Osmotic-sensitive (os-1) mutant alleles in Neurospora crassa exhibit resistance to dicarboximides, aromatic hydrocarbons and phenylpyrroles. We have previously reported that the os-1 mutants can be classified into two groups based on their resistance to fungicides and osmotic stress: type I, which are highly resistant to iprodione and fludioxonil but moderately sensitive to osmotic stress, and type II, which are highly sensitive to osmotic stress but moderately resistant to fungicides. To explain the mechanism of resistance to these fungicides, we cloned and sequenced the mutant os-1 genes that encode putative osmo-sensing histidine kinase. Within the os-1 gene product (Os1p), the type I strains, NM233t and Y256M209, carried a stop codon at amino acid position 308 and a frameshift at amino acid position 294, respectively. These mutation sites were located on the upstream of histidine kinase and the response regulator domains of Os1p, strongly suggesting that type I strains are null mutants. The null mutants, NM233t and Y256M209, were highly resistant to iprodione and fludioxonil; thus Os1p is essential for these fungicides to express their antifungal activity. The amino acid changes in Os1p, 625Pro from Leu, 578Val from Ala, and 580Arg from Gly were found in the type II strains, M16, M155-1 and P5990, respectively. Os1p is novel in having six tandem repeats of 90 amino acids in the N terminal. Each amino acid change of the type II strains was located on the fifth unit of six tandem repeats. Type II strains with single amino acid changes were more sensitive to osmotic stress than the null mutants (type I), indicating that the amino acid repeats of Os1p were responsible for an important function in osmo-regulation.

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Neurospora osmotic sensitive strains with os-1, os-2, os-4 and os-S mutations showed cross-resistance to dicarboximides, aromatic hydrocarbons and fludioxonil. The os-2, os-4, and os-5 mutant strains were highly resistant to them, while the os-1 mutant strain was moderately resistant and its growth was inhibited by fludioxonil (LD50: 0.087 ,ug/ml) and iprodione (LD50:14 ug/ml). Another osmotic sensitive mutation cut did not give resistance to these fungicides. The conidia of wild-type and cut strains swelled and burst without germination on the medium containing fludioxonil, and bursting of conidia was rescued by high osmotic pressure. Stimulation of glycerol biosynthesis by fludioxonil and iprodione observed in the wild-type strain was not induced in os-2, os-4 and os-5 mutant strains. The moderately resistant os-1 mutant strain accumulated substantial quantities of glycerol by 10 pg/ml of fludioxonil, but produced less glycerol by 10ug/ml of iprodione. Thus it was assumed that fungal toxicity of these fungicides might be due to abnormal glycerol accumulation.However, both fludioxonil and iprodione did not induce glycerol biosynthesis in the fungicide-sensitive cut mutant strain. In response to osmotic stress, as mutant strains accumulated less amount of glycerol than the wild-type strain, whereas cut mutant strain did not produce glycerol in the medium containing 4% NaCI. Despite its lack of ability of glycerol biosynthesis in cut mutant strain, wild-type and cut strains were not distinguishable in their modes of fungal toxicity of fludioxonil.These data suggest that dicarboximides, aromatic hydrocarbons and fludioxonij interfere with the osmotic signal transduction pathway resulting in stimulation of glycerol biosynthesis, but abnormal glycerol accumulation is not essential for their fungal toxicity.

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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.

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Fusarium graminearum was engineered for expression of enhanced green fluorescent protein gene (egfp) as a reporter regulated in a manner similar to Tri5, a key pathway gene in trichothecene biosynthesis. Using the transgenic fungus, it was found that the reporter gene was induced to express in aerial hyphae developed on trichothecene noninducing medium YG solidified by agar. Unexpectedly, the transcriptional activation of egfp was markedly suppressed by adding NaCl that does not significantly affect fungal growth. As suggested by these findings, wild-type F. graminearum that formed aerial hyphae on YG agar plates produced trichothecenes and the production was effectively suppressed by adding 1% NaCl to the agar. To evaluate the effects of abiotic stress on the expression of trichothecene biosynthesis (Tri) genes, a sensitive plate assay was established using GYEP medium (which very weakly induces trichothecene production) solidified with gellan gum. Using this assay, triazole fungicides were shown to cause transcriptional activation of egfp at sublethal concentrations. Indeed, trichothecene production significantly increased when F. graminearum was grown in rice medium (which moderately induces trichothecene) amended with low doses of tebuconazole. The real-time monitoring system described here may help predict the risks of trichothecene contamination by the fungus under various environmental conditions.