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Vacuolar Protein-Sorting Receptor MoVps13 Regulates Conidiation and Pathogenicity in Rice Blast Fungus Magnaporthe oryzae.

ABSTRACT: Magnaporthe oryzae (synonym Pyricularia oryzae) is a filamentous fungal pathogen that causes major yield losses in cultivated rice worldwide. However, the mechanisms of infection of M. oryzae are not well characterized. The VPS13 proteins play vital roles in various biological processes in many eukaryotic organisms, including in the organization of actin cytoskeleton, vesicle trafficking, mitochondrial fusion, and phagocytosis. Nevertheless, the function of the Vps13 protein in plant pathogenic fungi has not been explored. Here, we analysed the biological functions of the Vps13 protein in the development and pathogenicity of M. oryzae. Deletion mutants of MoVps13 significantly reduced the conidiation and decreased the rate of fungal infection on hosts. Moreover, the loss of MoVps13 resulted in defective cell wall integrity (CWI) and plasma membrane (PM) homeostasis when treated with chemicals for inducing cell wall stress (200 mg/mL Congo Red or 0.005% SDS) and sphingolipid synthesis inhibitors (2 μM myriocin or 2 μM amphotericin B). This indicated that MoVps13 is also involved in cell wall synthesis and sphingolipid synthesis. Through immunoblotting, autophagic flux detection, co-localization, and chemical drug sensitivity assays, we confirmed the involvement of Movps13 in ER-phagy and the response to ER stress. Additionally, we generated the C-terminal structure of MoVps13 with high accuracy using the alphaflod2 database. Our experimental evidence indicates that MoVps13 is an important virulence factor that regulates the pathogenicity of M. oryzae by controlling CWI, lipid metabolism and the ER-phagy pathway. These results have expanded our knowledge about pathogenic fungi and will help exploration for novel therapeutic strategies against the rice blast fungus.

SUBMITTER: Zhu X

PROVIDER: S-EPMC8708568 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:As the causal agent of the blast disease, Magnaporthe oryzae is one of the most destructive fungal pathogens of rice. Histone acetylation/deacetylation is important for remodeling of chromatin superstructure and thus altering gene expression. In this study, two genes encoding histone deacetylases, namely, MoRPD3 and MoHST4, were identified and functionally characterized in M. oryzae. MoHst4 was required for proper mycelial growth and pathogenicity, whereas overproduction of MoRpd3 led to loss of pathogenicity, likely due to a block in conidial cell death and restricted invasive growth within the host plants. Green fluorescent protein (GFP)-MoRpd3 localized to the nucleus and cytoplasm in vegetative hyphae and developing conidia. By comparative transcriptomics analysis, we identified potential target genes epigenetically regulated by histone deacetylases (HDACs) containing MoRpd3 or MoHst4, which may contribute to conidia formation and/or conidial cell death, which is a prerequisite for successful appressorium-mediated host invasion. Taken together, our results suggest that histone deacetylases MoRpd3 and MoHst4 differentially regulate mycelial growth, asexual development, and pathogenesis in M. oryzae. IMPORTANCE HDACs (histone deacetylases) regulate various aspects of growth, development, and pathogenesis in plant-pathogenic fungi. Most members of HDAC classes I to III have been functionally characterized, except for orthologous Rpd3 and Hst4, in the rice blast fungus Magnaporthe oryzae. In this study, we assessed the function of MoRpd3 and MoHst4 by reverse genetics and found that they differentially regulate M. oryzae vegetative growth, asexual development, and infection. Particularly, MoRpd3 negatively regulates M. oryzae pathogenicity, likely through suppression of conidial cell death, which we recently reported as being critical for appressorium maturation and functioning. Overall, this study broadens our understanding of fungal pathobiology and its critical regulation by histone modification(s) during cell death and in planta differentiation.

