Crystal Structure And Target Screening,Identification Of RxLR154 Interaction Protein From Phytophthora Capsici

Phytophthora capsici,which is an oomycete disease,can spreads through the soil and cause destructive diseases to many plants.With the increase in the cultivation of peppers,the diseases are becoming more and more serious,which can cause a large area of pepper death in a short time.At present,there are no effective measures to prevent and control the diseases,Moreover,the fungicides have not obvious effects.Then,screening for drug targets from the pathogenic mechanism of P.capsici is crucial to the prevention and treatment of the diseases.In this paper,the PcRxLR154 was amplified from the cDNA of Phytophthora capsici standard strain LT1534,and crystal structure of PcRxLR154 was analyzed by purification and crystallization.At the same time,the interaction protein of PcRxLR154 was screened by yeast two-hybrid.Pull-down validates the interaction between glycolate oxidase(GOX)and PcRxLR154;the interaction between OEE and PcRxLR154,The main findings are as follows:(1)PcRxLR154 was amplified by PCR from LT1534 and the pET22 b prokaryotic expression vector of RxLR154 was constructed.The protein was successfully expressed in E.coli by fusing the His tag.(2)The soluble protein was obtained by nickel column affinity chromatography.High-concentration and high-purity protein was obtained by ion exchange chromatography and gel exclusion chromatography.The crystallization conditions of PcRxLR154 were screened and optimized by using the Hampton Crystallization Kit,and obtain a single good quality protein crystal.In Shanghai,the light source received a set of 1.8? data by X-ray diffraction.(3)The yeast two-hybrid technique was used to screen the interaction proteins of PcRxLR154.A total of three suspected interaction proteins related to disease resistance were successfully screened and fully verified GOX,STPK and OEE,Separately.(4)Constructed the three interacting protein into a prokaryotic expression vector for protein expression.The Pull-down technique verified the interaction of the screened interacting proteins and PcRxLR154.Pull-down verified the in vitro interaction of PcRxLR154 with glycolate oxidase(GOX)and oxygen-enhancing protein(OEE).This study further reveals the interaction mechanism between pathogens and hosts,and provides new strategies and theoretical guidance for the development of disease control.