Functional Identification Of OsbHLH81 Gene In Rice Resistance To Bacterial Leaf Streak And Bacterial Blight

Plant diseases are a serious threat to food security in the world.Rice bacterial blight(BB)caused by Xanthomonas oryzae pv.oryzae,Xoo and bacterial leaf streak(BLS)caused by Xanthomonas oryzae pv.oryzicola,Xoc are the two main types of bacterial diseases of rice.In South China,Central China and East China,as well as in the Southeast Asian rice region and the African rice region,under the right conditions,the two major diseases can cause serious damage at the same time.At present,there have been identified major resistance genes against bacterial blight,but rice BLS resistance is a quantitative trait controlled by multiple genes.Practice has shown that the discovery of disease-resistant genes and the development of rice varieties with broad-spectrum resistance are important for combating these two bacterial diseases.Using differentially expressed cDNA library technology to study the expression of differentially expressed genes in high-resistance material Acc8558 and susceptibility material zhonghua 11 during pathogen infection and to analyze their correlation with identified QTLs,screened for genes associated with bacterial leaf streak resistance with priority for research AK066188(LOC_Os09g33580),this gene encodes an unknown function transcription factor bHLH81.The homologous gene UPA20 in pepper was proved to be the target gene of the avrBs3 of Xanthomonas campestris pv.vesicatoria,and the expression of UPA20 promoted cell enlargement and caused disease.UPA20 homologous gene BPEp in Arabidopsis can participate in the regulation of petal size.OsbHLH81 is a homologue of UPA20 in rice with a sequence similarity of 63%.It is hypothesized that the OsbHLH81 gene may have a similar mode of UPA20.In order to study the biological function of OsbHLH81 gene in resistance to bacterial diseases in rice,this study first demonstrated that OsbHLH81 gene has the typical characteristics of transcription factors through bioinformatics analysis,subcellular localization and transcriptional activation verification.OsbHLH81 can be induced by both Xoc and Xoo.In order to further study the role of OsbHLH81 in disease resistance response,inhibitory and overexpression vectors were constructed and positive transgenic lines were obtained through Agrobacterium-mediated genetic transformation.After inoculation with the pathogen Xoc and Xoo,it was found that inhibition of OsbHLH81 gene expression significantly enhanced rice resistance to bacterialleaf streak and bacterial blight compared to wild type,while over-expression of this gene weakened the resistance to two diseases.These results indicate that OsbHLH81 negatively controls rice resistance to stripe disease and bacterial blight.In addition,RNAi transgenic lines could activate the expression of OsAOC,OsAOS and OsPOX after inoculation with Xoc,but could not up-regulate the expression of PR gene.It was suggested that the OsbHLH81gene-mediated resistance to Xoc may be related to the Jasmonic acid signal pathway and not to the PR gene.In order to fully demonstrate the resistance results of RNAi transgenic materials,OsbHLH81 gene knockout mutants were obtained using CRISPR/Cas9 gene editing technolog,Inoculation experiments confirm that OsbHLH81 gene knockout mutants can increase resistance to bacterial streak disease...