The G-beta subunit MGB1 is involved in regulating multiple steps of infection-related morphogenesis in Magnaporthe grisea

Trimeric G-proteins transmit extracellular signals to various downstream effectors (e.g. MAP kinases) in eukaryotes. In the rice blast fungus Magnaporthe grisea, the Pmk1 MAP kinase is essential for appressorium formation and infectious growth. The pmk1 deletion mutant fails to form appressoria but still responds to exogenous cAMP for tip deformation. Since gene disruption mutants of three Galpha subunits still form appressoria and are phenotypically different from pmk1 mutants, it is likely that the Pmk1 pathway is activated by Gbeta in M. grisea. In this study, we isolated and characterized the MGB1 gene that encodes the G subunit in M. grisea. Mutants disrupted in MGB1 were reduced in conidiation. Conidia from mgb1 mutants were defective in appressorium formation and failed to penetrate or grow invasively on rice leaves. Exogenous cAMP induced appressorium formation in mgb1 mutants, but these appressoria were abnormal in shape and could not penetrate. The intracellular cAMP level was reduced in mgb1 mutants and the defects in conidiation and hyphal growth were partially suppressed with 1 mM cAMP. Transformants expressing multiple copies of MGB1 were able to form appressoria on hydrophilic surfaces. Our results suggest that MGB1 may be involved in the cAMP signalling for regulating conidiation, surface recognition and appressorium formation. The Pmk1 pathway may be the downstream target of MGB1 for regulating penetration and infectious hyphae growth in M. grisea.     

Two PAK kinase genes, CHM1 and MST20, have distinct functions in Magnaporthe grisea

In the rice blast fungus Magnaporthe grisea, the Pmk1 mitogen-activated protein (MAP) kinase is essential for appressorium formation and infectious growth. PMK1 is homologous to yeast Fus3 and Kss1 MAP kinases that are known to be regulated by the Ste20 PAK kinase for activating the pheromone response and filamentation pathways. In this study, we isolated and characterized two PAK genes, CHM1 and MST20, in M. grisea. Mutants disrupted in MST20 were reduced in aerial hyphae growth and conidiation, but normal in growth rate, appressorium formation, penetration, and plant infection. In chm1 deletion mutants, growth, conidiation, and appressorium formation were reduced significantly. Even though appressoria formed by chm1 mutants were defective in penetration, chm1 mutants were able to grow invasively on rice leaves and colonize through wounds. The chm1 mutants were altered in conidiogenesis and produced conidia with abnormal morphology. Hyphae of chm1 mutants had normal septation, but the length of hyphal compartments was reduced. On nutritionally poor oatmeal agar, chm1 mutants were unstable and produced sectors that differed from original chm1 mutants in growth rate, conidiation, or colony morphology. However, none of the monoconidial cultures derived from these spontaneous sectors were normal in appressorial penetration and fungal pathogenesis. These data suggest that MST20 is dispensable for plant infection in M. grisea, but CHM1 plays a critical role in appressorium formation and penetration. Both mst20 and chm1 deletion mutants were phenotypically different from the pmk1 mutant that is defective in appressorium formation and infectious hyphae growth. It is likely that MST20 and CHM1 individually play no critical role in activating the PMK1 MAP kinase pathway during appressorium formation and infectious hyphae growth. However, CHM1 appears to be essential for appressorial penetration and CHM1 and MST20 may have redundant functions in M. grisea.     

Magnaporthe oryzae nucleoside diphosphate kinase is required for metabolic homeostasis and redox-mediated host innate immunity suppression

The fungus Magnaporthe oryzae causes blast, the most devastating disease of cultivated rice. After penetrating the leaf cuticle, M. oryzae grows as a biotroph in intimate contact with living rice epidermal cells before necrotic lesions develop. Biotrophic growth requires maintaining metabolic homeostasis while suppressing plant defenses, but the metabolic connections and requirements involved are largely unknown. Here, we characterized the M. oryzae nucleoside diphosphate kinase-encoding gene NDK1 and discovered it was essential for facilitating biotrophic growth by suppressing the host oxidative burst—the first line of plant defense. NDK enzymes reversibly transfer phosphate groups from tri- to diphosphate nucleosides. Correspondingly, intracellular nucleotide pools were perturbed in M. oryzae strains lacking NDK1 through targeted gene deletion, compared to WT. This affected metabolic homeostasis: TCA, purine and pyrimidine intermediates, and oxidized NADP⁺, accumulated in Δndk1. cAMP and glutathione were depleted. ROS accumulated in Δndk1 hyphae. Functional appressoria developed on rice leaf sheath surfaces, but Δndk1 invasive hyphal growth was restricted and redox homeostasis was perturbed, resulting in unsuppressed host oxidative bursts that triggered immunity. We conclude Ndk1 modulates intracellular nucleotide pools to maintain redox balance via metabolic homeostasis, thus quenching the host oxidative burst and suppressing rice innate immunity during biotrophy.     

