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.     

Regulation of the MPG1 hydrophobin gene in the rice blast fungus Magnaporthe grisea

The hydrophobin-encoding gene MPG1 of the rice blast fungus Magnaporthe grisea is highly expressed during the initial stages of host plant infection and targeted deletion of the gene results in a mutant strain that is reduced in virulence, conidiation, and appressorium formation. The green fluorescent protein-encoding allele sGFP was used as a reporter to investigate regulatory genes that control MPG1 expression. The MAP kinase-encoding gene PMK1 and the wide domain regulators of nitrogen source utilization, NPR1 and NUT1, were required for full expression of MPG1 in response to starvation stress. The CPKA gene, encoding the catalytic subunit of protein kinase A, was required for repression of MPG1 during growth in rich nutrient conditions. During appressorium morphogenesis, high-level MPG1 expression was found to require the CPKA and NPR1 genes. Expression of a destabilized GFP allele indicated that de novo MPG1 expression occurs during appressorium formation. Three regions of the MPG1 promoter were identified which are required for high-level expression of MPG1 during appressorium formation and are necessary for the biological activity of the MPG1 hydrophobin during spore formation and plant infection.     

MAP kinase and protein kinase A-dependent mobilization of triacylglycerol and glycogen during appressorium turgor generation by Magnaporthe grisea

Magnaporthe grisea produces an infection structure called an appressorium, which is used to breach the plant cuticle by mechanical force. Appressoria generate hydrostatic turgor by accumulating molar concentrations of glycerol. To investigate the genetic control and biochemical mechanism for turgor generation, we assayed glycerol biosynthetic enzymes during appressorium development, and the movement of storage reserves was monitored in developmental mutants. Enzymatic activities for glycerol generation from carbohydrate sources were present in appressoria but did not increase during development. In contrast, triacylglycerol lipase activity increased during appressorium maturation. Rapid glycogen degradation occurred during conidial germination, followed by accumulation in incipient appressoria and dissolution before turgor generation. Lipid droplets also moved to the incipient appressorium and coalesced into a central vacuole before degrading at the onset of turgor generation. Glycogen and lipid mobilization did not occur in a Deltapmk1 mutant, which lacked the mitogen-activated protein kinase (MAPK) required for appressorium differentiation, and was retarded markedly in a DeltacpkA mutant, which lacks the catalytic subunit of cAMP-dependent protein kinase A (PKA). Glycogen and lipid degradation were very rapid in a Deltamac1 sum1-99 mutant, which carries a mutation in the regulatory subunit of PKA, occurring before appressorium morphogenesis was complete. Mass transfer of storage carbohydrate and lipid reserves to the appressorium therefore occurs under control of the PMK1 MAPK pathway. Turgor generation then proceeds by compartmentalization and rapid degradation of lipid and glycogen reserves under control of the CPKA/SUM1-encoded PKA holoenzyme.     

cAMP-pKA signaling regulates multiple steps of fungal infection cooperatively with Cmk1 MAP kinase in Colletotrichum lagenarium

In Colletotrichum lagenarium, RPK1 encoding the regulatory subunit of PKA is required for pathogenicity. From the rpkl mutant that forms small colonies, we isolated three growth-suppressor mutants. All rpk1-suppressor mutants are nonpathogenic and contain amino acid changes in the PKA catalytic subunit Cpkl. To assess the roles of cyclic AMP (cAMP) signaling in detail, we generated knockout mutants of CPK1 and the adenylate cyclase gene CAC1. The cpk1 and cac1 mutants are nonpathogenic on cucumber. Interestingly, both of the mutants germinated poorly, suggesting involvement of cAMP signaling in germination. Germination defect in the cpk1 and cac1 mutants is partially rescued by incubation of the conidia at lower concentrations. Germinating conidia of the cpk1 and cac1 mutants can form appressoria, but the appressoria formed by them are nonfunctional, like those of the rpk1 mutant. Cytological analysis indicates that the appressoria of the cpk1 mutant contain larger numbers of lipid bodies compared with the wild type, whereas lipid levels in the rpk1 mutants are lower, suggesting cAMP-mediated regulation of lipid metabolism for appressorium functionality. Furthermore, the cpk1 and cacl mutants have a defect in infectious growth in plant. In C. lagenarium, Cmkl mitogen-activated protein kinase (MAPK) regulates germination, appressorium formation, and infectious growth. These results suggest that cAMP signaling controls multiple steps of fungal infection in cooperative regulation with Cmkl MAPK in C. lagenarium.     

