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1.
Phenylpyrrole Fungicides: Mitotic Instability in Aspergillus nidulans and Resistance in Botrytis cinerea 总被引:1,自引:0,他引:1
The somatic recombinogenic activity of the phenylpyrrole fungicide fludioxonil, in diploid Aspergillus nidulans was found similar to that caused by aromatic hydrocarbon and dicarboximide fungicides (AHDFs), such as iprodione, chlozolinate and tolclofos–methyl. All these fungicides not only increased the number of mitotic recombinants but also provided similar appearance, small sectors, of white and yellow mitotic recombination products. Fludioxonil highly resistant strains (resistant factor approximately 5000) of Botrytis cinerea were isolated at high frequency (1.08 × 10−5 ). Study of cross-resistance patterns of mutant strains to other fungicides, revealed cross-resistance of fludioxonil with dicarboximides (iprodione, procymidone, and chlozolinate) and aromatic hydrocarbons, such as tolclofos–methyl, pentachloronitrobenzene (PCNB), tecnazene and chloroneb. The positive cross-resistance relationships found between phenylpyrroles and members of the AHDFs and their ability to increase mitotic instability in diploid A. nidulans , indicate that phenylpyrroles should be included with AHDFs. A study of fitness parameters in wild-type and representative fludioxonil-resistant mutants of B. cinerea , showed that the mutation(s) leading to fludioxonil resistance may or may not affect some fitness-determining characteristics, such as sensitivity to high osmolarity, growth rate, conidial germination and germ-tube elongation. Pathogenicity tests on cucumber seedlings showed that an osmosensitive representative strain of B. cinerea , resistant to fludioxonil, was as virulent as the wild-type strain. The phenylpyrrole fungicide was ineffective, even in high concentrations, to control grey mould caused by this isolate. 相似文献
2.
Ochiai N Fujimura M Oshima M Motoyama T Ichiishi A Yamada-Okabe H Yamaguchi I 《Bioscience, biotechnology, and biochemistry》2002,66(10):2209-2215
We investigated the effects of iprodione and fludioxonil on the pathogenic yeast Candida albicans. Growth of the wild-type IFO1385 strain of C. albicans was inhibited by both fungicides, while Saccharomyces cerevisiae was basically unaffected by them even at a concentration of 25 microg/ml. Both fungicides stimulated glycerol synthesis in C. albicans but not in S. cerevisiae. The antioxidant alpha-tocopherol acetate and the cytochrome P-450 inhibitor piperonyl butoxide antagonized the fungitoxicity of iprodione and fludioxonil in C. albicans. It is known that mutations within the histidine kinase NIK1/OS-1 gene confer resistance to iprodione and fludioxonil in Neurospora crassa, while the fungicide-insensitive S. cerevisiae has only one histidine kinase SLN1 gene in its genome. In contrast, C. albicans has three histidine kinase genes, namely CaSLN1, CaNIK1/COS1, and CaHK1, the null mutants of which were found to impair the hyphal formation. Iprodione and fludioxonil were found to suppress filamentation when the IFO1385 strain was incubated on a solid medium containing fetal bovine serum. These observations suggest that iprodione and fludioxonil interfere with the CaNIK1/COS1 signal transduction pathway, resulting in glycerol synthesis stimulation and the inhibition of hyphal formation. 相似文献
3.
Mutational analysis of a conserved signal-transducing element: the HAMP linker of the Escherichia coli nitrate sensor NarX
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The HAMP linker, a predicted structural element observed in sensor proteins from all domains of life, is proposed to transmit signals between extracellular sensory input domains and cytoplasmic output domains. HAMP (histidine kinase, adenylyl cyclase, methyl-accepting chemotaxis protein, and phosphatase) linkers are located just inside the cytoplasmic membrane and are projected to form two short amphipathic alpha-helices (AS-1 and AS-2) joined by an unstructured connector. The presumed helices are comprised of hydrophobic residues in heptad repeats, with only three positions exhibiting strong conservation. We generated missense mutations at these three positions and throughout the HAMP linker in the Escherichia coli nitrate sensor kinase NarX and screened the resulting mutants for defective responses to nitrate. Most missense mutations in this region resulted in a constitutive phenotype mimicking the ligand-bound state, and only one residue (a conserved Glu before AS-2) was essential for HAMP linker function. We also scanned the narX HAMP linker with an overlapping set of seven-residue deletions. Deletions in AS-1 and the connector resulted in constitutive phenotypes. Two deletions in AS-2 resulted in a novel reversed response phenotype in which the response to ligand was the opposite of that seen for the narX(+) strain. These observations are consistent with the proposed HAMP linker structure, show that the HAMP linker plays an active role in transmembrane signal transduction, and indicate that the two amphipathic alpha-helices have different roles in signal transduction. 相似文献
4.
