical analysis of mi RNAs induced by Magnaporthe oryzae obtained from previous Small RNA sequencing.The potential target genes and the circ RNAs induced by Magnaporthe oryzae were identified using Degradome Sequencing and Circ RNA Sequencing.The functions of some mi RNAs and their target genes were further verified by bioinformatics,biochemistry and molecular biology.A further comprehensive analysis of the intrinsic link between mi RNA,circ RNA and its corresponding target genes.We obtained the following important findings:(1)Targets of known/novel mi RNA which identified in previous study were predicted by Degradome Sequencing.A total of 1175 target genes of 236 known mi RNAs and 176 target genes of 23 novel mi RNAs were identified.The targets of known mi RNA GO and KEGG annotations show that tthey are mainly enriched in transcription-related regulation,hormone signaling networks,and in the synthesis,transport,and degradation of RNA.The targets of novel mi RNA GO and KEGG annotation functional annotations show that they are mainly enriched in enzyme activity regulation,processing and transport of ribosome,plant-pathogen interaction,oxidative phosphorylation,and peroxide reaction,etc.(2)Northern blot showed that except for mi RNA T42 which was detected only in H4,the other 11 mi RNAs could be expressed in both resistant and susceptible materials.q RTPCR of mi RNA before and after inoculation with Magnaporthe oryzae showed that 3 selected known mi RNAs were negatively correlated with the expression of their target genes;9 novel mi RNAs showed similar expression patterns in resistant and susceptible materials,and 8 novel mi RNAs showed different expression patterns in resistant and susceptible materials.Correlation analysis of mi RNA and its targets showed that 7 novel mi RNAs were negatively correlated with their targets in both resistant and susceptible materials,and 4 novel mi RNAs were negatively correlated with their targets in a single variety.The other 3 novel mi RNAs(T13,T31 and T37)have no correlation with their target genes in both materials.(3)A total of 2900 circ RNAs were predicted by circ RNA sequencing.Differential expression analysis showed that there were significant differences in the expression levels of 14,9 and 12 circ RNAs in R＿Mock vs R＿12hpi,R＿Mock vs R＿24hpi and R＿12hpi vs R＿24hpi,respectively,before and after innoculation.Similarly,in the susceptible samples,there were significant differences in the expression levels of 10,12,and 10 circ RNAs between S＿Mock vs S＿12hpi,S＿Mock vs S＿24hpi,and S＿12hpi vs R＿24hpi,respectively.The differential expression patterns can be roughly divided into two categories,one is cultivar-specific,such as circ2465,circ2466,circ192 and circ2595.The other type is pathogen-inducible.For example,after inoculation,the expression levels of circ72,circ519 and circ1577 in the disease-resistant samples increased,and circ236,circ448 and circ1278 in the susceptible samples were down-regulated.Further prediction of the circ RNA-mi RNA binding site revealed that a total of 14 circ RNA binding sites for 5 mi RNAs(T6,T34,T41,T42 and T43)were found.There is a certain mismatch in the corresponding binding sequences of circ RNA-mi RNA.(4)Combining the results of pre-prediction and degradome sequencing,two target genes of novel mi RNA T46 were obtained.One is the main blast resistance genes Pik2-H4 and the other is NADPH oxidoreductase,they are both negatively correlated with the expression level of T46 after after inoculation with Magnaporthe oryzae.Semi-quantitative and quantitative results showed that T46 expressed in roots,stems and leaves.Alignment analysis of Pik locus allele 3’UTR sequences revealed that they have no sequence difference,and they are all predicted to be cleaved by T46.Further studies have shown that overexpression of T46 leads to rice susceptibility.It is speculated that the expression level of T46 is increased in overexpression line,and the disease resistance gene Pik2-H4 is continuously spliced,resulting in degradation of R protein and plant susceptibility.T46 knockout line or target mimic plants can inhibit the cleavage of target genes by T46,so that the disease resistance gene Pik2-H4 can be continuously expressed and conferred resistance to plants;After T46 inoculation with Magnaporthe oryzae,GD0193(Avr Pik H4)and GD0193m(absent Avr Pik H4),the expression pattern did not differ.In the two cultivars,they were induced by Magnaporthe oryzae,and the expression decreased first and then increased,which indicated that there is no direct relationship between T46 and Avr Pikh.