Response Of Rice MiRNA To Rhizoctonia Solani Infection And Early Detection Of Sheath Blight

Rice sheath blight,caused by Rhizoctonia solani Kuhn,is one of the three major diseases of rice which causes serious agricultural and economic losses.To date,some functional genes have been identified to participate in the disease process of resistance to rice sheath blight,but rice variety or immune variety that are resistant to rice sheath blight disease has not been found.Therefore,the current treatment methods for R.solani are still based on pesticide control.Although the damage of rice sheath blight is serious,the research on resistance is progressing slowly partly because of the lack of standard resistance identification methods and the field conditions of small-scale farmland.The molecular mechanism of rice defense against the defense response of R.solani will sheld light on molecular breeding which leads to improve rice yield and quality.1.Comprehensive profile of microRNAs associated with host response to rice sheath blight infection.In this study,the rice sheath blight pathogen R.solani strain YN-7 was used to infect two different resistant cultivar YSBR1 and susceptible cultivar Xudao 3 treating with water/pathogens for 5,10 and 20 hour,respectively.Totally 12 small RNA libraries were constructed including water treatment Xudao3 named XC5,XC10 and XC20,pathogen treatment Xudao3 named XT5,XT10 and XT20;water treatment YSBR]named YC5,YC10 and YC20,pathogen treatment YSBR1 named YT5,YT10 and YT20.(1)The sRNA-seq data were analyzed by bio informatics.The results showed that there were complex and diverse miRNA populations in the tested rice varieties,which provided a meaningful database for discovering the possible molecular basis of plant-pathogen interactions in rice.(2)The expression levels of 4 known miRNAs(miR444b.2,miR531a,miR535-3p and miR166i-3p)and 2 unknown miRNAs(novel miR 1956 and novel miR135)were confirmed by Northern Blot and qRT-PCR analysis.(3)The target genes of these miRNAs were predicted by bioinformatics.The tobacco transient co-expression experiments indicated that LOC Os04g 16540 and LOC_Os11g07270 were regulated at the transcriptional level by OsamiR535-3p and OsamiR166i-3p,respectively.(4)Overexpression of miR535-3p and miR444b.2 enhanced the resistance of rice to sheath blight,and optimized the agricultural traits of rice including plant height,ear length and 1000-grain weight.In summary,in this study,we recruited two rice cultivars,susceptible Xudao 3 and resistant YSBRl,to present different levels of resistance to R.solani strain YN-7.The main objective of this study is to explore the potential miRNAs from sheath tissues upon infection by R.solani in different rice cultivars.Our results indicated the existence of a complex and diverse miRNA population in these rice cultivars.We also identified and validated several miRNAs responsive to the infection by R.solani,thereby providing a valuable resource to the discovery of possible molecular basis underlying rice-R.solani interaction.2.Rapid early detection of rice sheath blight(1)Based on the ITS region,a pair of specific primers from R.solani genome was used for the specific detection of pathogen biomass in rice plant.(2)A standard curve was created using an optimized Quantitative PCR(qPCR)system to quantify the pathogen.The determination coefficient R2 of the standard curve is 0.9963,which is completely suitable for the quantitative detection,and is 10000 times higher than the sensitivity of conventional PCR.(3)After inoculating of five different rice cultivars using R.solani strain YN-7,rice genomic DNA from 2 hpi,5 hpi,8 hpi,10 hpi and 20 hpi rice sheaths were extracted for detecting the biomass of the pathogen.Combinding pathogen hypha staining and qPCR,rice sheath blight could be detected at 10 hpi.The method is a strong specificity,high sensitivity and rapid detection for quantifying the R.solani biomass.It is developed for mastering the occurrence and prevalence of diseases and guiding disease prediction.