Doebber M Bordignon E Klare JP Holterhues J Martell S Mennes N Li L Engelhard M Steinhoff HJ 《The Journal of biological chemistry》2008,283(42):28691-28701
HAMP domains (conserved in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases) perform their putative function as signal transducing units in diversified environments in a variety of protein families. Here the conformational changes induced by environmental agents, namely salt and temperature, on the structure and function of a HAMP domain of the phototransducer from Natronomonas pharaonis (NpHtrII) in complex with sensory rhodopsin II (NpSRII) were investigated by site-directed spin labeling electron paramagnetic resonance. A series of spin labeled mutants were engineered in NpHtrII157, a truncated analog containing only the first HAMP domain following the transmembrane helix 2. This truncated transducer is shown to be a valid model system for a signal transduction domain anchored to the transmembrane light sensor NpSRII. The HAMP domain is found to be engaged in a "two-state" equilibrium between a highly dynamic (dHAMP) and a more compact (cHAMP) conformation. The structural properties of the cHAMP as proven by mobility, accessibility, and intra-transducer-dimer distance data are in agreement with the four helical bundle NMR model of the HAMP domain from Archaeoglobus fulgidus. 相似文献
5.
6.
Buschart A Gremmer K El-Mowafy M van den Heuvel J Mueller PP Bilitewski U 《Journal of biotechnology》2012,157(1):268-277
Signal transduction systems comprising histidine kinases are suggested as new molecular targets of antibiotics. The important human fungal pathogen Candida albicans possesses three histidine kinases, one of which is the type III histidine kinase CaNik1, which activates the MAP kinase Hog1. We established a screening system for inhibitors of this class of histidine kinases by functional expression of the CaNIK1 gene in S. cerevisiae. This transformant was susceptible to fungicides to which the wild type strain was resistant, such as fludioxonil and ambruticin. Growth inhibition correlated with phosphorylation of Hog1 and was dependent on an intact Hog1 pathway. At the N-terminus the histidine kinase CaNik1 has four amino acid repeats of 92 amino acids each and one truncated repeat of 72 amino acids. Within these repeats we identified 9 HAMP domains with a paired structure. We constructed mutants in which one or two pairs of these domains were deleted. S. cerevisiae transformants expressing the full-length CaNIK1 showed the highest sensitivity to the fungicides, any truncation reduced the susceptibility of the transformants to the fungicides. This indicates that the HAMP domains are decisive for the mode of action of the antifungal compounds. 相似文献
7.
Motoyama T Kadokura K Ohira T Ichiishi A Fujimura M Yamaguchi I Kudo T 《Fungal genetics and biology : FG & B》2005,42(3):200-212
We isolated and characterized a histidine kinase gene (HIK1) from the rice blast fungus, Pyricularia oryzae (Magnaporthe grisea). The deduced amino acid sequence of HIK1 showed highest similarity (85.7%) to a hybrid-type histidine kinase, Os-1/Nik-1 of Neurospora crassa. Disruption of HIK1 caused no defect in cell growth on normal media and in pathogenicity to rice plants. The Deltahik1 strain acquired resistance to three groups of fungicides (phenylpyrroles, dicarboximides, and aromatic hydrocarbons) similar to os-1 mutants of N. crassa. The Deltahik1 strain showed increased sensitivity to high concentrations of sugars although its salt sensitivity was not elevated, suggesting that the rice blast fungus can distinguish osmostresses caused by high sugar concentrations and high salt concentrations. In contrast, os-1 mutants of N. crassa are sensitive to high concentrations of both salts and sugars. These findings suggest that P. oryzae and N. crassa partially differ in their os (osmosensitive) signal transduction pathway. 相似文献
8.
