Akira Yoshimi

MpkA-Dependent and -Independent Cell Wall Integrity Signaling in Aspergillus nidulans

ABSTRACT Cell wall integrity signaling (CWIS) maintains cell wall biogenesis in fungi, but only a few transcription factors (TFs) and target genes downstream of the CWIS cascade in filamentous fungi are known. Because a mitogen-activated protein kinase (MpkA) is a key CWIS enzyme, the transcriptional regulation of mpkA and of cell wall-related genes (CWGs) is important in cell wall biogenesis. We cloned Aspergillus nidulans mpkA; rlmA, a TF gene orthologous to Saccharomyces cerevisiae RLM1 that encodes Rlm1p, a major Mpk1p-dependent TF that regulates the transcription of MPK1 besides that of CWGs; and Answi4 and Answi6, homologous to S. cerevisiae SWI4 and SWI6, encoding the Mpk1p-activating TF complex Swi4p-Swi6p, which regulates CWG transcription in a cell cycle-dependent manner. A. nidulans rlmA and mpkA cDNA functionally complemented S. cerevisiae rlm1Δ and mpk1Δ mutants, respectively, but Answi4 and Answi6 cDNA did not complement swi4Δ and swi6Δ mutants. We constructed A. nidulans rlmA, Answi4 and Answi6, and mpkA disruptants (rlmAΔ, Answi4Δ Answi6Δ, and mpkAΔ strains) and analyzed mpkA and CWG transcripts after treatment with a β-1,3-glucan synthase inhibitor (micafungin) that could activate MpkA via CWIS. Levels of mpkA transcripts in the mutants as well as those in the wild type were changed after micafungin treatment. The β-glucuronidase reporter gene controlled by the mpkA promoter was expressed in the wild type but not in the mpkAΔ strain. Thus, mpkA transcription seems to be autoregulated by CWIS via MpkA but not by RlmA or AnSwi4-AnSwi6. The transcription of most CWGs except α-1,3-glucan synthase genes (agsA and agsB) was independent of RlmA and AnSwi4-AnSwi6 and seemed to be regulated by non-MpkA signaling. The transcriptional regulation of mpkA and of CWGs via CWIS in A. nidulans differs significantly from that in S. cerevisiae.

Group III Histidine Kinase Is a Positive Regulator of Hog1-Type Mitogen-Activated Protein Kinase in Filamentous Fungi

ABSTRACT We previously reported that the group III histidine kinase Dic1p in the maize pathogen Cochliobolus heterostrophus is involved in resistance to dicarboximide and phenylpyrrole fungicides and in osmotic adaptation. In addition, exposure to the phenylpyrrole fungicide fludioxonil led to improper activation of Hog1-type mitogen-activated protein kinases (MAPKs) in some phytopathogenic fungi, including C. heterostrophus. Here we report, for the first time, the relationship between the group III histidine kinase and Hog1-related MAPK: group III histidine kinase is a positive regulator of Hog1-related MAPK in filamentous fungi. The phosphorylation pattern of C. heterostrophus BmHog1p (Hog1-type MAPK) was analyzed in wild-type and dic1-deficient strains by Western blotting. In the wild-type strain, phosphorylated BmHog1p was detected after exposure to both iprodione and fludioxonil at a concentration of 1 μg/ml. In the dic1-deficient strains, phosphorylated BmHog1p was not detected after exposure to 10 μg/ml of the fungicides. In response to osmotic stress (0.4 M KCl), a trace of phosphorylated BmHog1p was found in the dic1-deficient strains, whereas the band representing active BmHog1p was clearly detected in the wild-type strain. Similar results were obtained for Neurospora crassa Os-2p MAPK phosphorylation in the mutant of the group III histidine kinase gene os-1. These results indicate that group III histidine kinase positively regulates the activation of Hog1-type MAPKs in filamentous fungi. Notably, the Hog1-type MAPKs were activated at high fungicide (100 μg/ml) and osmotic stress (0.8 M KCl) levels in the histidine kinase mutants of both fungi, suggesting that another signaling pathway activates Hog1-type MAPKs in these conditions.

