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 rlm1⌬ and mpk1⌬ mutants, respectively, but Answi4 and Answi6 cDNA did not complement swi4⌬ and swi6⌬ mutants. We constructed A. nidulans rlmA, Answi4 and Answi6, and mpkA disruptants (rlmA⌬, Answi4⌬ Answi6⌬, and mpkA⌬ strains) and analyzed mpkA and CWG transcripts after treatment with a -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 -glucuronidase reporter gene controlled by the mpkA promoter was expressed in the wild type but not in the mpkA⌬ 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 ␣-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.
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.
The developmental time and thermal threshold for temperature-dependent sex determination (TSD), gender differences in temperature sensitivity, the fertility of thermally sex reversed fish, and the effect of temperature on the expression of two major sex determination/differentiation genes (DMY/DMRT1bY and DMRT1) were examined in the Hd-rR strain of medaka, Oryzias latipes. Fertilized eggs were exposed from either shortly after fertilization (8–16 cells; embryonic stages 5–6) or from middle embryogenesis (heart development stage; stage 36) until hatching to temperatures ranging from 17°C to 34°C. Secondary sexual characteristics, gonadal histology, progeny testing, sex-linked body coloration and gene expression were used to determine phenotypic and genotypic sex. Sex determination was unaffected by low or high temperatures in genotypic (XY) males. In contrast, genotypic (XX) females treated from stages 5–6 showed increasing rates of sex reversal into phenotypic males at temperatures above 27°C up to 100% at 34°C. Thermal manipulation of sex was ineffective after stage 36, indicating that gonadal fate in medaka is determined considerably earlier than histological differentiation (stage 39). High temperature induced DMRT1 expression in genotypic females, which was observed already from stage 36. Sex-reversed males had histologically normal testes, were capable of sexual courtship and, with the exception of fish from 34°C, sired viable progeny when mating with fertile females. These results clarify the pattern of TSD in medaka and provide important clues to understand the mechanism of sex determination in this species. They also suggest that a brief exposure to high temperature early in life could impair the fertility of medaka as adults.
Calcium signaling systems are widely employed in eukaryotes and are implicated in the regulation of diverse biological processes. Calcineurin is an important signaling component, which mediates ion homeostasis and virulence in several fungi. Based on intensive studies conducted on budding yeast, transcription factor Crz1p is thought to be a target of calcineurin. To provide insight into calcium signaling, a Crz1p homolog (CrzA) in a filamentous fungus Aspergillus nidulans was identified and its function with special reference to calcium response was characterized. A crzA gene disruption mutant exhibited sensitivity to high concentrations of Mn(2+) and Ca(2+), and mediated the expression of P-type calcium-ATPase homologous genes. Comprehensive transcriptional analysis with DNA microarrays indicated that CrzA regulates the expression of a vacuolar Ca(2+)/H(+) exchanger gene in response to external calcium stimuli. It is suggested that the calcineurin-CrzA pathway is the mediator of Ca(2+) homeostasis in A. nidulans. Moreover, a crzA/hogA double mutant showed hypersensitivity to osmotic stress, indicating the importance of calcium homeostasis for adaptation to osmotic stress, a universal stress in filamentous fungi.
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