Loss of growth polarity and mislocalization of septa in a Neurospora mutant altered in the regulatory subunit of cAMP-dependent protein kinase.

In filamentous fungi, growth polarity (i.e. hyphal extension) and formation of septa require polarized deposition of new cell wall material. To explore this process, we analyzed a conditional Neurospora crassa mutant, mcb, which showed a complete loss of growth polarity when incubated at the restrictive temperature. Cloning and DNA sequence analysis of the mcb gene revealed that it encodes a regulatory subunit of cAMP-dependent protein kinase (PKA). Unexpectedly, the mcb mutant still formed septa when grown at the restrictive temperature, indicating that polarized deposition of wall material during septation is a process that is, at least in part, independent of polarized deposition during hyphal tip extension. However, septa formed in the mcb mutant growing at the restrictive temperature are mislocalized. Both polarized growth and septation are actin-dependent processes, and a concentration of actin patches is observed at growing hyphal tips and sites where septa are being formed. In the mcb mutant growing at the restrictive temperature, actin patches are uniformly distributed over the cell cortex; however, actin patches are still concentrated at sites of septation. Our results suggest that the PKA pathway regulates hyphal growth polarity, possibly through organizing actin patches at the cell cortex.     

Apolar growth of Neurospora crassa leads to increased secretion of extracellular proteins

Protein secretion in filamentous fungi has been shown to be restricted to actively growing hyphal tips. To determine whether an increase in the amount of growing surface area of a fungus can lead to an increase in the amount of protein secretion, we examined secretion in a temperature-sensitive Neurospora crassa mcb mutant that shows a loss of growth polarity when incubated at restrictive temperature. Incubation of the mcb mutant at restrictive temperature results in a three- to fivefold increase in the level of extracellular protein and a 20-fold increase in carboxymethyl cellulase activity relative to a wild-type strain. A mutation in the cr-1 gene has been shown previously to suppress the apolar growth phenotype of the mcb mutant, and we find that the level of extracellular protein produced by a mcb ; cr-1 double mutant was reduced to that of the wild-type control. Immunolocalization of a secreted endoglucanase revealed that proteins are secreted mainly at hyphal tips in hyphae exhibiting polar growth and over the entire surface area of bulbous regions of hyphae that are produced after a shift of the mcb mutant to restrictive temperature. These results support the hypothesis that secretion of extracellular protein by a filamentous fungus can be significantly increased by mutations that alter growth polarity.     

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.     

Expression levels and subcellular localization of Bcy1p in Candida albicans mutant strains devoid of one BCY1 allele results in a defective morphogenetic behavior

We investigated expression, functionality and subcellular localization of C. albicans Bcy1p, the PKA regulatory subunit, in mutant strains having one BCY1 allele fused to a green fluorescent protein (GFP). DE-52 column chromatography of soluble extracts of yeast cells from strains bearing one BCY1 allele (fused or not to GFP) showed co-elution of Bcy1p and Bcy1p-GFP with phosphotransferase activity, suggesting that interaction between regulatory and catalytic subunits was not impaired by the GFP tag. Subcellular localization of Bcy1p-GFP supports our previous hypothesis on the nuclear localization of the regulatory subunit, which can thus tether the PKA catalytic subunit to the nucleus. Protein modeling of CaBcy1p, showed that the fusion of the GFP tag to Bcy1p C-terminus did not significantly disturb its proper folding. Bcy1p levels in mutant strains having one or both BCY1 alleles, led us to establish a direct correlation between the amount of protein and the number of alleles, indicating that deletion of one BCY1 allele is not fully compensated by overexpression of the other. The morphogenetic behavior of several C. albicans mutant strains bearing one or both BCY1 alleles, in a wild-type and in a TPK2 null genetic background, was assessed in N-acetylglucosamine (GlcNAc) liquid medium at 37 degrees C. Strains with one BCY1 allele tagged or not, behaved similarly, displaying pseudohyphae and true hyphae. In contrast, hyphal morphology was almost exclusive in strains having both BCY1 alleles, irrespective of the GFP insertion. It can be inferred that a tight regulation of PKA activity is needed for hyphal growth.     

