| Stripe rust is one of the important diseases threatening wheat production.Its pathogen Puccinia striiformis f.sp.tritici(Pst)overcome wheat cultivars with excellent agronomic characteristics through frequent virulence variation.As an important pathogenic factor of pathogens,effector proteins play an important role in the virulence variation of stripe rust fungus.Therefore,the identification of important effectors of stripe rust and the analysis its virulence mechanism in host plants are of great significance to the creation of disease-resistant materials and the development of new strategies for the control of stripe rust.Based on the genome sequencing of the stripe rust fungus CYR32,the secretome of stripe rust fungus was systematically analyzed completed by our research group,and the effector proteins was predicted to be located in different cell spaces such as cytoplasm,nucleus,chloroplast,mitochondria,and apoplasts,indicating that rust effectors may regulate plant immunity in a variety of ways.For this reason,based on large-scale screening of the effector proteins of Pst,four effector proteins located in the cytoplasm,nucleus and apoplasts were selected for in-depth research.The results of the study broadened our understanding on the mechanism of host immune regulation by effectors of biotrophic fungi.1.The cytoplasmic effectors Pst?4 and Pst?5 inhibit the wheat resistance to yellow rust by suppressing TaISP from entering chloroplasts.Previous studies in our lab found that the two effectors Pst?4 and Pst?5 share similarity in the number and distribution of Cysteine,as well as their expression characteristics in the infection process of Pst.And found that the two effectors play important role in Pst pathogenicity.In order to further investigate the mechanism of these two effectors in the host,Pst?4-RNAi and Pst?5-RNAi transgenic wheat were created and inoculated with the epidemic stripe rust race CYR32 in china.The expression of the two effector genes of Pst?4 and Pst?5 in the transgenic wheat was found significantly reduced by RT-q PCR;The production of uredium and relative biomass of stripe rust fungus of transgenic wheat were significantly less than that of wild-type wheat.Histological observations showed that the area of infection hyphae in transgenic wheat was significantly reduced,and the area of ROS accumulation around the infection sites was significantly increased.These results indicate that Pst?4 and Pst?5 play important roles in the pathogenicity of stripe rust.The interaction between Pst?4 and Pst?5 and chloroplast protein TaISP was identified by yeast two hybrid(Y2H)in previous studies of our laboratory.We verified the interaction between TaISP and Pst?4 or Pst?5 in the plant by Co-immunoprecipitation(Co-IP)technology.The results of Y2H and bimolecular fluorescence complementation(Bi FC)show that Pst?4 and Pst?5 interact with TaISP87-222(C-terminal)containing Rieske domain,but not with TaISP1-63(N-terminal)containing chloroplast transit peptide.In order to study the role of TaISP in plant immunity,we transient expressed TaISP in N.benthamiana and performed the ROS staining assay.The results showed that TaISP could promote the production of ROS in chloroplasts.Combining with transiently expressing TaISP in N.benthamiana and with reactive oxygen staining experiments,we found that TaISP promoted the production of reactive oxygen species(ROS)in chloroplasts.In order to study the effect of TaISP on wheat resistance to Pst,we created TaISP-RNAi transgenic wheat and inoculated with the avirulent race CYR23.The result show that uredium can produce in TaISP-RNAi wheat but not wild-type wheat.Through RT-q PCR technology,it was found that TaISP was effectively silenced,and the expression of pathogenesis-related genes TaPR1 and TaPR2was also reduced.Histological observation found that the infection area of Pst hyphae was decreased in TaISP-RNAi wheat,and the accumulation of ROS around the infection sites was also decreased.This indicates that TaISP may regulate the disease resistance of wheat by regulating the chloroplast ROS production and the retrograde signals from the chloroplast to the nucleus.Transient expression of TaISP increased the photosynthetic electron transfer rate(ETR)of N.benthamiana,indicating that TaISP is regulating the photosynthesis of plants.When co-expressed with TaISP and Pst?4 or Pst?5,ETR was significantly lower than that in N.benthamiana co-expressed with TaISP and e GFP,indicating that Pst?4 and Pst?5 can attenuate photosynthesis in plants by targeting TaISP.TaISP is a nuclear gene encoded chloroplast protein,which is synthesized in cytoplasm and then transported to chloroplasts.Previous studies of our lab have shown that Pst?4 and Pst?5 located in cytoplasm and nucleus.In order to analyze how the two effectors affect TaISP,we conducted co-localization experiments in N.benthamiana,it was found that when co-expressed with e GFP,TaISP-m Cherry mainly located in chloroplasts,while when co-expressed with e GFP-Pst?4 and e GFP-Pst?5,TaISP-m Cherry mainly located in cytoplasm,by contrast,TaISP1-63-m Cherry still mainly located in chloroplasts.To comfirm the results of co-localization experiments,the chloroplasts of these N.benthamiana plants