The Expression,Purification And Crystallization Of RxLR101012 And Screening The Interacting Protein Of RxLR101860 From Phytophthora Capsici

Phytophthora capsici is a worldwide soil borne disease.It’s widely distributed.P.capsici has a wide range of hosts,including pepper,pumpkin,cucumber,eggplant,tomato,and so on,up to more than 20 kinds of plants.The pathogens oospores hibernate spend the winter in soil or plant residues,and spreaded by winding,raining,irrigating and cultivation.After colonization,the pathogens germinate new sporangium and zoospores for the next infection.Resent years,we have made some progress on the machnisam of Phytophthora infection and it also attracts more and more attention due to its destructive ability.Phytophthora cause enormous economic losses on crop species as well as environmental damage in natural ecosystems.Phytopathogenic pathogens secrete a series of effectors into the host cells or remain stay in the periplasmic space or called extracellular space during the colonization and infection.Effectors could damage or disturb the normal metabolism of host cells,thereby promoting the pathogen secretion and transmission.Pathogenic effect of the oomycete could induce host cell necrosis or induce the host cells to produce defense response.Pathogenic oomycete have the function of inducing necrosis or induced host cells to produce defensive responses.There are two classes of cytoplasmic effects,one is the RxLR effector,which has conserved N-terminal sequence and their C-terminal have no conventional function domain.Most of the effector’s N-terminal contains an RxLR sequence and is therefore referred to as the RxLR effector.The other class of effectors contain a conserved sequence Lx LFLAK in their N-terminal,called the Crinklers(CRNs).In this study,we focus on cytoplasmic effectors RxLR.There is few studies on the effector expression,purification and protein crystallography of Phytophthora capsici,especially the crystal structure of the effector.In this study,we successfully cloned a RxLR effector(RxLR101012)from Phytophthora capsici,and based on prokaryotic expression and protein purification technology including affinity chromatography,ion exchange chromatography and gel exclusion chromatography,we got the protein solution with high purity,and we have got the initially protein crystal by using hanging drop and sitting drop method and didX-ray diffraction.By optimizing the conditions,the crystal diffracts to 3.8 ?.We made foundation for the optimization of protein crystal culture.The cytoplasmic effectors are secreted into host cell and then regulated the host’s immune response by binding to the target protein.In this study,we used yeast two hybrid technology to screen four interaction proteins of effector RxLR101860,and then verified by yeast two hybrid co-transformation.The whole gene of the interacting protein was cloned successfully,then made the BIFC vector and co-immunopreciptation vector.We will then do BIFC and Co-IP experiment.The screening and validation of the interaction protein of effector RxLR101860 provides a basic foundation for further study about the role of the effector in host immune defense and pathogenic mechanism of oomycete.Through this study,we got crystal structure of effector RxLR101012,accumulated experience for the study of optimization crystal structure and biological,further we obtained the target protein of effect RxLR101860,and provides a basic foundation for the interaction between pathogenic oomycetes and hosts.