MoOpy2 is essential for fungal development,pathogenicity, and autophagy in Magnaporthe oryzae

The development and pathogenicity of the fungus Magnaporthe oryzae, the causal agent of destructive rice blast disease, require it to perceive external environmental signals. Opy2, an overproduction-induced pheromone-resistant protein 2, is a crucial protein for sensing external signals in Saccharomyces cerevisiae. However, the biological functions of the homologue of Opy2 in M. oryzae are unclear. In this study, we identified that MoOPY2 is involved in fungal development, pathogenicity, and autophagy in M. oryzae. Deletion of MoOPY2 resulted in pleiotropic defects in hyphal growth, conidiation, germ tube extension, appressorium formation, appressorium turgor generation, and invasive growth, therefore leading to attenuated pathogenicity. Furthermore, MoOpy2 participates in the Osm1 MAPK pathway and the Mps1 MAPK pathway by interacting with the adaptor protein Mst50. The interaction sites of Mst50 and MoOpy2 colocalized with the autophagic marker protein MoAtg8 in the preautophagosomal structure sites (PAS). Notably, the ΔMoopy2 mutant caused cumulative MoAtg8 lipidation and rapid GFP-MoAtg8 degradation in response to nitrogen starvation, showing that MoOpy2 is involved in the negative regulation of autophagy activity. Taken together, our study revealed that MoOpy2 of M. oryzae plays an essential role in the orchestration of fungal development, appressorium penetration, autophagy and pathogenesis.     

Genetic analysis of a mutation on appressorium formation in Magnaporthe grisea

The rice blast fungus, Magnaporthe grisea, forms a dome-shaped and darkly pigmented infection structure, an appressorium, to penetrate its host. Differentiation and maturation of appressoria are critical steps for successful infection. A spontaneous developmental mutant (MG01) defective in appressorium formation was found in this fungus. The mutant did not form appressoria either on inductive hydrophobic surfaces or on rice leaves. The addition of cyclic AMP or 1,16-hexadecanediol was not effective in inducing appressorium formation in this mutant. This mutant did not cause lesions on rice when inoculated with conidial suspension by spraying or injecting into the leaf sheath. Genetic analysis of the mutant indicated that the phenotype is under single gene control, designated APP5. Crosses with previously described appressorium defective mutants (app1⁻ and app3⁻) of Magnaporthe grisea suggested that the mutations are at different loci. Bulked segregant analysis was employed to obtain DNA markers linked to the APP5 locus.     

Differential roles of the phospholipase C genes in fungal development and pathogenicity of Magnaporthe oryzae

Calcium plays a critical role in a variety of cellular processes in cells. However, relatively little is known about the biological effects of Ca2+ signaling on morphogenesis and pathogenesis in the rice blast fungus Magnaporthe oryzae compared to other signaling pathways. We have previously demonstrated that MoPLC1-mediated calcium regulation is important for infection-related development and pathogenicity in M. oryzae. In the present study, four genes encoding phospholipase C (PLC) isozymes (MoPLC2 to MoPLC5), which differ from MoPLC1 in their domain organization, were additionally identified. The C2 domain involved in Ca2+-dependent membrane binding is found only in MoPLC2 and MoPLC3. Detailed functional analysis using deletion mutants for MoPLC2 and MoPLC3 indicated that MoPLC2 and MoPLC3 play essential roles in development. The two deletion mutants for MoPLC2 and MoPLC3 showed reduced conidiation and a defect in appressorium-mediated penetration. Reintroduction of the genes restored defects of ΔMoplc2 and ΔMoplc3. Notably, ΔMoplc2 and ΔMoplc3 mutants developed multiple appressoria on separate germ tubes of a conidium, indicating that MoPLC2- and MoPLC3-regulated signaling suppresses a feedback loop of a pathway for appressorial development. The similarity in phenotypic defects between the two mutants indicates that both MoPLC2 and MoPLC3 are important for regulation of appropriate levels of signaling molecules in a similar manner. Comparative analysis indicated that the two MoPLCs-mediated signaling pathways have interrelated, but distinct, roles in the development of M. oryzae.     

