Endocytic protein Pal1 regulates appressorium formation and is required for full virulence of Magnaporthe oryzae

Endocytosis plays key roles during infection of plant-pathogenic fungi, but its regulatory mechanisms are still largely unknown. Here, we identified a putative endocytosis-related gene, PAL1, which was highly expressed in appressorium of Magnaporthe oryzae, and was found to be important for appressorium formation and maturation. Deletion of PAL1 significantly reduced the virulence of M. oryzae due to defects in appressorial penetration and invasive growth in host cells. The Pal1 protein interacted and colocalized with the endocytosis protein Sla1, suggesting it is involved in endocytosis. The Δpal1 mutant was significantly reduced in appressorium formation, which was recovered by adding exogenous cAMP and 3-isobutyl-1-methylxanthine (IBMX). Moreover, the phosphorylation level of Pmk1 in Δpal1 was also reduced, suggesting Pal1 functions upstream of both the cAMP and Pmk1 signalling pathways. As a consequence, the utilization of glycogen and lipid, appressorial autophagy, actin ring formation, localization of septin proteins, as well as turgor accumulation were all affected in the Δpal1 mutant. Taken together, Pal1 regulates cAMP and the Pmk1 signalling pathway for appressorium formation and maturation to facilitate infection of M. oryzae.     

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

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

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

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

A Genetic Screen for Pathogenicity Genes in the Hemibiotrophic Fungus Colletotrichum higginsianum Identifies the Plasma Membrane Proton Pump Pma2 Required for Host Penetration

We used insertional mutagenesis by Agrobacterium tumefaciens mediated transformation (ATMT) to isolate pathogenicity mutants of Colletotrichum higginsianum. From a collection of 7200 insertion mutants we isolated 75 mutants with reduced symptoms. 19 of these were affected in host penetration, while 17 were affected in later stages of infection, like switching to necrotrophic growth. For 16 mutants the location of T-DNA insertions could be identified by PCR. A potential plasma membrane H⁺-ATPase Pma2 was targeted in five independent insertion mutants. We genetically inactivated the Ku80 component of the non-homologous end-joining pathway in C. higginsianum to establish an efficient gene knockout protocol. Chpma2 deletion mutants generated by homologous recombination in the ΔChku80 background form fully melanized appressoria but entirely fail to penetrate the host tissue and are non-pathogenic. The ChPMA2 gene is induced upon appressoria formation and infection of A. thaliana. Pma2 activity is not important for vegetative growth of saprophytically growing mycelium, since the mutant shows no growth penalty under these conditions. Colletotrichum higginsianum codes for a closely related gene (ChPMA1), which is highly expressed under most growth conditions. ChPMA1 is more similar to the homologous yeast genes for plasma membrane pumps. We propose that expression of a specific proton pump early during infection may be common to many appressoria forming fungal pathogens as we found ChPMA2 orthologs in several plant pathogenic fungi.     

Phosphatase-associated protein MoTip41 interacts with the phosphatase MoPpe1 to mediate crosstalk between TOR and cell wall integrity signalling during infection by the rice blast fungus Magnaporthe oryzae

The type 2A (PP2A) and type 2A-like (PP4 and PP6) serine/threonine phosphatases participate in a variety of cellular processes, such as cell cycle progression, signal transduction and apoptosis. Previously, we reported that the PP6 catalytic subunit MoPpe1, which interacts with and is suppressed by type 2A associated protein of 42 kDa (MoTap42), an essential protein involved in the target of rapamycin (TOR) signalling pathway, has important roles in development, virulence and activation of the cell wall integrity (CWI) pathway in the rice blast fungus Magnaporthe oryzae. Here, we show that Tap42-interacting protein 41 (MoTip41) mediates crosstalk between the TOR and CWI signalling pathways; and participates in the TOR pathway via interaction with MoPpe1, but not MoTap42. The deletion of MoTIP41 resulted in disruption of CWI signalling, autophagy, vegetative growth, appressorium function and plant infection, as well as increased sensitivity to rapamycin. Further investigation revealed that MoTip41 modulates activation of the CWI pathway in response to infection by interfering with the interaction between MoTap42 and MoPpe1. These findings enhance our understanding of how crosstalk between TOR and CWI signalling modulates the development and pathogenicity of M. oryzae.     

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.     

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.     

H3K4 trimethylation by CclA regulates pathogenicity and the production of three families of terpenoid secondary metabolites in Colletotrichum higginsianum

The role of histone 3 lysine 4 (H3K4) methylation is poorly understood in plant pathogenic fungi. Here, we analysed the function of CclA, a subunit of the COMPASS complex mediating H3K4 methylation, in the brassica anthracnose pathogen Colletotrichum higginsianum. We show that CclA is required for full genome-wide H3K4 trimethylation. The deletion of cclA strongly reduced mycelial growth, asexual sporulation and spore germination but did not impair the morphogenesis of specialized infection structures (appressoria and biotrophic hyphae). Virulence of the ΔcclA mutant on plants was strongly attenuated, associated with a marked reduction in appressorial penetration ability on both plants and inert cellophane membranes. The secondary metabolite profile of the ΔcclA mutant was greatly enriched compared to that of the wild type, with three different families of terpenoid compounds being overproduced by the mutant, namely the colletochlorins, higginsianins and sclerosporide. These included five novel molecules that were produced exclusively by the ΔcclA mutant: colletorin D, colletorin D acid, higginsianin C, 13-epi-higginsianin C and sclerosporide. Taken together, our findings indicate that H3K4 trimethylation plays a critical role in regulating fungal growth, development, pathogenicity and secondary metabolism in C. higginsianum.     

