Identification And Characterization Of Promoters And Genes Of Rice In Response To Pathogen Infections

Rice is an important food crop,but various diseases often lead to a large-scale reduction in rice production,posing a serious threat to global food security.Bacterial blight and rice blast are the two most common diseases in rice growing areas worldwide.The use of rice's own resistance to control diseases is the most economical and effective method,and this depends on a comprehensive and profound understanding of rice disease resistance mechanisms.At present,the rice genomic data and a large number of transcriptomic data infected with Xanthomonas oryzae pv.oryzae(Xoo)and Magnaporthe oryzae(Mor)are available from public databases.It is of great significance to make full use of these big data resources to reveal the molecular mechanisms of rice in response to pathogen infectionsThis study is divided into four parts.In the first part,the pathogen-inducible promoters(PIPs)were identified from the rice genome,and the characteristics of the genes under the control of PIPs were analyzed.In the second part,the rice genes that frequently respond to the infections of Xoo and Mor were identified and analyzed.In the third part,members of the rice NLR gene family were re-identified and their transcriptional activities induced by pathogens were analyzed.In the fourth part,one identified rice NLR gene LOC_Os01g70080,which was most up-regulated by Xoo infection,was edited.The main results are as follows.1.Identification and characterization of rice genes driven by pathogen-inducible promoterA set of 882 rice genes regulated by PIPs that contained AS-1,G-box,GCC-box,and H-box cis-regulatory elements was identified.Of these genes,190 encode disease resistance/susceptibility related proteins,and 70 encode transcription factors.Analysis of the rice Microarray data infected with pathogens demonstrated that 357 transcripts regulated by PIPs were differentially expressed after pathogen infections.Of the 53 transcription factor encoding genes regulated by PIPs,48 were up-regulated or down-regulated by more than 1.1-fold in response to pathogen infections.Analysis of the RNA-seq data infected with pathogens showed that 327 transcripts regulated by PIPs were differentially expressed after pathogen infections.A total of 100 up-regulated genes and 37 down-regulated genes were found in common between the Microarray and RNA-Seq data by comparative analysis2.Identification and characterization of genes frequently responsive to Xoo and Mor infections in riceThrough a comprehensive analysis of the Microarray data from a variety of rice samples with inoculation of Xoo and Mor,we identified 12932 genes regulated by Xoo and 2709 genes regulated by Mor.GO enrichment analysis indicated that the up-regulated genes were most frequently located in mitochondria,and the down-regulated genes were most frequently located in chloroplast.Cytokinin-related processes were most frequently repressed by Xoo,while processes relevant to jasmonic acid and abscisic acid were most frequently activated by Xoo and Mor.Among genes responsive to Xoo and Mor infections,defense responses and diverse signaling pathways were the most frequently enriched resistance mechanisms.InterPro enrichment analysis showed the Zinc finger domain family,WRKY proteins,and Myb domain proteins were the most significant transcription factors regulated by Xoo and Mor.KEGG analysis demonstrated pathways including 'phenylpropanoid biosynthesis','biosynthesis of antibiotics','phenylalanine metabolism',and 'biosynthesis of secondary metabolites' were most frequently triggered by Xoo and Mor,whereas 'circadian rhythm-plant' was the most frequent pathway repressed by Xoo and Mor.3.Identification and transcriptional characterization of rice NLR genes responsive to the infections of Xoo and Mor430 regular NLR(Nucleotide-binding site and leucine-rich repeat)genes were identified from rice Nipponbare genome,including 192 CC-NBS-LRR genes and 238 X-NBS-LRR genes.Through the individual and integrative analysis based on 69 samples from rice Microarray after the infections of Xoo and Mor,we found that 397 rice NLR genes were expressed at low/medium level,while 10 NLR genes were expressed at high expression levels.A total of 400 NLR genes were discovered to be differentially expressed in at least one sample.Further analysis revealed that 46 NLR genes were frequently expressed in response to Xoo or Mor and 38 of them were also differentially expressed in RNA-seq data infected with pathogens.Six cis-regulatory elements(MYC,STRE,MYB,ABRE,G-box,and AS-1)exhibited high frequency enrichment in the promoter regions of these rice NLR genes.Ten NLR genes were selected for qRT-PCR detection.The results showed that the detection results of seven NLR genes were consistent with the analysis of Microarray and RNA-seq data.4.Construction of rice LOC_Os01g70080 gene editing vector and acquisition of transgenic plantsWe used CRISPR/Cas9 technology to construct the knockout expression vector of rice NLR gene LOC_Os01g70080,and obtained the genetically modified transgenic rice plants by genetic transformation.