Cutinase and hydrophobin interplay: A herald for pathogenesis?

Surface-penetrating phytopathogenic fungi frequently form appressoria. These are specialised infection structures pivotal to fungal ingress into the host. Recently, we demonstrated that one member of a family of cutinases in Magnaporthe grisea is involved in surface sensing, mediating appressorium differentiation and penetration peg formation and hence facilitates host penetration. Cutinase2 serves as an upstream activator of cAMP/PKA and DAG/PKC signalling cascades and is essential for full virulence. Here, we speculate on the role of rice blast hydrophobins as surface interactors facilitating fungal cutinase activity.Key words: rice blast fungus, appressorium, cutinase, hydrophobin, penetration, surface sensing, signalling     

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.     

A Rho3 homolog is essential for appressorium development and pathogenicity of Magnaporthe grisea

The small GTPase Rho3 is conserved in fungi and plays a key role in the control of cell polarity and exocytosis in yeast. In this report, we show that a Rho3 homolog, MgRho3, is dispensable for polarized hyphal growth in the rice blast fungus Magnaporthe grisea. However, MgRho3 is required for plant infection. Appressoria formed by the Mgrho3 deletion mutants are morphologically abnormal and defective in plant penetration. Conidia of the Mgrho3 deletion mutants are narrower than those of the wild-type strain and delayed in germination. Transformants expressing a dominant negative Mgrho3 allele exhibit similar phenotypes as the Mgrho3 deletion mutant, while transformants expressing a constitutively active allele of MgRho3 can produce normal conidia but remain defective in appressorium formation and plant infection. In contrast, overexpression of wild-type MgRho3 increases the infectivity of M. grisea. Our results reveal a new role for the conserved Rho3 as a critical regulator of developmental processes and pathogenicity of M. grisea.     

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.     

MoCDC14 is important for septation during conidiation and appressorium formation in Magnaporthe oryzae

As a typical foliar pathogen, appressorium formation and penetration are critical steps in the infection cycle of Magnaporthe oryzae. Because appressorium formation and penetration are closely co‐regulated with the cell cycle, and Cdc14 phosphatases have an antagonistic relationship with cyclin‐dependent kinases (CDKs) on proteins related to mitotic exit and cytokinesis, in this study, we functionally characterized the MoCDC14 gene in M. oryzae. The Mocdc14 deletion mutant showed significantly reduced growth rate and conidiation. It was also defective in septum formation and nuclear distribution. Septation was irregular in Mocdc14 hyphae and hyphal compartments became multi‐nucleate. Mutant conidia often showed incomplete septa or lacked any septum. During appressorium formation, the septum delimiting appressoria from the rest of the germ tubes was often formed far away from the neck of the appressoria or not formed at all. Unlike the wild‐type, some mutant appressoria had more than one nucleus at 24 h. In addition to appressoria, melanization occurred on parts of the germ tubes and conidia, depending on the irregular position of the appressorium‐delimiting septum. The Mocdc14 mutant was also defective in glycogen degradation during appressorium formation and appressorial penetration of intact plant cells. Similar defects in septum formation, melanization and penetration were observed with appressorium‐like structures formed at hyphal tips in the Mocdc14 mutant. Often a long fragment of mutant hyphae was melanized, together with the apical appressorium‐like structures. These results indicate that MoCDC14 plays a critical role in septation, nuclear distribution and pathogenesis in M. oryzae, and correct septum formation during conidiogenesis and appressorium formation requires the MoCdc14 phosphatase.     

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.     

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.     

Independent genetic mechanisms mediate turgor generation and penetration peg formation during plant infection in the rice blast fungus

The first barrier to infection encountered by foliar pathogens is the host cuticle. To traverse this obstacle, many fungi produce specialized infection cells called appressoria. MST12 is essential for appressorium-mediated penetration and infectious growth by the rice pathogen Magnaporthe grisea. In this study, we have characterized in detail the penetration defects of an mst12 deletion mutant. Appressoria formed by the mst12 mutant developed normal turgor pressure and ultrastructure but failed to form penetration pegs either on cellophane membranes or on plant epidermal cells. Deletion and site-directed mutagenesis analyses indicated that both the homeodomain and zinc finger domains, but not the middle region, of MST12 are essential for appressorial penetration and plant infection. The mst12 mutant appeared to be defective in microtubule reorganization associated with penetration peg formation. In mature appressoria, the mutant lacked vertical microtubules observed in the wild type. The mst12 mutant also failed to elicit localized host defence responses, including papilla formation and autofluorescence. Our data indicate that generation of appressorium turgor pressure and formation of the penetration peg are two independent processes. MST12 may play important roles in regulating penetration peg formation and directing the physical forces exerted by the appressorium turgor in mature appressoria.     

