Comparative Transcriptome Analyses Of Rice Gene Expression Changes Triggered By Rhizoctonia Solani AG1 IA Infection And Research Of Resistance-related Genes Of Rice

Rice,as an important crop to ensure grain production,plays an important role in maintaining food security.In recent years,rice sheath blight has become the second most important disease after rice blast in China.Due to the complexity of the disease resistance process and the diversity of identification of resistance indicators in rice,the mechanism of rice sheath blight resistance was restricted by traditional methods and the related work moves forward slowly.In this study,the moderately resistant cultivar Te Qing and the high susceptible cultivar Lemont were used as the materials for inoculating Rhizoctonia solani AG1 IA.RNA-seq technique was used to sequence the transcription of two cultivars at different time points of after AG1 IA infection.The molecular mechanism of rice resistance to sheath blight was studied by comparing the transcriptional differences at different time points between Te Qing and Lemont.Based on the comparative transcriptional analysis,we screened and identified the resistance related genes.The analysis of gene function further elucidates the mechanism of rice resistance to sheath blight and also provides a technical basis for the creation of resistant germplasm to sheath blight.The research results are as follows:1.We used an RNA-seq approach to analyze the gene expression changes induced by the R.solani AG1 IA in rice at 12,24,36,48 and 72 h.By comparing the transcriptomes of Te Qing(a moderately resistant cultivar)and Lemont(a susceptible cultivar)leaves,variable transcriptional responses under control and infection conditions were revealed.From these data,4,802 differentially expressed genes were identified.Gene ontology and pathway enrichment analyses suggested that most differentially expressed genes and related metabolic pathways in both rice genotypes were common and spanned most biological activities after AG1 IA inoculation.The main difference between the resistant and susceptible plants was in the timing of their responses to AG1 IA infection.Photosynthesis,photorespiration,and jasmonic acid and phenylpropanoid metabolism play important roles in disease resistance,and the relative response of disease resistance-related pathways in Te Qing leaves was more rapid than that of Lemont leaves at 12 h.Here,the transcription data include the most comprehensive list of genes and pathway candidates induced by AG1 IA that is available for rice and will serve as a resource for future studies into the molecular mechanisms of the responses of rice to AG1 IA.2.Based on comparison of transcriptional profiling,we previously reported that more than 11,947 common differentially expressed genes(TAM>10)were found between the Te Qing and Lemont rice genotypes.In the current study,we extended these findings by focusing on the analysis of gene co-expression in response to Rhizoctonia solani AG1 IA and identified gene modules within these networks using a weighted gene co-expression network analysis.We compared the different genes assigned to each module and the biological interpretations of the gene co-expression modules at different time points in the two rice genotypes to reveal their different responses to AG1 IA.Our results showed that different changes occurred in the two rice genotypes and that the modules in the two groups contained a number of candidate genes possibly involved in pathogenesis,such as VQ protein and other.Furthermore,gene co-expression networks provide comprehensive transcriptional information regarding rice gene expression in response to AG1 IA.The co-expression networks derived from our data provide ideas for follow-up experiments that will help advance our understanding of the translational regulation of rice gene expression changes in response to AG1 IA.3.Transcriptomic analysis showed that the rice variety Te Qing,which is resistant to R.solani AG1 IA,expressed Os RLCK5 at higher levels than the susceptible variety Lemont did,suggesting that Os RLCK5 may be involved in response to R.solani AG1 IA infection.In this study,the molecular characterization and role of Os RLCK5 in rice defense against R.solani AG1 IA infection were examined.Subcellular localization assays indicated that Os RLCK5 was localized to the plasma membrane and cytoplasm.A bimolecular fluorescence complementation assay suggested that Os RLCK5 directly interacts with Os RBCS2 and Os GRX20.RNAi-induced silencing of Os RLCK5 in Te Qing revealed that down-regulation of Os RLCK5 expression significantly impaired resistance to R.solani AG1 IA.Additionally,transgenic Os RLCK5 overexpression in Lemont significantly enhanced resistance to sheath blight and increased the accumulation of reactive oxygen species(ROS)in rice plants at 72 h after R.solani AG1 IA inoculation.Furthermore,ROS-scavenging enzymatic activity and certain defense-associated and jasmonic acid(JA)biosynthetic genes were upregulated in Os RLCK5-overexpressing plants.These findings suggested that Os RLCK5 positively contributes to rice resistance to R.solani AG1 IA through regulating ROS levels and the expression of defense-associated genes.4.Based on the Transcripts per million(TPM)value,comparative transcriptome analysis showed that the transcriptional expression of Os BURP16 in the middle resistant variety Te Qing from 24 h to 72 h was significantly higher than that in Lemont.In this study,we attempted to explore possible effects of Os BURP16 on rice sheath blight resistance through Os BURP16 overexpression in Lemont and interfering the expression of Os BURP16 in Te Qing.Transgenic results showed that Os BURP16 interference reduced the resistance of Te Qing and increased the resistance of Lemont to R.solani AG1 IA.In our study,subcellular localization showed that Os BURP16 also distributes in the nucleus.A series of genes interacting with Os BURP16 were screened by yeast two-hybrid.Interaction gene analysis showed that most of the genes were involved in the process of stress resistance in rice.Os BURP16 was widely involved in the plant related process and further affected the resistance of rice sheath blight.Quantitative analysis of defense-associated genes showed that PR10 and PAL was significantly increased than that in Lemont.Our results suggested that Os BURP16 may regulate plant resistance gene interaction and regulation of resistance-related gene expression through the interaction with related resistance genes and participate in regulation of the expression of resistance related gene and then affected the resistance of rice to sheath blight.