Junichiro Marui

The SskA and SrrA Response Regulators Are Implicated in Oxidative Stress Responses of Hyphae and Asexual Spores in the Phosphorelay Signaling Network of Aspergillus nidulans

Histidine-to-Aspartate (His-Asp) phosphorelay (or two-component) systems are common signal transduction mechanisms implicated in a wide variety of cellular responses to environmental stimuli in both prokaryotes and eukaryotes. For a model filamentous fungi, Aspergillus nidulans, in this study we first compiled a complete list of His-Asp phosphorelay components, including 15 genes for His-kinase (HK), four genes for response regulator (RR), and only one for histidine-containing phosphotransfer intermediate (HPt). For these RR genes, a set of deletion mutants was constructed so as to create a null allele for each. When examined these mutant strains under various conditions stressful for hyphal growth and asexual spore development, two of them (designated ΔsskA and ΔsrrA) showed a marked phenotype of hypersensitivity to oxidative stresses (particularly, to hydrogen peroxide). In this respect, expression of the vegetative-stage specific catB catalase gene was severely impaired in both mutants. Furthermore, conidia from ΔsskA were hypersensitive not only to treatment with H2O2, but also to treatment at aberrantly low (4 °C) and high (50 °C) temperatures, resulting in reduced germination efficiency. In this respect, not only the catA catalase gene specific for asexual development, but also a set of genes encoding the enzymes for synthesis of certain stress tolerant compatible solutes, such as trehalose and glycerol, were markedly downregulated in conidia from ΔsskA. These results together are indicative of the physiological importance of the His-Asp phosphorelay signaling network involving the SskA and SrrA response regulators.

Characterization of the NikA Histidine Kinase Implicated in the Phosphorelay Signal Transduction of Aspergillus nidulans, with Special Reference to Fungicide Responses

We recently compiled a complete list of phosphorelay signal transduction components in the model filamentous fungus Aspergillus nidulans. In this study, we characterized a histidine protein kinase (designated NikA) that is found in many fungi, with special reference to responses to potent fungicides (iprodione and fludioxonil). We provided evidence that not only NikA, but also two downstream response regulators (SskA and SrrA) are crucially implicated in the mode of action of these fungicides, and also that the further downstream HogA-MAPK cascade is exaggerated abnormally (or ectopically) in hyphae by the fungicides in a manner dependent on the NikA-SskA phosphorelay.

Characterization of a Tobacco TPK-type K+ Channel as a Novel Tonoplast K+ Channel Using Yeast Tonoplasts

The tonoplast K+ membrane transport system plays a crucial role in maintaining K+ homeostasis in plant cells. Here, we isolated cDNAs encoding a two-pore K+ channel (NtTPK1) from Nicotiana tabacum cv. SR1 and cultured BY-2 tobacco cells. Two of the four variants of NtTPK1 contained VHG and GHG instead of the GYG signature sequence in the second pore region. All four products were functional when expressed in the Escherichia coli cell membrane, and NtTPK1 was targeted to the tonoplast in tobacco cells. Two of the three promoter sequences isolated from N. tabacum cv. SR1 were active, and expression from these was increased ∼2-fold by salt stress or high osmotic shock. To determine the properties of NtTPK1, we enlarged mutant yeast cells with inactivated endogenous tonoplast channels and prepared tonoplasts suitable for patch clamp recording allowing the NtTPK1-related channel conductance to be distinguished from the small endogenous currents. NtTPK1 exhibited strong selectivity for K+ over Na+. NtTPK1 activity was sensitive to spermidine and spermine, which were shown to be present in tobacco cells. NtTPK1 was active in the absence of Ca2+, but a cytosolic concentration of 45 μm Ca2+ resulted in a 2-fold increase in the amplitude of the K+ current. Acidification of the cytosol to pH 5.5 also markedly increased NtTPK1-mediated K+ currents. These results show that NtTPK1 is a novel tonoplast K+ channel belonging to a different group from the previously characterized vacuolar channels SV, FV, and VK.

Transcriptional activator, AoXlnR, mediates cellulose-inductive expression of the xylanolytic and cellulolytic genes in Aspergillus oryzae

AoXlnR was isolated as a transcriptional activator of the major xylanase gene, xynF1, in Aspergillus oryzae. To investigate the spectrum of genes under the control of AoXlnR, expression of the xylanolytic and cellulolytic genes in an A. oryzae wild type strain, an AoxlnR disruptant and an AoXlnR overexpressed strain was analyzed by Northern blotting. AoXlnR mediated expression of at least four xylanolytic genes and four cellulolytic genes when induced by xylan and D‐xylose. Moreover, AoXlnR was newly found to mediate the cellulose‐inductive expression of the xylanolytic genes as well as the cellulolytic genes.

