Functional Analysis Of Genes Of Arginine Biosythesis Pathway And Metabolomics In Magnaporthe Oryzae

Magnaporthe oryzae as the causal agent of the rice blast disease causes huge yield losses in rice production globally every year. Owing to its economic importance and genetic tractability, M. oryzae has recently emerged as a model organism for the studying the mechanisms of plant-fungal pathogen interactions. For many plant pathogenic fungi, the ability to sense nutrition and adjust metabolism accordingly is crucial to successfully infect hosts. For the hemi-biotrophic ascomycete M. oryzae, long standing questions concerning which metabolites the fungi make themselves, and what they obtain from the plant, are largely unanswered.Part I Several studies provide evidence that nutritional requirements, such as amino acid biosynthesis or nutrient availabilities, are important for fungal infection cycles and pathogenicity. Arginine as one of the most versatile amino acids in eukaryote cells, which plays very important roles in a multitude of processes such as protein synthesis, nitrogen metabolism, nitric oxide and urea biosynthesis. In this study, we delineate the putative arginine biosynthetic pathway and systematically analysis the function of MoARG1, MoARG5,6 and MoARG7 in M. oryzae. The main results are showed as follows:1) According to the pathway in S. cerevisiae and other organisms, using the BlastP procedure, all the genes involved arginine biosynthesis were searched in the M. oryzae genome, and the resulting orthologous genes of M. oryzae were cloned respectively [MoARG1 （MGG₁5868.7）, MoARG2 （MGG₀1507.7）, MoARG3 （MGG₀1102.7）, MoARG4 （MGG₁7278.7）, MoARG5,6 （MGG₀2690.7）, MoARG7 （MGG₀4210.7）, MoARG8 （MGG₁1934.7） and MoargE （MGG₀1970.7）]. By amino acid sequence alignment and phylogenetic analysis, the results indicate that the arginine biosynthesis pathway is conserved among fungal kingdom.2) MoARGl, MoARG5,6 and MoARG7 encode three different synthetases, which catalyze the seventh step, second-third step and fifth step in arginine biosynthesis of M. oryzae, respectively. MoARGl was localized in the cytoplasm of the hyphae, conidia, and appressoria. MoARG5,6 and MoARG7 were both localized in the mitochondria. The expression level of target genes during the appressorial development was up-regulated, which indicated that MoARGl, MoARG5,6 and MoARG7 involved the arginine biosynthesis in the development of appressorium.3) The deletion of target genes was performed, the resulting AMoargl and AMoarg5,6 mutants were arginine auxotrophy, while AMoarg7 was a leaky arginine auxotrophy. All Arg-mutants were significantly impaired in aerial growth, melanin pigmentation, conidiation, sexual reproduction, pathogenicity and the degree of defect was AMoarg5,6> AMoargl> AMoarg7 in contrast to Guy11. Supplement of exogenous arginine could partly restore the phenotypic defects of mutants.4) Penetration and invasive growth assays on barley epidermal cells were carried out and found that all Arg-mutants could penetrate plant leaf cuticles but with attenuated in penetration rate, invasive hyphae growth delayed and restricted in host cells, which may be the reason for the severely attenuated pathogenicity during infection.5) NO fluorescence detection was performed and found that the amount of nitric oxide was rised largely during the conidial germination and appressorial maturation of M. oryzae. Additionally, we explored the connection between NO and arginine in M. oryzae. NO unable restore the defects of muants and all Arg-mutants still could generating NO, these results suggested that NO generation may not via arginine/NO mode, or maybe exist other alternative branch routes in M. oryzae.PartⅡ Metabolomics, as a new research technique, it can detect the small molecule metabolites （MW<1000） of biological samples effectively. In this study, using the methods of metabolomics, the metabolites of mycelial, conidia and appressoria in Guyl 1 （wild-type strain） and AMoatgl （autophagy-deficient mutant） were detected. The main results are showed as follows:1) Basing on two technology platform LC-MS and GC-MS, according to the methods of metabolomics in plants and microorganisms and some procedures such as sample preparation, metabolites extraction, instrument parameters and program were modified, then a suitable method for metabolomics analysis in M. oryzae was set up.2) Compared with wild-type strain Guyll, most of metabolites in ΔMoatgl （mycelium, conidia and appressoria） contents decreased in different degree. The significantly different metabolites included sugars, amino acids, polyols and lipid. The results of appressorial metabolomics showed that glycerol content decreased significantly among metabolites, which was consistent with the phenotypic defects of ΔMoatgl in turgor generation, appressorial penetration and pathogenicity.