Mechanism Research On Phytophthora Infestans RxLR Effector Promoting Late Blight Disease By Interaction With Host Protein

Potato late blight, the agent of Phytophthora infestans, is easy to lead to disease spread and epidemic under suitable conditions, and can be infected for several times, it is one of the most destructive oomycete diseases. The most effective measure for controlling the disease is to use resistant cultivars in the agricultural production, but the virulence diversity of P. infestans often leads cultivars to lost resistance.Pathogens interact with host during their infection, accordingly the plants produce immunity response, which involve a complex network of cross-linked signalling and regulatory processes. The first line of inducible defence in plants involves the recognition of conserved microbial molecules（Pathogen associated molecular patterns; PAMPs）, or molecules generated as a result of cellular damage（DAMPs）, leading to the activation of defence pathways. This can be referred to as pattern-triggered immunity（PTI）. Host-adapted pathogens have evolved to manipulate or suppress PTI to promote susceptibility. One of the strategies employed for PTI suppression involves the secretion of effectors which may function in the apoplastic interface between the pathogen and host or be translocated into host cells. Plant pathogens deliver proteins called effector that suppress or alter processes in their hosts to create an environment conducive to colonisation. Attention has focussed on identifying the targets of effectors and how their manipulation facilitates disease.This thesis focused on an effector of PexRD41/PITG₀4089 localized in the nuclear of cell, which is an RxLR effector secreted by Phytophthora infestans. The research results are the following:（1）Agrobacterium-mediated transient expression Pi04089 tagged at the N-terminus with GFP, significantly enhanced P. infestans lesion size compared to an unfused GFP control. GFP-Pi04089 expressed transiently in N. benthamiana leaves was stable as an intact fusion protein and predominantly located in the nucleus, forming a ring around the nucleolus. Pi04089 is up-regulated in P. infestans strain at 24 and 48 hours post-inoculation. This corresponding to the early stages of the biotrophic phase of infection, which normally extends to 72 hpi in this isolate.（2）To determine whether the nuclear location of Pi04089 is important to the function of the effector a nuclear export signal（NES） was added to the N-terminus of the GFP fusion form（NESGFP-Pi04089）. As anticipated, the NESGFP-Pi04089 showed greatly reduced fluorescence in the plant cell nucleus, and correspondingly increased cytoplasmic fluorescence. Transient expression of NESGFP-Pi04089 did not support enhanced leaf colonisation of P. infestans compared to the unmodified GFP-Pi04089.（3）To search for possible host target proteins of Pi04089 a yeast-2-hybrid library created from potato c DNA infected for 15 h and 72 h. Pi04089 interacts with a putative RNA binding protein with 3 K-homology（KH） domains（named StKRBP1） by Yeast-two-Hybrid technique. Furthermore, StKRBP1 was confirmed to specific interact with this effector in planta by co-immunoprecipitation assay.（4）A bimolecular fluorescence complementation（BiFC） assay was performed. YFP fluorescence generated by co-expression of YN-04089 with YC-StKRBP1 was restricted to the nucleus, and especially emphasised the nuclear speckles.When co-expressed with GFP-Pi04089 the effector fusion co-located with the RFP-StKRBP1 and was no longer visible as a ring around the nucleolus. Co-expression of Pi04089 with StKRBP1 results in increased protein levels of the latter. Interestingly, such increased StKRBP1 protein accumulation is also seen in the first 24 hours after inoculation of P. infestans spores onto leaves, which shows KRBP1 is a positive regulator of infection.（5）Three GxxG conserve motifs in StKRBP1 were mutated to GDDG to create StKRBP1 mut by VIGS technology. The mutated form no longer interacted with Pi04089 either in yeast using Y2 H or in planta using co-immunoprecipitation. The StKRBP1 mut was tagged with mRFP and GFP and was found to locate to the nucleoplasm but not to form the nuclear speckles seen with the wild-type form. When co-expressed with GFP-04089 the mRFP-StKRBP1 mut did not alter the location of the effector fusion.（6）Transient expression of cMyc-tagged StKRBP1 mut did not result in increased pathogen colonisation compared to the EV control, the mutant KRBP1 protein no longer supported enhanced leaf colonisation by P. infestans indicating that nucleotide binding is likely required for this activity.Pi04089 is beneficial to the pathogen. StKRBP1 can thus be regarded as a susceptibility factor. The effector Pi04089 may facilitate, modify or enhance its activity to promote late blight disease.