Biological Function Analysis Of Sporulation-associated Gene SNT2 In Magnaporthe Oryzae

Magnaporthe oryzae is one of the most important phytopathogens that affect global rice production.Besides,it is capable of infecting barley,wheat,corn and other cereal plants,causing serious losses of food production worldwide.Therefore,it is of great interest to study the pathogenic mechanism,identify important pathogenic related genes,in order to develop new strategies for the prevention of rice blast fungus.M oryzae infects plant in the form of spore,and the ability in producing spores determines the epidemicity of rice blast disease.Thus,it has been of great interest in identifying the genetic factors and pathways that affect spore production and differentiation in M.oryzae.SNT2 is one of the important sporulation-related genes in the fungal phytopathogen Fusarium oxysporum.Previous studies have shown that deletion of gene SNT2 impairs sporulation,pathogenicity and increases autophagosome abundance in F.oxysporum severely.In the model fungus Saccharomyces cerevisiae,SNT2 encodes a transcriptional factor which tends to enrich in the promoters of genes involved in oxidative stress responses.SNT2 gene is therefore considered as a key regulator in regulation of oxidative stress responses in S.cerevisiae.It also exits SNT2 homolog in M.genome,but its biological functions remain unclear.In this study,we charaterized the biological functions of SNT2 in M oryzae,and main results are listed as follows:1.Protein sequence analysis revealed that the SNT2 from M oryzae contains the conserved SNT2 protein domains,composing a typical BAH(bromo-adjacent domain)domain and three PHD(plant homeodomain)domains and a ELM(Egl-27 and MTA1 homology2)domain,is highly similar to those homologs from other fungi in amino acid sequence.2.In order to analyze the subcellular localization of proteins SNT2,?we constructed a green fluorescent protein(GFP)fusion expression vector,in which the GFP was labeled in C terminal of SNT2 protein.The GFP-SNT2 fusion expression vector was introduced into M.oryzae by PEG-mediated propoplast fungal transformation method,after which transformants stably expressing SNT2-GFP fusion proteins were screened in the presence of herbicide glufosinate ammonium sulphateum.Fluorescence microscopy analysis revealed in mycelium,conidium and appressorium SNT2-GFP fusion proteins were mainly localized in the nuclei.3.Using Aku70 as a starting strain,we generated ?ku70?snt2 gene knock-out mutants by the split-marker method.PCR analysis indicated that eight independent mutants of Aku70Asnt2 mutants were producted through homologous recombination.Phenotypic analysis showed that,compared to the wild type strain ?ku70,the gene SNT2 knock-out mutants Aku70Asnt2 was severely impaired in both conidiation on complete medium(CM),and asexual reproduction when mating with the opposite stran TH3.When grown on CM agar medium,ability of the vegetative growth for the mutants was decreased,and the ablility in withstanding osmotic stress(NaCl and Sorbital),cell wall stress(SDS,CFW,Congo Red)was also reduced.In addition,the mutants were highly sensitive to oxidative stress(H2O2),indicating the importance of SNT2 gene in regulating oxidative stress responses in M.oryzae.Collectively,these results suggest that the gene SNT2 is critical for sporulation and sexual reproduction of M oryzae and plays important roles in regulation of oxidative stress responses.