Target Screening,Identification And Co-crystallization Of RxLR145 And RxLR197 Interaction Protein From Phytophthora Capsici

Phytophthora capsici,which is a kind of destructive soil-borne oomycete disease,infecting a lot of agricultural crops..When Phytophthora capsici infects the host,it secretes a large amount of Rx LR effectors to interfere with the plant and promote the occurrence of plant diseases.In recent years,the Phytophthora blight of capsicum is becoming increasingly serious and causing huge blow to the pepper industry.At present,there are no effective measures to control the disease.Therefore,it is extremely important to screen potential targets based on the pathogenic mechanism of Phytophthora capsici.Based on the three-dimensional structure of PcRx LR145 and PcRx LR197 effector proteins,this paper explored the pathogenic mechanisms of these two effectors in the host plant using CHIP-Seq,RNA-Seq and other technical means.Gathering with pull-down,micro thermal surge(MST)and any other experimental methods,the suspected positive interaction targets wereverified.Thus to determine the interaction targets of these effectors and explain their pathogenic mechanisms.Subsequently,we expected to analyze the complex structures of effectors and their interaction targets using crystallographic methods.The main findings are as follows:(1)CHIP-Seq experiments were conducted on transgenic Arabidopsis thaliana containing PcRx LR145.The results showed that PcRx LR145 could bind to DNA sequences containing GAA repeats and GAGA repeats at the 5’ terminal in the Arabidopsis thaliana genome.Meanwhile,the expression of these genes could be regulated by PcRx LR145 proved by fluorescence quantitative PCR.(2)Microscale thermophoresis(MST)experiments verified that PcRx LR145 binds to DNA fragments containing GAA and GAGA repeats to binding capacity extent of n M level.(3)Complex crystals containing PcRx LR145 and GAA/GAGA repeating DNA fragments were cultured and the complexes were analyzed.However,only PcRx LR145 was detected in the crystals,which may due to the DNA was too flexible and being low-affinity in the crystals.(4)Pull-down experiment demonstrated that PcRx LR197 interacts with RPM1 interaction protein(RIN4)and heat shock protein 90(HSP90).In addition,the interaction of capsicum serine threonine protein kinase(STPK)and RIN4,GME and HSP90 were also proved in this study.(5)The crystals of PcRx LR197 and its interaction protein RIN4 were co-cultivated to analyzed the three-dimensional structure,which is growing now.This study was aimed to further reveal the interaction mechanism between the Phytophthora capsici effectosrand the host with stuctural biology,the pathogenic mechanisms of Phytophthora capsici.