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.

Use of the Aspergillus oryzae actin gene promoter in a novel reporter system for exploring antifungal compounds and their target genes

Demand for novel antifungal drugs for medical and agricultural uses has been increasing because of the diversity of pathogenic fungi and the emergence of drug-resistant strains. Genomic resources for various living species, including pathogenic fungi, can be utilized to develop novel and effective antifungal compounds. We used Aspergillus oryzae as a model to construct a reporter system for exploring novel antifungal compounds and their target genes. The comprehensive gene expression analysis showed that the actin-encoding actB gene was transcriptionally highly induced by benomyl treatment. We therefore used the actB gene to construct a novel reporter system for monitoring responses to cytoskeletal stress in A. oryzae by introducing the actB promoter::EGFP fusion gene. Distinct fluorescence was observed in the reporter strain with minimum background noise in response to not only benomyl but also compounds inhibiting lipid metabolism that is closely related to cell membrane integrity. The fluorescent responses indicated that the reporter strain can be used to screen for lead compounds affecting fungal microtubule and cell membrane integrity, both of which are attractive antifungal targets. Furthermore, the reporter strain was shown to be technically applicable for identifying novel target genes of antifungal drugs triggering perturbation of fungal microtubules or membrane integrity.

Mitogen-activated protein kinases MpkA and MpkB independently affect micafungin sensitivity in Aspergillus nidulans

The transcriptional regulation of the MAPK mpkA and cell wall-related genes in Aspergillus nidulans differs from that of their counterparts in Saccharomyces cerevisiae. The A. nidulans MAPK MpkB is putatively orthologous to the yeast MAPKs Kss1p and Fus3p. To investigate MpkB and its contribution to cell wall integrity in A. nidulans, we constructed mpkB-disruptant (mpkB∆) strains. We previously showed that mpkA∆ strains exhibited reduced colony growth and increased sensitivity to the β-1,3-glucan synthase inhibitor micafungin. Like mpkA∆ strains, mpkB∆ strains exhibited slight growth retardation and increased sensitivity to micafungin. Although MpkB-dependent signaling modulated the transcription of some cell wall-related genes, the sugar composition of cell wall fractions was similar among wild-type, mpkA∆, and mpkB∆ strains. To elucidate the relationship between MpkA and MpkB pathways, we compared conditional mutants of mpkB with those with mpkA deletion. Sensitivity testing suggested that MpkA and MpkB additively contribute to micafungin activity in A. nidulans. Graphical Abstract Phenotypic analyses of the A. nidulans mpkA∆, mpkB∆, and conditional-mpkB with mpkA∆ strains revealed that the MpkA and MpkB additively contribute to micafungin activity. (24 words, 147 characters)

Culture-independent bacterial community analysis of the salty-fermented fish paste products of Thailand and Laos

A bacterial community analysis, using a culture-independent method (polymerase chain reaction-denaturing gradient gel electrophoresis), detected 17 species of bacteria including species of the genera Tetragenococcus, Lactobacillus, Pediococcus, Weissella Halanaerobium, Clostridium, and Sphingomonas in a traditional salty-fermented fish paste known as pla-ra or pa-daek in Thailand and Laos, which is used as a storage-stable multi-purpose seasoning. The representative genus of lactic acid bacteria seemed to vary in the 10 products collected from Thailand and Laos. Tetragenococci were common in products from central Thailand and Vientiane in Laos which had salinities of not less than 11% and pH values ranging from 5.6 to 6.1. However, lactobacilli were common in products from northern Thailand which had the lowest salinities (8.3–8.6%) and pH values (4.5–4.8) of all the samples examined. Two Lactobacillus and one Tetragenococcus species were detected in one product from northeastern Thailand containing 10% salt. These results suggest that salinity in pla-ra/pa-daek is an important determinant of the representative genus of lactic acid bacteria such as, Tetragenococcus or Lactobacillus. Additionally, differences in the acidity between these two groups seemed to be related to the production of d-/l-lactic acid in the lactic acid bacteria in each product. This is the first study to report a correlation between bacterial community structure and taste components in pla-ra/pa-daek products from various regions. This scientific work on a traditional fermented food will be useful in helping local producers meet differing consumer preferences in various regions.