Yanhan Dong

The bZIP transcription factor MoAP1 mediates the oxidative stress response and is critical for pathogenicity of the rice blast fungus Magnaporthe oryzae.

Saccharomyces cerevisiae Yap1 protein is an AP1-like transcription factor involved in the regulation of the oxidative stress response. An ortholog of Yap1, MoAP1, was recently identified from the rice blast fungus Magnaporthe oryzae genome. We found that MoAP1 is highly expressed in conidia and during invasive hyphal growth. The Moap1 mutant was sensitive to H2O2, similar to S. cerevisiae yap1 mutants, and MoAP1 complemented Yap1 function in resistance to H2O2, albeit partially. The Moap1 mutant also exhibited various defects in aerial hyphal growth, mycelial branching, conidia formation, the production of extracellular peroxidases and laccases, and melanin pigmentation. Consequently, the Moap1 mutant was unable to infect the host plant. The MoAP1-eGFP fusion protein is localized inside the nucleus upon exposure to H2O2, suggesting that MoAP1 also functions as a redox sensor. Moreover, through RNA sequence analysis, many MoAP1-regulated genes were identified, including several novel ones that were also involved in pathogenicity. Disruption of respective MGG_01662 (MoAAT) and MGG_02531 (encoding hypothetical protein) genes did not result in any detectable changes in conidial germination and appressorium formation but reduced pathogenicity, whereas the mutant strains of MGG_01230 (MoSSADH) and MGG_15157 (MoACT) showed marketed reductions in aerial hyphal growth, mycelial branching, and loss of conidiation as well as pathogenicity, similar to the Moap1 mutant. Taken together, our studies identify MoAP1 as a positive transcription factor that regulates transcriptions of MGG_01662, MGG_02531, MGG_01230, and MGG_15157 that are important in the growth, development, and pathogenicity of M. oryzae.

A two-component histidine kinase, MoSLN1, is required for cell wall integrity and pathogenicity of the rice blast fungus, Magnaporthe oryzae

A two-component signal transduction system is a common mechanism for environmental sensing in bacteria. The functions of the two-component molecules have been also well characterized in the lower eukaryotic fungi in recent years. In Saccharomyces cerevisiae, the histidine kinase Sln1p is a major component of the two-component signaling pathways and a key regulator of the osmolarity response. To determine the function of MoSLN1, a Sln1 homolog of Magnaporthe oryzae, we cloned the MoSLN1 gene and generated specific mutants using gene knock-out strategy. Disruption of MoSLN1 resulted in hypersensitivity to various stresses, reduced sensitivity to cell wall perturbing agent Calcofluor white, and loss of pathogenicity, mainly due to a penetration defect. Additionally, we showed that MoSLN1 is involved in oxidative signaling through modulation of intra- and extracellular peroxidase activities. These results indicate that MoSLN1 functions as a pathogenicity factor that plays a role in responses to osmotic stress, the cell wall integrity, and the activity of peroxidases.

Global genome and transcriptome analyses of Magnaporthe oryzae epidemic isolate 98-06 uncover novel effectors and pathogenicity-related genes, revealing gene gain and lose dynamics in genome evolution.

Genome dynamics of pathogenic organisms are driven by pathogen and host co-evolution, in which pathogen genomes are shaped to overcome stresses imposed by hosts with various genetic backgrounds through generation of a variety of isolates. This same principle applies to the rice blast pathogen Magnaporthe oryzae and the rice host; however, genetic variations among different isolates of M. oryzae remain largely unknown, particularly at genome and transcriptome levels. Here, we applied genomic and transcriptomic analytical tools to investigate M. oryzae isolate 98-06 that is the most aggressive in infection of susceptible rice cultivars. A unique 1.4 Mb of genomic sequences was found in isolate 98-06 in comparison to reference strain 70-15. Genome-wide expression profiling revealed the presence of two critical expression patterns of M. oryzae based on 64 known pathogenicity-related (PaR) genes. In addition, 134 candidate effectors with various segregation patterns were identified. Five tested proteins could suppress BAX-mediated programmed cell death in Nicotiana benthamiana leaves. Characterization of isolate-specific effector candidates Iug6 and Iug9 and PaR candidate Iug18 revealed that they have a role in fungal propagation and pathogenicity. Moreover, Iug6 and Iug9 are located exclusively in the biotrophic interfacial complex (BIC) and their overexpression leads to suppression of defense-related gene expression in rice, suggesting that they might participate in biotrophy by inhibiting the SA and ET pathways within the host. Thus, our studies identify novel effector and PaR proteins involved in pathogenicity of the highly aggressive M. oryzae field isolate 98-06, and reveal molecular and genomic dynamics in the evolution of M. oryzae and rice host interactions.

