Isolation Of Sheath Blight Disease Resistance Genes Based On The Genetic And Transcriptomic Approaches In Rice

Rice is one of the most important creal crops,cultured in worldwide,and most of area growth rice in China.Rice sheath blight is a fungal disease caused by Rhizocatonia solani,which is one of the three major rice diseases and can cause severe yield loss to 50%.Chemical control is still the main method for rice sheath disease.Therefore,screening and utilizing durable and stable resistant sources,and study the mechanism of rice resistance to sheath blight,will be the premise to breeding resistance to sheath blight and the important way to control the disease.This study was based on rice Ds insert mutants to screening resistance genes against sheath blight disease.Further,the Ds insertioan sites were cloned by using i PCR approach in the strongly resistant to subsequent mechasnim characterization.In addition,the transcriptome analysis was performed using Rzhicotonia solani AG1-IA inoculated rice leaves to isolate the genes associated with sheath blight disease.Based on those analyses,the following results were obtained:1.The starter line for expanding group that was homozygous of Ds-s line with a single Ds insertion in chromosome 1.The Ds-s plants exhibited a similar response to rice sheath blight as the wild-type control plants Dongjin.After expanding the Ds insertion population via tissue culture regeneration methods from the start line,Southern blot analysis used to analyze porlymorphism of the Ds element in 300(Regeneration 1)R1 heterozygote lines generated from Ds-s.R2 homozygoute plants were generated by selfifing of 300 R1 lines.Southern blot analysis indicated that Ds was actively transposed in R1 plants.2.300 R2 mutant plants were inoculated with R.sonali AG1-IA to isolate the resistant mutants.Out of 300 R2 lines,0.67% showed susceptibility,and 0.67% exhibited strong resistance to rice sheath blight.In addition,7.6% displayed resistant symptoms,and the remaining 91% lines showed no difffferences compared to the rice sheath blight susceptible wild type control plants.3.The Ds flanking sequences were cloned in sheath blight disease strong resistant lines.The sequencing results indicated that the Ds was inserted at the second intron of PHYB gene,and Southern blot analysis indicated that a single Ds insertion was identifified in the phy B mutants.The RT-PCR results showed that no visible transcript was detected in the phy B mutants.4.RT-PCR results was performed to examine phy B defense mechanism.The results showed that Ethylene signaling genes(EIL1,EIL2,EIN2,and ERF063),Jasmonic acid synthesis gene(LOX2),and Salicylic acid signaling gene PBZ1 were up-regulated in the phy B mutants compared with the wild-type control,suggesting that PHYB somehow regulates hormone signaling.Further genetic study using EIL1 RNAi and EIL1 overexpression plants revealed that EIL1 positively regulates the resistance of rice to rice sheath blight.These results suggest that PHYB negatively regulates the resistance of rice to rice sheath blight via the suppression of ethylene signaling.5.To further screen the resistant genes of rice sheath blight,the transcriptome analysis was performed using R.solani AG1-IA inoculated rice leaves.After 48 hours of the inoculation,6838 genes were differentially expressed in rice leaves(>2 folds,P<0.05).Among them,3802 genes were up-regulated while 3036 were down-regulated compared to control group.Nine genes of all differentially expressed genes were randomly selected and their expression was verified via q RT-PCR.6.The results were similar with transcriptome data.Further,the differentially expressed genes were classified via GO and KEGG,and Mapman analyses.GO terms include binding and catalytic activity,cell part and metabolic process were significantly enriched,and 30 KEGG pathways include ribosome,carbon metabolism,biosynthesis of amino acid.Mapman analysis demonstrated that phytohormone and metabolic pathways were significantly altered.WRKY53 was identifed from transcriptome results,Os WRKY53 transgenic lines were tested with rice sheath blight showed that Os WRKY53 negatively regulate rice resistance to rice sheath blight.In addition,many differentially expressed genes contain R.solani responsive cis-elements in their promoter region.Overall,this study use genetic and transcriptomic approaches to isolate genes resistance to sheath blight in rice,which provided a theoretical basis for future studies on resistance mechanism of rice.