Cui W Beever RE Parkes SL Weeds PL Templeton MD 《Fungal genetics and biology : FG & B》2002,36(3):187-198
A two-component histidine protein kinase gene, homologous to os-1 from Neurospora crassa, was cloned and sequenced from a single ascospore isolate of Botryotinia fuckeliana. A series of nine spontaneous mutants resistant to dicarboximide fungicides was selected from this strain and characterized with respect to fungicide resistance and osmotic sensitivity. Genetic crosses of the mutants with an authentic Daf1 strain showed that the phenotypes mapped to this locus. Single point mutations (seven transitions, one transversion, and one short deletion) were detected in the alleles of the nine mutants sequenced. The mutational changes were shown to cosegregate with the dicarboximide resistance and osmotic sensitivity phenotypes in progeny obtained from crossing selected resistant strains with a sensitive strain. All mutations detected are predicted to result in amino acid changes in the coiled-coil region of the putative Daf1 histidine kinase, and it is proposed that dicarboximide fungicides target this domain. 相似文献
9.
The HAMP linker, a common structural element between a sensor and a transmitter module in various sensor proteins, plays an essential role in signal transduction. Here, by in vivo complementation experiments with Tar-EnvZ hybrid receptor mutants in which the HAMP linker forms a heterodimer with Tar and EnvZ-type subunits, we found that mutations at one linker only affect the function of EnvZ in the same subunit. However, the same mutations affect the EnvZ function of both subunits when only a Tar or EnvZ-type HAMP linker is used. These results suggest that intersubunit interactions in the HAMP linker normally mediate signal transduction through both subunits in a sensor dimer, whereas the signal is asymmetrically transduced through the linker in a heterodimer. This is the first demonstration that two HAMP linkers in a sensor dimer are functionally coupled for normal signal transduction; however, this functional coupling can be reduced when the HAMP linkers lose their symmetric nature. 相似文献
10.
Genetic analysis of the HAMP domain of the Aer aerotaxis sensor localizes flavin adenine dinucleotide-binding determinants to the AS-2 helix 总被引:7,自引:4,他引:3
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HAMP domains are signal transduction domains typically located between the membrane anchor and cytoplasmic signaling domain of the proteins in which they occur. The prototypical structure consists of two helical amphipathic sequences (AS-1 and AS-2) connected by a region of undetermined structure. The Escherichia coli aerotaxis receptor, Aer, has a HAMP domain and a PAS domain with a flavin adenine dinucleotide (FAD) cofactor that senses the intracellular energy level. Previous studies reported mutations in the HAMP domain that abolished FAD binding to the PAS domain. In this study, using random and site-directed mutagenesis, we identified the distal helix, AS-2, as the component of the HAMP domain that stabilizes FAD binding. AS-2 in Aer is not amphipathic and is predicted to be buried. Mutations in the sequence coding for the contiguous proximal signaling domain altered signaling by Aer but did not affect FAD binding. The V264M residue replacement in this region resulted in an inverted response in which E. coli cells expressing the mutant Aer protein were repelled by oxygen. Bioinformatics analysis of aligned HAMP domains indicated that the proximal signaling domain is conserved in other HAMP domains that are not involved in chemotaxis or aerotaxis. Only one null mutation was found in the coding sequence for the HAMP AS-1 and connector regions, suggesting that these are not active signal transduction sites. We consider a model in which the signal from FAD is transmitted across a PAS-HAMP interface to AS-2 or the proximal signaling domain. 相似文献
11.
Two-component response regulators Ssk1p and Skn7p additively regulate high-osmolarity adaptation and fungicide sensitivity in Cochliobolus heterostrophus
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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. 相似文献
12.
Kishii R Falzon L Yoshida T Kobayashi H Inouye M 《The Journal of biological chemistry》2007,282(36):26401-26408
The HAMP domain plays an essential role in signal transduction not only in histidine kinase but also in a number of other signal-transducing receptor proteins. Here we expressed the EnvZ HAMP domain (Arg(180)-Thr(235)) with the R218K mutation (termed L(RK)) or with L(RK) connected with domain A (Arg(180)-Arg(289)) (termed LA(RK)) of EnvZ, an osmosensing transmembrane histidine kinase in Escherichia coli, by fusing it with protein S. The L(RK) and LA(RK) proteins were purified after removing protein S. The CD analysis of the isolated L protein revealed that it consists of a random structure or is unstructured. This suggests that the EnvZ HAMP domain by itself is unable to form a stable structure and that this structural fragility may be important for its role in signal transduction. Interestingly the substitution of Ala(193) in the EnvZ HAMP domain with valine or leucine in Tez1A1, a chimeric protein of Tar and EnvZ, caused a constitutive OmpC phenotype. The CD analysis of LA(RK)(A193L) revealed that this mutated HAMP domain possesses considerable secondary structures and that the thermostability of this entire LA(RK)(A193L) became substantially lower than that of LA(RK) or just domain A, indicating that the structure of the HAMP domain with the A193L mutation affects the stability of downstream domain A. This results in cooperative thermodenaturation of domain A with the mutated HAMP domain. These results are discussed in light of the recently solved NMR structure of the HAMP domain from a thermophilic bacterium (Hulko, M., Berndt, F., Gruber, M., Linder, J. U., Truffault, V., Schultz, A., Martin, J., Schultz, J. E., Lupas, A. N., and Coles, M. (2006) Cell 126, 929-940). 相似文献
13.
