Take and utilization of exogenous methionine is not impaired in Dstr3 strains compared to Guy11.Results MoSTR3 gene replacement mutants are unable to convert homocysteine to methionineThe predicted pathway for methionine biosynthesis in M. oryzae is shown in Figure 1. We chose to analyse this pathway in M. oryzae because the genes and enzymes involved have been extensively studied using classical and molecular genetics in the yeast Saccharomyces cerevisiae and the filamentous fungi Aspergillus nidulans and 1676428 Neurospora crassa ([16?8]; and references therein). metG in A. nidulans [17], met-2 in N. crassa [19] and STR3 in Saccharomyces cerevisiae [20] are orthologous genes encoding cystathionine betalyase (EC:4.4.1.8) that converts cystathionine to homocysteine during the de novo biosynthesis of methionine. When these genes are deleted, the resulting mutant strains are strict methionine auxotrophs. Wild type growth and get ML-281 development is restored in strains lacking a functional cystathionine beta-lyase when grown on media supplemented with methionine, indicating STR3 proteins have no additional roles in the cell unrelated to methionine biosynthesis. We used this information to determine which gene to target in order to abolish methionine metabolism. Figure 2 shows that STR3 orthologues are widely distributed across fungal taxa. While the Ascomycota carry similar STR3 orthologues, as shown in Figure 2, STR3 orthologues identified in all the Basidiomycota we examined (except for Postia placenta) carry extra C-terminal sequences (,450 aa) compared to the ascomycete STR3 orthologues. This extra region has weak sequence similarity (30?0 identity) with mevalonate kinase. Mevelonate kinase proteins are found in a wide variety of eukaryotes and prokaryotes, but among fungi they exist only in the Ascomycota (e.g., XP_723495.1 from Candida albicans, XP_661473.1 from A. nidulans and ERG12 from S. cerevisiae [21]) where, at least in S. cerevisiae, they function in the biosynthesis of isoprenoids and sterols [21]. Interestingly, our preliminary analysis indicates that this mevalonate kinase sequence does not exist in basidiomycetes as aFigure 1. Methionine metabolism in Magnaporthe oryzae. De novo biosynthesis of methionine requires homocysteine derived from cysteine ?via cystathionine ?and involves cystathionine beta-lyase (MoStr3). Homocysteine might also result from O-acetyl-L-homoserine. O-acetyl-L-homoserine is derived from aspartate in a pathway involving a number of enzymatic steps that have been omitted for clarity [16]. This scheme is based on the predicted methionine and cysteine metabolic pathway map for M. oryzae at the Kyoto Encyclopedia of Genes and Genomes. doi:10.1371/journal.pone.0047392.gNutrient Conditions during Rice InfectionFigure 2. Maximum likelihood phylogeny of STR3 orthologs. The maximum likelihood phylogeny was reconstructed with RAxML, as described in Materials and Methods. Nodes with black circles indicate that these clusters are well supported ( 70 bootstrap support). Purple branches and species names indicate sequences with a fused C-terminal mevalonate kinase BIBS39 custom synthesis domain. Species that have known STR3 orthologs prior to this study are shown in green. Protein sequences were obtained from the Fungal Genome Collection (FGC). For those species not present within FGC, sequences were obtained from the NCBI database and their accession numbers are given in parentheses. Asterisks indicate sequences retrieved from the Saccharomyces Genom.Take and utilization of exogenous methionine is not impaired in Dstr3 strains compared to Guy11.Results MoSTR3 gene replacement mutants are unable to convert homocysteine to methionineThe predicted pathway for methionine biosynthesis in M. oryzae is shown in Figure 1. We chose to analyse this pathway in M. oryzae because the genes and enzymes involved have been extensively studied using classical and molecular genetics in the yeast Saccharomyces cerevisiae and the filamentous fungi Aspergillus nidulans and 1676428 Neurospora crassa ([16?8]; and references therein). metG in A. nidulans [17], met-2 in N. crassa [19] and STR3 in Saccharomyces cerevisiae [20] are orthologous genes encoding cystathionine betalyase (EC:4.4.1.8) that converts cystathionine to homocysteine during the de novo biosynthesis of methionine. When these genes are deleted, the resulting mutant strains are strict methionine auxotrophs. Wild type growth and development is restored in strains lacking a functional cystathionine beta-lyase when grown on media supplemented with methionine, indicating STR3 proteins have no additional roles in the cell unrelated to methionine biosynthesis. We used this information to determine which gene to target in order to abolish methionine metabolism. Figure 2 shows that STR3 orthologues are widely distributed across fungal taxa. While the Ascomycota carry similar STR3 orthologues, as shown in Figure 2, STR3 orthologues identified in all the Basidiomycota we examined (except for Postia placenta) carry extra C-terminal sequences (,450 aa) compared to the ascomycete STR3 orthologues. This extra region has weak sequence similarity (30?0 identity) with mevalonate kinase. Mevelonate kinase proteins are found in a wide variety of eukaryotes and prokaryotes, but among fungi they exist only in the Ascomycota (e.g., XP_723495.1 from Candida albicans, XP_661473.1 from A. nidulans and ERG12 from S. cerevisiae [21]) where, at least in S. cerevisiae, they function in the biosynthesis of isoprenoids and sterols [21]. Interestingly, our preliminary analysis indicates that this mevalonate kinase sequence does not exist in basidiomycetes as aFigure 1. Methionine metabolism in Magnaporthe oryzae. De novo biosynthesis of methionine requires homocysteine derived from cysteine ?via cystathionine ?and involves cystathionine beta-lyase (MoStr3). Homocysteine might also result from O-acetyl-L-homoserine. O-acetyl-L-homoserine is derived from aspartate in a pathway involving a number of enzymatic steps that have been omitted for clarity [16]. This scheme is based on the predicted methionine and cysteine metabolic pathway map for M. oryzae at the Kyoto Encyclopedia of Genes and Genomes. doi:10.1371/journal.pone.0047392.gNutrient Conditions during Rice InfectionFigure 2. Maximum likelihood phylogeny of STR3 orthologs. The maximum likelihood phylogeny was reconstructed with RAxML, as described in Materials and Methods. Nodes with black circles indicate that these clusters are well supported ( 70 bootstrap support). Purple branches and species names indicate sequences with a fused C-terminal mevalonate kinase domain. Species that have known STR3 orthologs prior to this study are shown in green. Protein sequences were obtained from the Fungal Genome Collection (FGC). For those species not present within FGC, sequences were obtained from the NCBI database and their accession numbers are given in parentheses. Asterisks indicate sequences retrieved from the Saccharomyces Genom.