Cell Cycle–Mediated Regulation of Plant Infection by the Rice Blast Fungus

To gain entry to plants, many pathogenic fungi develop specialized infection structures called appressoria. Here, we demonstrate that appressorium morphogenesis in the rice blast fungus Magnaporthe oryzae is tightly regulated by the cell cycle. Shortly after a fungus spore lands on the rice (Oryza sativa) leaf surface, a single round of mitosis always occurs in the germ tube. We found that initiation of infection structure development is regulated by a DNA replication-dependent checkpoint. Genetic intervention in DNA synthesis, by conditional mutation of the Never-in-Mitosis 1 gene, prevented germ tubes from developing nascent infection structures. Cellular differentiation of appressoria, however, required entry into mitosis because nimA temperature-sensitive mutants, blocked at mitotic entry, were unable to develop functional appressoria. Arresting the cell cycle after mitotic entry, by conditional inactivation of the Blocked-in-Mitosis 1 gene or expression of stabilized cyclinB-encoding alleles, did not impair appressorium differentiation, but instead prevented these cells from invading plant tissue. When considered together, these data suggest that appressorium-mediated plant infection is coordinated by three distinct cell cycle checkpoints that are necessary for establishment of plant disease.     

Endosomal sorting complexes required for transport-0 (ESCRT-0) are essential for fungal development,pathogenicity, autophagy and ER-phagy in Magnaporthe oryzae

Magnaporthe oryzae is an important plant pathogen that causes rice blast. Hse1 and Vps27 are components of ESCRT-0 involved in the multivesicular body (MVB) sorting pathway and biogenesis. To date, the biological functions of ESCRT-0 in M. oryzae have not been determined. In this study, we identified and characterized Hse1 and Vps27 in M. oryzae. Disruption of MoHse1 and MoVps27 caused pleiotropic defects in growth, conidiation, sexual development and pathogenicity, thereby resulting in loss of virulence in rice and barley leaves. Disruption of MoHse1 and MoVps27 triggered increased lipidation of MoAtg8 and degradation of GFP-MoAtg8, indicating that ESCRT-0 is involved in the regulation of autophagy. ESCRT-0 was determined to interact with coat protein complex II (COPII), a regulator functioning in homeostasis of the endoplasmic reticulum (ER homeostasis), and disruption of MoHse1 and MoVps27 also blocked activation of the unfolded protein response (UPR) and autophagy of the endoplasmic reticulum (ER-phagy). Overall, our results indicate that ESCRT-0 plays critical roles in regulating fungal development, virulence, autophagy and ER-phagy in M. oryzae.     

A copper-transporting ATPase BcCCC2 is necessary for pathogenicity of Botrytis cinerea

Copper is an essential trace element that serves as a cofactor for numerous enzymes. In eukaryotes, copper-transporting ATPases deliver copper to various copper-containing proteins in the trans-golgi network. This study identified a copper-transporting ATPase gene BcCcc2 in a fungus pathogenic to plants, Botrytis cinerea. We investigated the biological roles of BcCCC2 by generating null mutants for BcCcc2. Melanization, conidiation and the formation of sclerotia were severely affected in ∆BcCcc2 mutants. Moreover, a pathogenicity assay using tomato leaves and carnation petals revealed the mutants to be nonpathogenic. Further analysis indicated that they formed fewer appressoria and infection cushions than the wild-type. These structures were aberrant in morphology and in many cases had a significantly reduced ability to penetrate the plant epidermis. An assay also indicated that ∆BcCcc2 mutants were defective in infection through wounds. BcCCC2 is necessary not only for penetrating a host but also for fungal growth within plant tissues. Our results also imply that B. cinerea requires copper-containing proteins for infection that are inactive in the absence of the copper-transporting ATPase BcCCC2.     

