Interactome Analysis Of RAR1/SGT1/HSP90,NPR1 And XA21 Node In The Defense Response Of Rice And Wheat

In the long history of battle and co-evolution between plant and pathogens,plant has evoluted a complicated defense system,which comprised and intercrossed by multi-signal pathways.The protein existing in the intercross of multi-signal transduction ways were called hub or node proteins that are the key regulators of plant defense response.It is facilitate to elucidate the signal transduction pathways that node protein involved in and to dissect the regulating roles of node proteins in plant defense mechanism by studying and expanding the interactome of node proteins,which set up a better understanding of plant defense mechanism for plant resistance improvements.Base on the published rice interactome network in defense response,we compared and analyzed conservation of interactome of three nodes,including RAR1/SGT1/HSP90,NPR1 and XA21 in wheat by Y2H(yeast-two-hybrid)and BiFC(bimolecular fluoresence complementation)assays.In order to indentify the novel components involved in the XA21-mediated pathway and test the possibility that NPR1 is the target of effectors,we screened a wheat cDNA library using wheat TaXA21 and NPR1 as bait,respectively.Four novel XA21 ineractors were yielded from screening by Y2H and BiFC validation and the interactions between their rice homolgous protein and rice XA21 were indentified.The interplay relationship was detected between transcription factor TGA2,stripe rust effector PNPI fished by wheat NPR1 from library screening,and wheat NPR1 by Y3H(yeast-three-hybrid)assays.The main results and conclusions are as following:1.The interactions among components of RAR1/SGT1/HSP90 complex were completely conserved in rice and wheat by Y2H assay and interaction pairs(wRAR1-wSGT1、wRAR1-wHSP90.2、wRAR1-wHSP90.3、wSGT1-wHSP90.2、wSGT1-wHSP90.3)were validated in rice protoplast by BiFC assay.Y2H results were revealed that the interactome of NPR1 node with 8 known interactors(rTGA2.1,rTGA2.2,rTGA2.3,rNRR,rNRRH1,rNRRH2,wNRRH3 and rLG2)was also completely conserved in wheat.And the interactions between wheat NPR1 and wTGA2.1,wTGA2.2,wTGA2.3,wNRRl,wNRR2 and wNRRH1 were observed in nucleus by BiFC assay.2.We isolated two homolgous protein from wheat,TaXA21-like1 and TaXA21-like2.Among 11 known rice XA21 interactors(XBs),only wXB3,wXB15 and wXB24 interact with both TaXA21-likel and TaXA21-like2,and only the interaction between TaXA21 and wXB 15,wXB24 were validated by BiFC,which suggested that the conservation of interactome of rice XA21 node was low in wheat and probably this is because of the evolution history of XA21 gene experienced in rice and wheat.3.Based on Y2H library screening by TaXA21-likel as bait and Y2H assay validation,we indentified four novel TaXA21 interacrors,including TaARG,TaPR2,TmSKL1 and TaCIPK.Additional test showed that interaction between TaXA21-likel and TaSGT1.4.We then cloned the rice homologs of the novel wheat interactors and confirmed that they all interact with rice XA21,which expanded the interactom of XA21 node.Including all validated wheat interactor of TaXA21,we constructed interactome of TaXA21 at least 9 proteins.The transcriptional profiles of seven of these 9 interactors were in responded in wheat after stripe rust inoculation.5.We indentified the interaction between wheat NPR1 and stripe rust effector PNPI from wheat Y2H cDNA library screening using wheat NPR1 as bait.The protein sequence of stripe rust effector PNPI comprised signal peptide by online SignalP prediction and the proteins were completed conserved in many stripe rust and stem rust isolates.The interaction tests between the truncated donmains of wheat NPR1 revealed that the ANK-repeat functional domain can interact with effector PNPI directly by Y2H assay,which demonstrated that NPR1 was targeted by pathogen effectors.The interplay relationship test among wheat NPR1,TGA2 and effector PNPI showed that the effector PNPI is competitively to bind wheat NPR1 and is stronger competition than TGA2,which basically set up an interplay model among wheat NPR1,TGA2 and effector PNPI.