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.     

The Late Endosomal HOPS Complex Anchors Active G-Protein Signaling Essential for Pathogenesis in Magnaporthe oryzae

In Magnaporthe oryzae, the causal ascomycete of the devastating rice blast disease, the conidial germ tube tip must sense and respond to a wide array of requisite cues from the host in order to switch from polarized to isotropic growth, ultimately forming the dome-shaped infection cell known as the appressorium. Although the role for G-protein mediated Cyclic AMP signaling in appressorium formation was first identified almost two decades ago, little is known about the spatio-temporal dynamics of the cascade and how the signal is transmitted through the intracellular network during cell growth and morphogenesis. In this study, we demonstrate that the late endosomal compartments, comprising of a PI3P-rich (Phosphatidylinositol 3-phosphate) highly dynamic tubulo-vesicular network, scaffold active MagA/Gα_S, Rgs1 (a GAP for MagA), Adenylate cyclase and Pth11 (a non-canonical GPCR) in the likely absence of AKAP-like anchors during early pathogenic development in M. oryzae. Loss of HOPS component Vps39 and consequently the late endosomal function caused a disruption of adenylate cyclase localization, cAMP signaling and appressorium formation. Remarkably, exogenous cAMP rescued the appressorium formation defects associated with VPS39 deletion in M. oryzae. We propose that sequestration of key G-protein signaling components on dynamic late endosomes and/or endolysosomes, provides an effective molecular means to compartmentalize and control the spatio-temporal activation and rapid downregulation (likely via vacuolar degradation) of cAMP signaling amidst changing cellular geometry during pathogenic development in M. oryzae.     

Requirements for the adenylyl cyclases in the development of Dictyostelium

It has been suggested that all intracellular signaling by cAMP during development of Dictyostelium is mediated by the cAMP-dependent protein kinase, PKA, since cells carrying null mutations in the acaA gene that encodes adenylyl cyclase can develop so as to form fruiting bodies under some conditions if PKA is made constitutive by overexpressing the catalytic subunit. However, a second adenylyl cyclase encoded by acrA has recently been found that functions in a cell autonomous fashion during late development. We have found that expression of a modified acaA gene rescues acrA- mutant cells indicating that the only role played by ACR is to produce cAMP. To determine whether cells lacking both adenylyl cyclase genes can develop when PKA is constitutive we disrupted acrA in a acaA- PKA-C(over) strain. When developed at high cell densities, acrA- acaA- PKA-C(over) cells form mounds, express cell type-specific genes at reduced levels and secrete cellulose coats but do not form fruiting bodies or significant numbers of viable spores. Thus, it appears that synthesis of cAMP is required for spore differentiation in Dictyostelium even if PKA activity is high.     

A catalytic subunit of cyclic AMP-dependent protein kinase, PKAC-1, regulates asexual differentiation in Neurospora crassa