were isolated for Western blotting analysis.The results show that the content of TaISP-m Cherry protein was decreased when co-expressed with e GFP-Pst?4 and e GFP-Pst?5,while the content of TaISP1-63-m Cherry was not significantly affected by the two effectors.In order to verify whether this mechanism exists in the host wheat,we used the Type three secretion system(T3SS)of bacteria to express Pst?4 and Pst?5 in the leaves of wheat,and isolated chloroplasts for Western blotting.The results showed that the content of TaISP in the chloroplasts decreased.And we found more TaISP protein accumulation in chloroplasts of Pst?4-RNAi and Pst?5-RNAi wheat after CYR32 inoculation.These results indicated that Pst?4 and Pst?5 suppressed TaISP from entering chloroplasts.In order to improve the disease resistance and photosynthesis of wheat at the same time,we created TaISP overexpression transgenic wheat,and inoculated with the virulent seed CYR32.The results showed that the resistance of TaISP-overexpressing wheat to stripe rust was significantly improved.This study revealed that effectors Pst?4 and Pst?5 regulate host immunity by inhibiting the transport of TaISP from cytoplasm to chloroplast,thereby promoting the pathogenicity of Pst.2.The effector Pst?9 is recognized by a receptor-like kinase on the cell membrane and stimulates plant immunityThe secreted protein Pst?9 contained signal peptide and a nuclear localization signal.We verified that the secretory function of signal peptide of Pst?9 by yeast experiment.Results of RT-q PCR showed that Pst?9 was upregulated in the process of stripe rust infection in wheat.This indicated that Pst?9 is a secreted protein during Pst infection of wheat.In order to study the role of Pst?9 in the pathogenicity of stripe rust,a transgenic Pst?9-RNAi was created.The inoculation of the avirulent race CYR23 showed that the relative biomass of stripe rust in Pst?9-RNAi wheat increased.To study the location of Pst?9 function in plant cells,we performed the subcellular localization experiments in N.benthamiana.The results show that the full-length Pst?9-e GFP mainly located near the cell membrane and in the nucleus,while Pst?9nosp-e GFP located in the nucleus.In order to investigate whether Pst?9 can accumulate in the apoplast,we performed the plasmolysis assay.The results showed that Pst?9-e GFP containing signal peptide,Pst?9SP-e GFP(Pst?9 signal peptide fused e GFP),and PR1SP-Pst?9-e GFP(PR1 signal peptide replaced the original Pst?9 signal peptide)could accumulate in apoplast,while Pst?9nosp-e GFP and e GFP could not.These results indicate that Pst?9 can accumulate in both the plasmid and the nucleus.To study the function of Pst?9 in plant cell.We transient expressed different forms of Pst?9.We found that Pst?9-PVX and the PR1SP-Pst?9-PVX could induce cell death of N.benthamiana,but Pst?9nosp-PVX and Pst?9SP-GFP-PVX could not,which indidate that the signal peptide is required for cell death induction of Pst?9.To confirm that conclusion,we replaced the native signal peptide of Pst?9 by the signal peptide of PR1 gene,and found that PR1SP-Pst?9-PVX induced cell death but PR1SP-GFP-PVX did not.Combined with the results of subcellular localization,we think that apoplast accumulated Pst?9 can induce cell death.The homologous genes of Pst?9,PSTG?07677,PSTG?16598,PSTG?13167,PSTG?03196 of stripe rust and PTTG?10718 of leaf rust were transient expressed in N.benthamiana by PVX vector,and found that the homologies can also induce cell death of N.benthamiana.These results infer that these effectors can stimulate plant defenses in apoplast.To investigate whether Pst?9 affects the immune response of plants,we detected defense-related genes in N.benthamiana expressing Pst?9-GFP by RT-q PCR.The results showed that Pst?9-GFP stimulated the expression of N.benthamiana defense genes Nb PR1,Nb PR2 and Nb WRKY12,while Pst?9nosp-GFP inhibited the expression of these genes.In addition,transient expression of Pst?9-GFP improved resistance of N.benthamiana to Sclerotinia sclerotiorum,while Pst?9nosp-GFP promoted its expansion.In order to study whether Pst?9 can be used to improve the disease resistance of wheat,we infiltrated the proteins that produced from E.coli into wheat leaves and caused no obvious cell death.However,compared to the wheat injected with GFP control,the uredium production of wheat that infiltrated with Pst?9nosp protein was reduced after inoculation with CYR32.RT-q PCR results show that the expression of TaPR1 and TaPR2was decreased in wheat with Pst?9nosp infiltration but without Pst inoculation,while their expression in wheat that inoculated with Pst and infiltrated with Pst?9nosp was increased.In order to improve the disease resistance of wheat,a transgenic wheat Pst?9-OE overexpressing Pst?9 was created.Compared with wild-type wheat,the uredium production in Pst?9-OE wheat was decreased,and the expression of defense genes TaPR1and TaPR2 increased after inoculation with CYR32,while the non-inoculated Pst?9-OE wheat did not exhibit cell death,and the levels of TaPR1 and TaPR2 expression is significantly lower than that of wild-type wheat.This indicates that overexpression of Pst?9can enhance defense response when plant facing attack of pathogen.Which