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     

Peroxisomal fission is induced during appressorium formation and is required for full virulence of the rice blast fungus

Peroxisomes are involved in various metabolic processes and are important for virulence in different pathogenic fungi. How peroxisomes rapidly emerge in the appressorium during fungal infection is poorly understood. Here, we describe a gene, PEF1, which can regulate peroxisome formation in the appressorium by controlling peroxisomal fission, and is required for plant infection in the rice blast fungus Magnaporthe oryzae. Targeted deletion of PEF1 resulted in a reduction in virulence and a delay in penetration and invasive growth in host cells. PEF1 was particularly expressed during appressorial development, and its encoding protein was co‐localized with peroxisomes during appressorial development. Compared with the massive vesicle‐shaped peroxisomes formed in the wild‐type appressorium, the Δpef1 mutant could only form stringy linked immature peroxisomes, suggesting that PEF1 was involved in peroxisomal fission during appressorium formation. We also found that the Δpef1 mutant could not utilize fatty acids efficiently, which can improve significantly the expression level of PEF1 and induce peroxisomal fission. As expected, the Δpef1 mutant showed reduced intracellular production of reactive oxygen species (ROS) during appressorium formation and induced ROS accumulation in host cells during infection. Taken together, PEF1‐mediated peroxisomal fission is important for fungal infection by controlling the number of peroxisomes in the appressorium.     

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

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

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.     

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 cAMP-dependent protein kinase A pathway perturbs autophagy and plays important roles in development and virulence of Sclerotinia sclerotiorum

Previous research has demonstrated that sclerotia production is suppressed by exogenous cyclic AMP (cAMP) in Sclerotinia sclerotiorum and enhanced upon deletion of the adenylate cyclase gene. This study focuses on further functionally characterizing the cAMP-dependent protein kinase A (PKA) signaling pathway in S. sclerotiorum. Here, we demonstrate functions for two components of cAMP signaling: the catalytic, SsPKA, and the regulatory, SsPKAR, subunits of cAMP-dependent PKA. Growth and virulence were greatly reduced by disruption of either Sspka2 or SspkaR in addition to deficiencies in appressorium development. Surprisingly, disruption of both Sspka2 (dSspka2) and SspkaR (dSspkaR) display an up-regulation of autophagy without nutrient starvation suggesting that properly regulated PKA activity is required for control of autophagy. SsPKAR is demonstrated to be required for carbohydrate metabolism and mobilization, which are required for appressorium development and sclerotium initiation. A closer examination of dSspkaR during Nicotiana benthamiana infection revealed that an oxalic acid (OA)-independent necrosis protein(s) or metabolite(s) may be involved in the lesion development in dSspkaR-N. benthamiana interaction. In summary, these data demonstrate that the cAMP-dependent PKA signaling is essential for multiple forms of S. sclerotiorum development as well as virulence which rely on optimal regulation of autophagy.     

The autophagy genes atg8 and atg1 affect morphogenesis and pathogenicity in Ustilago maydis

Autophagy is a complex degradative process in which cytosolic material, including organelles, is randomly sequestered within double‐membrane vesicles termed autophagosomes. In Saccharomyces cerevisiae, the autophagy genes ATG1 and ATG8 are crucial for autophagy induction and autophagosome assembly, respectively, and their deletion has an impact on the autophagic potential of the corresponding mutant strains. We were interested in the role of autophagy in the development and virulence of U. maydis. Using a reverse genetic approach, we showed that the U. maydis ATG8 orthologue, atg8, is associated with autophagy‐dependent processes. Deletion of atg8 abolished autophagosome accumulation in the vacuoles of carbon‐starved cells and drastically reduced the survival of U. maydisΔatg8 mutant strains during these conditions. In addition, atg8 deletion had an impact on the budding process during saprobic haploid growth. The infection of maize with compatible Δatg8 strains resulted in fewer galled plants, and fungal gall colonization was strongly reduced, as reflected by the very low hyphal density in these tissues. Δatg8 infections resulted in the formation of very few teliospores. To corroborate the role of autophagy in U. maydis development, we also deleted the ATG1 orthologue, atg1. Deletion of atg1 yielded phenotypes similar to the Δatg8 strains during saprobic growth, but of lower magnitude. The Δatg1 strains were only slightly less pathogenic than the wild‐type and teliospore production was not affected. Surprisingly, atg1 deletion in the Δatg8 background exacerbated those phenotypes already observed in the Δatg8 and Δatg1 single‐mutant strains, strongly suggesting an additive phenotype. In particular, the double mutant was completely suppressed for plant gall induction.     

Signaling via cAMP in fungi: interconnections with mitogen-activated protein kinase pathways

The cAMP signal transduction pathway controls a wide variety of processes in fungi. For example, considerable progress has been made in describing the involvement of cAMP pathway components in the control of morphogenesis in Saccharomyces cerevisiae, Ustilago maydis, and Magnaporthe grisea. These morphological processes include the establishment of filamentous growth in S. cerevisiae and U. maydis, and the differentiation of an appressorial infection structure in M. grisea. The discovery that appressorium formation requires cAMP signaling provides an immediate connection to fungal virulence. This connection may have broader implications among fungal pathogens because recent work indicates that cAMP signaling controls the expression of virulence traits in the human pathogen Cryptococcus neoformans. In this fungus, cAMP also influences mating, as has been found for Schizosaccharomyces pombe and as may occur in U. maydis. Finally, cAMP and mitogen-activated protein kinase pathways appear to function coordinately to control the response of certain fungi, e.g., Saccharomyces cerevisiae and Schizosaccharomyces pombe, to environmental stress. There are clues that interconnections between these pathways may be common in the control of many fungal processes. Received: 5 June 1998 / Accepted: 11 September 1998