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.     

MoVam7, a conserved SNARE involved in vacuole assembly, is required for growth, endocytosis, ROS accumulation, and pathogenesis of Magnaporthe oryzae

Soluble NSF attachment protein receptor (SNARE) proteins play a central role in membrane fusion and vesicle transport of eukaryotic organisms including fungi. We previously identified MoSce22 as a homolog of Saccharomyces cerevisiae SNARE protein Sec22 to be involved in growth, stress resistance, and pathogenicity of Magnaporthe oryzae. Here, we provide evidences that MoVam7, an ortholog of S. cerevisiae SNARE protein Vam7, exerts conserved functions in vacuolar morphogenesis and functions in pathogenicity of M. oryzae. Staining with neutral red and FM4-64 revealed the presence of abnormal fragmented vacuoles and an absence of the Spitzenkörper body in the ΔMovam7 mutant. The ΔMovam7 mutant also exhibited reduced vegetative growth, poor conidiation, and failure to produce the infection structure appressorium. Additionally, treatments with cell wall perturbing agents indicated weakened cell walls and altered distributions of the cell wall component chitin. Furthermore, the ΔMovam7 mutant showed a reduced accumulation of reactive oxygen species (ROS) in the hyphal apex and failed to cause diseases on the rice plant. In summary, our studies indicate that MoVam7, like MoSec22, is a component of the SNARE complex whose functions in vacuole assembly also underlies the growth, conidiation, appressorium formation, and pathogenicity of M. oryzae. Further studies of MoVam7, MoSec22, and additional members of the SNARE complex are likely to reveal critical mechanisms in vacuole formation and membrane trafficking that is linked to fungal pathogenicity.     

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.     

Infection-associated nuclear degeneration in the rice blast fungus Magnaporthe oryzae requires non-selective macro-autophagy

Background

The rice blast fungus Magnaporthe oryzae elaborates a specialized infection structure called an appressorium to breach the rice leaf surface and gain access to plant tissue. Appressorium development is controlled by cell cycle progression, and a single round of nuclear division occurs prior to appressorium formation. Mitosis is always followed by programmed cell death of the spore from which the appressorium develops. Nuclear degeneration in the spore is known to be essential for plant infection, but the precise mechanism by which it occurs is not known.

Methodology/Principal Findings

In yeast, nuclear breakdown requires a specific form of autophagy, known as piecemeal microautophagy of the nucleus (PMN), and we therefore investigated whether this process occurs in the rice blast fungus. Here, we report that M. oryzae possesses two conserved components of a putative PMN pathway, MoVac8 and MoTsc13, but that both are dispensable for nuclear breakdown during plant infection. MoVAC8 encodes a vacuolar membrane protein and MoTSC13 a peri-nuclear and peripheral ER protein.

Conclusions/Significance

We show that MoVAC8 is necessary for caffeine resistance, but dispensable for pathogenicity of M. oryzae, while MoTSC13 is involved in cell wall stress responses and is an important virulence determinant. By functional analysis of ΔMoatg1 and ΔMoatg4 mutants, we demonstrate that infection-associated nuclear degeneration in M. oryzae instead occurs by non-selective macroautophagy, which is necessary for rice blast disease.     

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.     

Screening of a synthetic peptide combinatorial library to identify inhibitors of the appressorium formation in Magnaporthe oryzae

The rice blast disease caused by Magnaporthe oryzae is one of the most devastating diseases of cultivated rice. One of the most important stages in the infective cycle of M. oryzae is the formation of the dome-shaped structure called appressorium. The purpose of the present study was to identify novel peptides to control the rice blast disease by blocking the appressorium formation through screening of a synthetic peptide combinatorial library. As result of the screening, a set of 29 putative bioactive peptides were identified, synthesized and assayed in comparison with the previously identified peptide PAF104. The peptides MgAPI24, MgAPI40 and MgAPI47 showed improved inhibitory activity on the M. oryzae appressorium formation. Our data show that these peptides have a differential effect on two developmental structures: appressoria and appressorium-like structures. Antimicrobial assays against M. oryzae and other non-target microorganisms showed a weak or no toxicity of these peptides, demonstrating their specific activity blocking the appressorium formation. Therefore, the outcome of this research would be useful in the development of novel target-oriented peptides to use in plant protection.     

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.     

New findings on phosphodiesterases,MoPdeH and MoPdeL,in Magnaporthe oryzae revealed by structural analysis

The cyclic adenosine monophosphate (cAMP) signalling pathway mediates signal communication and sensing during infection‐related morphogenesis in eukaryotes. Many studies have implicated cAMP as a critical mediator of appressorium development in the rice blast fungus, Magnaporthe oryzae. The cAMP phosphodiesterases, MoPdeH and MoPdeL, as key regulators of intracellular cAMP levels, play pleiotropic roles in cell wall integrity, cellular morphology, appressorium formation and infectious growth in M. oryzae. Here, we analysed the roles of domains of MoPdeH and MoPdeL separately or in chimeras. The results indicated that the HD and EAL domains of MoPdeH are indispensable for its phosphodiesterase activity and function. Replacement of the MoPdeH HD domain with the L1 and L2 domains of MoPdeL, either singly or together, resulted in decreased cAMP hydrolysis activity of MoPdeH. All of the transformants exhibited phenotypes similar to that of the ΔMopdeH mutant, but also revealed that EAL and L1 play additional roles in conidiation, and that L1 is involved in infectious growth. We further found that the intracellular cAMP level is important for surface signal recognition and hyphal autolysis. The intracellular cAMP level negatively regulates Mps1‐MAPK and positively regulates Pmk1‐MAPK in the rice blast fungus. Our results provide new information to better understand the cAMP signalling pathway in the development, differentiation and plant infection of the fungus.     

