Structure Solution Of The RxLR-type Effector RxLR19781 From Phytophthora Capsici

The oomycete Phytophthora capsici is an important plant pathogen that often attacks a variety of crops including pepper,tomato,eggplant and melon.During the interaction between P.capsici and host plants,P.capsici secretes a large number of effectors to suppress the immune defense reaction of the host plants and to promote the infection and colonization by the pathogen.Therefore,understanding the molecular mechanisms unnderlying the Phytophthora-plant interactions is important to elucidate the pathogenesis,and the structure provides a new perspective to help us understand the effect of molecular interactions with plants.In previous studies,we found that Rx LR19781 is an effector of P.capsici.The PVX expression vector and Agrobacterim-mediated transient expression in Nicotiana benthaminan were employed to analyze the function of Rx LR19781.The results showed that Rx LR19781can suppress N.benthaminan programmed cell death triggered by Bax and CRN4.So we selected this protein for structural research.In this paper,in order to solve the structure of Rx LR19781,we cloned the RxLR19781into pET28a and expressed RxLR19781 induced by IPTG in Rosetta?DE3?.Recombinant protein was purified through Ni²⁺affinity column,Ion exchange chromatography and gel filtration chromatography.Crystals were grown by the sitting drop vapor diffusion method.The crystal structure of RxLR19781 was determined by MAD method.The main results and conclusions are as follows:?1?The Rx LR19781 effector was screened and cloned successfully from the genome of P.capsici.The molecular characters of Rx LR19781 was consistant with RxLR effectors family:1-20 amino acids of N terminus are signal peptide and followed by the characteristic amino-terminal motif Arg-X-Leu-Arg?where X is any amino acid?with rapidly evolving C-terminal effector domains..?2?We constructed the RxLR19781-pET28a expression plasmid and it was properly expressed in E.coli through His-tag.?3?By Ni²⁺affinity chromatography purified we found RxLR19781-pET28a expressed target protein soluble.Three methods were used in protein purification that contained Ni²⁺affinity chromatography,ion exchange chromatography and gel filtration chromatography,and then we got high purity stable uniform protein for crystal growth.?4?Through continuous Hampton kits for protein crystal growth,we obtained high resolution diffraction data using sitting drop.Moreover,we got the crystal of selenomethionine-substituted Rx LR19781,and the diffraction data and the crystal phase information were obtained subsequently.We finally got the Rx LR19781 crystal structure.The study provides important experimental data for elucidating the interaction mechanism between oomycete effectors and plants,and offer novel opportunities to develop durable disease control measures.