Pleiotropic Function of the Putative Zinc-Finger Protein MoMsn2 in Magnaporthe oryzae

The mitogen-activated protein kinase MoOsm1-mediated osmoregulation pathway plays crucial roles in stress responses, asexual and sexual development, and pathogenicity in Magnaporthe oryzae. Utilizing an affinity purification approach, we identified the putative transcriptional activator MoMsn2 as a protein that interacts with MoOsm1 in vivo. Disruption of the MoMSN2 gene resulted in defects in aerial hyphal growth, conidial production, and infection of host plants. Quantitative reverse transcription-polymerase chain reaction analysis showed that the expression of several genes involved in conidiophore formation was reduced in ΔMomsn2, suggesting that MoMsn2 might function as a transcriptional regulator of these genes. Subsequently, MoCos1 was identified as one of the MoMsn2 targets through yeast one-hybrid analysis in which MoMsn2 binds to the AGGGG and CCCCT motif of the MoCOS1 promoter region. Phenotypic characterization showed that MoMsn2 was required for appressorium formation and penetration and pathogenicity. Although the ΔMomsn2 mutant was tolerant to the cell-wall stressor Calcofluor white, it was sensitive to common osmotic stressors. Further analysis suggests that MoMsn2 is involved in the regulation of the cell-wall biosynthesis pathway. Finally, transcriptome data revealed that MoMsn2 modulates numerous genes participating in conidiation, infection, cell-wall integrity, and stress response. Collectively, our results led to a model in which MoMsn2 mediates a series of downstream genes that control aerial hyphal growth, conidiogenesis, appressorium formation, cell-wall biosynthesis, and infection and that also offer potential targets for the development of new disease management strategies.

MoLys2 is necessary for growth, conidiogenesis, lysine biosynthesis, and pathogenicity in Magnaporthe oryzae.

Amino acid biosyntheses are complex but essential processes in growth and differentiation of eukaryotic cells. In the budding yeast Saccharomyces cerevisiae, the lysine biosynthesis via the α-aminoadipate (AA) pathway involves several steps, including reduction of AA to AA 6-semialdehyde by AA reductase ScLys2. In filamentous fungus Penicillium chrysogenum, disruption of the LYS2 gene blocked the lysine biosynthesis but promoted the production of the secondary metabolite penicillin. In comparison, little is known about the function of AA reductase Lys2 in phytopathogenic fungi. We here characterized the functions of MoLys2, a homolog of ScLys2, from the rice blast fungus Magnaporthe oryzae. Our results showed that the ΔMolys2 mutants were auxotrophic for lysine. The ΔMolys2 mutants also exhibited drastic reduction in pathogenicity on rice, inducing small disease lesions. Microscopic examination of the lesions revealed that the invasive hyphae of ΔMolys2 mutants were mostly restricted to the primary infected leaf sheath cells. In addition, exogenous lysine restored the production of conidia and near wild-type appressoria differentiation, and rescued the defect of pathogenicity in conidia infection of detached barely and rice leaf sheath. Our results indicated that MoLys2 is necessary for lysine biosynthesis that affects growth, conidiogenesis, and pathogenicity of the fungus. This study does implicate the potential for targeting lysine biosynthesis for the development of novel fungicides against M. oryzae.

MoYcp4 is required for growth, conidiogenesis and pathogenicity in Magnaporthe oryzae

The general transcriptional repressor Tup1 proteins play important regulatory roles in the growth and development of fungi. In this report, we characterized MoTup1, a protein homologous to Tup1 of Saccharomyces cerevisiae, from M. oryzae. Disruption of MoTUP1 resulted in severe mycelial growth reduction and a defect in conidiogenesis. We found that MoTup1 is required for the maintenance of cell wall integrity by regulating the expression of the genes involved in cell wall biosynthesis. Pathogenicity assays indicated that the ΔMotup1 mutants lost the ability to invade both rice and barley hosts. Moreover, observation of rice epidermis penetration showed that the hyphal tips of the mutants could still form appressorium-like structures, but were unable to invade host cells. Taken together, our results demonstrate that M. oryzae MoTup1 is an important regulatory factor in fungal growth, development and pathogenesis on hosts.

Comparative proteomic analyses reveal that the regulators of G-protein signaling proteins regulate amino acid metabolism of the rice blast fungus Magnaporthe oryzae.

