Mechanism Of S-nitrosylation Regulated Pathogenic Process In Magnaporthe Oryzae

S-nitrosylation is a protein redox modification,which is involved in the regulation of various biological activities in eukaryotes.The level of intracellular S-nitrosylation is regulated by S-nitrosylglutathione reductase（GSNOR）.In this study,we propose to reveal the biological significance of S-nitrosylation by analyzing the function of ADH5,a homologous protein encoding GSNOR in Magnaporthe oryzae.S-nitrosylation target proteins were identified by proteomics analysis and though the research on the regulation mechanism of target protein of S-nitrosylation,the molecular mechanism of S-nitrosylation in pathogenicity was revealed in Magnaporthe oryzae The results are as follows:（1）Firstly,the functional characteristics of S-nitrosoglutathione reductase ADH5protein in Magnaporthe oryzae were analyzed.ADH5 encodes a protein with 381 amino acids,containing an ADH＿N ethanol dehydrogenase domain at the N-terminal and an ADH＿zinc＿N zinc finger domain at the C-terminal.The ADH5 protein of Magnaporthe oryzae was relatively conserved in different species,and shared the highest homology with Fusarium graminis and a relatively high homology with several pathogenic fungi.The expression level of ADH5 gene was relatively high during appressorium maturation and invasive hyphae,suggesting that ADH5 may play an important role at these stages.The ADH5 gene was knocked out by split-PCR method,and two mutants with the same phenotype were obtained.Then complementary experiments were performed on the knockouts.The level of S-nitrosylation could be detected by biotin switch assay,and it was found that the level of S-nitrosylation was significantly increased inΔadh5 compared that in the wild type,indicating that ADH5 negatively regulates the level of S-nitrosylation.NO detected by NO fluorescence probe（DAF-FM）was found to highly accumulated in mycelium and appressorium inΔadh5,suggesting that ADH5 may be involved in the regulation of NO level in cells.Subcellular localization revealed that ADH5 protein was mainly located in cytoplasm and mitochondria,which was consistent with its role in redox modification.（2）Secondly,the biological function of S-nitrosoglutathione reductase ADH5 in Magnaporthe oryzae was revealed.Biological phenotypic analysis ofΔadh5 showed defects in mycelia growth,sporulation and conidia formation.Pathogenicity analysis showed that the pathogenicity ofΔadh5 was significantly reduced in both barley and its spread ability in host cells was weakened.In order to further understand the reasons for the weakened virulence ofΔadh5,we observed the infection process in barely leaves such as the appressorium and the infection hyphae of the knockouts.The results showed that appressorium formation inΔadh5 was not affected,but the appressorium turgor was significantly impaired.Furthermore the utilization of glycogen and liposome was slowed down,causing the failure to form functional appressorium inΔadh5 and thus weakened the ability to penetrate the host cells slowed down inΔadh5.Further analysis showed that the expansion of the infectious hyphae in the host cells slowed down inΔadh5 and led to the production of a large amount of reactive oxygen species（ROS）in the host cells,indicating that theΔadh5 may not be able to remove the ROS produced by the host plant cells and limited the expansion of the infectious hyphae.Environmental pressure sensitivity experiments show that the knockouts were extremely sensitive to hydrogen peroxide,which is consistent with the above results.At the same time,the deletion of ADH5 gene affected the utilization of different carbon sources by Magnaporthe oryzae,so it might also affect the nutrition utilization of Magnaporthe oryzae from host cells,thus reduced the expansion of infectedhyphae.In conclusion,ADH5 is involved in the regulation of functional appressorium formation of Magnaporthe oryzae and the process of host penetration,as well as the process of active oxygen detoxification and carbon source utilization of infectious hyphae in host cells.（3）Thirdly,we identified a large of target proteins of S-nitrosylation in Magnaporthe oryzae by establishing the S-nitrosylation proteomics technique.Combined with iodo TMT labeling and tandem mass spectrometry,a large-scale identification technique for S-nitrosylation modified target proteins of Magnaporthe oryzae was established.Using this technique,741 modification sites in 483 target proteins modified by S-nitrosylation were successfully identified from different developmental stages of Magnaporthe oryzae and samples treated with reactive oxygen species.In order to further explore the regulation mechanism of S-nitrosylation in M.oryzae,29 S-nitrosylation proteins related to pathogenicity of M.oryzae were found by searching the published pathogenicity related proteins of M.oryzae,which were mainly involved in appressorium formation and infectious hyphae expansion.The four Septin proteins were all predicted to be S-nitrosylated proteins.SEP6 was selected as the representative for S-nitrosylation verification and functional analysis.SEP6 directly interacted with ADH5 through yeast two-hybrid hybridization and immunoprecipitation,and the level of S-nitrosylation of SEP6 was significantly increased inΔadh5.After mutating the C144 site of SEP6 to serine,it was found that the level of S-nitrosylation in SEP6 was reduced,indicating that this site was indeed the modification site of SEP6.The pathogenicity,growth rate,sporulation,infection rate and wound extension ofΔsep6/SEP6^C144S were significantly decreased,which was consistent with theΔsep6 phenotype.It was found that SEP6^C144S could not located in Septin ring by subcellular localization experiments,and GFP-SEP6 could not locate in Septin ring inΔadh5,indicating that ADH5 regulates the normal localization of Septin protein by regulating the S-nitrosylation homeostasis of SEP6 to influence its biological function.In conclusion,this study revealed the pathogenic mechanism of ADH5-mediated S-nitrosylation in M.oryzae by studying the characteristics of ADH5 protein,its biological function in M.oryzae,as well as the proteomics analysis of S-nitrosylation and the verification of target protein.