The Research On Interactions Between Wheat Stripe Rust Resistance Gene Yr10 And Host Target

Stripe rust,caused by Puccinia striiformis f.sp.tritici(Pst),is an important fungal disease in wheat production,which is widely popular in the global wheat producing area.Wheat stripe rust is a biotrophic obligate parasite that can only parasitize,grow and reproduce on living host plants in nature.Controlling the prevalence of wheat stripe rust has become a difficult problem,and the cultivation of lasting disease-resistant varieties becomes a focus of research.The resistant variety containing resistance gene Yr10 is resistant during the whole growth period,and still be effective to the most rust pathotypes in China.Rapid virulence variation of pathogen results in “resistance loss” of wheat cultivars,which severely impacts the security of wheat production and foods.It is urgent to reveal the mechanism of resistance against stripe rust for wheat resistance reasonable utilization and disease control.Through the research on interaction between wheat stripe rust resistance gene Yr10 and host target,it provides a theoretical basis for the research and cultivation of resistant varieties.A downstream target gene was obtained by screening the yeast two-hybrid library(Y2H).Subcellular localization of Yr10 and Ta NF-YC showed the first interaction compartment.Histological observation showed the important issues occurred during the compatible(AVS-CYR32))and incompatible(AVS+Yr10-CYR32)interactions.The functional analysis of target gene suggested it play a role in the resistant pathway mediated by Yr10.It is an important theoretical basis for exploring the Yr10 resistant mechanism.The results of the study were as follows:(1)The Y2 H was used to screening the downstream target of Yr10.A target transcriptional factor designated as Ta NF-YC was obtained.The positive protein interactions was detected between Yr10 and Ta NF-YC by using the Y2 H assay,Bi FC analysis,Pull-down assays and Co-IP assays.And the LRR domains of Yr10 and Ta NF-YC interact with each other.The transcriptional auto-activation of Ta NF-YC was demonstrated.(2)To examine the subcellular distribution of Yr10 and Ta NF-YC,we expressed them in tobacco transient expression system and wheat protoplast.It showed Yr10 accumulated in the cytosolic compartment and Ta NF-YC targeted in both the nucleus and cytoplasm.It indicated that the first interactions were in the cytoplasm.(3)Histological studies identified the basic information on infection strategies used by the pathogen and defense responses from the host during the interactions between wheat and CYR32.The content of reactive oxygen began to burst and continued to increase in the incompatible interaction,which was significantly higher compared with compatible interaction at different time points after 48 hours post interaction(hpi).The area of necrosis in AVS+Yr10 also showed significantly larger than AVS from 48 hpi to 120 hpi,while the growth and development of stripe rust was inhibited from 48 hpi to 120 hpi,indicating that resistance gene Yr10 began to play a significant role in disease resistance at 48 hpi.(4)The expression of Ta NF-YC was induced after inoculated CYR32 both in incompatible and compatible interactions by q RT-PCR analysis.However,the expression of Ta NF-YC is much higher in the incompatible interaction than that in compatible interaction and peaked at 72 hpi.It indicated Ta NF-YC was involved in the Yr10 resistant pathway.Ta NF-YC was also induced by exogenous hormones including ABA,SA,JA and ET.It implied that Ta NF-YC has a function in different signaling pathway.The functional analysis of Ta NF-YC gene was studied by the barley stripe mosaic virus(BSMV)induced gene silencing.Necrotic area in wheat leaves were reduced after inoculation with CYR32 after silencing by comparation with the control.The active oxygen area in Ta NF-YC silenced leaves were significantly reduced at 48 hpi,72 hpi and 120 hpi.Fungal growth increased after silencing Ta NF-YC,and the infection areas were significantly larger than the control at 72 hpi and 120 hpi after silencing.Similarly,cell necrosis can also be promoted by the transient expression of Ta NF-YC in tobaccos.Taken together,it indicates that Ta NF-YC played a role in promoting disease resistance through the interaction with Yr10,the defense was through active reactive oxygen species accumulation and triggering HR.