The role of actin, fimbrin and endocytosis in growth of hyphae in Aspergillus nidulans

Filamentous fungi are ideal systems to study the process of polarized growth, as their life cycle is dominated by hyphal growth exclusively at the cell apex. The actin cytoskeleton plays an important role in this growth. Until now, there have been no tools to visualize actin or the actin-binding protein fimbrin in live cells of a filamentous fungus. We investigated the roles of actin (ActA) and fimbrin (FimA) in hyphal growth in Aspergillus nidulans . We examined the localization of ActA::GFP and FimA::GFP in live cells, and each displayed a similar localization pattern. In actively growing hyphae, cortical ActA::GFP and FimA::GFP patches were highly mobile throughout the hypha and were concentrated near hyphal apices. A patch-depleted zone occupied the apical 0.5 μm of growing hypha. Both FimA::GFP and Act::GFP also localize transiently to septa. Movement and later localization of both was compromised after cytochalasin treatment. Disruption of fimA resulted in delayed polarity establishment during conidium germination, abnormal hyphal growth and endocytosis defects in apolar cells. Endocytosis was severely impaired in apolar fimA disruption cells. Our data support a novel apical recycling model which indicates a critical role for actin patch-mediated endocytosis to maintain polarized growth at the apex.     

Cross-talk between cAMP and calcium signalling in Aspergillus niger

Very little is known about cross-talk between cAMP and calcium signalling in filamentous fungi. The aim of this study was to analyse the influence of cAMP and protein kinase A (PKA)-dependent phosphorylation on calcium signalling in Aspergillus niger. For this purpose, cytosolic free calcium ([Ca2+]c) was measured in living hyphae expressing codon-optimized aequorin. The calcium signature following mechanical perturbation was analysed after applying dibutryl-cAMP or IBMX which increased intracellular cAMP, or H7 which inhibited phosphorylation by PKA. Calcium signatures were also measured in mutant strains in which phosphorylation by PKA was increased or lacking. The results indicated that calcium channels were activated by cAMP-mediated, PKA-dependent phosphorylation. Further evidence for cross-talk between cAMP and calcium signalling came from the analysis of a mutant in which the catalytic subunit of PKA was under the control of an inducible promoter. The consequence of PKA induction was a transient increase in [Ca2+]c which correlated with a polar-apolar transition in hyphal morphology. A transient increase in [Ca2+]c was not observed in this mutant when the morphological shift was in the opposite direction. The [Ca2+]c signatures in response to mechanical perturbation by polarized and unpolarized cells were markedly different indicating that these two cell types possessed different calcium signalling capabilities. These results were consistent with PKA-dependent phosphorylation increasing [Ca2+]c to induce a polar to apolar shift in hyphal morphology.     

The regulatory subunit of protein kinase A promotes hyphal growth and plays an essential role in Yarrowia lipolytica

The gene encoding the regulatory subunit (RKA1) of the cAMP-dependent protein kinase (PKA) of Yarrowia lipolytica was isolated to analyze the role of the PKA pathway in the dimorphic transition of the fungus. The gene encoded a protein of 397 amino acids that exhibits significant homology to fungal PKA regulatory subunits. Attempts to disrupt the gene by double homologous recombination, or the Pop-in Pop-out technique, were unsuccessful. The gene could be mutated only in merodiploids constructed with an autonomous replicating plasmid. Loss of the plasmid occurred with growth under nonselective conditions in the whole population of merodiploids carrying the mutation in the plasmid, but in merodiploids with the mutation at the chromosome, a resistant population prevailed. These data suggest that RKA1 is essential in Y. lipolytica. cAMP addition inhibited the dimorphic transition of the parental strain, but merodiploids carrying several copies of RKA1 were more resistant to cAMP. These results, and the observation that RKA1 was upregulated in mycelial cells, indicate that an active PKA pathway promotes yeast-like growth and opposes mycelial development. This behavior is in contrast to that of Candida albicans, where the PKA pathway favors hyphal growth.     

