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.     

Septin-Dependent Assembly of the Exocyst Is Essential for Plant Infection by Magnaporthe oryzae

Magnaporthe oryzae is the causal agent of rice blast disease, the most devastating disease of cultivated rice (Oryza sativa) and a continuing threat to global food security. To cause disease, the fungus elaborates a specialized infection cell called an appressorium, which breaches the cuticle of the rice leaf, allowing the fungus entry to plant tissue. Here, we show that the exocyst complex localizes to the tips of growing hyphae during vegetative growth, ahead of the Spitzenkörper, and is required for polarized exocytosis. However, during infection-related development, the exocyst specifically assembles in the appressorium at the point of plant infection. The exocyst components Sec3, Sec5, Sec6, Sec8, and Sec15, and exocyst complex proteins Exo70 and Exo84 localize specifically in a ring formation at the appressorium pore. Targeted gene deletion, or conditional mutation, of genes encoding exocyst components leads to impaired plant infection. We demonstrate that organization of the exocyst complex at the appressorium pore is a septin-dependent process, which also requires regulated synthesis of reactive oxygen species by the NoxR-dependent Nox2 NADPH oxidase complex. We conclude that septin-mediated assembly of the exocyst is necessary for appressorium repolarization and host cell invasion.     

Cells in cells: morphogenetic and metabolic strategies conditioning rice infection by the blast fungus Magnaporthe oryzae

The rice blast fungus Magnaporthe oryzae is a global food security threat due to its destruction of cultivated rice. Of the world's rice harvest, 10–30 % is lost each year to this pathogen, and changing climates are likely to favor its spread into new areas. Insights into how the fungus might be contained could come from the wealth of molecular and cellular studies that have been undertaken in order to shed light on the biological underpinnings of blast disease, aspects of which we review herein. Infection begins when a three-celled spore lands on the surface of a leaf, germinates, and develops the specialized infection structure called the appressorium. The mature appressorium develops a high internal turgor that acts on a thin penetration peg, forcing it through the rice cuticle and into the underlying epidermal cells. Primary then invasive hyphae (IH) elaborate from the peg and grow asymptomatically from one living rice cell to another for the first few days of infection before host cells begin to die and characteristic necrotic lesions form on the surface of the leaf, from which spores are produced to continue the life cycle. To gain new insights into the biology of rice blast disease, we argue that, conceptually, the infection process can be viewed as two discrete phases occurring in markedly different environments and requiring distinct biochemical pathways and morphogenetic regulation: outside the host cell, where the appressorium develops in a nutrient-free environment, and inside the host cell, where filamentous growth occurs in a glucose-rich, nitrogen-poor environment, at least from the perspective of the fungus. Here, we review the physiological and metabolic changes that occur in M. oryzae as it transitions from the surface to the interior of the host, thus enabling us to draw lessons about the strategies that allow M. oryzae cells to thrive in rice cells.     

Spatial Uncoupling of Mitosis and Cytokinesis during Appressorium-Mediated Plant Infection by the Rice Blast Fungus Magnaporthe oryzae

To infect plants, many pathogenic fungi develop specialized infection structures called appressoria. Here, we report that appressorium development in the rice blast fungus Magnaporthe oryzae involves an unusual cell division, in which nuclear division is spatially uncoupled from the site of cytokinesis and septum formation. The position of the appressorium septum is defined prior to mitosis by formation of a heteromeric septin ring complex, which was visualized by spatial localization of Septin4:green fluorescent protein (GFP) and Septin5:GFP fusion proteins. Mitosis in the fungal germ tube is followed by long-distance nuclear migration and rapid formation of an actomyosin contractile ring in the neck of the developing appressorium, at a position previously marked by the septin complex. By contrast, mutants impaired in appressorium development, such as Δpmk1 and ΔcpkA regulatory mutants, undergo coupled mitosis and cytokinesis within the germ tube. Perturbation of the spatial control of septation, by conditional mutation of the SEPTATION-ASSOCIATED1 gene of M. oryzae, prevented the fungus from causing rice blast disease. Overexpression of SEP1 did not affect septation during appressorium formation, but instead led to decoupling of nuclear division and cytokinesis in nongerminated conidial cells. When considered together, these results indicate that SEP1 is essential for determining the position and frequency of cell division sites in M. oryzae and demonstrate that differentiation of appressoria requires a cytokinetic event that is distinct from cell divisions within hyphae.     

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.     

Large-Scale Gene Disruption in Magnaporthe oryzae Identifies MC69, a Secreted Protein Required for Infection by Monocot and Dicot Fungal Pathogens

To search for virulence effector genes of the rice blast fungus, Magnaporthe oryzae, we carried out a large-scale targeted disruption of genes for 78 putative secreted proteins that are expressed during the early stages of infection of M. oryzae. Disruption of the majority of genes did not affect growth, conidiation, or pathogenicity of M. oryzae. One exception was the gene MC69. The mc69 mutant showed a severe reduction in blast symptoms on rice and barley, indicating the importance of MC69 for pathogenicity of M. oryzae. The mc69 mutant did not exhibit changes in saprophytic growth and conidiation. Microscopic analysis of infection behavior in the mc69 mutant revealed that MC69 is dispensable for appressorium formation. However, mc69 mutant failed to develop invasive hyphae after appressorium formation in rice leaf sheath, indicating a critical role of MC69 in interaction with host plants. MC69 encodes a hypothetical 54 amino acids protein with a signal peptide. Live-cell imaging suggested that fluorescently labeled MC69 was not translocated into rice cytoplasm. Site-directed mutagenesis of two conserved cysteine residues (Cys36 and Cys46) in the mature MC69 impaired function of MC69 without affecting its secretion, suggesting the importance of the disulfide bond in MC69 pathogenicity function. Furthermore, deletion of the MC69 orthologous gene reduced pathogenicity of the cucumber anthracnose fungus Colletotrichum orbiculare on both cucumber and Nicotiana benthamiana leaves. We conclude that MC69 is a secreted pathogenicity protein commonly required for infection of two different plant pathogenic fungi, M. oryzae and C. orbiculare pathogenic on monocot and dicot plants, respectively.     

