Functional Analysis Of Two Putative Citrate Synthase Genes In Magnaporthe Oryzae

The rice blast fungus?Magnaporthe oryzae?is an ideal model system to study the intereaction between a fungal pathogen and the host?rice?.Energy and metabolism is vital to spore germination and appressoria formation.However the contribution of glyoxylate and TCA cycles to the pathogneicity of M.oryzae is unknow.Citrate synthases are key enzymes in both glyoxylate and TCA cycles and may play an important role in the growth,development and pathogenicity of M.oryzae.In this study,we identified two putative citrate synthase genes MGG 07202 and MGG₀2617 from the M.oryzae genome database.Bioinformatics analysis showed that both genes encode hydrophilic proteins,without signal peptide and transmembrane domains.Both have the Citrate_Synt domain and the mitochondrial transit peptide,suggesting that they are located in the mitochondria.Further analysis revealed that MGG₀7202 is homologous to Citl in yeast and CitA in Aspergillus nidulans,while MGG₀2617 is homologous to Cit3 in yeast and McsA in A.nidulans.Public gene expression data showed that MGG₀7202 gene expression is higher than MGG₀2617 in spore germination and reaches the highest level at the time point of 6 h after spore germination.To investigate the functions of MGG₀7202 and MGG₀2617,we carried out gene konckout and complementation experiments in M.oryzae.The data showed that the MGG₀7202 deletion mutants?? 7202 strains?displayed some defects in growth,conidiation,appressorium formation and pathogenicity as well as in degradation of glycogen and lipid during spore germination suggesting that the metabolic reaction catalyzed by this enzyme is important for proper development and pathogenecity in M.oryzae.In contrast,the MGG₀2617 deletion mutants??2617 strains?showed no defects in growth rate,but their sporulation and pathogenicity were significantly reduced.The A24617 strains were non-pathogenic on the intact leaves of barley and rice but exhibited weak pathogenicity in wounded leaves.To investigate further the roles of these two putative citrate synthases in carbon metabolism in M.oryzae,we determined the growth rate of A 2617 and A 7202 mutants in MM medium with different carbon sources.Our results showed that in MM medium with glucose,maltose,glucose,sucrose or lactose,the growth of A7202 strains was inhibited,but the growth of A2617 strains was not.Both A2617 and A7202 mutants can grow on MM with pyruvic acid and citric acid,suggesting the presence of residual citrate synthase activity in each mutant.However,the growth of each mutant in MM with pyruvic acid as the sole carbon resource is slower than the wild type strain,indicating that both citrate synthases are important for the synthesis of citrate from pyruvic acid.High concentrations of propionate can inhibit the growth of both mutants,while at low concentrations of propionate,the growth ?617 mutants can be restored,but the ?7202 mutants remain defective in growth,which is similar to the AMcsA mutant of A.nidulans,implying that MGG₀7202 may also have the activity of methyl citrate synthase.In this study,our functional characterization of the two putative citrate synthase genes in M.oryzae has laid the foundation for further eluciding the roles of energy and metabolism in the development of fungal pathogens and their interactions with plant hosts.