MoWhi2 regulates appressorium formation and pathogenicity via the MoTor signalling pathway in Magnaporthe oryzae

Magnaporthe oryzae causes rice blast disease, which seriously threatens the safety of food production. Understanding the mechanism of appressorium formation, which is one of the key steps for successful infection by M. oryzae, is helpful to formulate effective control strategies of rice blast. In this study, we identified MoWhi2, the homolog of Saccharomyces cerevisiae Whi2 (Whisky2), as an important regulator that controls appressorium formation in M. oryzae. When MoWHI2 was disrupted, multiple appressoria were formed by one conidium and pathogenicity was significantly reduced. A putative phosphatase, MoPsr1, was identified to interact with MoWhi2 using a yeast two-hybridization screening assay. The knockout mutant ΔMopsr1 displayed similar phenotypes to the ΔMowhi2 strain. Both the ΔMowhi2 and ΔMopsr1 mutants could form appressoria on a hydrophilic surface with cAMP levels increasing in comparison with the wild type (WT). The conidia of ΔMowhi2 and ΔMopsr1 formed a single appressorium per conidium, similar to WT, when the target of rapamycin (TOR) inhibitor rapamycin was present. In addition, compared with WT, the expression levels of MoTOR and the MoTor signalling activation marker gene MoRS3 were increased, suggesting that inappropriate activation of the MoTor signalling pathway is one of the important reasons for the defects in appressorium formation in the ΔMowhi2 and ΔMopsr1 strains. Our results provide insights into MoWhi2 and MoPsr1-mediated appressorium development and pathogenicity by regulating cAMP levels and the activation of MoTor signalling in M. oryzae.     

The farnesyltransferase β-subunit RAM1 regulates localization of RAS proteins and appressorium-mediated infection in Magnaporthe oryzae

Post-translational farnesylation can regulate subcellular localization and protein–protein interaction in eukaryotes. The function of farnesylation is not well identified in plant pathogenic fungi, particularly during the process of fungal infection. Here, through functional analyses of the farnesyltransferase β-subunit gene, RAM1, we examine the importance of protein farnesylation in the rice blast fungus Magnaporthe oryzae. Targeted disruption of RAM1 resulted in the reduction of hyphal growth and sporulation, and an increase in the sensitivity to various stresses. Importantly, loss of RAM1 also led to the attenuation of virulence on the plant host, characterized by decreased appressorium formation and invasive growth. Interestingly, the defect in appressoria formation of the Δram1 mutant can be recovered by adding exogenous cAMP and IBMX, suggesting that RAM1 functions upstream of the cAMP signalling pathway. We found that two Ras GTPases, RAS1 and RAS2, can interact with Ram1, and their plasma membrane localization was regulated by Ram1 through their C-terminal farnesylation sites. Adding a farnesyltransferase inhibitor Tipifarnib can result in similar defects as in Δram1 mutant, including decreased appressorium formation and invasive growth, as well as mislocalized RAS proteins. Our findings indicate that protein farnesylation regulates the RAS protein-mediated signaling pathways required for appressorium formation and host infection, and suggest that abolishing farnesyltransferase could be an effective strategy for disease control.     

Identification of rice Allene Oxide Cyclase mutants and the function of jasmonate for defence against Magnaporthe oryzae

Two photomorphogenic mutants of rice, coleoptile photomorphogenesis 2 (cpm2) and hebiba, were found to be defective in the gene encoding allene oxide cyclase (OsAOC) by map‐based cloning and complementation assays. Examination of the enzymatic activity of recombinant GST–OsAOC indicated that OsAOC is a functional enzyme that is involved in the biosynthesis of jasmonic acid and related compounds. The level of jasmonate was extremely low in both mutants, in agreement with the fact that rice has only one gene encoding allene oxide cyclase. Several flower‐related mutant phenotypes were observed, including morphological abnormalities of the flower and early flowering. We used these mutants to investigate the function of jasmonate in the defence response to the blast fungus Magnaporthe oryzae. Inoculation assays with fungal spores revealed that both mutants are more susceptible than wild‐type to an incompatible strain of M. oryzae, in such a way that hyphal growth was enhanced in mutant tissues. The level of jasmonate isoleucine, a bioactive form of jasmonate, increased in response to blast infection. Furthermore, blast‐induced accumulation of phytoalexins, especially that of the flavonoid sakuranetin, was found to be severely impaired in cpm2 and hebiba. Together, the present study demonstrates that, in rice, jasmonate mediates the defence response against blast fungus.     

