Characterization And Functional Analysis On MoSFa1 And MoHB1 Of Magnaporthe Oryzae

Rice blast is one of the most destructive rice diseases,caused by Magnaporthe oryzae,and sustainable control of rice blast disease is still one of the last important problems in rice production.Research on gene function of this fungus will help us to better understand its biological characteristic,from which will be benefited to develop some targeted control measures.Basing on the released genomic data and the perfect performance of genetic manipulation of M.oryzae,this fungus gradually has become a model to study molecular biology and pathogenicity mechanism,and research on the interaction with rice has been an important component to understand the interaction of plant pathogenic fungi and their hosts.Nitric oxide?NO?is a pleiotropic molecule in organisms.And it is involved in a number of physiological and biological processes as an important signaling molecule.On the other hand,an excess of NO can also cause cells in nitrosative stress.During the interaction of pathogen and plant,plant-derived NO burst in the infected tissues can be induced by the recognition of effectors or pathogen-associated molecular patterns?PAMPs?.Rapid accumulation of NO can trigger plant defense responses,also create a potent antimicrobial environment that conduces to the restriction of pathogen growth.Simultaneously,pathogen-derived NO is discovered and involved in conidiation,formation of infection structure and virulence.Therefore,maintaining the intracellular NO homeostasis has important significance to realize the normal physiological and biological function of pathogen.Microbes generally evolve defense mechanisms to avoid NO excessive accumulation,e.g.S-nitrosoglutathione reductase?GSNOR?and flavohemoglobin,which can convert NO to less toxic molecules.It is well known that S-?hydroxymethyl?glutathione dehydrogenase?EC 1.1.1.284?with GSNOR activity specifically reduces GSNO to ammonia?NH3?and glutathione disulphide?GSSG?in the presence of NADH and GSH,which is the spontaneous reaction product of GSH and NO.This enzyme belongs to a class III alcohol dehydrogenase,and also is involved in function in the glutathione-dependent oxidation of formaldehyde.This enzyme is conserved from bacteria to humans.In yeast,deletion of its homolog results in increased hypersensitivity to NO stress and abnormal accumulation of cellular S-nitrosothiols?SNOs?.Flavohemoglobin in microbes functions as a NO dioxygenase,specifically oxidating NO to produce NO₃^- under aerobic conditions,flavohemoglobin,but also consumes NO to produce nontoxic N2O under anaerobic conditions.Deletion of its homologs in microbes result in increased hypersensitivity to NO stress.In some pathogens,growth of flavohemoglobin-null mutant is inhibited and their virulence is reduced or even lost.Critically,NO burst in rice cells has been discovered after treatment with compatible or incompatible blast fungus elicitors,and endogenous NO in M.oryzae is crucial to the development and initiation of infection.Presumably,maintaining the intracellular NO homeostasis in M.oryzae could be important during the development or interaction with hosts.Therefor,we firstly focused on GSNOR and flavohemoglobin in M.oryzae in present study.The findings were mainly as follows:Protein sequence of Saccharomyces cerevisiae SFA1 was used to search its homolog from the Magnaporthe comparative database with the BlastP program.A predicted gene MGG₀6011 exhibited the highest similarity to SFA1.The known active residues,which is coordinated to protein structure and function of S-?hydroxymethyl?glutathione dehydrogenase,were strictly conserved in MGG 06011 encoding protein.MGG₀6011 restored the growth of the?sfal mutant on formaldehyde-containing medium and the integral ability of GSNO reduction,which was deemed as the homologue of SFA1 in M.oryzae with the functions in formaldehyde detoxification and GSNO reduction,and was named MoSFAI1.Subcellular localization of GFP and MoSFA1 fusion proteins showed that MoSFA1 protein was distributed in cytoplasm.MoSFA1 expression was developmentally regulated during conidia germination,appressorium development and in planta.MoSFAl gene deletion in M.oryzae lead to a lethal penotype in formaldehyde-containing medium,and growth of mutants were inhibited by