Cross regulation between Candida albicans catalytic and regulatory subunits of protein kinase A

In the pathogen Candida albicans protein kinase A (PKA) catalytic subunit is encoded by two genes TPK1 and TPK2 and the regulatory subunit by one gene, BCY1. PKA mediates several cellular processes such as cell cycle regulation and the yeast to hyphae transition, a key factor for C. albicans virulence. The catalytic isoforms Tpk1p and Tpk2p share redundant functions in vegetative growth and hyphal development, though they differentially regulate glycogen metabolism, the stress response pathway and pseudohyphal formation. In Saccharomyces cerevisiae it was earlier reported that BCY1 overexpression not only increased the amount of TPK3 mRNA but also its catalytic activity. In C. albicans a significant decrease in Bcy1p expression levels was already observed in tpk2Δ null strains. In this work we showed that the upregulation in Bcy1p expression was observed in a set of strains having a TPK1 or TPK2 allele reintegrated in its own locus, as well as in strains expressing the TPKs under the control of the constitutive ACT1 promoter. To confirm the cross regulation event between Bcy1p and Tpkp expression we generated a mutant strain with the lowest PKA activity carrying one TPK1 and a unique BCY1 allele with the aim to obtain two derived strains in which BCY1 or TPK1 were placed under their own promoters inserted in the RPS10 neutral locus. We found that placing one copy of BCY1 upregulated the levels of Tpk1p and its catalytic activity; while TPK1 insertion led to an increase in BCY1 mRNA, Bcy1p and in a high cAMP binding activity. Our results suggest that C. albicans cells were able to compensate for the increased levels of either Tpk1p or Tpk2p subunits with a corresponding elevation of Bcy1 protein levels and vice versa, implying a tightly regulated mechanism to balance holoenzyme formation.     

The heterotrimeric G‐protein beta subunit Gpb1 controls hyphal growth under low oxygen conditions through the protein kinase A pathway and is essential for virulence in the fungus Mucor circinelloides

Mucor circinelloides, a dimorphic opportunistic pathogen, expresses three heterotrimeric G‐protein beta subunits (Gpb1, Gpb2 and Gpb3). The Gpb1‐encoding gene is up‐regulated during mycelial growth compared with that in the spore or yeast stage. gpb1 deletion mutation analysis revealed its relevance for an adequate development during the dimorphic transition and for hyphal growth under low oxygen concentrations. Infection assays in mice indicated a phenotype with considerably reduced virulence and tissue invasiveness in the deletion mutants (Δgpb1) and decreased host inflammatory response. This finding could be attributed to the reduced filamentous growth in animal tissues compared with that of the wild‐type strain. Mutation in a regulatory subunit of cAMP‐dependent protein kinase A (PKA) subunit (PkaR1) resulted in similar phenotypes to Δgpb1. The defects exhibited by the Δgpb1 strain were genetically suppressed by pkaR1 overexpression, indicating that the PKA pathway is controlled by Gpb1 in M. circinelloides. Moreover, during growth under low oxygen levels, cAMP levels were much higher in the Δgpb1 than in the wild‐type strain, but similar to those in the ΔpkaR1 strain. These findings reveal that M. circinelloides possesses a signal transduction pathway through which the Gpb1 heterotrimeric G subunit and PkaR1 control mycelial growth in response to low oxygen levels.     

Protein kinase A encoded by TPK2 regulates dimorphism of Candida albicans

External signals induce the switch from a yeast to a hyphal growth form in the fungal pathogen Candida albicans. We demonstrate here that the catalytic subunit of a protein kinase A (PKA) isoform encoded by TPK2 is required for internal signalling leading to hyphal differentiation. TPK2 complements the growth defect of a Saccharomyces cerevisiae tpk1-3 mutant and Tpk2p is able to phosphorylate an established PKA-acceptor peptide (kemptide). Deletion of TPK2 blocks morphogenesis and partially reduces virulence, whereas TPK2 overexpression induces hyphal formation and stimulates agar invasion. The defective tpk2 phenotype is suppressed by overproduction of known signalling components, including Efg1p and Cek1p, whereas TPK2 overexpression reconstitutes the cek1 but not the efg1 phenotype. The results indicate that PKA activity of Tpk2p is an important contributing factor in regulating dimorphism of C. albicans.     