A cyclic AMP (cAMP)-dependent protein kinase pathway has been shown to regulate growth, morphogenesis and virulence in filamentous fungi. However, the precise mechanisms of regulation through the pathway remain poorly understood. In Neurospora crassa, the cr-1 adenylate cyclase mutant exhibits colonial growth with short aerial hyphae bearing conidia, and the mcb mutant, a mutant of the regulatory subunit of cAMP-dependent protein kinase (PKA), shows the loss of growth polarity at the restrictive temperature. In the present study, we isolated mutants of the catalytic subunit of the PKA gene pkac-1 through the process of repeat-induced point mutation (RIP). PKA activity of the mutants obtained through RIP was undetectable. The genome sequence predicts two distinct catalytic subunit genes of PKA, named pkac-1 (NCU06240.1, AAF75276) and pkac-2 (NCU00682.1), as is the case in most filamentous fungi. The results suggest that PKAC-1 works as the major PKA in N. crassa. The phenotype of the pkac-1 mutants included colonial growth, short aerial hyphae, premature conidiation on solid medium, inappropriate conidiation in submerged culture, and increased thermotolerance. This phenotype of pkac-1 mutants resembled to that of cr-1 mutants, except that the addition of cAMP did not rescue the abnormal morphology of pkac-1 mutants. The loss of growth polarity at the restrictive temperature in the mcb mutant was suppressed by pkac-1 mutation. These results suggest that the signal transduction pathway mediated by PKAC-1 plays an important role in regulation of aerial hyphae formation, conidiation, and hyphal growth with polarity.     

The cAMP-dependent protein kinase catalytic subunit is required for appressorium formation and pathogenesis by the rice blast pathogen Magnaporthe grisea.

Magnaporthe grisea, the causal agent of rice blast disease, differentiates a specialized infection cell, an appressorium, that is required for infection of its host. Previously, cAMP was implicated in the endogenous signaling pathway leading to appressorium formation. To obtain direct evidence for the role of cAMP in appressorium formation, the gene encoding the catalytic subunit of the cAMP-dependent protein kinase (cpkA) was cloned, sequenced, and disrupted. Polymerase chain reaction primers designed after highly conserved regions in the same gene from other organisms were used to amplify genomic DNA fragments. The cloned amplification products were used to identify genomic clones. DNA blot analysis indicated that cpkA is present as a single copy in the genome. cpkA consists of 1894 bp, including three short introns sufficient to encode a protein of 539 amino acids with a predicted molecular mass of 60.7 kD. The deduced peptide shares > 45% identity with other catalytic subunits and contains all functional motifs and residues with the addition of a glutamine-rich region at the N terminus. Two transformants, L5 and T-182, in which cpkA had been replaced with a hygromycin resistance gene cassette, were unable to produce appressoria, could not be induced to form appressoria by cAMP, and were nonpathogenic on susceptible rice, even when leaves were abraded. These results were confirmed by analysis of 57 progeny from a cross between transformant L5 and the wild-type laboratory strain 70-6. Other aspects of growth and development, including vegetative growth as well as asexual and sexual competence, were unaffected when measured in vitro. These results provide direct evidence that the cAMP-dependent protein kinase is necessary for infection-related morphogenesis and pathogenesis in a phytopathogenic fungus.     

ras2 Controls morphogenesis,pheromone response,and pathogenicity in the fungal pathogen Ustilago maydis

Ustilago maydis, a pathogen of maize, is a useful model for the analysis of mating, pathogenicity, and the morphological transition between budding and filamentous growth in fungi. As in other fungi, these processes are regulated by conserved signaling mechanisms, including the cyclic AMP (cAMP)/protein kinase A (PKA) pathway and at least one mitogen-activated protein kinase (MAP kinase) pathway. A current challenge is to identify additional factors that lie downstream of the cAMP pathway and that influence morphogenesis in U. maydis. In this study, we identified suppressor mutations that restored budding growth to a constitutively filamentous mutant with a defect in the gene encoding a catalytic subunit of PKA. Complementation of one suppressor mutation unexpectedly identified the ras2 gene, which is predicted to encode a member of the well-conserved ras family of small GTP-binding proteins. Deletion of the ras2 gene in haploid cells altered cell morphology, eliminated pathogenicity on maize seedlings, and revealed a role in the production of aerial hyphae during mating. We also used an activated ras2 allele to demonstrate that Ras2 promotes pseudohyphal growth via a MAP kinase cascade involving the MAP kinase kinase Fuz7 and the MAP kinase Ubc3. Overall, our results reveal an additional level of crosstalk between the cAMP signaling pathway and a MAP kinase pathway influenced by Ras2.     