Project description:Rice blast disease caused by Magnaporthe oryzae is a devastating disease of cultivated rice worldwide. Infections by this fungus lead to a significant reduction in rice yields and threats to food security. To gain better insight into growth and cell death in M. oryzae during infection, we characterized two predicted M. oryzae metacaspase proteins, MoMca1 and MoMca2. These proteins appear to be functionally redundant and can complement the yeast Yca1 homologue. Biochemical analysis revealed that M. oryzae metacaspases exhibited Ca2+-dependent caspase activity in vitro Deletion of both MoMca1 and MoMca2 in M. oryzae resulted in reduced sporulation, delay in conidial germination, and attenuation of disease severity. In addition, the double ?Momca1mca2 mutant strain showed increased radial growth in the presence of oxidative stress. Interestingly, the ?Momca1mca2 strain showed an increased accumulation of insoluble aggregates compared to the wild-type strain during vegetative growth. Our findings suggest that MoMca1 and MoMca2 promote the clearance of insoluble aggregates in M. oryzae, demonstrating the important role these metacaspases have in fungal protein homeostasis. Furthermore, these metacaspase proteins may play additional roles, like in regulating stress responses, that would help maintain the fitness of fungal cells required for host infection.IMPORTANCE Magnaporthe oryzae causes rice blast disease that threatens global food security by resulting in the severe loss of rice production every year. A tightly regulated life cycle allows M. oryzae to disarm the host plant immune system during its biotrophic stage before triggering plant cell death in its necrotrophic stage. The ways M. oryzae navigates its complex life cycle remain unclear. This work characterizes two metacaspase proteins with peptidase activity in M. oryzae that are shown to be involved in the regulation of fungal growth and development prior to infection by potentially helping maintain fungal fitness. This study provides new insights into the role of metacaspase proteins in filamentous fungi by illustrating the delays in M. oryzae morphogenesis in the absence of these proteins. Understanding the mechanisms by which M. oryzae morphology and development promote its devastating pathogenicity may lead to the emergence of proper methods for disease control.

Project description:BACKGROUND: Magnaporthe oryzae, the causal agent of blast disease of rice, is the most destructive disease of rice worldwide. The genome of this fungal pathogen has been sequenced and an automated annotation has recently been updated to Version 6 http://www.broad.mit.edu/annotation/genome/magnaporthe_grisea/MultiDownloads.html. However, a comprehensive manual curation remains to be performed. Gene Ontology (GO) annotation is a valuable means of assigning functional information using standardized vocabulary. We report an overview of the GO annotation for Version 5 of M. oryzae genome assembly. METHODS: A similarity-based (i.e., computational) GO annotation with manual review was conducted, which was then integrated with a literature-based GO annotation with computational assistance. For similarity-based GO annotation a stringent reciprocal best hits method was used to identify similarity between predicted proteins of M. oryzae and GO proteins from multiple organisms with published associations to GO terms. Significant alignment pairs were manually reviewed. Functional assignments were further cross-validated with manually reviewed data, conserved domains, or data determined by wet lab experiments. Additionally, biological appropriateness of the functional assignments was manually checked. RESULTS: In total, 6,286 proteins received GO term assignment via the homology-based annotation, including 2,870 hypothetical proteins. Literature-based experimental evidence, such as microarray, MPSS, T-DNA insertion mutation, or gene knockout mutation, resulted in 2,810 proteins being annotated with GO terms. Of these, 1,673 proteins were annotated with new terms developed for Plant-Associated Microbe Gene Ontology (PAMGO). In addition, 67 experiment-determined secreted proteins were annotated with PAMGO terms. Integration of the two data sets resulted in 7,412 proteins (57%) being annotated with 1,957 distinct and specific GO terms. Unannotated proteins were assigned to the 3 root terms. The Version 5 GO annotation is publically queryable via the GO site http://amigo.geneontology.org/cgi-bin/amigo/go.cgi. Additionally, the genome of M. oryzae is constantly being refined and updated as new information is incorporated. For the latest GO annotation of Version 6 genome, please visit our website http://scotland.fgl.ncsu.edu/smeng/GoAnnotationMagnaporthegrisea.html. The preliminary GO annotation of Version 6 genome is placed at a local MySql database that is publically queryable via a user-friendly interface Adhoc Query System. CONCLUSION: Our analysis provides comprehensive and robust GO annotations of the M. oryzae genome assemblies that will be solid foundations for further functional interrogation of M. oryzae.

Project description: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.

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Vacuolar Protein-Sorting Receptor MoVps13 Regulates Conidiation and Pathogenicity in Rice Blast Fungus Magnaporthe oryzae.

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