Preliminary Analysis Of Expression,Purification And Crystallization Of Effector-RXLR23 From Phytophthora Capsici

Phytophthora capsici is a kind of highly dynamic and destructive pathogen and can infect peppers,tomatoes,cucumbers and other species in Cucurbitaceae and Solanaceae.With incidence and severity of pepper blight caused by Phytophthora capsici increasing in recent decades,more and more people have paid attention on the infected mechanism.As the impact of crop loss by P.capsici has increased in recent years,there are new ways and resources to study pathogenic mechanism while many pathogenic genes are found.The interaction between host and pathogen is explained by gene-for-gene hypothesis.The plant immunity system can be activated by the directly or indirectly recognition between the protein encoded by the host resistance genes(R genes)and the protein encoded by the pathogen avirulence genes(Avr genes).Avr genes are also known as effector genes.Thanks to Phytophthora capsici genome published by JGI,more and more effector genes have been predicted but structural biology about effectors is poorly understood.Recently,transport mechanisms of oomycete effectors become one of the hottest issues during the interaction between pathogen and host.Most of effectors are intracellular protein but how these effectors are transported into host is still unclear.We attempt to study the transport mechanism to reveal host resistance mechanism that will make great foundation for biological control and breeding resistant cultivar.In this study,we cloned several effector genes from Phytophthora capsici genome.We successfully obtain high purity and concentration protein through vitro prokaryotic expression and ultimately get an effector crystal structure.The crystal diffracts to 3.2 ? resolution.The main results are as follows:1.Via bioinformatics analysis and forecasting,we cloned more than 50 Avr genes from c DNA of Phytophthora capsici.Three genes can be successfully expressed in prokaryotic expression system and proteins can be easily purified.From results of amino acid sequences alignment,their N-terminal is conserved Rx LR-deer motif while C-terminal keeps the diversity of sequence.Our major work is focused on RXLR23.2.I have obtained stable and high purity RXLR23 recombinant protein by affinity chromatography,ion exchange chromatography and gel filtration chromatography.Because the structural homology identity of RXLR23 is <30%,we can't get its structure through molecular replacement instead by using single-wavelength anomalous dispersion.The approach of expression and purification of selenomethionine protein is the same as the native protein.3.We have got the initially protein crystals by using hanging drop and sitting drop method.After optimizing with the hanging drop method,we finally get high quality protein crystals for X-ray diffraction.Solved by using single-wavelength anomalous dispersion(SAD),RXLR23 is a dimmer that functions in plants.4.The current report is that some effectors can interact with phosphatidylinositol-3-phosphate(PI3P)which is located in the host cell membrane and then directly enter into the host cell.We attempt to explain the binding sites between RXLR23 and PI3 P.Finally,we obtain complex crystal of RXLR23 binding with PI3 P by protein purification and X-ray crystallography.5.In the crystal optimization process,initial crystal resolution is only about 8 ?.After using truncation,dehydration,replacement of antifreeze and other methods,crystal diffracts to 3.2 ? and belongs to the trigonal space group P3221.In our study,we preliminarily obtained the crystal structure of RXLR23 and the complexes crystal of PI3 P and RXLR23.We can observe spatial structure of RXLR23 and try to explain how effectors play role in biological functions.Thereby,structure of RXLR23 will provide important theoretical basis for the interaction between pathogen and host.