Linkage of autophagy to fungal development,lipid storage and virulence in Metarhizium robertsii

Autophagy is a highly conserved process that maintains intracellular homeostasis by degrading proteins or organelles in all eukaryotes. The effect of autophagy on fungal biology and infection of insect pathogens is unknown. Here, we report the function of MrATG8, an ortholog of yeast ATG8, in the entomopathogenic fungus Metarhizium robertsii. MrATG8 can complement an ATG8-defective yeast strain and deletion of MrATG8 impaired autophagy, conidiation and fungal infection biology in M. robertsii. Compared with the wild-type and gene-rescued mutant, Mratg8Δ is not inductive to form the infection-structure appressorium and is impaired in defense response against insect immunity. In addition, accumulation of lipid droplets (LDs) is significantly reduced in the conidia of Mratg8Δ and the pathogenicity of the mutant is drastically impaired. We also found that the cellular level of a LD-specific perilipin-like protein is significantly lowered by deletion of MrATG8 and that the carboxyl terminus beyond the predicted protease cleavage site is dispensable for MrAtg8 function. To corroborate the role of autophagy in fungal physiology, the homologous genes of yeast ATG1, ATG4 and ATG15, designated as MrATG1, MrATG4 and MrATG15, were also deleted in M. robertsii. In contrast to Mratg8Δ, these mutants could form appressoria, however, the LD accumulation and virulence were also considerably impaired in the mutant strains. Our data showed that autophagy is required in M. robertsii for fungal differentiation, lipid biogenesis and insect infection. The results advance our understanding of autophagic process in fungi and provide evidence to connect autophagy with lipid metabolism.     

Autophagy vitalizes the pathogenicity of pathogenic fungi

Plant pathogenic fungi utilize a series of complex infection structures, in particular the appressorium, to gain entry to and colonize plant tissue. As a consequence of the accumulation of huge quantities of glycerol in the cell the appressorium generates immense intracellular turgor pressure allowing the penetration peg of the appressorium to penetrate the leaf cuticle. Autophagic processes are ubiquitous in eukaryotic cells and facilitate the bulk degradation of macromolecules and organelles. The study of autophagic processes has been extended from the model yeast Saccharomyces cerevisiae to pathogenic fungi such as the rice blast fungus Magnaporthe oryzae. Significantly, null mutants for the expression of M. oryzae autophagy gene homologs lose their pathogenicity for infection of host plants. Clarification of the functions and network of interactions between the proteins expressed by M. oryzae autophagy genes will lead to a better understanding of the role of autophagy in fungal pathogenesis and help in the development of new strategies for disease control.     

MHP1, a Magnaporthe grisea hydrophobin gene, is required for fungal development and plant colonization

Fungal hydrophobins are implicated in cell morphogenesis and pathogenicity in several plant pathogenic fungi including the rice blast fungus Magnaporthe grisea. A cDNA clone encoding a hydrophobin (magnaporin, MHP1) was isolated from a cDNA library constructed from rice leaves infected by M. grisea. The MHP1 codes for a typical fungal hydrophobin of 102 amino acids containing eight cysteine residues spaced in a conserved pattern. Hydropathy analysis of amino acids revealed that MHP1 belongs to the class II group of hydrophobins. The amino acid sequence of MHP1 exhibited about 20% similarity to MPG1, an M. grisea class I hydrophobin. Expression of MHP1 was highly induced during plant colonization and conidiation, but could hardly be detected during mycelial growth. Transformants in which MHP1 was inactivated by targeted gene replacement showed a detergent wettable phenotype, but were not altered in wettability with water. mhp1 mutants also exhibited pleiotropic effects on fungal morphogenesis, including reduction in conidiation, conidial germination, appressorium development and infectious growth in host cells. Furthermore, conidia of mhp1 mutants were defective in their cellular organelles and rapidly lose viability. As a result, mhp1 mutants exhibited a reduced ability to infect and colonize a susceptible rice cultivar. These phenotypes were recovered by re-introduction of an intact copy of MHP1. Taken together, these results indicate that MHP1 has essential roles in surface hydrophobicity and infection-related fungal development, and is required for pathogenicity of M. grisea.