Structure Analysis And PcUE1 Enzyme Activity Of Phytophthora Capsici Effector Molecule RxLR98344

Phytophthora capsici is a soil-borne plant pathogen with a variety of hosts.Pathogens secrete a large number of effectors during host plant infections,including CRN and RxLR effectors,but their mechanism of action in pathogens remains largely unknown.Therefore,this study investigated the RxLR effectors and dehydrogenases of Phytophthora capsici.The infested crops of Phytophthora capsici include pepper,eggplant,tomato,cucumber and other solanaceous crops,which have been reported throughout the country.Phytophthora disease has a rapid onset rate and a wide range of incidence,causing great losses and impacts on the yield and quality of vegetables.At present,the main prevention and control measures for the oomycetes of Phytophthora capsici are chemical control and the control of disease-resistant varieties.The chemical agents used in chemical control are single,and the resistance to pathogens is enhanced.Therefore,the structural biology of ozon protein provides a theoretical basis for screening new drug targets and designing low-toxicity and high-efficiency bactericidal drugs.This article mainly analyzes the mechanism and function of the pathogenic mechanism by analyzing the structure of the RxLR effector protein of Capsicum annuum.The target gene is linked to the pET28a vector by using the gene cloned gene RxLR98344,and the most commonly used and highly efficient prokaryotic expression system is used.E.coli Rosetta(DE3)as a host,IPTG induction of target protein expression;by nickel affinity chromatography,gelfiltration chromatography and other steps for protein purification;the use of meteorological diffusion method sit drop method and hanging drop method two methods of screening purposes protein crystals;Finally,data were collected by X-ray diffraction,and the use of kinetic UV spectrophotometer and high performance liquid chromatography for enzyme activity assay analysis.Thiamin pyrophosphate(ThDP)is often used as a coenzyme in plants or animals to help its receptor proteins catalyze and transport substrates,and plays an integral role in the growth and metabolism of plants or animals.PcUE1 is the first ThDP-dependent enzyme found in the oomycete of Phytophthora capsici.At present,we have obtained the tertiary structure of PcUE1,and its structural characteristics show that PcUE1 has high similarity with NAD~+dependent alcohol dehydrogenase in other bacteria.After further analysis,it was found that the tertiary structure catalytic center conformation of PcUE1 is different from that of ThDP-dependent enzyme,and no combination of NAD~+cofactor was observed in the tertiary structure of PcUE1.According to its three-dimensional structure conformation and characteristics,we speculated 18 possible substrates,and used kinetic UV spectrophotometer and high performance liquid chromatography for enzyme activity determination.The main findings are as follows:(1)The target gene of RxLR98344 was cloned from the SD33 genome of the Phytophthora capsici.The N-terminal of the RxLR98344 effector molecule contains the RxLR-DEER conserved region and the C-terminal is the effector region,which conforms to the sequence characteristics of the RxLR effector molecule.A prokaryotic expression vector of RxLR98344 pET28a fused with a His-tag was constructed and successfully expressed in E.coli.(2)The RxLR98344 protein was purified by nickel affinity chromatography,cation exchange chromatography and molecular sieve chromatography to obtain a relatively pure and relatively homogeneous mother protein solution and selenoprotein solution;Using the Hampton Crystallization Kit to screen protein crystals,RxLR98344 maternal protein data with a resolution of 2.6?was obtained by X-ray diffraction;selenoprotein crystals are undergoing further collection of selenium data.(3)RxLR98344 cannot cause cell death of Nicotiana benthamiana and cannot cause HR reaction.The results of coexpression of RxLR98344 with INF1,Bax,and CRN4,respectively,showed that RxLR98344 was able to inhibit Bax and not to inhibit cell necrosis caused by INF1 and CRN4.(4)By analyzing the three-dimensional structure of the PcUE1 mutant and mass spectrometry analysis,it was judged whether it combined with NAD~+cofactor.In addition,according to the structural conformational characteristics,we predicted 18 kinds of substrates,and carried out the determination of enzyme activity by kinetic UV spectrophotometry and high performance liquid chromatography.It was found that PcUE1 may act as an NADH-dependent dehydrogenase and participate in the reversible oxidation-reduction reaction.The primary substrates for its reduction are m-nitroacetophenone and p-nitroacetophenone.