Group III histidine kinase is a positive regulator of Hog1-type mitogen-activated protein kinase in filamentous fungi 总被引:1,自引:0,他引:1
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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 microg/ml. In the dic1-deficient strains, phosphorylated BmHog1p was not detected after exposure to 10 microg/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 microg/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. 相似文献
14.
The PAS and HAMP domain superfamilies are signal transduction modules found in all kingdoms of life. The Aer receptor, which contains both domains, initiates rapid behavioural responses to oxygen (aerotaxis) and other electron acceptors, guiding Escherichia coli to niches where it can generate optimal cellular energy. We used intragenic complementation to investigate the signal transduction pathway from the Aer PAS domain to the signalling domain. These studies showed that the HAMP domain of one monomer in the Aer dimer stabilized FAD binding to the PAS domain of the cognate monomer. In contrast, the signal transduction pathway was intra-subunit, involving the PAS and signalling domains from the same monomer. The minimal requirements for signalling were investigated in heterodimers containing a full-length and truncated monomer. Either the PAS or signalling domains could be deleted from the non-signalling subunit of the heterodimer, but removing 16 residues from the C-terminus of the signalling subunit abolished aerotaxis. Although both HAMP domains were required for aerotaxis, signalling was not disrupted by missense mutations in the HAMP domain from the signalling subunit. Possible models for Aer signal transduction are compared. 相似文献
15.
In order to circumvent deleterious effects of hypo- and hyperosmotic conditions in its environment, Corynebacterium glutamicum has developed a number of mechanisms to counteract osmotic stress. The first response to an osmotic upshift is the activation of uptake mechanisms for the compatible solutes betaine, proline, or ectoine, namely BetP, EctP, ProP, LcoP and PutP. BetP, the most important uptake system responds to osmotic stress by regulation at the level of both protein activity and gene expression. BetP was shown to harbor three different properties, i.e. catalytic activity (betaine transport), sensing of appropriate stimuli (osmosensing) and signal transduction to the catalytic part of the carrier protein which adapts its activity to the extent of osmotic stress (osmoregulation). BetP is comprised of 12 transmembrane segments and carries N- and C-terminal domains, which are involved in osmosensing and/or osmoregulation. Recent results on molecular properties of these domains indicate the significance of particular amino acids within the terminal 25 amino acids of the C-terminal domain of BetP for the process of osmosensing and osmoregulation. 相似文献
16.
Mitogen activated protein kinase (MAPK) signal transduction pathways are ubiquitous among eukaryotic organisms with evolutionary conserved modules. Although generally classified as osmotic and cell wall integrity pathways, functional divergences have been observed for HOG1- and SLT2-related MAPK pathways. Here we show that the osmotic signal transduction cascade is involved in cell wall integrity in the phytopathogenic ascomycete Botrytis cinerea. The deletion mutants of the upstream histidine kinase Bos1 and of the MAPK Sak1 showed modified tolerance to cell wall degrading enzymes and cell wall interfering agents, as well as increased staining of β1-3 glucan and chitin compared to the wild-type. The Sak1 MAPK was phosphorylated upon cell wall challenging. Sak1 interfered with the phosphorylation status of the SLT2 type MAPK Bmp3 hinting to cross talk between both MAPK pathways. All signal transduction components interfered with the expression of melanin biosynthesis genes in dark and bright, suggesting a coordinated control of melanin biosynthesis. 相似文献
17.
