Fine Mapping Of Resistance Gene YrSm139-1B To Stripe Rust And Functional Verification Of Resistance-related Gene TaYRG1 In Wheat

Stripe rust in common wheat（Triticum aestivum L.）,caused by Puccinia striiformis f.sp.tritici（Pst）,is a serious fungal disease in wheat.Deployment of resistant cultivars is an effective way to control the disease and to minimize crop losses,but the deployed resistances usually fail sometime after introduction due to changes in the pathogen population.Therefore,we should strengthen the development of disease resistance genes and to analyze the interaction mechanism between wheat and pathogens.This is important for the identification and cloning of resistance genes.In our previous study,three pairs of near-isogenic lines（NILs）for Yr SM139-1B locus from the cross of Shaanmai 139（SM139）×Hu 901-19,named as 1013R/S,185R/S and1812R/S,were used for constructing the F₂ fine mapping population.In order to construct high density map of Yr SM139-1B,three pairs of NILs were tested by SLAF sequencing（SALF-Seq）and BSR sequencing（BSR-Seq）analysis.Based on the SLAF-Seq and BSR-Seq data,the new markers flanking the region of Yr SM139-1B,were designed to identify the recombination lines.The linked markers were screened to map the resistance gene Yr SM139-1B using NILs.The follow main results were obtained:1.Fine mapping of stripe rust resistance gene Yr SM139-1B and associated candidate genes analysis（1）Based on the analysis of SLAP-Seq,98 SNPs were detected between the three pairs of NILs which were distributed from 180.9 to 684.2 Mb（Ref Seq v1.0）.By using these SPNs,27 AS-PCR markers were designed to construct the linkage map.Moreover,12 AS-PCR markers were selected to genotype the F2 fine mapping population（600 susceptible lines）.Finally,Yr SM139-1B flanked by SLAF75 and SLAF82,was mapped in a 0.9 c M interval in the genetic map corresponding to a 1Mb（238-239 Mb）interval in IWGSC Ref Seq v1.0.（2）According to the BSR-Seq analysis,none of candidate genes related to stripe rest resistance,were detected in the 238-239 Mb region（IWGSC Ref Seq v1.0）.Thus,we hypothesized that Yr SM139-1B could derive from Triticum dicoccoides.Based on the collinear comparison analysis between the genome of hexaploid wheat（IWGSC Ref Seq v1.0）and Triticum dicoccoides in the target region,suggested that the 1Mb（238-239 Mb）interval in IWGSC Ref Seq v1.0 corresponding to a 22 Mb（216.2-238.5）in Triticum dicoccoides.Combing gene annotation and q RT-PCR identified potential candidate genes,we will continue to reduce the target candidate gene region by fine mapping work.These results provide insight for researching the gene function and mechanisms in the strip rust resistance.2.Functional verification of wheat related disease resistance gene Ta YRG1When characterizing the resistance-related genes in SM139,a suppression subtractive hybridization（SSH）-generated clone was annotated to a disease-resistant homolog.An EST-STS marker-derived from this clone was found to be linked with the stripe rust resistance locus Yr SM139-1B.Sequence analysis showed that the co-localized EST-STS marker overlaps with a NB-ARC gene.An EST sequence was isolated as the initial probe and we obtained the full-length sequences via RACE,this gene was designated Ta YRG1 and associated with distinct alternative splicing events in wheat infected by Pst.In the current study,we investigated the expression and function of this NB-ARC gene in stripe rust resistance.Surprisingly,we found that this R gene,encoding a non-toll and interleukin-1 receptor（non-TIR）NB-ARC gene,generates five natural splice variants in SM139 infected with the stripe rust pathogen CYR32.The native splice variant,Ta YRG1.6,encodes internal motif-deleted polypeptides with the same N-and C-termini as Ta YRG1.1,resulting in gain-of-function.Following inoculation with Pst CYR32,the expression of Ta YRG1 transcripts were upregulated,this indicated that expression of the NB-ARC homolog Ta YRG1 was induced by the stripe rust pathogen.To test the function of Ta YRG1 in stripe rust resistance,we knocked down its expression in wheat by virus-induced gene silencing（VIGS）using BSMV.The result indicated that VIGS-induced downregulation of wheat Ta YRG1 enhances disease susceptibility.Ta YRG1.6 induced a robust HR observed as clear necrosis of the infiltrated region at 3 d post-agroinfiltration in N.benthamiana.Furthermore,molecular genetics analyses indicated that Ta YRG1.6 enhanced resistance to Pst in wheat.Ta YRG1.6 interacts with a dynamin-related protein（Ta Drp1）via yeast two-hybrid and Bi FC assay.Proteome profiling suggested that the Ta YRG1.6-Ta Drp1-DNM complex in the membrane trafficking systems may trigger cell death by mobilizing lipid and kinase signaling in the endocytosis pathway.Our findings reveal a unique mechanism by which Ta YRG1 activates cell death and enhances disease resistance by reconfiguring protein structure through alternative splicing（AS）.