Magnaporthe grisea cutinase2 mediates appressorium differentiation and host penetration and is required for full virulence

The rice blast fungus Magnaporthe grisea infects its host by forming a specialized infection structure, the appressorium, on the plant leaf. The enormous turgor pressure generated within the appressorium drives the emerging penetration peg forcefully through the plant cuticle. Hitherto, the involvement of cutinase(s) in this process has remained unproven. We identified a specific M. grisea cutinase, CUT2, whose expression is dramatically upregulated during appressorium maturation and penetration. The cut2 mutant has reduced extracellular cutin-degrading and Ser esterase activity, when grown on cutin as the sole carbon source, compared with the wild-type strain. The cut2 mutant strain is severely less pathogenic than the wild type or complemented cut2/CUT2 strain on rice (Oryza sativa) and barley (Hordeum vulgare). It displays reduced conidiation and anomalous germling morphology, forming multiple elongated germ tubes and aberrant appressoria on inductive surfaces. We show that Cut2 mediates the formation of the penetration peg but does not play a role in spore or appressorium adhesion, or in appressorial turgor generation. Morphological and pathogenicity defects in the cut2 mutant are fully restored with exogenous application of synthetic cutin monomers, cAMP, 3-isobutyl-1-methylxanthine, and diacylglycerol (DAG). We propose that Cut2 is an upstream activator of cAMP/protein kinase A and DAG/protein kinase C signaling pathways that direct appressorium formation and infectious growth in M. grisea. Cut2 is therefore required for surface sensing leading to correct germling differentiation, penetration, and full virulence in this model fungus.     

Hydrophobicity of contact surface induces appressorium formation in Magnaporthe grisea

Abstract Infection by Magnaporthe grisea , the causal agent of rice blast, requires the formation of a melanized, dome-shaped infection cell, called an appressorium. Little is known about the signals and mechanisms regulating this important developmental process. We have previously observed a correlation between hydrophobicity of the contact surface and appressorium formation. To evaluate this thigmotropic response more precisely, we measured appressorium formation on the surfaces of silicon wafers modified to create various degrees of hydrophobicity. We also examined the effects of artificial ridges created on polystyrene surfaces. Hydrophobic surfaces induced a high level of appressorium formation, whereas hydrophilic surfaces did not. Tips of germ-tubes did not respond to ridges of any particular height, but formed appressoria in a random manner. These results indicate that hydrophobicity of the substratum is a primary determinant and is sufficient to induce appressorium formation in M. grisea .     

Cellular localization and role of kinase activity of PMK1 in Magnaporthe grisea

A mitogen-activated protein (MAP) kinase gene, PMK1, is known to regulate appressorium formation and infectious hyphal growth in the rice blast fungus Magnaporthe grisea. In this study, we constructed a green fluorescent protein gene-PMK1 fusion (GFP-PMK1) to examine the expression and localization of PMK1 in M. grisea during infection-related morphogenesis. The GFP-PMK1 fusion encoded a functional protein that complemented the defect of the pmk1 deletion mutant in appressorium formation and plant infection. Although a weak GFP signal was detectable in vegetative hyphae, conidia, and germ tubes, the expression of GFP-Pmk1 was increased in appressoria and developing conidia. Nuclear localization of GFP-Pmk1 proteins was observed in a certain percentage of appressoria. A kinase-inactive allele and a nonphosphorylatable allele of PMK1 were constructed by site-directed mutagenesis. Expression of these mutant PMK1 alleles did not complement the pmk1 deletion mutant. These data confirm that kinase activity and activation of PMK1 by the upstream MAP kinase kinase are required for appressorium formation and plant infection in M. grisea. When overexpressed with the RP27 promoter in the wild-type strain, both the kinase-inactive and nonphosphorylatable PMK1 fusion proteins caused abnormal germ tube branching. Overexpression of these PMK1 mutant alleles may interfere with the function of native PMK1 during appressorium formation.     