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.     

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.     

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.     

Transcriptome analysis reveals new insight into appressorium formation and function in the rice blast fungus <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Background  

Rice blast disease is caused by the filamentous Ascomycetous fungus Magnaporthe oryzae and results in significant annual rice yield losses worldwide. Infection by this and many other fungal plant pathogens requires the development of a specialized infection cell called an appressorium. The molecular processes regulating appressorium formation are incompletely understood.     

Autophagic cell death and its importance for fungal developmental biology and pathogenesis

In order to cause disease in plants, many fungal pathogens develop a specialized structure called an appressorium. We have recently shown that the rice blast fungus Magnaporthe grisea undergoes a regulated form of programmed cell death during appressorium development involving autophagy. Significantly, this form of cell death is a prerequisite for plant infection and fungal pathogenesis and part of a growing body of evidence implicating autophagy as a key process in fungal developmental biology.     

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.     

Differential Gene Expression Reflects Morphological Characteristics and Physiological Processes in Rice Immunity against Blast Pathogen Magnaporthe oryzae

The rice blast fungus Magnaporthe oryzae is a serious pathogen that jeopardises the world’s most important food-security crop. Ten common Malaysian rice varieties were examined for their morphological, physiological and genomic responses to this rice blast pathogen. qPCR quantification was used to assess the growth of the pathogen population in resistant and susceptible rice varieties. The chlorophyll content and photosynthesis were also measured to further understand the disruptive effects that M. oryzae has on infected plants of these varieties. Real-time PCR was used to explore the differential expression of eight blast resistance genes among the ten local varieties. Blast disease has destructive effects on the growth of rice, and the findings of our study provide evidence that the Pikh, Pi9, Pi21, and Osw45 genes are involved in defence responses in the leaves of Malaysian rice at 31 h after inoculation with M. oryzae pathotype P7.2. Both the chlorophyll content and photosynthesis were reduced, but the levels of Pikh gene expression remained constant in susceptible varieties, with a developed pathogen population and mild or severe symptoms. The Pi9, Pi21, and Osw45 genes, however, were simultaneously upregulated in infected rice plants. Therefore, the presence of the Pikh, Pi9, Pi21, and Osw45 genes in the germplasm is useful for improving the resistance of rice varieties.     

NTPDase Specific Inhibitors Suppress Rice Infection by Magnaporthe oryzae

Evolutionarily conserved ecto‐nucleoside triphosphate diphosphohydrolases (referred to ‘NTPDases’ below) are important ecto‐nucleotidases that are able to hydrolyse NTPs and NDPs in the environment to the monophosphate form. NTPDases are found in a variety of eukaryotic organisms including medical pathogens. However, pathogenic roles of these NTPDases in medical and plant pathogens are still very obscure. Here, we demonstrate that conidial germination, appressorium formation and pathogenicity of rice blast fungus Magnaporthe oryzae that had been pretreated with NTPDase‐specific inhibitors were significantly reduced, suggesting that NTPDases of M. oryzae play an important role in its infection. Our findings may provide a new avenue for powerful fungicide development and the control of rice blast.     

Characterizing Roles for the Glutathione Reductase,Thioredoxin Reductase and Thioredoxin Peroxidase-Encoding Genes of Magnaporthe oryzae during Rice Blast Disease

Understanding how pathogenic fungi adapt to host plant cells is of major concern to securing global food production. The hemibiotrophic rice blast fungus Magnaporthe oryzae, cause of the most serious disease of cultivated rice, colonizes leaf cells asymptomatically as a biotroph for 4–5 days in susceptible rice cultivars before entering its destructive necrotrophic phase. During the biotrophic growth stage, M. oryzae remains undetected in the plant while acquiring nutrients and growing cell-to-cell. Which fungal processes facilitate in planta growth and development are still being elucidated. Here, we used gene functional analysis to show how components of the NADPH-requiring glutathione and thioredoxin antioxidation systems of M. oryzae contribute to disease. Loss of glutathione reductase, thioredoxin reductase and thioredoxin peroxidase-encoding genes resulted in strains severely attenuated in their ability to grow in rice cells and that failed to produce spreading necrotic lesions on the leaf surface. Glutathione reductase, but not thioredoxin reductase or thioredoxin peroxidase, was shown to be required for neutralizing plant generated reactive oxygen species (ROS). The thioredoxin proteins, but not glutathione reductase, were shown to contribute to cell-wall integrity. Furthermore, glutathione and thioredoxin gene expression, under axenic growth conditions, was dependent on both the presence of glucose and the M. oryzae sugar/ NADPH sensor Tps1, thereby suggesting how glucose availability, NADPH production and antioxidation might be connected. Taken together, this work identifies components of the fungal glutathione and thioredoxin antioxidation systems as determinants of rice blast disease that act to facilitate biotrophic colonization of host cells by M. oryzae.