SNP treatment,a NO donor.SNOs in MoSFAl deletion mutants were abnormally accumulated,and SNOs accumulation was induced by exogenous NO.MoSFA1 was required for vegetative growth,melanization of mycelium,conidiation.MoSFA1 deletion attenuates the virulence in the integral rice leaf,but not in the wounded leaf.This finding implies that MoSFA1 significantly contributes to virulence in penetration or biotrophic phases,not in necrotrophic phase.Conidial germination and appressorium development of MoSFA1 deletion mutants were normal,compared with that of the wild type,but appressorium turgor and infection rate in barley cells were reduced and growth of infection hyphae were inhibited in host cells.Futhermore,we found that the accumulation of H₂O₂ at infected barley cells by MoSFA1 deletion mutants was more than that by the wild type,and growth of MoSFA1 deletion mutants were significantly inhibited by different oxidants,and loss of MoSFA1 results in the significant reduction of of SOD and POD activities,and of reduced GSH content in cells.Howere,the expression levels of PR genes in the challenged rice tissue by MoSFA1 deletion mutant were not different from those in challenged rice tissue by the wild type.These findings suggest that MoSFAl in M.oryzae is involved in antioxidant system,and in compromising the host defense-related oxidative burst in compatible interaction.Additionally,MoSFA1 also is involved in N utilization.MoSFAl deletion mutants grow slower in minimal medium with nitrate and nitrite as the sole nitrogen source,but the expressions of NR and NiR were dramatically upregulated than of the wild type.One possible explanation is that the related proteins involved in N assimilation could be abnormally S-nitrosylated in muatants,which cause the changes of protein structures and activities.Protein sequence of S.cerevisiae YHB1 was used to search its homolog from the Magnaporthe comparative database with the BlastP program.A predicted gene MGG₀0198 exhibited the highest similarity to YHB1.The known active residues,which is coordinated to protein structure and function of flavohemoglobin,were strictly conserved in MGG₀0198 encoding protein.MGG₀0198 restored the growth of the?yhb1 mutant in GSNO-containing medium,which was deemed as the homologue of YHB1 in M.oryzae,named MoHB1.MoHB1 expression was developmentally regulated during conidia germination and appressorium development during germination.Its expression was enhanced quickly upon exposure to exogenous NO.Growth of MoHB1 gene deletion mutants were significantly inhibited by different NO donors and H₂O₂ treatments.And inhibition effect of MoHB1 deletion mutants on NO stress was more than that of MoSFAl deletion mutants,but effect on H₂O₂ stress was just the opposite.But MoHBl was not required for vegetative growth,conidiation and virulence.MoHB1 deletion resulted in an upregulated expression of MoSFA1 during conidia germination and appressorium development.MoHB1 and MoSFA1 double deletion resulted in more sensitivity to NO donors and oxidants,but double deletion mutant further reduced vilurence compared with MoSFAl deletion mutant.In summary,we characterized the GSNOR and flavohemoglobin encoded genes MoSFAl and MoHBl in M.oryzae,analyzed their functions in the response to NO and oxidative stress,development,and pathogenicity.Our data showed that MoSFAl gene deletion lead to hypersensitivity to NO stress,abnormal accumulation of SNOs in deletion mutants,which implies that the mutants have an enhanced levels of S-nitrosylated proteins.MoSFA1 gene deletion results in the reduction of antoxidant system.Finally,MoSFA1 gene deletion results in the defects in development,virulence and N utilization.MoHB1 gene deletion lead to hypersensitivity to NO and H₂O₂ stress.But compared with MoSFA1,MoHBl plays more important roles in response of NO stress.MoHB1 and MoSFA1 double deletion leads to enhanced sensitivity to NO and oxidative stress,but no further attenuation of virulence.These results imply that NO-reducing pathways mediated by MoSFAl contribute to the development and virulence of M.oryzae,and MoHB1 contribute to dealing with the exogenous NO stress.The findings in this work will contribute to the understanding of NO function and pathogenesis of M.oryzae,and lay a theoretical basis for the development of targeted fungicides.