Genetic and functional characterization of the Escherichia coli BarA-UvrY two-component system: point mutations in the HAMP linker of the BarA sensor give a dominant-negative phenotype
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Tomenius H Pernestig AK Méndez-Catalá CF Georgellis D Normark S Melefors O 《Journal of bacteriology》2005,187(21):7317-7324
The BarA-UvrY two-component system family is strongly associated with virulence but is poorly understood at the molecular level. During our attempts to complement a barA deletion mutant, we consistently generated various mutated BarA proteins. We reasoned that characterization of the mutants would help us to better understand the signal transduction mechanism in tripartite sensors. This was aided by the demonstrated ability to activate the UvrY regulator with acetyl phosphate independently of the BarA sensor. Many of the mutated BarA proteins had poor complementation activity but could counteract the activity of the wild-type sensor in a dominant-negative fashion. These proteins carried point mutations in or near the recently identified HAMP linker, previously implicated in signal transduction between the periplasm and cytoplasm. This created sensor proteins with an impaired kinase activity and a net dephosphorylating activity. Using further site-directed mutagenesis of a HAMP linker-mutated protein, we could demonstrate that the phosphoaccepting aspartate 718 and histidine 861 are crucial for the dephosphorylating activity. Additional analysis of the HAMP linker-mutated BarA sensors demonstrated that a dephosphorylating activity can operate via phosphotransfer within a tripartite sensor dimer in vivo. This also means that a tripartite sensor can be arranged as a dimer even in the dephosphorylating mode. 相似文献
18.
A gene for a putative two-component histidine kinase, which is homologous to os-1 from Neurospora crassa, was cloned and sequenced from the plant-pathogenic fungus Cochliobolus heterostrophus. The predicted protein possessed the conserved histidine kinase domain, the response regulator domain, and six tandem repeats of 92-amino-acids at the N-terminal end that are found in histidine kinases from other filamentous fungi. Introduction of the histidine kinase gene complemented the deficiency of the C. heterostrophus dic1 mutant, suggesting that the Dic1 gene product is a histidine kinase. Dic1 mutants are resistant to dicarboximide and phenylpyrrole fungicides, and they are sensitive to osmotic stress. We previously classified dic1 alleles into three types, based on their phenotypes. To explain the phenotypic differences among the dic1 mutant alleles, we cloned and sequenced the mutant dic1 genes and compared their sequences with that of the wild-type strain. Null mutants for Dic1, and mutants with a deletion or point mutation in the N-terminal repeat region, were highly sensitive to osmotic stress and highly resistant to both fungicides. A single amino acid change within the kinase domain or the regulator domain altered the sensitivity to osmotic stress and conferred moderate resistance to the fungicides. These results suggest that this predicted protein, especially its repeat region, has an important function in osmotic adaptation and fungicide resistance.Communicated by C. A. M. J. J. van den Hondel 相似文献
19.
Chen Wang Jiayan Sang Jiawei Wang Mingyan Su Jennifer S. Downey Qinggan Wu Shida Wang Yongfei Cai Xiaozheng Xu Jun Wu Dilani B. Senadheera Dennis G. Cvitkovitch Lin Chen Steven D. Goodman Aidong Han 《PLoS biology》2013,11(2)
Two-component systems (TCSs) are important for the adaptation and survival of bacteria and fungi under stress conditions. A TCS is often composed of a membrane-bound sensor histidine kinase (SK) and a response regulator (RR), which are relayed through sequential phosphorylation steps. However, the mechanism for how an SK is switched on in response to environmental stimuli remains obscure. Here, we report the crystal structure of a complete cytoplasmic portion of an SK, VicK from Streptococcus mutans. The overall structure of VicK is a long-rod dimer that anchors four connected domains: HAMP, Per-ARNT-SIM (PAS), DHp, and catalytic and ATP binding domain (CA). The HAMP, a signal transducer, and the PAS domain, major sensor, adopt canonical folds with dyad symmetry. In contrast, the dimer of the DHp and CA domains is asymmetric because of different helical bends in the DHp domain and spatial positions of the CA domains. Moreover, a conserved proline, which is adjacent to the phosphoryl acceptor histidine, contributes to helical bending, which is essential for the autokinase and phosphatase activities. Together, the elegant architecture of VicK with a signal transducer and sensor domain suggests a model where DHp helical bending and a CA swing movement are likely coordinated for autokinase activation. 相似文献
20.
Hagiwara D Matsubayashi Y Marui J Furukawa K Yamashino T Kanamaru K Kato M Abe K Kobayashi T Mizuno T 《Bioscience, biotechnology, and biochemistry》2007,71(3):844-847
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. 相似文献