The mitogen-activated protein kinase gene MAF1 is essential for the early differentiation phase of appressorium formation in Colletotrichum lagenarium

Colletotrichum lagenarium, the causal agent of cucumber anthracnose, invades host plants by forming a specialized infection structure called an appressorium. In this fungus, the mitogen-activated protein kinase (MAPK) gene CMK1 is involved in several steps of the infection process, including appressorium formation. In this study, the goal was to investigate roles of other MAPKs in C. lagenarium. The MAPK gene MAF1, related to Saccharomyces cerevisiae MPK1 and Magnaporthe grisea MPS1, was isolated and functionally characterized. The maf1 gene replacement mutants grew normally, but there was a significant reduction in conidiation and fungal pathogenicity. The M. grisea mps1 mutant forms appressoria, but conidia of the C. lagenarium maf1 mutants produced elongated germ tubes without appressoria on both host plant and glass, on which the wild type forms appressoria, suggesting that MAF1 has an essential role in appressorium formation on inductive surfaces. On a nutrient agar, wild-type conidia produced elongated germ tubes without appressoria. The morphological phenotype of the wild type on the nutrient agar was similar to that of the maf1 mutants on inductive surfaces, suggesting repression of the MAF1-mediated appressorium differentiation on the nutrient agar. The cmk1 mutants failed to form normal appressoria but produced swollen, appressorium-like structures on inductive surfaces, which is morphologically different from the maf1 mutants. These findings suggest that MAF1 is required for the early differentiation phase of appressorium formation, whereas CMK1 is involved in the maturation of appressoria.     

Fatty Acids and Their Derivatives as Modulators of Appressorium Formation in Magnaporthe grisea

Appressorium formation in germinating conidia of Magnaporthe grisea was inhibited on inductive and on noninductive surfaces by monounsaturated fatty acids with chain lengths of 16, 18, or 20 carbon atoms. On a noninductive surface, the inhibition was only observed upon stimulation with 1,16-hexadecanediol or oleyl alcohol, but not upon stimulation with 8-(4-chlorophenylthio)-adenosine-3′,5′-monophosphate. In the C₁₈-series, fatty acids with a double bond in position 9 were the most active ones. At 1 μg/ml of oleic or elaidic acid, less than 30% of the germinated conidia formed appressoria. The mode of inhibition was competitive to the inducing agent. On an inductive surface, compared to a noninductive surface the concentrations of oleic and elaidic acid needed for inhibition of appressorium formation were one order of magnitude higher. Methyl esters of inhibitory fatty acids and acids with two double bonds were not active. Like oleyl alcohol, elaidyl alcohol and petroselinyl alcohol stimulated infection structure formation on the noninductive surface.     

The adenylate cyclase gene MAC1 of Magnaporthe grisea controls appressorium formation and other aspects of growth and development.

Magnaporthe grisea, the causal agent of rice blast disease, differentiates a specialized infection structure called an appressorium that is crucial for host plant penetration. Previously, it was found that cAMP regulates appressorium formation. To further understand the cellular mechanisms involved in appressorium formation, we have cloned a gene (MAC1) encoding adenylate cyclase, a membrane-bound enzyme that catalyzes the production of cAMP from ATP, by using a polymerase chain reaction-based strategy. The entire gene was isolated and subcloned from a large insert bacterial artificial chromosome library. Sequence characterization showed that MAC1 has a high degree of identity with other adenylate cyclase genes from several filamentous fungi as well as yeasts. Gene deletion resulted in reduced vegetative growth, conidiation, and conidial germination. Transformants lacking MAC1 were unable to form appressoria on an inductive surface and were unable to penetrate susceptible rice leaves. mac1- transformants were also sterile and produced no perithecia. Appressorium formation was restored in the presence of exogenous cAMP derivatives. These results confirm that cell signaling involving cAMP plays a central role in the development and pathogenicity of M. grisea.     

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.     