Complexity of roles and regulation of the PMK1-MAPK pathway in mycelium development,conidiation and appressorium formation in Magnaporthe oryzae

MST50, MST11, MST7, PMK1 and GAS1/GAS2 genes are the important components in the PMK1-MAPK signal transduction pathway in fungi. Mutants with deletion of these five genes of Magnaporthe oryzae, a pathogen of the rice blast, were constructed. A cDNA array containing 4108 unique genes of M. oryzae was developed and used to analyze the gene expression profiles of these mutants against the wild type to dissect the gene expression regulation networks responsible for conidiation and appressorium formation. With this approach, differentially regulated genes by these five components were identified. The vast majority of the regulated genes were mutant-specific, while only a small proportion were in common for all of the mutants, suggesting that each of these genes has its own regulon. Functional groups and expression patterns of the regulated genes showed that (1) gene members in the PMK1-MAPK pathway are associated with multiple signaling pathways; (2) the regulation of PMK1-mediated signaling pathways is very complex and likely involved in other signaling networks; (3) glucose metabolism and signals are required in mycelium development; and (4) appressorium formation likely shares the mechanisms responsible for sexual conjugation and meiosis, which is affected by carbohydrate metabolism.     

MoGrr1, a novel F-box protein,is involved in conidiogenesis and cell wall integrity and is critical for the full virulence of <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

The production of asexual spores plays a critical role in rice blast disease. However, the mechanisms of the genes involved in the conidiogenesis pathway are not well understood. F-box proteins are specific adaptors to E3 ubiquitin ligases that determine the fate of different substrates in ubiquitin-mediated protein degradation and play diverse roles in fungal growth regulation. Here, we identify a Saccharomyces cerevisiae Grr1 homolog, MoGrr1, in Magnaporthe oryzae. Targeted disruption of Mogrr1 resulted in defects in vegetative growth, melanin pigmentation, conidial production, and resistance to oxidative stress, and these mutants consequently exhibited attenuated virulence to host plants. Microscopy studies revealed that the inability to form conidiophores is responsible for the defect in conidiation. Although the Mogrr1 mutants could develop melanized appressoria from hyphal tips, the appressoria were unable to penetrate into plant tissues due to insufficient turgor pressure within the appressorium, thereby attenuating the virulence of the mutants. Quantitative RT-PCR results revealed significantly decreased expression of chitin synthase-encoding genes, which are involved in fungal cell wall integrity, in the Mogrr1 mutants. The Mogrr1 mutants also displayed reduced expression of central components of the MAP kinase and cAMP signaling pathways, which are required for appressorium differentiation. Furthermore, domain complementation analysis indicated that two putative protein-interacting domains in MoGrr1 play essential roles during fungal development and pathogenicity. Taken together, our results suggest that MoGrr1 plays essential roles in fungal development and is required for the full virulence of M. oryzae.     

Rice blast fungus (Magnaporthe oryzae) infects Arabidopsis via a mechanism distinct from that required for the infection of rice

Magnaporthe oryzae is a hemibiotrophic fungal pathogen that causes rice (Oryza sativa) blast. Although M. oryzae as a whole infects a wide variety of monocotyledonous hosts, no dicotyledonous plant has been reported as a host. We found that two rice pathogenic strains of M. oryzae, KJ201 and 70-15, interacted differentially with 16 ecotypes of Arabidopsis (Arabidopsis thaliana). Strain KJ201 infected all ecotypes with varying degrees of virulence, whereas strain 70-15 caused no symptoms in certain ecotypes. In highly susceptible ecotypes, small chlorotic lesions appeared on infected leaves within 3 d after inoculation and subsequently expanded across the affected leaves. The fungus produced spores in susceptible ecotypes but not in resistant ecotypes. Fungal cultures recovered from necrotic lesions caused the same symptoms in healthy plants, satisfying Koch's postulates. Histochemical analyses showed that infection by the fungus caused an accumulation of reactive oxygen species and eventual cell death. Similar to the infection process in rice, the fungus differentiated to form appressorium and directly penetrated the leaf surface in Arabidopsis. However, the pathogenic mechanism in Arabidopsis appears distinct from that in rice; three fungal genes essential for pathogenicity in rice played only limited roles in causing disease symptoms in Arabidopsis, and the fungus seems to colonize Arabidopsis as a necrotroph through the secretion of phytotoxic compounds, including 9,12-octadecadienoic acid. Expression of PR-1 and PDF1.2 was induced in response to infection by the fungus, suggesting the activation of salicylic acid- and jasmonic acid/ethylene-dependent signaling pathways. However, the roles of these signaling pathways in defense against M. oryzae remain unclear. In combination with the wealth of genetic and genomic resources available for M. oryzae, this newly established pathosystem allows comparison of the molecular and cellular mechanisms underlying pathogenesis and host defense in two well-studied model plants.