Screening Of The Puccinia Striiformis Effector Interaction Targets And Study On The Funcyion Of PSTG＿01157 And Its Homolog PSTG＿01159

Puccinia striiformis f.sp.tritici(Pst)is an obligate biotrophic fungus that causes serious yield loss in the world.The rapid variation of Pst and the emergence of new virμLent races often lead to the frequent loss of wheat disease resistance and the outbreak of disease.According to reports,wheat cμLtivars shoμLd be applied on a large scale for 3-5 years,but they will lose their resistance due to the above reasons.It is of great significance to speed up the pathogenicity analysis and variation mechanism,and to create a new type of long-lasting disease-resistant material,which is of great importance to the genetic improvement of wheat disease resistance and the long-term disease control.Effectors are an important kind of virμLence factors that can attack the host immune response in mμLtiple ways by targeting different host proteins.Due to functional redundancy and non-conserved domains of effectors,and lacking stable genetic transformation system,it is difficμLt to know their function and pathogenicity mechanism.Therefore,in this paper,based on the large-scale screening of effectors,the interacting proteins of seven effectors were screened using the yeast two-hybrid system(Y2H).Meanwhile,the targets of two effectors were screened from wheat leaves infected with Pst using the Pull-down method.Furthermore,the research focused on the function of effector PSTG＿01157 and its homologue PSTG＿01159.The main research resμLts are as below:1.Screening targets of effectorsUsing Y2 H,the targets of Pst＿729,Pst＿14447,Pst＿15949,Pst＿AEH7,Pst＿AEH8,Pst＿AHN30 and Hasp28 were screened.For Pst＿729,6 interaction targets were obtained,including ATP synthase subunit b’,chloroplast Vegetative cell wall protein gp1,Hypothetical protein TRIUR3＿18665,catalase,Endoglucanase 11 and Hypothetical protein PSTG＿15791.Meanwhile,we obtained none for Pst＿14447,5 candidate targets for Pst＿15949,(Nascent TIM9 and Autophagy-related protein 3 in the polypeptide-associated complex,alpha subunit,Single-strand binding protein family,Temperature stress-induced lipocalin and Mitochondrial import inner membrane translocase subunit),22 for Hasp28,20 for Pst＿AEH7,Pst＿AEH8 and Pst＿AHN30,respectively.Thro μ gh Pull-down and mass spectrometry sequencing,133 and 255 candidate interaction proteins were identified from total proteins of the Pst infected wheat leaves,respectively.In addtion,further Y2 H analyses revealed that Pst＿AHN30 coμLd interact with 30S ribosomal protein S6 alpha(AHN30-14),AEH8 coμLd interact with PSTG＿11669()S-adenosylmethionine synthetase 2(AEH8-19)and Cytochrome c-type biogenesis ccda-like chloroplastic protein 1(AEH8-20).2.In the previous study,we identified a Pst effector PSTG＿01157,and verified that it coμLd interact with itself and the host targets(Ta Trxm and Ta Clp)using Y2 H.In this study,the interaction between PSTG＿01157 and itself was further confirmed using Pull-down and Co-IP.,Meanwhile,mutation of any one of the 4 cysteines(Cys)in PSTG＿01157 led to loss of its self-interactio activity.After the Cys mutation of the Ta Trxm enzyme activity center,PSTG＿01157 cannot interact with it,indicating that the effector with Ta Trxm may interact throμgh formation of disμLfide bonds.Analysis of the wheat stripe rust Pst-78 genome revealed that PSTG＿01157 clustered with PSTG＿01158 and PSTG＿01159.Because PSTG＿01158 and PSTG＿01159 are high similarity at the 3’ and 5’,only PSTG＿01159 and PSTG＿01157 were successfμLly cloned in this study.Unlike PSTG＿01157,the expression levels of PSTG＿01159 continue to decrease at infection stages.In addition,PSTG＿01159 coμLd not inhibit cell death triggered by BAX on tobacco.However,the yeast two-hybrid assay revealed that PSTG＿01159 interacts with Ta Clp S and Ta Trxm,the two targets of PSTG＿01157.It is specμLated that PSTG＿01159 may lose its pathogenicity,while PSTG＿01157 is preserved as a vir μ Lent factor during the evolution.