Cloning And Functional Verification Of Wheat Sharp Eyespot Resistance Gene TaFBW2-7A

Wheat Sharp Eyespot is a soil-borne disease of wheat which is mainly caused by Rhizoctonia cerealis.This disease occurs widely all over the world and seriously affects the yield and quality of wheat.With the popularization of high-throughput sequencing and the continuous development of molecular breeding,finding resistance genes and cultivating resistant varieties have become the most effective and safe way to control Wheat Sharp Eyespot.Using the genome sequencing information of Rhizoctonia cerealis epidemic strain R0301,combined with transcriptome data and gene polymorphism analysis,we explored a key toxic gene RcNRPS-inpB,which encodes non-ribosomal peptide synthase(NRPS)protein.And the function of the NRPS gene was verified by virus induced gene silencing(VIGS)technology.Using the NRPS gene as the bait protein,the interaction protein was screened in the yeast library by yeasttwo hybrid assay.Interaction between the NRPS and TaFBW2 was confirmed by luciferase experiment.The VIGS,phenotypic identification of mutants and gene editing materials showed that this gene significantly improved the resistance to Wheat Sharp Eyespot.The main results of this study are as follows:1.18 NRPS were predicted by using the genome sequencing information of epidemic strain R0301 in Wheat Sharp Eyespot.Combined with the transcriptome data induced by R0301,the results showed that seven NRPS genes responded to Rhizoctonia cerealis induction,of which five NRPS genes had an adenylate(A)domain.Sequencing in 50 Rhizoctonia cerealis strains with different pathogenicity,the results showed that the polymorphism of a gene(EMV0011169)in R0301 was significantly related to the pathogenicity of the strain,which was named as RcNRPS-inpB.2.The results of VIGS showed that the expression of RcNRPS-inpB gene decreased significantly in plants.Compared with the wild type,the disease index(DI)decreased significantly after this gene was silenced.3.Using yeast-two hybrid assay and RcNRPS-inpB as a bait protein,a protein annotated as FBW2 was screened from wheat c DNA library,which was located on wheat 7A chromosome and named as TaFBW2-7A.Furthermore,strong interaction between RcNRPS-inpB and TaFBW2-7A was confirmed by luciferase experiment.The interaction analysis of different segments showed that the interaction site between RcNRPS-inpB and TaFBW2-7A occurred in the LRR functional domain of TaFBW2-7A.4.After inoculating R0301 with resistant and susceptible materials to Wheat Sharp Eyespot,the expression level of TaFBW2-7A was analyzed at different periods.The results showed that the expression level of TaFBW2-7A was inhibited after inoculating,while the expression level of TaFBW2-7A in resistant materials was significantly higher than that in susceptible materials.It suggests that TaFBW2-7A may positively regulate Wheat Sharp Eyespot resistance.5.Using VIGS technology to silence wheat gene TaFBW2-7A and inoculate sheath blight strain R0301,it was found that the expression of TaFBW2-7A gene decreased significantly in wheat plants after the gene was silenced.Compared with imported BSMV:γ0 and wild type,the disease index of sheath blight was significantly increased,and the resistance of sheath blight was significantly decreased.6.A tetraploid Kronos mutant with early termination of TaFBW2-7A was screened.The identification results of resistance to sharp eyespot showed that the resistance of TaFBW2-7A mutant plants to sharp eyespot was significantly lower than that of wild-type Kronos.Physiological index tests showed that compared with wild-type Kronos,the activities of peroxidase(POD),catalase(CAT)and superoxide dismutase(SOD)of TaFBW2-7A mutant plants decreased significantly,indicating that this gene may indirectly affect wheat resistance to Wheat Sharp Eyespot by activating antioxidant system and maintaining ROS homeostasis.7.The TaFBW2-7A gene silenced plants had been constructed and obtained by CRISPR-cas9 gene editing technology.The phenotypic identification of T1 generation plants were carried out in greenhouse.The results showed that the resistance to sharp eyespot of editing mutant plants were more susceptible than that of wild-type fielder,which further confirmed that TaFBW2-7A gene positively regulated Wheat Sharp Eyespot resistance.8.At present,the overexpression vector of TaFBW2-7A gene has been constructed and is been transformed to common wheat.