Development Of A Screening System For MTI-suppressing Effectors In Nicotiana Benthamiana And Functional Characterization Of SFI5

As a consequence of the continuous coevolutionary arms race between plants and pathogens,plants have developed an efficient and complex immune system.One important branch of this system involves the detection by cell surface-distributed pattern recognition receptors of highly conserved microbe-associated molecular patterns（MAMPs）,leading to the activation of defense responses.This can be referred to as MAMP-triggered immunity（MTI）.Successful pathogens secret during infection a large amount of toxic effectors for subverting MTI from hosts.Thus,identifying the targets of effectors in host cells would help deeper understanding of the innate immune of plants and host-pathogen interaction.Phytophthora infestans is a pathogenic oomycete that widely causes late blight disease in solanaceous plants,including potato,tomato and tobacco.During the biotrophic stage of infection,P.infestans transfers a class of Rx LR domain-containing effectors into host cells to disturb the immune system.This thesis focused on a P.infestans-derived Rx LR effector,called SFI5,which localizes on plasma membrane and is able to repress flg22-induced MTI in host cells.With the aid of Agrobacterium-mediated transient expression approach,functional characterization of SFI5 was studied in Nicotiana benthamiana.The results obtained are as follows:1.Establishment of a rapid method for identifying MTI-suppressing effectors via the Agrobacterium-mediated transient expression in N.benthamiana.Learning from the ROS assay in tomato protoplasts,it was found that flg22 and chitin were both able to induce oxidative production in N.benthamiana leaves.Furthermore,it was shown that flg22 or chitin-triggered ROS was failed to be detected in N.benthamiana leaves transiently expressing Avr Pto-GFP,but was detective in GFP-expressing leaves.These results suggested that ROS burst assay in N.benthamiana leaves expressing exogenous proteins can fast identify MTI-suppressing effecotors.2.The N-terminal region from Phe63 to Lys84 is important for the effector activity of SFI5.a series of N-terminal deletion mutants of SFI5 were transiently expressed in N.benthamiana leaves followed by flg22-induced ROS burst assay,it revealed that the truncated variants containing full-length of effector domain（SFI5 28-241aa and SFI5 63-241aa/SFI5 ED）were able to suppress flg22-triggered oxidative burst,while other variants（SFI5 84-241aa、SFI5 102-241aa and SFI5 178-241aa）lost the ability to repress ROS production elicited by flg22.Subcellular localization observation showed that all these variants are mainly enriched on plasma membrane.These results suggested that loss of N-terminal region（63-84aa）significantly destroy effector activity of SFI5,but has no effect on its plasma membrane localization.3.The potential ATP/GTP-binding motif（P-loop motif）is critical for the effector activity of SFI5.Based on a bioinformatics analysis used for motif scanning,a predicted P-loop motif appeared was found in the N-terminal region（63-84aa）of SFI5 and its significance was examined by replacement of the conserved Lys⁸² with Ala（SFI5 ED-K82A）.The results showed that mutation of the Lys82 residue dramatically attenuated the capability of SFI5 ED to inhibit oxidative burst mediated by flg22,but did not influence the plasma membrane localization.Patho-assay conducted with P.infestans in N.benthamiana also proved that transient expression of SFI5 ED-K82A did not enhance host susceptibility to P.infestans infection,suggesting that the Lys⁸² residue in the P-loop motif serves as a determinant for toxic function of SFI5.4.SFI5 may target Nb PHB1 and Nb MPB2Cb in N.benthamiana.We performed immunoprecipitation assays followed by LC-MS/MS analysis with N.benthamiana leaves expressing SFI5 ED or SFI5 ED-K82A.A number of candidate proteins associated with SFI5 were detected.The interaction between SFI5 and Nb PHB1 or Nb P2C was further confirmed by co-immunoprecipitation assays.Subcellular localization analysis presented that GFP-Nb PHB1 was exhibited in spot-like particles in cytoplasm,while GFP-Nb MPB2C displayed plasma membrane localization.Thus,more experiments need to be performed to uncover the relationship between SFI5 and its targets.5.SFI5 may interrupt flg22-induced expression of Nb WRKY1 and Nb MPB2Cb in N.benthamiana.It was shown that the transcriptional levels of Nb WRKY1 and Nb MPB2Cb were evidently upregulated upon flg22-elicitation,while the expression levels of Nb P2C were not affected by flg22.In the presence of SFI5 ED,flg22-promoted expression of Nb WRKY1 and Nb MPB2Cb was strongly diminish,indicating that SFI5 may toxic function by interfering with expression of Nb WRKY1 and Nb MPB2Cb mediated by flg22-induced MTI.In summary,in this study,a transient expression system of Tobacco was constructed to optimize the expression and detection system of toxic proteins.It was revealed that the P-loop motif at the N-terminus of the SFI5 effector domain has an important effect on its toxic function.Each of its target proteins and regulatory genes in the host provides molecular basis for further analysis of SFI5 toxicity mechanism in the future.