Methionine Biosynthesis is Essential for Infection in the Rice Blast Fungus Magnaporthe oryzae

Methionine is a sulfur amino acid standing at the crossroads of several biosynthetic pathways. In fungi, the last step of methionine biosynthesis is catalyzed by a cobalamine-independent methionine synthase (Met6, EC 2.1.1.14). In the present work, we studied the role of Met6 in the infection process of the rice blast fungus, Magnaporthe oryzae. To this end MET6 null mutants were obtained by targeted gene replacement. On minimum medium, MET6 null mutants were auxotrophic for methionine. Even when grown in presence of excess methionine, these mutants displayed developmental defects, such as reduced mycelium pigmentation, aerial hypha formation and sporulation. They also displayed characteristic metabolic signatures such as increased levels of cysteine, cystathionine, homocysteine, S-adenosylmethionine, S-adenosylhomocysteine while methionine and glutathione levels remained unchanged. These metabolic perturbations were associated with the over-expression of MgCBS1 involved in the reversed transsulfuration pathway that metabolizes homocysteine into cysteine and MgSAM1 and MgSAHH1 involved in the methyl cycle. This suggests a physiological adaptation of M. oryzae to metabolic defects induced by the loss of Met6, in particular an increase in homocysteine levels. Pathogenicity assays showed that MET6 null mutants were non-pathogenic on both barley and rice leaves. These mutants were defective in appressorium-mediated penetration and invasive infectious growth. These pathogenicity defects were rescued by addition of exogenous methionine and S-methylmethionine. These results show that M. oryzae cannot assimilate sufficient methionine from plant tissues and must synthesize this amino acid de novo to fulfill its sulfur amino acid requirement during infection.     

An S-(Hydroxymethyl)Glutathione Dehydrogenase Is Involved in Conidiation and Full Virulence in the Rice Blast Fungus Magnaporthe oryzae

Magnaporthe oryzae is a hemibiotrophic fungal pathogen that causes rice blast disease. A compatible interaction requires overcoming plant defense responses to initiate colonization during the early infection process. Nitric oxide (NO) plays important roles in defense responses during host-pathogen interactions. Microbes generally protect themselves against NO-induced damage by using enzymes. Here, we characterized an S-(hydroxymethyl)- glutathione dehydrogenase gene in M. oryzae, MoSFA1, the homologs of which are involved in NO metabolism by specifically catalyzing the reduction of S-nitrosoglutathione (GSNO) in yeasts and plants. As expected from the activities of S-(hydroxymethyl)glutathione dehydrogenase in formaldehyde detoxification and GSNO reduction, MoSFA1 deletion mutants were lethal in formaldehyde containing medium, sensitive to exogenous NO and exhibited a higher level of S-nitrosothiols (SNOs) than that of the wild type. Notably, the mutants showed severe reduction of conidiation and appressoria turgor pressure, as well as significantly attenuated the virulence on rice cultivar CO-39. However, the virulence of MoSFA1 deletion mutants on wounded rice leaf was not affected. An infection assay on barley leaf further revealed that MoSFA1 deletion mutants exhibited a lower infection rate, and growth of infectious hyphae of the mutants was retarded not only in primary infected cells but also in expansion from cell to cell. Furthermore, barley leaf cell infected by MoSFA1 deletion mutants exhibited a stronger accumulation of H₂O₂ at 24 and 36 hpi. MoSFA1 deletion mutants displayed hypersensitivity to different oxidants, reduced activities of superoxide dismutases and peroxidases, and lower glutathione content in cells, compared with the wild type. These results imply that MoSFA1-mediated NO metabolism is important in redox homeostasis in response to development and host infection of M. oryzae. Taken together, this work identifies that MoSFA1 is required for conidiation and contributes to virulence in the penetration and biotrophic phases in M. oryzae.     