Multiple upstream signals converge on the adaptor protein Mst50 in Magnaporthe grisea

Rice blast fungus (Magnaporthe grisea) forms a highly specialized infection structure for plant penetration, the appressorium, the formation and growth of which are regulated by the Mst11-Mst7-Pmk1 mitogen-activated protein kinase cascade. We characterized the MST50 gene that directly interacts with both MST11 and MST7. Similar to the mst11 mutant, the mst50 mutant was defective in appressorium formation, sensitive to osmotic stresses, and nonpathogenic. Expressing a dominant active MST7 allele in mst50 complemented its defects in appressorium but not lesion formation. The sterile alpha-motif (SAM) domain of Mst50 was essential for its interaction with Mst11 and for appressorium formation. Although the SAM and Ras-association domain (RAD) of Mst50 were dispensable for its interaction with Mst7, deletion of RAD reduced appressorium formation and virulence on rice (Oryza sativa) seedlings. The interaction between Mst50 and Mst7 or Mst11 was detected by coimmunoprecipitation assays in developing appressoria. Mst50 also interacts with Ras1, Ras2, Cdc42, and Mgb1 in yeast two-hybrid assays. Expressing a dominant active RAS2 allele in the wild-type strain but not in mst50 stimulated abnormal appressorium formation. These results indicate that MST50 functions as an adaptor protein interacting with multiple upstream components and plays critical roles in activating the Pmk1 cascade for appressorium formation and plant infection in M. grisea.     

The Cyclase-Associated Protein Cap1 Is Important for Proper Regulation of Infection-Related Morphogenesis in Magnaporthe oryzae

Surface recognition and penetration are critical steps in the infection cycle of many plant pathogenic fungi. In Magnaporthe oryzae, cAMP signaling is involved in surface recognition and pathogenesis. Deletion of the MAC1 adenylate cyclase gene affected appressorium formation and plant infection. In this study, we used the affinity purification approach to identify proteins that are associated with Mac1 in vivo. One of the Mac1-interacting proteins is the adenylate cyclase-associated protein named Cap1. CAP genes are well-conserved in phytopathogenic fungi but none of them have been functionally characterized. Deletion of CAP1 blocked the effects of a dominant RAS2 allele and resulted in defects in invasive growth and a reduced intracellular cAMP level. The Δcap1 mutant was defective in germ tube growth, appressorium formation, and formation of typical blast lesions. Cap1-GFP had an actin-like localization pattern, localizing to the apical regions in vegetative hyphae, at the periphery of developing appressoria, and in circular structures at the base of mature appressoria. Interestingly, Cap1, similar to LifeAct, did not localize to the apical regions in invasive hyphae, suggesting that the apical actin cytoskeleton differs between vegetative and invasive hyphae. Domain deletion analysis indicated that the proline-rich region P2 but not the actin-binding domain (AB) of Cap1 was responsible for its subcellular localization. Nevertheless, the AB domain of Cap1 must be important for its function because CAP1 ^ΔAB only partially rescued the Δcap1 mutant. Furthermore, exogenous cAMP induced the formation of appressorium-like structures in non-germinated conidia in CAP1 ^ΔAB transformants. This novel observation suggested that AB domain deletion may result in overstimulation of appressorium formation by cAMP treatment. Overall, our results indicated that CAP1 is important for the activation of adenylate cyclase, appressorium morphogenesis, and plant infection in M. oryzae. CAP1 may also play a role in feedback inhibition of Ras2 signaling when Pmk1 is activated.     

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.     

The crucial role of the Pls1 tetraspanin during ascospore germination in Podospora anserina provides an example of the convergent evolution of morphogenetic processes in fungal plant pathogens and saprobes

Pls1 tetraspanins were shown for some pathogenic fungi to be essential for appressorium-mediated penetration into their host plants. We show here that Podospora anserina, a saprobic fungus lacking appressorium, contains PaPls1, a gene orthologous to known PLS1 genes. Inactivation of PaPls1 demonstrates that this gene is specifically required for the germination of ascospores in P. anserina. These ascospores are heavily melanized cells that germinate under inducing conditions through a specific pore. On the contrary, MgPLS1, which fully complements a ΔPaPls1 ascospore germination defect, has no role in the germination of Magnaporthe grisea nonmelanized ascospores but is required for the formation of the penetration peg at the pore of its melanized appressorium. P. anserina mutants with mutation of PaNox2, which encodes the NADPH oxidase of the NOX2 family, display the same ascospore-specific germination defect as the ΔPaPls1 mutant. Both mutant phenotypes are suppressed by the inhibition of melanin biosynthesis, suggesting that they are involved in the same cellular process required for the germination of P. anserina melanized ascospores. The analysis of the distribution of PLS1 and NOX2 genes in fungal genomes shows that they are either both present or both absent. These results indicate that the germination of P. anserina ascospores and the formation of the M. grisea appressorium penetration peg use the same molecular machinery that includes Pls1 and Nox2. This machinery is specifically required for the emergence of polarized hyphae from reinforced structures such as appressoria and ascospores. Its recurrent recruitment during fungal evolution may account for some of the morphogenetic convergence observed in fungi.     

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.     

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.     

Involvement of a Magnaporthe grisea serine/threonine kinase gene, MgATG1, in appressorium turgor and pathogenesis

We isolated an MgATG1 gene encoding a serine/threonine protein kinase from the rice blast fungus Magnaporthe grisea. In the DeltaMgatg1 mutant, in which the MgATG1 gene had been deleted, autophagy was blocked; the mutant also showed fewer lipid droplets in its conidia, lower turgor pressure of the appressorium, and such defects in morphogenesis as delayed initiation and slower germination of conidia. As a result of lower turgor pressure of the appressorium, the DeltaMgatg1 mutant lost its ability to penetrate and infect the two host plants, namely, rice and barley. However, normal values of the parameters and infective abilities were restored on reintroducing an intact copy of the MgATG1 gene into the mutant. Autophagy is thus necessary for turnover of organic matter during the formation of conidia and appressoria and for normal development and pathogenicity 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.