A mitogen-activated protein kinase kinase required for induction of cytokinesis and appressorium formation by host signals in the conidia of Colletotrichum gloeosporioides

Differentiation of fungal conidia of phytopathogens into the infection structure, appressorium, requires contact with a hard surface and host signals. The molecular signaling involved in the induction of this differentiation is poorly understood. We report the cloning of a mitogen-activated protein kinase kinase (MEK), CgMEK, from Colletotrichum gloeosporioides and its role in the induction of these developmental processes involved in pathogenesis. Disruption of CgMEK1 resulted in the loss of its ability to form appressoria in response to the host's signals and a loss of virulence. Results of confocal microscopic examination of germinating conidia of the gene-disrupted mutants were similar to those for wild-type conidia treated with an MEK inhibitor, suggesting that CgMEK1 is involved in two developmental processes in the differentiation into appressorium: (1) polarized cell division, with the preferential increase in F-actin in one of the daughter nuclei after nuclear division and the formation of septum; and (2) differentiation of the germ tube into an appressorium. CgMEK1 is required for the differentiation.     

Evidence that the cAMP pathway controls emergence of both primary and appressorial germ tubes of barley powdery mildew

Development of conidia of barley powdery mildew involves the formation of a primary germ tube (PGT), an appressorial germ tube (AGT), and an appressorium. Previously, it was found that cyclic AMP (cAMP) was involved in these developmental processes. Comparison of development on the host surface with two types of cellulose membrane revealed that frequency of PGT emergence was surface independent. On one type of cellulose, where the frequencies of both AGT and appressorial differentiation were similar to that on the host surface, cAMP levels and protein kinase A (PKA) activities had a biphasic pattern with peaks at 15 min and 4 h after inoculation (prior to PGT and AGT emergence, respectively). The effect of manipulating cAMP levels was tested on another type of cellulose membrane, which stimulated a lower degree of AGT and appressorial formation than the host surface. Cholera toxin and forskolin, activators of adenylyl cyclase, significantly increased PGT emergence, but cAMP did not. Cholera toxin, forskolin, and cAMP increased the frequency of AGT and appressorial formation, but in a time-dependent manner.     

A novel gene MGA1 is required for appressorium formation in Magnaporthe grisea

Insertional mutagenesis is an effective way to study the infection mechanism of fungal pathogens. In an attempt to identify the genes involved in appressorium formation from Magnaporthe grisea, we carried out Agrobacterium tumefaciens mediated transformation (ATMT) of the fungus. Analysis of the region flanking the T-DNA integration site in one of the appressorium mutants showed insertion in a gene coding a 78 amino acid protein (MGA1), showing no significant homology to any of the known proteins. The mutant mga1 caused neither foliar nor root infection. Complementation of the mutated gene with the full length wild type gene restored appressorium formation as well as rice infection demonstrating the involvement of this gene in pathogenicity of M. grisea. In an indirect immunolocalisation assay, the MGA1 expression was seen predominantly in germ tube and appressoria. The mutant was impaired in glycogen and lipid mobilization required for appressorium formation. The glycerol content in the mycelia of the mutant under hyperosmotic stress conditions was less as compared to wild type and was thus unable to tolerate the hyperosmotic stress induced by sorbitol. We hypothesize that MGA1 plays a crucial role in signal transduction leading to the metabolism of glycogen and lipids, which is a part of appressorium differentiation process.     

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.     

Proteome analysis of rice blast fungus (Magnaporthe grisea) proteome during appressorium formation

We used two-dimensional gel electrophoresis (2-DE) to identify the proteins that are induced in the rice blast fungus Magnaporthe grisea during appressorium formation. Proteins were extracted from conidia that had germinated on hydrophilic glass plates or from germinated and appressoria-forming conidia on leaf wax-coated hydrophobic glass plates after 4, 8, and 12 h of incubation. Differentially expressed protein spots during appressorium formation were confirmed from gels after 2-DE analysis where proteins had been labeled with (35)S methionine and stained with silver. Internal amino acid sequencing identified five proteins among several proteins induced during appressorium formation. Two denoted as M. grisea proteasome homolgues (MgP1 and MgP5) were 20S proteasome alpha subunits. The remaining three were scytalone dehydratase (SCD), and serine carboxypeptidase Y (CPY). None of the five have been reported previously in the rice blast fungus apart from SCD. We further investigated the role the alpha subunit of 20S proteasome plays in appressorium formation. We confirmed by Western blot analysis that MgP5 is highly expressed during appressorium formation and found that it is also markedly induced by nitrogen- and carbon-starvation, in particular by the former. These observations suggest that the 20S proteasome may be involved in remobilizing storage proteins, which then help to build the appressorium. Thus, fungal proteome analysis may provide important clues about developmental changes such as the generation of the appressorium.     