A screen in Saccharomyces cerevisiae identified CaMCM1, an essential gene in Candida albicans crucial for morphogenesis

Morphogenesis in Saccharomyces cerevisiae and the pathogenic yeast Candida albicans is governed in part by the same molecular circuits. In S. cerevisiae, FLO11/MUC1 expression has been shown to be modulated by multiple signalling pathways required for pseudohyphal development. We have established a screen in S. cerevisiae to identify regulators of fungal development in C. albicans based on FLO11::lacZ expression as a reporter. This screen identified both known components of the mitogen-activated protein kinase (MAPK) cascade and the cAMP cascade that are important for hyphal development in C. albicans, as well as genes not yet known to be involved in morphogenesis. The Candida homologue of MCM1 is one of the novel factors identified in this screen as being important for morphogenesis. CaMcm1p levels do not vary significantly in different cell types and respond to an autoregulatory feedback mechanism, arguing that CaMcm1p activity is regulated by post-translational modifications. Both overexpression and repression of this essential gene led to the induction of hyphae. Moreover, we found that the expression of HWP1, a hyphae-specific gene, was induced by repression of CaMCM1. The changes in morphology and HWP1 expression were not the result of a change in expression levels of NRG1 or TUP1, known repressors of hyphal development. Thus, CaMcm1p is a component of a hitherto unknown regulatory mechanism of hyphal growth.     

Continuous cultivations of a Penicillium chrysogenum strain expressing the expandase gene from Streptomyces clavuligerus: Growth yields and morphological characterization

The growth stoichiometry of a Penicillium chrysogenum strain expressing the expandase gene from Streptomyces clavuligerus was determined in glucose-limited chemostat cultivations using a chemically defined medium. This strain produces adipoyl-7-aminodeacetoxycephalosporanic acid (ad-7-ADCA) when it is fed with adipic acid. The biomass yield and maintenance coefficients for the strain were similar to those found for penicillin-producing strains of Penicillium chrysogenum. The maximum specific growth rate in the chemostat was found to be 0.11 h(-1). Metabolic degradation of adipate was found to take place in significant amounts only at dilution rates below 0.03 h(-1). After three to five residence times, adipate degradation and ad-7-ADCA production disappeared, and this allowed determination of the biomass yield coefficient on adipate. The morphology was measured at different dilution rates and the mean total hyphal length and mean number of tips both increased with an increase in dilution rate from 0.015 to 0.065 h(-1). Both variables decreased when the dilution rate was increased above 0.065 h(-1). A correlation between mean total hyphal length and productivity of ad-7-ADCA was found.     

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.     

Ras1-induced hyphal development in Candida albicans requires the formin Bni1

Formins are downstream effector proteins of Rho-type GTPases and are involved in the organization of the actin cytoskeleton and actin cable assembly at sites of polarized cell growth. Here we show using in vivo time-lapse microscopy that deletion of the Candida albicans formin homolog BNI1 results in polarity defects during yeast growth and hyphal stages. Deletion of the second C. albicans formin, BNR1, resulted in elongated yeast cells with cell separation defects but did not interfere with the ability of bnr1 cells to initiate and maintain polarized hyphal growth. Yeast bni1 cells were swollen, showed an increased random budding pattern, and had a severe defect in cytokinesis, with enlarged bud necks. Induction of hyphal development in bni1 cells resulted in germ tube formation but was halted at the step of polarity maintenance. Bni1-green fluorescent protein is found persistently at the hyphal tip and colocalizes with a structure resembling the Spitzenk?rper of true filamentous fungi. Introduction of constitutively active ras1G13V in the bni1 strain or addition of cyclic AMP to the growth medium did not bypass bni1 hyphal growth defects. Similarly, these agents were not able to suppress hyphal growth defects in the wal1 mutant which is lacking the Wiskott-Aldrich syndrome protein (WASP) homolog. These results suggest that the maintenance of polarized hyphal growth in C. albicans requires coordinated regulation of two actin cytoskeletal pathways, including formin-mediated secretion and WASP-dependent endocytosis.     

Novel Mechanism Coupling Cyclic AMP-Protein Kinase A Signaling and Golgi Trafficking via Gyp1 Phosphorylation in Polarized Growth

