| Phytophthora sojae infects soybean and results in soybean root rot,causing billions of dollars of losses worldwide every year.At present,the safe and effective strategy in agriculture is to rely on disease-resistant cultivars to control the occurrence of the disease.But Phytophthora is prone to mutation and invalidation of disease-resistant cultivars,leading to a large-scale outbreak of crop blight.Studying the interaction mechanism between plant pathogens and host not only helps us to understand the pathogenic mechanism of plant pathogens,but also identifies new components of plant immune system,which provides new clues for improving durable disease resistance of crops.In our laboratory,we focused on soybean and P.sojae,and a series of studies were carried out in the early stage.It was found that hundreds of RxLR effector molecules secreted by P.sojae could function through programmed transcription and functional interaction,but the virulence mechanism of most effector is still unclear.In this study,the structures of two RxLR effectors PsAvh240 and PsAvr3b were solved and their functions were analyzed.We also investigated the virulence mechanism of PsAvh240.The main research contents are as follows:Structural and functional analysis of P.sojae RxLR effector PsAvh240.We found that PsAvh240 could significantly promote the infection of P.sojae when we used P.sojae to infect soybean hairy roots expressing PsAvh240.Subcellular localization experiments revealed that PsAvh240 was located at the cell periphery.We next confirmed that PsAvh240 localizes in plant plasma membrane by plasmolysis,co-localization with plasma membrane protein Remorin and detection of different subcellular components.In order to study the virulence mechanism of PsAvh240,we solved the crystal structure of PsAvh240 by structural biology experiments.The structure of PsAvh240 contains two typical WY motif structural units,but these two structural unitsare coupled tightly together through a long a helix,thus maintaining the structural stability,which is obviously different from the previously reported connection mode of WY motif.PsAvh240 packed as a dimer in the crystal.We confirmed that PsAvh240 is also a dimer in solution,and the dimerization form is required for it to perform virulence function.We identified the key region that determine the localization of PsAvh240 on plant plasma membranes by deletion mutants,and proved that the plasma membrane localization of PsAvh240 is necessary for its virulence function.Our results show that different regions of PsAvh240 determine its localization on plant plasma membrane and self-dimerization,which is necessary for the virulence function of PsAvh240.Study of the virulence mechanism of P.sojae RxLR effector PsAvh240.The target protein of PsAvh240 in plants was identified by in coimmunoprecipitation assay combined with liquid chromatography-tandem mass spectrometry(LC-MS/MS).A soybean aspartic protease GmAP1 can interact with PsAvh240 on plant plasma membrane in planta,and the region determined the plasma membrane localization of PsAvh240 is necessary for the interaction.We cloned three other genes with high similarity to GmAP1 from soybean,namely GmAP2,GmAP3 and GmAP4.Coimmunoprecipitation results showed that PsAvh240 could only interact with GmAP2,but could not interact with GmAP3 and GmAP4.GmAP2 shares 97%amino acid sequence similarity with GmAP1,while GmAP3 and GmAP4 share 76%amino acid sequence similarity with GmAP1.GmAP1 contains a functional signal peptide.After expressing GmAPl in Nicotiana benthamiana,GmAPl can be detected in apoplast fluid,indicating that GmAP1 is an apoplastic protein.Overexpression of GmAPl in soybean can enhance soybean resistance to P.sojae,while silence of GmAP1 and GmAP2 in soybean can improve soybean susceptibility to P.sojae,suggesting that GmAPl plays a positive role in resistance to P.sojae infection.When PsAvh240 and GmAP1 were co-expressed in N.benthamiana,the protein level of GmAP1 detected in apoplast fluid decreased significantly,while the amount of GmAPl protein increased significantly in plant cells,suggesting that PsAvh240 inhibited the secretion of GmAP1.Mutants that could not inhibit the secretion of GmAP1 could not promote the infection of P.sojae,suggesting that the function of PsAvh240 to inhibit the secretion of GmAP1 depended on their interaction.We found that PsAvh240 specifically inhibited the secretion of GmAP1 and GmAP2,but not GmAP3 and GmAP4.In addition,PsAvh240 could not inhibit the secretion of a plant serine protease P69B.Our study reveals a new virulence mechanism of pathogen effector,that is,PsAvh240 can promote infection by inhibiting the secretion of host aspartic protease.Structural analysis of P.sojae RxLR effector PsAvr3b.We expressed PsAvr3bP6497,PsAvr3bP7076 and GmCYPl through Escherichia coli expression system respectively.After that,affinity chromatography,ion exchange chromatography and gel filtration chromatography were used to obtain higher purity protein.Finally,we obtained the crystals of PsAvr3bP6497,PsAvr3bP7076 and PsAvr3bP6497 treated with GmCYP1.Selenomethionine protein derivatives was expressed and the phase was resolved by single-wavelength anomalous diffraction method,and the three-dimensional structure of PsAvr3bP6497 treated with GmCYP1 was obtained.However,the crystal of PsAvr3bP7076 has low diffraction resolution and we can not solve its structure,which need to further screening and optimization.The crystal structure shows that PsAvr3bP6497 cantains a WY motif-like structure and a typical Nudix hydrolase domain structure.Although the structure of WY motif-like is significantly different from that of typical WY motif units,it still has two "W"and "Y" amino acids and forms hydrophobic core through their interaction to maintain the stability of the structure composed of three a helixes.The structure of Nudix hydrolase domain of PsAvr3bP6497 is highly homologous to that of Arabidopsis AtNUDT7.Our results show that PsAvr3bP6497 is an RxLR effector with special structure containing WY-Nudix. |
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