Michael J. Kershaw

MPG1 Encodes a Fungal Hydrophobin Involved in Surface Interactions during Infection-Related Development of Magnaporthe grisea.

The rice blast fungus expresses a pathogenicity gene, MPG1, during appressorium formation, disease symptom development, and conidiation. The MPG1 gene sequence predicts a small protein belonging to a family of fungal proteins designated hydrophobins. Using random ascospore analysis and genetic complementation, we showed that MPG1 is necessary for infection-related development of Magnaporthe grisea on rice leaves and for full pathogenicity toward susceptible rice cultivars. The protein product of MPG1 appears to interact with hydrophobic surfaces, where it may act as a developmental sensor for appressorium formation. Ultrastructural studies revealed that MPG1 directs formation of a rodlet layer on conidia composed of interwoven ~5-nm rodlets, which contributes to their surface hydrophobicity. Using combined genetic and biochemical approaches, we identified a 15-kD secreted protein with characteristics that establish it as a class I hydrophobin. The protein is able to form detergent-insoluble high molecular mass complexes, is soluble in trifluoroacetic acid, and exhibits mobility shifts after treatment with performic acid. The production of this protein is directed by MPG1.

The glyoxylate cycle is required for temporal regulation of virulence by the plant pathogenic fungus Magnaporthe grisea

We describe the isolation and characterization of ICL1 from the rice blast fungus Magnaporthe grisea, a gene that encodes isocitrate lyase, one of the principal enzymes of the glyoxylate cycle. ICL1 shows elevated expression during development of infection structures and cuticle penetration, and a targeted gene replacement showed that the gene is required for full virulence by M. grisea. In particular, we found that the prepenetration stage of development, before entry into plant tissue, is affected by loss of the glyoxylate cycle. There is a delay in germination, infection‐related development and cuticle penetration in Δicl1 mutants. Recent reports have shown the importance of the glyoxylate cycle in the virulence of the human pathogenic fungus Candida albicans and the bacterial pathogen Mycobacterium tuberculosis. Our results indicate that the glyoxylate cycle is also important in this plant pathogenic fungus, demonstrating the widespread utility of the pathway in microbial pathogenesis.

Genome-wide functional analysis reveals that infection-associated fungal autophagy is necessary for rice blast disease

To cause rice blast disease, the fungus Magnaporthe oryzae elaborates specialized infection structures called appressoria, which use enormous turgor to rupture the tough outer cuticle of a rice leaf. Here, we report the generation of a set of 22 isogenic M. oryzae mutants each differing by a single component of the predicted autophagic machinery of the fungus. Analysis of this set of targeted deletion mutants demonstrated that loss of any of the 16 genes necessary for nonselective macroautophagy renders the fungus unable to cause rice blast disease, due to impairment of both conidial programmed cell death and appressorium maturation. In contrast, genes necessary only for selective forms of autophagy, such as pexophagy and mitophagy, are dispensable for appressorium-mediated plant infection. A genome-wide analysis therefore demonstrates the importance of infection-associated, nonselective autophagy for the establishment of rice blast disease.

Functional Analysis of Lipid Metabolism in Magnaporthe grisea Reveals a Requirement for Peroxisomal Fatty Acid β-Oxidation During Appressorium-Mediated Plant Infection

The rice blast fungus Magnaporthe grisea infects plants by means of specialized infection structures known as appressoria. Turgor generated in the appressorium provides the invasive force that allows the fungus to breach the leaf cuticle with a narrow-penetration hypha gaining entry to the underlying epidermal cell. Appressorium maturation in M. grisea involves mass transfer of lipid bodies to the developing appressorium, coupled to autophagic cell death in the conidium and rapid lipolysis at the onset of appressorial turgor generation. Here, we report identification of the principal components of lipid metabolism in M. grisea based on genome sequence analysis. We show that deletion of any of the eight putative intracellular triacylglycerol lipase-encoding genes from the fungus is insufficient to prevent plant infection, highlighting the complexity and redundancy associated with appressorial lipolysis. In contrast, we demonstrate that a peroxisomally located multifunctional, fatty acid beta-oxidation enzyme is critical to appressorium physiology, and blocking peroxisomal biogenesis prevents plant infection. Taken together, our results indicate that, although triacylglycerol breakdown in the appressorium involves the concerted action of several lipases, fatty acid metabolism and consequent generation of acetyl CoA are necessary for M. grisea to complete its prepenetration phase of development and enter the host plant.

Complementation of the mpg1 mutant phenotype in Magnaporthe grisea reveals functional relationships between fungal hydrophobins.

