Mechanism Dissection Of Phytophthora Infestans RxLR Effector PITG₂2926 Targets StMAP3Kβ2 To Regulate Potato Immunity

Potato is invaded by various diseases,among which late blight,caused by Phytophthora infestans,is the most devastating disease in potato production.In order to fend off pathogen infection,plants have evolved sophisticated innate immune system to activate the defense responses,including the PAMPs-triggered immunity(PTI)and Effector-triggered immunity(ETI).P.infestans delivers effectors into host cells to suppress the host immunity and promote colonization.Yeast two-hybrid was used to identity potato candidate targets of 70 RxLR effectors in previous study.Among these,the target of effector Pi22926 is a MAPK protein kinase StMAP3Kβ2.In this study,Agrobacterium-mediated transformation,transient expression,Yeast two-hybrid,co-immunoprecipitation and virus-induced gene silencing(VIGS)techniques are used to investigate how Pi22926 interacts with StMAP3Kβ2 to regulate plant immune response.The main results are listed as fllows:1.Pi22926 was constitutively up-regulated during the early stage of P.infestans infection.Transient overexpression or stable overexpression of Pi22926 in Nicotiana benthamiana and potato enhanced P.infestans colonization;Pi22926 specially suppresses cell death signalling pathway triggered by co-expression of Avr4/Cf4 or AvrPto/Pto.This indicates that Pi22926 has virulent function.2.Yeast two-hybrid and co-immunoprecipitation showed that Pi22926 interacted with a potato mitogen activated protein kinase kinase kinase StMAP3Kβ2 and its kinase domain StMAP3Kβ2(KD),but it failed to interact with the inactive form in which the active site lysine in the ATP binding site(Lys 430)was substituted with an arginine.This confirmed that the intact active kinase domain of StMAP3Kβ2 was necessary and sufficient for the specific interaction with Pi22926.This indicates that Pi22926 specifically targets key site of kinase domain of StMAP3Kβ2 to suppress kinase activity.In addition,Pi22926 failed to interact with another MAP3 K StMAP3Kε,indicating that Pi22926 specifically interacts with StMAP3Kβ2.3.Pi22926 localized predominantly in the nucleus and nucleolus.StMAP3Kβ2localized mainly in the cytoplasm with weak fluorescence in the nucleoplasm.Additional split YFP experiments showed that YN-Pi22926 and YC-StMAP3Kβ2 co-localized only in nucleoplasm.This implies that the interaction of these proteins occurs in the nucleoplasm.4.Transient expression of StMAP3Kβ2 in N.benthamiana induced cell death;Silencing of NbMAP3Kβ2 in N.benthamiana contributed to P.infestans colonization.In agreement,overexpression StMAP3Kβ2 inhibted P.infestans growth.This result indicates that MAP3Kβ2 is a positive regulator of plant immunity.5.Pi22926 or PexRD2 only suppressed cell death triggered respectively by expression of StMAP3Kβ2 or StMAP3Kε,indicating that Pi22926 specifically suppressed StMAP3Kβ2 induced cell death.6.Silencing of NbMAP3Kβ2 or NbMAP3Kε both partially inhibited cell death induced by Avr4/Cf4 and Avrpto/Pto.This suggests that MAP3Kβ2 and MAP3Kε function in the same signaling pathway;VIGS of NbMAP3Kβ2 did not compromise cell death triggered by expression of MAP3Kε.Similarly,VIGS of NbMAP3Kε did not attenuate cell death triggered by MAP3Kβ2.This demonstrates that StMAP3Kβ2 and StMAP3Kε act in parallel in Avr4/Cf4 and Avrpto/Pto cell death induction pathway;VIGS of MAPKs signal pathway compenents MEK2 and SIPK inhibited the cell death triggered by expression of the intact KD of StMAP3Kβ2.This indicates that StMAP3Kβ2 act upstream of MEK2 and SIPK.MAPK cascades are well established as key signaling pathways in regulating plant immune response.This study and previous study results demonstrate that P.infestans effectors Pi22926 and PexRD2 specifically target two different MAP3 Ks which act parallel in Avr4/Cf4 signaling pathway to suppress plant immune response.This study provides the new evidence of how these two effectors coordinately inhibit the same cell death signaling pathway mediated by Avr4/Cf4(and Avrpto/Pto)to regulate plant immunity.