Crystal Structure And Function Analysis Of Effector NLP12 From Phytophthora Capsici

When Phytophthora capsici infects host plants,it secretes NLPs effectors to interfere with plant defense and achieve the purpose of invasion.The epidemic caused by P.capsici has caused huge economic losses in the planting industry.Currently,the prevention and treatment of the disease are mainly based on chemical control.The widespread use of broad-spectrum chemical agents has also brought serious ecological problems.At present,there are still many unknown researches on the pathogenic mechanism of extracytoplasmic effector molecules NLPs.Protein structural biology can provide theoretical support for the research on the pathogenic mechanism of NLPs and the design of target drugs.In this study,the effector molecule Pc NLP12 was amplified from the c DNA of P.capsici SD33,and the protein structure of Pc NLP12 was analyzed by prokaryotic expression,purification,and crystallization.Five key amino acids were identified through protein structure analysis.Preliminary functional testing of Pc NLP12 and five mutants was performed using Agrobacteriummediated transient expression technology;at the same time,the mutants were purified and crystallized for protein structure analysis.The main results are as follows:Pc NLP12 was cloned from P.capsici SD33,a NLP12-p ET22 b prokaryotic expression vector was constructed,and the target protein was fused with the C-terminal his-tag of the vector.After transformation,the target protein could be expressed in large quantities in E.coli.After the nickel column affinity chromatography,anion exchange chromatography and gel exclusion chromatography,the fusion protein with high purity and stable properties were obtained.High-quality protein single crystals were obtained by screening using the Crystal Research Kit of Hampton Research Company.After X-ray diffraction,a set of data with a maximum resolution of 1.7(?) was received.The molecular structure of Pc NLP12 was analyzed by molecular replacement.The structure shows that Pc NLP12 contains a negatively charged core domain.This domain consists of two sets of antiparallel β-sheets.The core structure is composed of three α-helixes at the top of the core structure and a relatively flat surface at the bottom.The loops of different lengths constitute a relatively uneven surface and have a flexible N-terminal region.The key amino acid sites of Pc NLP12 were site-directed and mutated respectively.All mutants were purified and crystallized from prokaryotic expression on the one hand.After optimization,all high-quality protein single crystals were obtained.On the other hand,transient expression technology was used for inoculation experiments.Pc NLP12 bacterial solution can cause the necrosis of Benthian tobacco leaves and may promote the infection of INFI,all mutant bacterial solutions can not cause the necrosis of Benthian tobacco leaves.Through the comparative analysis of protein structure analysis,11 potential interaction targets with Pc NLP12 were selected,which laid a solid foundation for future work.In this study,the structure of NLP12 protein was analyzed and its functions and mutants were studied,providing theoretical support for studying the pathogenic mechanism and disease prevention of Phytophthora capsici.