| Wheat stripe rust,caused by Puccinia striiformis f.sp.tritici(Pst),is one of the main diseases of wheat production,resulting in huge yield loss.Due to the frequent variation of Pst virulence,the race-specific resistance of wheat cultivars was lost.Therefore,deploying wheat cultivars with durable and non-race-specific resistance is the one of important ways to control wheat stripe rust.High-temperature resistance to Pst in wheat is non-race-specific and effective in disease prevention and yield protection,which can be divided into hightemperature adult-plant resistance and high-temperature all-stage resistance.The high yield wheat cultivar Xiaoyan6(XY6)was developed by Academician Li Zhensheng’s team using chromosome engineering.XY6 has maintained resistance in fields since 1975 with typical high-temperature all-stage resistance to Pst.However,the molecular mechanism of the XY6-Pst interaction remains unclear.In this study,34 secreted proteins of Pst were identified based on the transcriptome data of Pst in response to the high-temperature resistance of XY6.Two secreted proteins,Pst CEP1 and PSTG＿01766,were found to inhibit programmed cell death(PCD)induced by BAX in Nicotiana benthamiana.We performed functional studies on the effector proteins of Pst.The main results are as follows:1.Pst effector protein Pst CEP1 targets ferredoxin Ta Fd1 and inhibits the hightemperature resistance of wheat by inhibiting the reactive oxygen species(ROS)production or interfering the stability of photosynthetic system.Pst CEP1 is a secreted protein with N-terminus signal peptide and cytoplasm localization.Pst CEP1 could inhibit PCD induced by BAX and INF1.The transcriptional expression of Pst CEP1 was up-regulated when the high-temperature resistance was activated in response to Pst infection.The number of haustorial mother cells and the hyphal length were significantly reduced,and the pathogenicity of Pst decreased under different temperature treatments after silencing Pst CEP1.Bacterial type three secretion system(TTSS)-mediated overexpression of Pst CEP1 inhibited callose deposition and ROS production,and enhanced the pathogenicity of Pst in the normalhigh-normal temperature(NHN)treatment.Yeast-two-hybrid(Y2H)assay showed that Pst CEP1 interacts with Ta Fd1,a homology protein of rice Os Fd1.Silencing of Ta Fd1 showed that the stability of photosynthetic system of wheat was affected,resulting in appearing chlorosis symptoms on the leaves and reducing high-temperature resistance.2.Wheat apoplastic thaumatin-like protein Ta TLP1 recognizes an effector protein PSTG＿11208 of Pst and triggers the immune response.PSTG＿11208 may evade plant immunity through the interaction with Pst CEP1.Y2 H,bimolecular fluorescence complementation(Bi FC),and pull-down assays showed that Pst CEP1 interacts with PSTG＿11208.Silencing PSTG＿11208 decreased the high-temperature resistance in wheat,while transient overexpression of PSTG＿11208 enhanced the resistance under different temperature treatments.In addition,overexpression of Pst CEP1 inhibited the resistance enhancement caused by overexpression of PSTG＿11208.Furthermore,Ta TLP1 may recognize pathogen invasion by interacting with PSTG＿11208 and initiates the downstream defense response by the pathogenesis-related protein Ta PR1.Silencing Ta TLP1 and Ta PR1 separately or co-silencing of these two genes reduced the high-temperature resistance to Pst in wheat.3.Pst effector protein PSTG＿01766 inhibits the guard model based on an NBS-LRR immune receptor Ta RPM1,a receptor-like cytoplasmic protein kinase Ta RIPK,and a papain-like cysteine protease Ta PLCP1 in response to the high-temperature resistance in wheat.Transient overexpression of PSTG＿01766 in N.benthamiana suppressed PCD induced by BAX and the expression level of salicylic acid(SA)signaling pathways-related gene.Silencing PSTG＿01766 decreased the pathogenicity of Pst under different temperature treatments,while overexpressing PSTG＿01766 enhanced the pathogenicity of Pst under the NHN treatment.Y2 H,Bi FC,pull-down,and co-immunoprecipitation assays indicated that PSTG＿01766 targets Ta PLCP1.The expression of Ta PLCP1 was induced by exogenous SA treatment.Silencing Ta PLCP1 decreased the high-temperature resistance of wheat.In the previous studies,Ta RIPK and Ta RPM1 were upregulated in the functioning of the hightemperature resistance and were induced by exogenous SA treatment.Silencing Ta RIPK or Ta RPM1 decreased the expression level of the SA-related defense gene Ta PR2.In this study,we demonstrated that Ta RIPK interacts with and phosphorylates Ta PLCP1,and PSTG＿01766inhibits the phosphorylation.A guard model was proposed based on these results.Ta RPM1 initiates the downstream immune response by monitoring the changes in the phosphorylation level of Ta PLCP1 induced by Ta RIPK during the occurrence of high-temperature resistance in wheat.Pst effector protein PSTG＿01766 responds to the high-temperature resistance of wheat by competitive interaction with Ta PLCP1.In addition,by the LC-MS/MS assay,we detected the endogenous hormone concentration of XY6 leaves in the functioning of the hightemperature resistance,indicating a significant increase in the concentration of SA.Therefore,we speculated that the SA signaling pathway plays an important role in the guard model. |
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