Colletotrichum trifolii mutants disrupted in the catalytic subunit of cAMP-dependent protein kinase are nonpathogenic

Colletotrichum trifolii is the fungal pathogen of alfalfa that causes anthracnose disease. For successful plant infection, this fungus must undergo a series of morphological transitions following conidial attachment, including germination and subsequent differentiation, resulting in appressorium formation. Our previous studies with pharmacological effectors of signaling pathways have suggested the involvement of cyclic AMP (cAMP)-dependent protein kinase (PKA) during these processes. To more precisely evaluate the role of PKA in C. trifolii morphogenesis, the gene encoding the catalytic (C) subunit of PKA (Ct-PKAC) was isolated, sequenced, and inactivated by gene replacement. Southern blot analysis with C. trifolii genomic DNA suggested that Ct-PKAC is a single-copy gene. Northern (RNA) blot analysis with total RNA from different fungal growth stages indicated that the expression of this gene was developmentally regulated. When Ct-PKAC was insertionally inactivated by gene replacement, the transformants showed a small reduction in growth relative to the wild type and conidiation patterns were altered. Importantly, PKA-deficient strains were unable to infect intact alfalfa (host) plants, though only a slight delay was observed in the timing for conidial germination and appressorial formation in the Ct-PKAC disruption mutants. Moreover, these mutants were able to colonize host tissues following artificial wounding, resulting in typical anthracnose disease lesions. Coupled with microscopy, these data suggest that the defect in pathogenicity is likely due to a failure in penetration. Our results demonstrate that PKA has an important role in regulating the transition between vegetative growth and conidiation, and is essential for pathogenic development in C. trifolii.     

Proper regulation of cyclic AMP-dependent protein kinase is required for growth, conidiation, and appressorium function in the anthracnose fungus Colletotrichum lagenarium

Colletotrichum lagenarium, the casual agent of anthracnose of cucumber, forms specialized infection structures, called appressoria, during infection. To evaluate the role of cAMP signaling in C. lagenarium, we isolated and functionally characterized the regulatory subunit gene of the cAMP-dependent protein kinase (PKA). The RPK1 gene encoding the PKA regulatory subunit was isolated from C. lagenarium by polymerase chain reaction-based screening. rpk1 mutants, generated by gene replacement, exhibited high PKA activity during vegetative growth, whereas the wild-type strain had basal level activity. The rpk1 mutants showed significant reduction in vegetative growth and conidiation. Furthermore, the rpk1 mutants were nonpathogenic on cucumber plants, whereas they formed lesions when inoculated through wounds. A suppressor mutant showing restored growth and conidiation was isolated from a rpk1 mutant culture. The rpkl-suppressor mutant did not show high PKA activity, unlike the parental rpk1 mutant, suggesting that high PKA activity inhibits normal growth and conidiation. The suppressor mutant, however, was nonpathogenic on cucumber and failed to form lesions, even when inoculated through wounds. The rpk1 and suppressor mutants formed melanized appressoria on the host leaf surface but were unable to generate penetration hyphae. These results suggest that proper regulation of the PKA activity by the RPK1-encoded regulatory subunit is required for growth, conidiation, and appressorium function in C. lagenarium.     

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.     

Infection-related development in the rice blast fungus Magnaporthe grisea

Recent developments have been made in the identification of signal transduction pathways and gene products involved in the infection-related development of the rice blast fungus, Magnaporthe grisea. It has been established that cAMP-dependent and MAP kinase-mediated signaling are both critical for appressorium morphogenesis and function. These signaling pathways may act downstream of hydrophobin-mediated surface sensing by the growing germ tube. Several genes have been identified that are required for invasive growth of M. grisea including genes that allow adaptation of fungal metabolism to growth within plant tissues.