infer that Pst?9may entered to host cells from the apoplast to inhibit the defense response.In order to study whether Pst?9 in the nucleus inhibited the disease resistance of wheat,we created transgenic wheat overexpressing Pst?9nosp.After inoculated with avirulent race CYR23,Pst?9nosp-OE produced uredium,the fungus biomass was also significantly increased,and the expression levels of TaPR1 and TaPR2 were significantly lower than that of wild-type wheat.These results suggested that Pst?9nosp in the nucleus could inhibit the immunity and resistance of the wheat to Pst.To study the mechanism of inhibition of plant immunity by Pst?9nosp in the nucleus,the interaction target of Pst?9 Ta DSK2a was screened by Y2H,and the interaction between Pst?9nosp and Ta DSK2a was determined in plants by Luciferase complementation and Co-IP.Subcellular localization show that Ta DSK2a locates in cytoplasm,nucleus and autophagosome.Their interaction was found to occur in the nucleus and the autophagosome by BIFC experiment.In addition,we found that transient expression of Ta DSK2a promoted N.benthamiana cell death under weak light condition,while Pst?9nosp inhibited Ta DSK2a induced cell death.In order to investage the mechanism of Pst?9-induced cell death,we first silenced the co-receptor SOBIR1 in N.benthamiana by TRV2-mediated VIGS.It was found that Pst?9could not induce cell death in SOBIR1 silenced plants,but it could induce cell death in N.benthamiana that silenced control GFP and EDS1,indicating that cell death caused by Pst?9 is the receptor dependent.In order to acquire the receptors recognizing Pst?9,we screen its receptors using TRV2-mediated VIGS and Arabidopsis mutants.The results showed that Pst?9 mutant could not induce cell death in Arabidopsis mutant of AT1G34110gene and Niben101scf09649g02016 silenced tobacco.At the same time,the LRR structure of Niben101SCF09649G02016 was found to interact with Pst?9nosp by Y2H.Both genes encode RLK receptor kinases,and the amino acid sequence similarity is 62.11%.In addition,there is a homologous gene TRAESCS7D02G3547001.1 in wheat,and the similarity with AT1G34110 on amino acid sequence is 67.1%.The results preliminarily proved that the receptor-like kinase was the receptor of Pst?9,and it was provisional named as RPst9.In this study,it was found that Pst?9 targeted wheat autophagy receptor Ta DSK2a and suppressed host immunity when it was localized to the host nucleus,while when accumulated in apoplast,Pst?9 was recognized by the receptor-like kinase RPST9 on the plant cell membrane and stimulated the immune response when it was localized to the plasmid.3.Rust effector Pst?R22 impairs the interaction between TaEDS1 and TaPAD4and suppress Sr35/Avr Sr35 induced cell deathIn previous studies of our lab,an effector protein Pst?R22 containing the Lipase domain was identified,and it was found to play a role in the pathogenicity of Pst,and interacts with the important plant immune-node protein TaEDS1 in the cytoplasm.In order to further understand the mechanism of Pst?R22 affects plant immunity through interaction with TaEDS1,we created TaEDS1-RNAi transgenic wheat,and analyzed the role of TaEDS1 in wheat disease resistance by inoculation with avirulent Pst CYR23.The results showed that TaEDS1-silenced wheat produced uredium,and the relative biomass of Pst was significantly increased in TaEDS1-silenced wheat.Results of RT-q PCR showed that the expression level of TaPR1 was also significantly decreased in TaEDS1-RNAi wheat,which verified the important role of TaEDS1 in wheat immune and disease resistance of wheat aganist Pst.It has been reported that EDS1-PAD4 complex mediates PTI,here we confirmed that TaEDS1 interacts with TaPAD4 in plants using Co-IP.In addition,Co-IP showed that Pst?R22 could interact with TaPAD4 in plants.In order to identify the role of TaPAD4 in wheat resistance to stripe rust,we silenced TaPAD4 using VIGS and inoculated with CYR32.The results showed that the uredium production was increased TaPAD4-silenced wheat.Results of RT-q PCR showed that the relative biomass of Pst was increased and the expression of TaPR1 and TaPR2 were significantly decreased in TaPAD4-silenced wheat.These results indicated that TaPAD4positively regulates the wheat immunity and resistance to stripe rust.In order to investigate whether Pst?R22 could affect the interaction between TaEDS1and TaPAD4,we used Bi FC and luciferase complementation assay to analyze the effect of Pst?R22 on the interaction between TaEDS1-TaPAD4 in plant.The results showed that pre-expression of Pst?R22 attenuated the interaction between TaEDS1 and TaPAD4.Previous studies of our lab have found that Pst?R22 can inhibit plant PTI.In order to investigate whether Pst?R22 can also inhibit plant ETI,we studied the effect of Pst?R22 on plant ETI by analyzing whether effectors can inhibit SR35/AVRSR35-induced cell death.The results showed that the homologous secretory proteins of Pst?R22,the PSTG?01470,PSTG?12835,PGTG?01618,PGTG?01889 and PSTG?08403 could inhibit the cell death induced by SR35/AVRSR35 in different degrees.It is speculated that these secreted proteins may inhibit ETI mediated by wheat CNL resistance gene. |
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