The MET13 Methylenetetrahydrofolate Reductase Gene Is Essential for Infection-Related Morphogenesis in the Rice Blast Fungus Magnaporthe oryzae

Methylenetetrahydrofolate reductases (MTHFRs) play a key role in the biosynthesis of methionine in both prokaryotic and eukaryotic organisms. In this study, we report the identification of a novel T-DNA-tagged mutant WH672 in the rice blast fungus Magnaporthe oryzae, which was defective in vegetative growth, conidiation and pathogenicity. Analysis of the mutation confirmed a single T-DNA insertion upstream of MET13, which encodes a 626-amino-acid protein encoding a MTHFR. Targeted gene deletion of MET13 resulted in mutants that were non-pathogenic and significantly impaired in aerial growth and melanin pigmentation. All phenotypes associated with Δmet13 mutants could be overcome by addition of exogenous methionine. The M. oryzae genome contains a second predicted MTHFR-encoding gene, MET12. The deduced amino acid sequences of Met13 and Met12 share 32% identity. Interestingly, Δmet12 mutants produced significantly less conidia compared with the isogenic wild-type strain and grew very poorly in the absence of methionine, but were fully pathogenic. Deletion of both genes resulted in Δmet13Δmet12 mutants that showed similar phenotypes to single Δmet13 mutants. Taken together, we conclude that the MTHFR gene, MET13, is essential for infection-related morphogenesis by the rice blast fungus M. oryzae.     

Polyubiquitin Is Required for Growth, Development and Pathogenicity in the Rice Blast Fungus Magnaporthe oryzae

Protein ubiquitination, which is highly selective, regulates many important biological processes including cellular differentiation and pathogenesis in eukaryotic cells. Here, we integrated pharmacological, molecular and proteomic approaches to explore the role of ubiquitination in Magnaporthe oryzae, the leading fungal disease of rice world-wide. Inhibition of ubiquitin-mediated proteolysis using the 26S proteasome inhibitor, Bortezomib, significantly attenuated conidia germination, appressorium formation and pathogenicity in M. oryzae. Gene expression analysis revealed that many genes associated with protein ubiquitination were developmentally regulated during conidia germination. Only a few, including a polyubiquitin encoding gene, MGG_01282, were more abundantly expressed during appressorium formation and under nitrogen starvation. Targeted gene deletion of MGG_01282, in addition to a significant reduction in protein ubiquitination as determined by immuno blot assays, resulted in pleiotropic effects on M. oryzae including reduced growth and sporulation, abnormal conidia morphology, reduced germination and appressorium formation, and the inability to cause disease. Mutants were also defective in sexual development and were female sterile. Using mass spectrometry, we identified 63 candidate polyubiquitinated proteins under nitrogen starvation, which included overrepresentation of proteins involved in translation, transport and protein modification. Our study suggests that ubiquitination of target proteins plays an important role in nutrient assimilation, development and pathogenicity of M. oryzae.     

Different Chitin Synthase Genes Are Required for Various Developmental and Plant Infection Processes in the Rice Blast Fungus Magnaporthe oryzae

Chitin is a major component of fungal cell wall and is synthesized by chitin synthases (Chs). Plant pathogenic fungi normally have multiple chitin synthase genes. To determine their roles in development and pathogenesis, we functionally characterized all seven CHS genes in Magnaporthe oryzae. Three of them, CHS1, CHS6, and CHS7, were found to be important for plant infection. While the chs6 mutant was non-pathogenic, the chs1 and chs7 mutants were significantly reduced in virulence. CHS1 plays a specific role in conidiogenesis, an essential step for natural infection cycle. Most of chs1 conidia had no septum and spore tip mucilage. The chs6 mutant was reduced in hyphal growth and conidiation. It failed to penetrate and grow invasively in plant cells. The two MMD-containing chitin synthase genes, CHS5 and CHS6, have a similar expression pattern. Although deletion of CHS5 had no detectable phenotype, the chs5 chs6 double mutant had more severe defects than the chs6 mutant, indicating that they may have overlapping functions in maintaining polarized growth in vegetative and invasive hyphae. Unlike the other CHS genes, CHS7 has a unique function in appressorium formation. Although it was blocked in appressorium formation by germ tubes on artificial hydrophobic surfaces, the chs7 mutant still produced melanized appressoria by hyphal tips or on plant surfaces, indicating that chitin synthase genes have distinct impacts on appressorium formation by hyphal tip and germ tube. The chs7 mutant also was defective in appressorium penetration and invasive growth. Overall, our results indicate that individual CHS genes play diverse roles in hyphal growth, conidiogenesis, appressorium development, and pathogenesis in M. oryzae, and provided potential new leads in the control of this devastating pathogen by targeting specific chitin synthases.     