cAMP regulation of "pathogenic" and "saprophytic" fungal spore germination

We report on the elucidation of two separate pathways of spore germination in a plant pathogenic fungus Colletotrichum gloeosporioides f. sp. aeschynomene. Conidia of the fungus can germinate either from one side or from both sides, depending on external conditions. In shake culture that includes an extract made up from fresh peas, the unicellular conidium divides and one of the two cells develops a germ tube. On a solid surface this germ tube differentiates an appressorium. In rich medium without pea extract, germination is highly similar to Aspergillus spore germination: the conidium swells, forms a single germ tube and then divides and forms a second germ tube. Conidia that germinate in a rich medium do not form appressoria even on a solid surface and are non-pathogenic. In rich medium, cAMP stimulates germination in rich liquid cultures and induces appressoria formation on a hard surface. In pea extract cAMP induces swelling and formation of irregular germ tubes and appressoria. Our results suggest that plant surface signals induce pathogenic-specific spore germination in a cAMP-independent manner. cAMP is required for saprophytic germination and for appressorium formation.     

Autophagy during conidiation, conidial germination and turgor generation in Magnaporthe grisea

Autophagy is a ubiquitous and evolutionarily conserved process found in all eukaryotic cells that allows for the degradation and recycling of old proteins and organelles. Starvation can induce autophagy, and autophagic pathway is an essential process for cellular function under starvation. In Magnaporthe grisea, starvation is one of the key induced factors for the germ tube tip to differentiate into an appressorium. Considering the importance of the rice blast fungus as a primary model for host-pathogen interaction, the role of autophagy in fungal development, appressorium turgor generation and pathogenicity of M. grisea via its role in organelle and protein turnover is a very significant subject.     

A novel gene,CBP1, encoding a putative extracellular chitin-binding protein,may play an important role in the hydrophobic surface sensing of Magnaporthe grisea during appressorium differentiation

The conidial germ tube of the rice blast fungus, Magnaporthe grisea, differentiates a specialized cell, an appressorium, required for penetration into the host plant. Formation of the appressorium is also observed on artificial solid substrata such as polycarbonate. A novel emerging germ tube-specific gene, CBP1 (chitin-binding protein), was found in a cDNA subtractive differential library. CBP1 coded for a putative extracellular protein (signal peptide) with two similar chitin-binding domains at both ends of a central domain with homology to fungal chitin deacetylases and with a C-terminus domain rich in Ser/Thr related extracellular matrix protein such as agglutinin. The consensus sequence of the chitin-binding domain found in CBP1 has never been reported in fungi and is similar to the chitin-binding motif in plant lectins and plant chitinases classes I and IV. CBPI was disrupted in order to identify its function. Null mutants of CBP1 failed to differentiate appressoria normally on artificial surface but succeeded in normally differentiating appressoria on the plant leaf surface. Since the null mutant Cbp1- showed abnormal appressorium differentiation only on artificial surfaces and was sensitive to the chemical inducers, CBP1 seemed to play an important role in the recognition of physical factors on solid surfaces.     

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.     

Early Expression of the Calmodulin Gene, Which Precedes Appressorium Formation in Magnaporthe grisea, Is Inhibited by Self-Inhibitors and Requires Surface Attachment