Dynamics of cell wall components of Magnaporthe grisea during infectious structure development.

Oligosaccharides derived from cell wall of fungal pathogens induce host primary immune responses. To understand fungal strategies circumventing the host plant immune responses, cell wall polysaccharide localization was investigated using fluorescent labels during infectious structure differentiation in the rice blast fungus Magnaporthe grisea. α‐1,3‐glucan was labelled only on appressoria developing on plastic surfaces, whereas it was detected on both germ tubes and appressoria on plant surfaces. Chitin, chitosan and β‐1,3‐glucan were detected on germ tubes and appressoria regardless of the substrate. Major polysaccharides labelled at accessible surface of infectious hyphae were α‐1,3‐glucan and chitosan, but after enzymatic digestion of α‐1,3‐glucan, β‐1,3‐glucan and chitin became detectable. Immunoelectron microscopic analysis showed α‐1,3‐glucan and β‐1,3‐glucan intermixed in the cell wall of infectious hyphae; however, α‐1,3‐glucan tended to be distributed farther from the fungal cell membrane. The fungal cell wall became more tolerant to chitinase digestion upon accumulation of α‐1,3‐glucan. Accumulation of α‐1,3‐glucan was dependent on the Mps1 MAP kinase pathway, which was activated by a plant wax derivative, 1,16‐hexadecanediol. Taken together, α‐1,3‐glucan spatially and functionally masks β‐1,3‐glucan and chitin in the cell wall of infectious hyphae. Thus, a dynamic change of composition of cell wall polysaccharides occurs during plant infection in M. grisea.

Two-Component Response Regulators Ssk1p and Skn7p Additively Regulate High-Osmolarity Adaptation and Fungicide Sensitivity in Cochliobolus heterostrophus

ABSTRACT Filamentous ascomycetous fungi possess many histidine kinases and two conserved response regulators, Ssk1p and Skn7p, in their two-component signaling systems. We previously reported that the fungus unique group III histidine kinase regulates high-osmolarity adaptation and iprodione/fludioxonil fungicide sensitivity by controlling the phosphorylation of Hog1-type mitogen-activated protein kinase (MAPK) in filamentous ascomycetes. Here, we have characterized the response regulator genes ChSsk1 and ChSkn7 in the southern corn leaf blight fungus Cochliobolus heterostrophus. Both ChSsk1- and ChSkn7-disrupted mutants showed little sensitivity to high-osmolarity stress and moderate resistance to the iprodione/fludioxonil fungicides. The phosphorylation of Hog1-type MAPK BmHog1p induced by high-osmolarity stress and fungicide treatments was only regulated by ChSsk1p, indicating that ChSkn7p has roles in high-osmolarity adaptation and fungicide sensitivity that are independent from the activation of BmHog1p. The Chssk1 Chskn7 double mutants clearly showed higher sensitivity to osmolar stress and higher resistance to fungicides than the single mutants. The dose responses of the double mutants fit well with those of the group III histidine kinase-deficient strain. These results suggest that in filamentous ascomycetes, the Ssk1- and Skn7-type response regulators control high-osmolarity adaptation and fungicide sensitivity additively with differential mechanisms under the regulation of the group III histidine kinase. This study provides evidence that filamentous fungi have a unique two-component signaling system that is different from that of yeast and is responsible for high-osmolarity adaptation and fungicide sensitivity.

Transcriptional profiling for Aspergillusnidulans HogA MAPK signaling pathway in response to fludioxonil and osmotic stress.

