Study On The Function Of Rhizoctonia Solani Small RNA In Infecting Rice And The Function Of Os-lsiRNA On Rice Blast Resistance

Rice is one of the three major food crops in the world.Nearly half of the world’s population regard rice as their staple food.Therefore,the safety of rice production is crucial to food security.Rice sheath blight and rice blast are both major diseases of rice.They are caused by Rhizoctonia solani and Magnaporthe oryzae,respectively,which seriously affect rice yield.Therefore,it is particularly important to study the disease resistance mechanism of rice for ensuring the safety of our country’s food safety.Small RNAs are widely involved in the interaction between plants and pathogens.In this study,laser microdissection technology was used to enrich R.solani mycelium inside rice.High-throughput sequencing was used to dig small RNAs that may involved in the infection process.And further experiments will be done to study the mechanism.Besides,our lab’s previous research found some rice long small interfering RNA（IsiRN A）response to M.oryzae infection.To further verify the role of Os-IsiRNA in rice endogenous immunity,we constructed Os-IsiR76113 silent mutant and the predicted target gene,Os12g28260,silent mutant and do futher research..We hoped that this study can provide new ideas for rice sheath blight disease and rice blast disease control,and provide a new theoretical basis for the control of rice diseases.The main research contents are summarized as follows:First,study on the function of R.solani small RNA in the process of infecting rice.Rice stems were infected with R.solani,and the infection status at different time periods were observed through a microscope.Then we determined 60 hours post-inoculation（hpi）as the best sampling time.Laser microdissection technology was used to isolate the R.solani mycelium inside rice,and the total RNA of the cut samples was extracted.The mycelium cultured on the PDA plate was used as a control group,the mycelium from plant surface and the laser-cut mycelium from inside plant stem after 60 hpi were used as two treatment groups.Through high-throughput sequencing,144 kinds of small RNAs were obtained.Compared with the control group,a total of 32 kinds of small RNA expression trends changed.Among them,9 sRNAs were up-regulated and 15 were down-regulated in plant surface group;and the same as this,9 sRNAs were up-regulated and 15 were down-regulated in Lacer-cut group.unconservative_KB317713.1₁4989（milR14989）)was up-regulated in both treatment groups,and two potential targets on rice,Os01g0869800 and Os01g0843800,were found through target prediction.Fluorescence quantitative PCR detection found that the transcription level of Os01g0869800 decreased significantly 48-60 hpi with R.solani;the transcription level of Os01g0843800 decreased significantly at 60 hpi.Os01g0869800 encodes Photosystem Ⅱ subunitS 1（PsbS1）,which is participates in rice photosynthesis.Os01g0843800 encodes a laccase precursor protein 5（LAC5）and some studies have shown that laccase is involved in plant lignin synthesis and cotton broad-spectrum resistance.This suggests that small RNAs of R.solani could play an important role in the process of infection.Second,the function analysis of rice long small interfering RNA（lsiR76113）against rice blast fungus.Previous research found that under the stress of M.oryzae,the expression level of some rice endogenous lsiRNAwill change.Among them,the expression level of OslsiR76113 decreases,whose predicted target gene is Os12g28260,encoding cyclic nucleotide-gated ion channel 5（CNGC5）.It has been reported that CNGCs play an important role in plant’s biotic and abiotic stress response and ionic homeostasis.Therefore,we speculated that Os-lsiR76113 may participate in the regulation of rice resistance to M.oryzae by affecting CNGC5.We constructed Os-lsiR76113 silent mutant（STTM-lsiR76113）and CNGC5,the predicted target gene of lsiR76113,mutant（oscngc5）transgenic rice and performed functional verification.We found that oscngc5 mutant showed more susceptible to M.oryzae than wild type,indicating that CNGC5 may positively regulate rice resistant to rice blast.These results suggest that rice downregulates lsiR76113 expression level after the infection by M.oryzae,and enhances the expression level of the target CNGC5,thereby improving the disease resistance of rice.