Preliminary Study On The Functional Domain Of Wheat Stripe Rust Resistance Gene Yr10

Stripe rust is an important wheat disease caused by Puccinia striiformis f.sp.tritici?Pst?.The disease is prevalent in wheat producing areas worldwide and causes significant losses to wheat production.P.striiformis is a biotrophic fungus producing five spore types,and reproducing asexually and sexually between two different hosts.Asexual reproduction of Pst in wheat produces uredospore,teliospores,and basidiospore.Pycniospores and aceiospores are produced in the alternate host of Berberis through sexual reproduction.P.striiformis are also characterized as physiological races with a different pathogenicity in wheat.Different races of stripe rust are virulent or aviruent to different stripe rust resistance genes?designated as Yr gene?of wheat.Emerging of new virulent races leads to the phenomenon of“loss-of-resistance”in originally resistant wheat varieties.Therefore,wheat rust-resistance breeding is a long-term and an indispensable task,which heavily relies on the discovery of new genes resistant to stripe rust.Knowledge of Yr genes,including the locations and the sequences of the Yr genes are a key to molecular breeding.There are currently about 80 Yr gene loci that are designated,but only six Yr genes have been cloned.Yr10,a gene identified in a common wheat cultivar‘Moro'.Yr10 confers resistance to stripe rust starting at the seedling stage,is also called all-stage resistance.In most parts of China,Yr10 is effective against the local Pst races.Therefore,Yr10 also have important use value in disease resistance breeding.Yr10 encodes a typical CC-NBS-LRR class of disease-resistant protein,but little study about its mode of action has been reported.Therefore,it is necessary to carry out a research on understanding the molecular mechanism of the gene,and to provide a knowledge basis for the rational use of Yr10 in rust breeding programs.The main findings of this study are as follows:1.The reported Yr10 sequence?AF149112?is incomplete.We cloned a complete Yr10gene containing four exons and three introns.The complete Yr10 coding region is 2,541 bp in length and encodes 846 aa,named Yr10-1.In addition,another Yr10 with 8-bp lack in sequence is also obtained;the coding region is 2,604 bp in length and encodes 867 aa,named Yr10-2.The two encoded proteins Yr10-1 and Yr10-2 differ only in the end of the C-terminus.Yr10-1 is a typical CC-NBS-LRR disease-resistant protein with a molecular weight of about 95.8 kDa and a theoretical isoelectric point of 6.82,which is a hydrophilic protein.Bioinformatics predicts that the protein has no transmembrane helix and signal peptides,localizes to the cytoplasm,participates in defense response and responds to salicylic acid signals,and has the function of binding ADP/ATP.Phylogenetic tree analysis showed that Yr10-1 is closely related to TuRPM1 of the disease resistance protein in Triticum urartu.2.Localization of Yr10-1 in Nicotiana benthamiana by Agrobacterium-mediated transient expression system was found in the cytoplasm and the protein induced cell death in infiltrated leaves.Artificially altering the subcellular localization of Yr10-1 did not induce cell death when the protein localized in the nucleus of the cells.Similarly,cell death in wheat was observed when a cytoplasmic Yr10-1 was transiently expressed via the Agrobacterium-mediated transient expression system.The infiltrated leaves showed an enhanced resistance to CYR32 and reduced the amount of uredospores.The results indicated that the localization of Yr10-1 in the cytoplasm was required for cell death and resistance against CYR32.3.The key domain that induces cell death in Yr10-1 is the N-terminal CC domain,and the core region is located between 1-116.The CC domain could also induce H₂O₂ in N.benthamiana.The conserved“EDVID”motif in the CC domain plays an important role in inducing cell death in N.benthamiana.The CC domain induced cell death only when it located in the cytoplasm,which was the same as the full length of Yr10-1.The interaction between the CC domains was demonstrated by Co-immunoprecipitation and bimolecular fluorescence complementation techniques.