In filamentous fungi, the His-Asp phosphorelay signaling system and HOG pathway are involved in the action of the fungicides, fludioxonil, and iprodione, as well as osmotic and oxidative stress responses. Aspergillusnidulans response regulators (RRs), SskA and SrrA, and histidine kinase (HK), NikA, are involved in the growth inhibitory effects of these fungicides. To gain further insights into the molecular basis for these signaling systems, we performed DNA microarray analyses of fludioxonil and osmotic stress responses in A.nidulans. A global expression analysis revealed that a large number of genes were modulated by fludioxonil treatment in an SskA-dependent manner, whereas SrrA hardly contributed to this modulation. The fludioxonil up-regulated or down-regulated genes (FUGs or FDGs, respectively) are also dependent on the HogA MAPK cascade. We found that the SskA-HogA pathway regulates expression of atfA gene encoding a transcription factor involved in conidia stress tolerance. From the results of microarray analyses, AtfA-dependent FUGs largely overlapped with HogA-dependent FUGs, suggesting that AtfA functions downstream of the HogA MAPK. A series of microarray analyses showed that the inferred SskA-HogA-AtfA pathway is implicated in the transcriptional response to osmotic stress as well as fludioxonil. The srrAatfA null double mutant turns off the SrrA and SskA-HogA-AtfA pathways and showed sensitivity to osmotic stress but no resistance to fludioxonil. Our data revealed that the growth inhibitory effect of fludioxonil depends on factors other than AtfA in spite of the fact that AtfA functions downstream of the HogA MAPK cascade. The complexity of the stress response in the His-Asp phosphorelay system followed by the HogA MAPK cascade is discussed.

NikA/TcsC Histidine Kinase Is Involved in Conidiation, Hyphal Morphology, and Responses to Osmotic Stress and Antifungal Chemicals in Aspergillus fumigatus

The fungal high osmolarity glycerol (HOG) pathway is composed of a two-component system (TCS) and Hog1-type mitogen-activated protein kinase (MAPK) cascade. A group III (Nik1-type) histidine kinase plays a major role in the HOG pathway of several filamentous fungi. In this study, we characterized a group III histidine kinase, NikA/TcsC, in the life-threatening pathogenic fungus, Aspergillus fumigatus. A deletion mutant of nikA showed low conidia production, abnormal hyphae, marked sensitivity to high osmolarity stresses, and resistance to cell wall perturbing reagents such as congo red and calcofluor white, as well as to fungicides such as fludioxonil, iprodione, and pyrrolnitrin. None of these phenotypes were observed in mutants of the SskA response regulator and SakA MAPK, which were thought to be downstream components of NikA. In contrast, in response to fludioxonil treatment, NikA was implicated in the phosphorylation of SakA MAPK and the transcriptional upregulation of catA, dprA, and dprB, which are regulated under the control of SakA. We then tested the idea that not only NikA, but also the other 13 histidine kinases play certain roles in the regulation of the HOG pathway. Interestingly, the expression of fos1, phkA, phkB, fhk5, and fhk6 increased by osmotic shock or fludioxonil treatment in a SakA-dependent manner. However, deletion mutants of the histidine kinases showed no significant defects in growth under the tested conditions. Collectively, although the signal transduction network related to NikA seems complicated, NikA plays a crucial role in several aspects of A. fumigatus physiology and, to a certain extent, modulates the HOG pathway.

Functional analysis of the α-1,3-glucan synthase genes agsA and agsB in Aspergillus nidulans: agsB is the major α-1,3-glucan synthase in this fungus.

Although α-1,3-glucan is one of the major cell wall polysaccharides in filamentous fungi, the physiological roles of α-1,3-glucan remain unclear. The model fungus Aspergillus nidulans possesses two α-1,3-glucan synthase (AGS) genes, agsA and agsB. For functional analysis of these genes, we constructed several mutant strains in A. nidulans: agsA disruption, agsB disruption, and double-disruption strains. We also constructed several CagsB strains in which agsB expression was controlled by the inducible alcA promoter, with or without the agsA-disrupting mutation. The agsA disruption strains did not show markedly different phenotypes from those of the wild-type strain. The agsB disruption strains formed dispersed hyphal cells under liquid culture conditions, regardless of the agsA genetic background. Dispersed hyphal cells were also observed in liquid culture of the CagsB strains when agsB expression was repressed, whereas these strains grew normally in plate culture even under the agsB-repressed conditions. Fractionation of the cell wall based on the alkali solubility of its components, quantification of sugars, and 13C-NMR spectroscopic analysis revealed that α-1,3-glucan was the main component of the alkali-soluble fraction in the wild-type and agsA disruption strains, but almost no α-1,3-glucan was found in the alkali-soluble fraction derived from either the agsB disruption strain or the CagsB strain under the agsB-repressed conditions, regardless of the agsA genetic background. Taken together, our data demonstrate that the two AGS genes are dispensable in A. nidulans, but that AgsB is required for normal growth characteristics under liquid culture conditions and is the major AGS in this species.