The rice blast fungus Magnaporthe oryzae encodes eight regulators of G‐protein (GTP‐binding protein) signaling (RGS) proteins MoRgs1–MoRgs8 that orchestrate the growth, asexual/sexual production, appressorium differentiation, and pathogenicity. To address the mechanisms by which MoRgs proteins function, we conducted a 2DE proteome study and identified 82 differentially expressed proteins by comparing five ∆Morgs mutants with wild‐type Guy11 strain. We found that the abundances of eight amino acid (AA) biosynthesis or degradation associated proteins were markedly altered in five ∆Morgs mutants, indicating one of the main collective roles for the MoRgs proteins is to influence AA metabolism. We showed that MoRgs proteins have distinct roles in AA metabolism and nutrient responses from growth assays. In addition, we characterized MoLys20 (Lys is lysine), a homocitrate synthase, whose abundance was significantly decreased in the ∆Morgs mutants. The ∆Molys20 mutant is auxotrophic for lys and exogenous lys could partially rescue its auxotrophic defects. Deletion of MoLYS20 resulted in defects in conidiation and infection, as well as pathogenicity on rice. Overall, our results indicate that one of the critical roles for MoRgs proteins is to regulate AA metabolism, and that MoLys20 may be directly or indirectly regulated by MoRgs and participated in lys biosynthesis, thereby affecting fungal development and pathogenicity.

Orotate phosphoribosyl transferase MoPyr5 is involved in uridine 5′-phosphate synthesis and pathogenesis of Magnaporthe oryzae

Orotate phosphoribosyl transferase (OPRTase) plays an important role in de novo and salvage pathways of nucleotide synthesis and is widely used as a screening marker in genetic transformation. However, the function of OPRTase in plant pathogens remains unclear. In this study, we characterized an ortholog of Saccharomyces cerevisiae Ura5, the OPRTase MoPyr5, from the rice blast fungus Magnaporthe oryzae. Targeted gene disruption revealed that MoPyr5 is required for mycelial growth, appressorial turgor pressure and penetration into plant tissues, invasive hyphal growth, and pathogenicity. Interestingly, the ∆Mopyr5 mutant is also involved in mycelial surface hydrophobicity. Exogenous uridine 5′-phosphate (UMP) restored vegetative growth and rescued the defect in pathogenicity on detached barley and rice leaf sheath. Collectively, our results show that MoPyr5 is an OPRTase for UMP biosynthesis in M. oryzae and indicate that UTP biosynthesis is closely linked with vegetative growth, cell wall integrity, and pathogenicity of fungus. Our results also suggest that UMP biosynthesis would be a good target for the development of novel fungicides against M. oryzae.

MoMyb1 is required for asexual development and tissue-specific infection in the rice blast fungus Magnaporthe oryzae.

BackgroundThe Myb super-family of proteins contain a group of functionally diverse transcriptional activators found in plant, animal and fungus. Myb proteins are involved in cell proliferation, differentiation and apoptosis, and have crucial roles in telomeres. The purpose of this study was to characterize the biological function of Myb1 protein in the rice blast fungus Magnaporthe oryzae.ResultsWe identified the Saccharomyces cerevisiae BAS1 homolog MYB1 in M. oryzae, named MoMyb1. MoMyb1 encodes a protein of 322 amino acids and has two SANT domains and is well conserved in various organisms. Targeted gene deletion of MoMYB1 resulted in a significant reduction in vegetative growth and showed defects in conidiation and conidiophore development. Quantitative RT-PCR analysis revealed that the transcription levels of several conidiophore-related genes were apparently decreased in the ΔMomyb1 mutant. Inoculation with mycelia mats displayed that the virulence of the ΔMomyb1 mutant was not changed on rice leaves but was non-pathogenic on rice roots in comparison to the wild type Guy11. In addition, ∆Momyb1 mutants showed increased resistance to osmotic stresses but more sensitive to cell wall stressor calcofluor white (CFW). Further analysis revealed that MoMyb1 has an important role in the cell wall biosynthesis pathway.ConclusionThis study provides the evidence that MoMyb1 is a key regulator involved in conidiogenesis, stress response, cell wall integrity and pathogenesis on rice roots in the filamentous phytopathogen M. oryzae.

Correction to: MoMyb1 is required for asexual development and tissue-specific infection in the rice blast fungus Magnaporthe oryzae

Following the publication of this article [1], the authors noticed that they mistakenly introduced duplicate images in Figure 6A during the preparation of figures. They apologize for any confusion that brought to the readers and have corrected the figure. This correction does not change any statement or conclusion drawn from the data.