The cyclic AMP (cAMP)-protein kinase A (PKA) signaling activates virulence expression during hyphal development in the fungal human pathogen Candida albicans. The hyphal growth is characterized by Golgi polarization toward the hyphal tips, which is thought to enhance directional vesicle transport. However, how the hypha-induction signal regulates Golgi polarization is unknown. Gyp1, a Golgi-associated protein and the first GTPase-activating protein (GAP) in the Rab GAP cascade, critically regulates membrane trafficking from the endoplasmic reticulum to the plasma membrane. Here, we report a novel pathway by which the cAMP-PKA signaling triggers Golgi polarization during hyphal growth. We demonstrate that Gyp1 plays a crucial role in actin-dependent Golgi polarization. Hyphal induction activates PKA, which in turn phosphorylates Gyp1. Phosphomimetic mutation of four PKA sites identified by mass spectrometry (Gyp1^4E) caused strong Gyp1 polarization to hyphal tips, whereas nonphosphorylatable mutations (Gyp1^4A) abolished it. Gyp1^4E exhibited enhanced association with the actin motor Myo2, while Gyp1^4A showed the opposite effect, providing a possible mechanism for Golgi polarization. A GAP-dead Gyp1 (Gyp1^R292K) showed strong polarization similar to that seen with Gyp1^4E, indicating a role for the GAP activity. Mutating the PKA sites on Gyp1 also impaired the recruitment of a late Golgi marker, Sec7. Furthermore, proper PKA phosphorylation and GAP activity of Gyp1 are required for virulence in mice. We propose that the cAMP-PKA signaling directly targets Gyp1 to promote Golgi polarization in the yeast-to-hypha transition, an event crucial for C. albicans infection.     

Regulation of aflatoxin synthesis by FadA/cAMP/protein kinase A signaling in Aspergillus parasiticus

Analysis of fadA and pkaA mutants in the filamentous fungus Aspergillus nidulans demonstrated that FadA (Galpha) stimulates cyclic AMP (cAMP)-dependent protein kinase A (PKA) activity resulting, at least in part, in inhibition of conidiation and sterigmatocystin (ST) biosynthesis. In contrast, cAMP added to the growth medium stimulates aflatoxin (AF) synthesis in Aspergillus parasiticus. Our goal was to explain these conflicting reports and to provide mechanistic detail on the role of FadA, cAMP, and PKA in regulation of AF synthesis and conidiation in A. parasiticus. cAMP or dibutyryl-cAMP (DcAMP) were added to a solid growth medium and intracellular cyclic nucleotide levels, PKA activity, and nor-1 promoter activity were measured in A. parasiticus D8D3 (nor1::GUS reporter) and TJYP1-22 (fadAGA2R, activated allele). Similar to Tice and Buchanan [34], cAMP or DcAMP stimulated AF synthesis (and conidiation) associated with an AflR-dependent increase in nor-1 promoter activity. However, treatment resulted in a 100-fold increase in intracellular cAMP/DcAMP accompanied by a 40 to 80 fold decrease in total PKA activity. ThefadAG42R allele in TJYP1-22 decreased AF synthesis and conidiation, increased basal PKA activity 10 fold, and decreased total PKA activity 2 fold. In TJYP1-22, intracellular cAMP increased 2 fold without cAMP or DcAMP treatment; treatment did not stimulate conidiation or AF synthesis. Based on these data, we conclude that: (1) FadA/PKA regulate toxin synthesis and conidiation via similar mechanisms in Aspergillus spp.; and (2) intracellular cAMP levels, at least in part, mediate a PKA-dependent regulatory influence on conidiation and AF synthesis.     

Characterization of a hyperactive Cyr1 mutant reveals new regulatory mechanisms for cellular cAMP levels in Candida albicans

The adenylyl cyclase Cyr1 plays a pivotal role in regulating virulence traits in the human fungal pathogen Candida albicans. Although a diverse range of signals are known to activate Cyr1, it remains unclear how low activity is maintained in the absence of stimuli. To uncover negative regulatory elements, we designed a genetic screen to identify mutations in Cyr1 that increase its catalytic activity. We found such a mutant carrying a single Glu1541 to Lys substitution in a conserved motif C-terminal to the catalytic domain. This E1541K mutation caused constitutive filamentous growth, hypersensitivity to stress, resistance to farnesol and overproduction of riboflavin. The mutant phenotype depends on Cap1 and Ras1, two known positive regulators of Cyr1, and the filamentous growth requires Hgc1, a key promoter of hyphal growth. Strikingly, expressing a truncated version of the mutant protein lacking the entire region N-terminal to the catalytic domain in cyr1Δ cells caused a fivefold increase in the cellular cAMP level. Such cells exhibited dramatic enlargement, cytokinetic defects, G1 arrest and impaired hyphal development. Thus, our studies have revealed novel regulatory elements in Cyr1 that normally repress Cyr1 activity to prevent the toxicity of unregulated high cAMP levels.