Fungal conidia contain chemicals that inhibit germination and appressorium formation until they are well dispersed in a favorable environment. Recently, such self-inhibitors were found to be present on the conidia of Magnaporthe grisea, and plant surface waxes were found to relieve this self-inhibition. To determine whether the self-inhibitors suppress the expression of early genes involved in the germination and differentiation of conidia, the calmodulin gene was chosen as a representative early gene, because it was found to be expressed early in Colletotrichum gloeosporioides and Colletotrichum trifolii differentiation. After calmodulin cDNA and genomic DNA from M. grisea were cloned, the promoter of the calmodulin gene was fused to a reporter gene, that for green fluorescent protein (GFP), and transformed into the M. grisea genome. Confocal microscopic examination and quantitation of expression of GFP green fluorescence showed (i) that the expression of the calmodulin gene decreased significantly when self-inhibition of M. grisea appressorium formation occurred because of high conidial density or addition of exogenous self-inhibitors and (ii) that the expression level of this gene was restored when self-inhibition was relieved by the addition of plant surface waxes. The increase in fluorescence correlated with the percentage of conidia that formed appressoria. The induction of calmodulin was also confirmed by RNA blotting. Concanavalin A inhibited surface attachment of conidia, GFP expression, and appressorium formation without affecting germination. The high correlation between GFP expression and appressorium formation strongly suggests that calmodulin gene expression and appressorium formation require surface attachment.     

Appressorium morphogenesis and cell cycle progression are linked in the grass powdery mildew fungus Blumeria graminis

Conidial germination and differentiation – the so-called prepenetration processes – of the barley powdery mildew fungus (Blumeria graminis f. sp. hordei) are essential prerequisites for facilitating penetration of the host cuticle. Although the cell cycle is known to be pivotal to cellular differentiation in several phytopathogenic fungi there is as yet no information available concerning the relationship between cell cycle and infection structure development in the obligate biotroph B. graminis. The timing of specific developmental events with respect to nuclear division and morphogenesis was followed on artificial and host leaf surfaces by 4′,6-diamidino-2-phenylindole (DAPI) staining in combination with a pharmacological approach applying specific cell cycle inhibitors. It was found that the uninucleate conidia germinated and then underwent a single round of mitosis 5–6 h after inoculation. During primary germ tube formation the nucleus frequently migrated close to the site of primary germ tube emergence. This nuclear repositioning was distinctly promoted by very-long-chain aldehydes that are common host cuticular wax constituents known to induce conidial differentiation. The subsequent morphogenesis of the appressorial germ tube preceded mitosis that was spatially uncoupled from subsequent cytokinesis. Blocking of S-phase with hydroxyurea did not inhibit formation of the appressorial germ tube but prevented cytokinesis and appressorium maturation. Benomyl treatment that arrests the cell cycle in mitosis inhibited nuclear separation, cytokinesis, and formation of mature appressoria. Thus, we conclude that a completed mitosis is not a prerequisite for the formation and swelling of the appressorial germ tube, which normally provides the destination for one of the daughter nuclei, while appressorium maturation depends on mitosis.     

Divergent cAMP signaling pathways regulate growth and pathogenesis in the rice blast fungus Magnaporthe grisea.

cAMP is involved in signaling appressorium formation in the rice blast fungus Magnaporthe grisea. However, null mutations in a protein kinase A (PKA) catalytic subunit gene, CPKA, do not block appressorium formation, and mutations in the adenylate cyclase gene have pleiotropic effects on growth, conidiation, sexual development, and appressorium formation. Thus, cAMP signaling plays roles in both growth and morphogenesis as well as in appressorium formation. To clarify cAMP signaling in M. grisea, we have identified strains in which a null mutation in the adenylate cyclase gene (MAC1) has an unstable phenotype such that the bypass suppressors of the Mac1(-) phenotype (sum) could be identified. sum mutations completely restore growth and sexual and asexual morphogenesis and lead to an ability to form appressoria under conditions inhibitory to the wild type. PKA assays and molecular cloning showed that one suppressor mutation (sum1-99) alters a conserved amino acid in cAMP binding domain A of the regulatory subunit gene of PKA (SUM1), whereas other suppressor mutations act independently of PKA activity. PKA assays demonstrated that the catalytic subunit gene, CPKA, encodes the only detectable PKA activity in M. grisea. Because CPKA is dispensable for growth, morphogenesis, and appressorium formation, divergent catalytic subunit genes must play roles in these processes. These results suggest a model in which both saprophytic and pathogenic growth of M. grisea is regulated by adenylate cyclase but different effectors of cAMP mediate downstream effects specific for either cell morphogenesis or pathogenesis.