Crystal Structure of Manganese Lipoxygenase of the Rice Blast Fungus Magnaporthe oryzae

Lipoxygenases (LOX) are non-heme metal enzymes, which oxidize polyunsaturated fatty acids to hydroperoxides. All LOX belong to the same gene family, and they are widely distributed. LOX of animals, plants, and prokaryotes contain iron as the catalytic metal, whereas fungi express LOX with iron or with manganese. Little is known about metal selection by LOX and the adjustment of the redox potentials of their protein-bound catalytic metals. Thirteen three-dimensional structures of animal, plant, and prokaryotic FeLOX are available, but none of MnLOX. The MnLOX of the most important plant pathogen, the rice blast fungus Magnaporthe oryzae (Mo), was expressed in Pichia pastoris. Mo-MnLOX was deglycosylated, purified to homogeneity, and subjected to crystal screening and x-ray diffraction. The structure was solved by sulfur and manganese single wavelength anomalous dispersion to a resolution of 2.0 Å. The manganese coordinating sphere is similar to iron ligands of coral 8R-LOX and soybean LOX-1 but is not overlapping. The Asn-473 is positioned on a short loop (Asn-Gln-Gly-Glu-Pro) instead of an α-helix and forms hydrogen bonds with Gln-281. Comparison with FeLOX suggests that Phe-332 and Phe-525 might contribute to the unique suprafacial hydrogen abstraction and oxygenation mechanism of Mo-MnLOX by controlling oxygen access to the pentadiene radical. Modeling suggests that Arg-525 is positioned close to Arg-182 of 8R-LOX, and both residues likely tether the carboxylate group of the substrate. An oxygen channel could not be identified. We conclude that Mo-MnLOX illustrates a partly unique variation of the structural theme of FeLOX.     

Genetic and Physical Mapping of Telomeres in the Rice Blast Fungus, Magnaporthe Grisea

Telomeric restriction fragments were genetically mapped to a previously described linkage map of Magnaporthe grisea, using RFLPs identified by a synthetic probe, (TTAGGG)(3). Frequent rearrangement of telomeric sequences was observed in progeny isolates creating a potential for misinterpretation of data. Therefore a consensus segregation data set was used to minimize mapping errors. Twelve of the 14 telomeres were found to be genetically linked to existing RFLP markers. Second-dimensional electrophoresis of restricted chromosomes confirmed these linkage assignments and revealed the chromosomal location of the two unlinked telomeres. We were thus able to assign all 14 M. grisea telomeres to their respective chromosome ends. The Achilles' cleavage (AC) technique was employed to determine that chromosome 1 markers 11 and CH5-120H were ~1.8 Mb and 1.28 Mb, respectively, from their nearest telomeres. RecA-AC was also used to determine that unlinked telomere 6 was ~530 kb from marker CH5-176H in strain 2539 and 580 kb in Guy11. These experiments indicated that large portions of some chromosome ends are unrepresented by genetic markers and provided estimates of the relationship of genetic to physical distance in these regions of the genome.     

Karyotypic Variation within Clonal Lineages of the Rice Blast Fungus, Magnaporthe grisea

We have analyzed the karyotype of the rice blast fungus, Magnaporthe grisea, by using pulsed-filed gel electrophoresis. We tested whether the electrophoretic karyotype of an isolate was related to its pathotype, as determined by infection assays, or its genetic lineage, as determined by DNA fingerprinting. Highly reproducible electrophoretic karyotypes were obtained for a collection of U.S. and Chinese isolates representing a diverse collection of pathotypes and genetic lineages. Chromosomes ranged in size from 3 to 10 Mb. Although chromosome number was largely invariant, chromosome length polymorphisms were frequent. Minichromosomes were also found, although their presence was not ubiquitous. They ranged in number from 1 to 3 and in size from 470 kb to 2.2 Mb. Karyotypes were sufficiently variable as to obscure the obvious relatedness of isolates on the basis of pathogenicity assays or genetic lineage analysis by DNA fingerprinting. We documented that the electrophoretic karyotype of an isolate can change after prolonged serial transfer in culture and that this change did not alter the isolate's pathotype. The mechanisms bringing about karyotype variability involve deletions, translocations, and more complex rearrangements. We conclude that karyotypic variability in the rice blast fungus is a reflection of the lack of sexuality in wild populations which leads to the maintenance of neutral genomic rearrangements in clones of the fungus.