The functional relationship between fungal hydrophobins was studied by complementation analysis of an mpg1− gene disruption mutant in Magnaporthe grisea. MPG1 encodes a hydrophobin required for full pathogenicity of the fungus, efficient elaboration of its infection structures and conidial rodlet protein production. Seven heterologous hydrophobin genes were selected which play distinct roles in conidiogenesis, fruit body development, aerial hyphae formation and infection structure elaboration in diverse fungal species. Each hydrophobin was introduced into an mpg1− mutant by transformation. Only one hydrophobin gene, SC1 from Schizophyllum commune, was able partially to complement mpg1− mutant phenotypes when regulated by its own promoter. In contrast, six of the transformants expressing hydrophobin genes controlled by the MPG1 promoter (SC1 and SC4 from S.commune, rodA and dewA from Aspergillus nidulans, EAS from Neurospora crassa and ssgA from Metarhizium anisopliae) could partially complement each of the diverse functions of MPG1. Complementation was always associated with partial restoration of a rodlet protein layer, characteristic of the particular hydrophobin being expressed, and with hydrophobin surface assembly during infection structure formation. This provides the first genetic evidence that diverse hydrophobin‐encoding genes encode functionally related proteins and suggests that, although very diverse in amino acid sequence, the hydrophobins constitute a closely related group of morphogenetic proteins.

NADPH oxidases regulate septin-mediated cytoskeletal remodeling during plant infection by the rice blast fungus

The rice blast fungus Magnaporthe oryzae infects plants with a specialized cell called an appressorium, which uses turgor to drive a rigid penetration peg through the rice leaf cuticle. Here, we show that NADPH oxidases (Nox) are necessary for septin-mediated reorientation of the F-actin cytoskeleton to facilitate cuticle rupture and plant cell invasion. We report that the Nox2–NoxR complex spatially organizes a heteroligomeric septin ring at the appressorium pore, required for assembly of a toroidal F-actin network at the point of penetration peg emergence. Maintenance of the cortical F-actin network during plant infection independently requires Nox1, a second NADPH oxidase, which is necessary for penetration hypha elongation. Organization of F-actin in appressoria is disrupted by application of antioxidants, whereas latrunculin-mediated depolymerization of appressorial F-actin is competitively inhibited by reactive oxygen species, providing evidence that regulated synthesis of reactive oxygen species by fungal NADPH oxidases directly controls septin and F-actin dynamics.

Regulation of the MPG1 hydrophobin gene in the rice blast fungus Magnaporthe grisea.

The hydrophobin-encoding gene MPG1 of the rice blast fungus Magnaporthe grisea is highly expressed during the initial stages of host plant infection and targeted deletion of the gene results in a mutant strain that is reduced in virulence, conidiation, and appressorium formation. The green fluorescent protein-encoding allele sGFP was used as a reporter to investigate regulatory genes that control MPG1 expression. The MAP kinase-encoding gene PMK1 and the wide domain regulators of nitrogen source utilization, NPR1 and NUT1, were required for full expression of MPG1 in response to starvation stress. The CPKA gene, encoding the catalytic subunit of protein kinase A, was required for repression of MPG1 during growth in rich nutrient conditions. During appressorium morphogenesis, high-level MPG1 expression was found to require the CPKA and NPR1 genes. Expression of a destabilized GFP allele indicated that de novo MPG1 expression occurs during appressorium formation. Three regions of the MPG1 promoter were identified which are required for high-level expression of MPG1 during appressorium formation and are necessary for the biological activity of the MPG1 hydrophobin during spore formation and plant infection.

Structure-function analysis of a CVNH-LysM lectin expressed during plant infection by the rice blast fungus Magnaporthe oryzae

The rice blast fungus Magnaporthe oryzaes genome encodes a hypothetical protein (MGG_03307) containing a type III CVNH lectin, in which a LysM domain is inserted between individual repeats of a single CVNH domain. At present, no structural or ligand binding data are available for any type III CVNH and functional studies in natural source organisms are scarce. Here, we report NMR solution structure and functional data on MGG_03307. The structure of the CVNH/LysM module revealed that intact and functionally competent CVNH and LysM domains are present. Using NMR titrations, carbohydrate specificities for both domains were determined, and it was found that each domain behaves as an isolated unit without any interdomain communication. Furthermore, live-cell imaging revealed a predominant localization of MGG_03307 within the appressorium, the specialized fungal cell for gaining entry into rice tissue. Our results suggest that MGG_03307 plays a role in the early stages of plant infection.

Four conserved intramolecular disulphide linkages are required for secretion and cell wall localization of a hydrophobin during fungal morphogenesis

Hydrophobins are morphogenetic proteins produced by fungi during assembly of aerial hyphae, sporulation, mushroom development and pathogenesis. Eight cysteine residues are present in hydrophobins and form intramolecular disulphide bonds. Here, we show that expressing eight cysteine–alanine substitution alleles of the MPG1 hydrophobin gene from Magnaporthe grisea causes severe defects in development of aerial hyphae and spores. Immunolocalization revealed that Mpg1 hydrophobin variants, lacking intact disulphide bonds, retain the capacity to self‐assemble, but are not secreted to the cell surface. This provides the first genetic evidence that disulphide bridges in a hydrophobin are dispensable for aggregation, but essential for secretion.

The emerging role of autophagy in plant pathogen attack and host defence.

Autophagy is emerging as an important process in plant infection by pathogenic fungi, which develop differentiated infection cells to breach the plant cuticle. Conversely, autophagic processes are also important in the defence responses of plants that are able to perceive and react to invading pathogens. The pivotal role of autophagy in both fungal pathogenesis and disease resistance is linked to its function in the regulation of programmed cell death which is a key component of plant immunity responses and fungal infection-related development.