Novel Reporter Gene Expression Systems for Monitoring Activation of the Aspergillus nidulans HOG Pathway

The Aspergillus nidulans high-osmolarity glycerol response (AnHOG) pathway is involved in osmoadaptation. We found that fludioxonil, a fungicide, causes improper activation of HogA mitogen-activated protein kinase (MAPK) in A. nidulans. Here we present novel reporter systems for monitoring activation of the AnHOG pathway. The promoter region of gfdB (glycerol-3-phosphate dehydrogenase), whose expression depends on the presence of HogA, was fused to a β-glucuronidase uidA gene (GUS) to construct the reporter, which was introduced into A. nidulans wild type and hogAΔ. Increased GUS activity was detected in the wild type only when it was treated with high osmolarity or fludioxonil, while reporter activity was scarcely stimulated in the hogAΔ mutant. These results indicate that the reporter activity is controlled via HogA activation. Furthermore, we present possible applications of the reporter systems in screening new antifungal compounds.

Increased enzyme production under liquid culture conditions in the industrial fungus Aspergillus oryzae by disruption of the genes encoding cell wall α-1,3-glucan synthase.

Under liquid culture conditions, the hyphae of filamentous fungi aggregate to form pellets, which reduces cell density and fermentation productivity. Previously, we found that loss of α-1,3-glucan in the cell wall of the fungus Aspergillus nidulans increased hyphal dispersion. Therefore, here we constructed a mutant of the industrial fungus A. oryzae in which the three genes encoding α-1,3-glucan synthase were disrupted (tripleΔ). Although the hyphae of the tripleΔ mutant were not fully dispersed, the mutant strain did form smaller pellets than the wild-type strain. We next examined enzyme productivity under liquid culture conditions by transforming the cutinase-encoding gene cutL1 into A. oryzae wild-type and the tripleΔ mutant (i.e. wild-type-cutL1, tripleΔ-cutL1). A. oryzae tripleΔ-cutL1 formed smaller hyphal pellets and showed both greater biomass and increased CutL1 productivity compared with wild-type-cutL1, which might be attributable to a decrease in the number of tripleΔ-cutL1 cells under anaerobic conditions. Graphical abstract Summary of the growth characteristic and enzyme productivity in A. oryzae ags tripleΔ strain.

Substantial decrease in cell wall α-1,3-glucan caused by disruption of the kexB gene encoding a subtilisin-like processing protease in Aspergillus oryzae.

Disruption of the kexB encoding a subtilisin-like processing protease in Aspergillus oryzae (ΔkexB) leads to substantial morphological defects when the cells are grown on Czapek-Dox agar plates. We previously found that the disruption of kexB causes a constitutive activation of the cell wall integrity pathway. To understand how the disruption of the kexB affects cell wall organization and components, we analyzed the cell wall of ΔkexB grown on the plates. The results revealed that both total N-acetylglucosamine content, which constitutes chitin, and chitin synthase activities were increased. Whereas total glucose content, which constitutes β-1,3-glucan and α-1,3-glucan, was decreased; this decrease was attributed to a remarkable decrease in α-1,3-glucan. Additionally, the β-1,3-glucan in the alkali-insoluble fraction of the ΔkexB showed a high degree of polymerization. These results suggested that the loss of α-1,3-glucan in the ΔkexB was compensated by increases in the chitin content and the average degree of β-1,3-glucan polymerization. Summary of the alteration in the cell wall composition of the A. oryzae ∆kexB.