Identification And Utilization Of Plant Interactors Of Phytophthora Cactorum Virulence Protein SCR96

Plant blight diseases caused by Phytophthora spp.are widely distributed,and often damage many kinds of important crops and forest trees,threatening the global food safety and natural ecosystem.The pathogens secrete apoplastic and cytoplasmic effectors to manipulate the immune systems of host plants.PcF/SCR(small cysteine-rich)effectors are one class of apoplastic effectors secreted by Phytophthora and other oomycetes,which can cause plant cell death.However,the action mechanism of these effectors is still unclear.Our previous studies revealed that SCR96 functions as a virulence factor of P.cactorum and an elicitor of the immune response in both Nicotiana benthamiana and tomato(Solanum lycopersicum).In this thesis,the following studies were performed to reveal the action mechanism of SCR96 involving in the pathogenesis,therefore providing the guidance for the disease management.?To facilitate the heterologous expression,detection and purification of small PcF/SCR effectors,three common affinity tags(6His,Flag and HA)and fusion ends were analyzed.It was found that C-terminal 6His is the best choice for the expression and bioactivity analysis of SCR96.?To elucidate the action mechanism of SCR96,its key interactor proteins in N.benthamiana were screened using both Y2H against the N.benthamiana infection library and Co-IP coupled with mass spectrometry of the N.benthamiana plants transiently expressing SCR96,and validated by Y2H,BiFC and GST pull-down.It turned out that three proteins,a glycine-rich protein NbGRP,a wound-inducing protein NbWIP2,and a protease inhibitor NbPI are the interactors of SCR96 in N.benthamiana.?To elucidate the functions of three plant proteins,their transient expression and VIGS were performed in N.benthamiana.It was found that NbGRP is a positive regulator but NbWIP2 a negative regulator of the plant disease resistance against P.capsici infection.However,NbPI had no significant effect on the resistance of N.benthamiana against P.capsici infection.?To further elucidate the function of NbGRP in plant defence for future utilization,the transgenic plants of N.benthamiana mediated by Agrobacterium tumefaciens infection were made.PCR and Western blot confirmed the positive transgenic lines.Prokaryotic expression and purification of NbGRP was achieved by cloning it into pRSFDuet-1 containing 6His tag.New Zealand white rabbits were immunized using the purified recombinant protein to produce the polyclonal antiserum.It was found that the polyclonal antiserum can be used to specifically detect the target protein with high sensitivity,and therefore is suitable for the detection of NbGRP protein in other studies.In summary,in this thesis,a suitable affinity tag,i.e.C-terminal 6His,was determined for SCR96 expression and analysis.More importantly,its interactors in plants were characterized.Furthermore,the transgenic N.benthamiana plants and polyclonal antiserum of the positive resistance regulator NbGRP were obtained for further studies.The results of this study not only provide experimental data for elucidating the function of Phytophthora apoplastic effectors and therefore understanding of the pathogenicity mechanism of Phytophthora infection,but also provide important resistance gene resources for the management of plant Phytophthora diseases.