Noriyuki Ochiai

Putative Homologs of SSK22 MAPKK Kinase and PBS2 MAPK Kinase of Saccharomyces cerevisiae Encoded by os-4 and os-5 Genes for Osmotic Sensitivity and Fungicide…

We cloned and characterized Neurospora NcSSK22 and NcPBS2 genes, similar to yeast SSK22 mitogen-activated protein (MAP) kinase kinase kinase and the PBS2 MAP kinase kinase genes, respectively. Disruptants of the NcSSK22 gene were sensitive to osmotic stress and resistant to iprodione and fludioxonil. Their phenotypes were similar to those of osmotic-sensitive (os) mutants os-1, os-2, os-4, and os-5. The os-4 mutant strain transformed with the wild-type NcSSK22 gene grew on a medium containing 4% NaCl and was sensitive to iprodione and fludioxonil. In contrast, the NcPBS2 gene complemented the osmotic sensitivity and fungicide resistance of the os-5 mutant strain. We sequenced the NcPBS2 gene of the os-5 mutant strain (NM216o) and found five nucleotides deleted within the kinase domain. This result suggests that the gene products of os-4 and os-5 are components of the MAP kinase cascade, which is probably regulated upstream by two-component histidine kinase encoded by the os-1/nik1 gene.

Effects of Iprodione and Fludioxonil on Glycerol Synthesis and Hyphal Development in Candida albicans

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 μg/ml. Both fungicides stimulated glycerol synthesis in C. albicans but not in S. cerevisiae. The antioxidant α-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.

Reduced Contamination by the Fusarium Mycotoxin Zearalenone in Maize Kernels through Genetic Modification with a Detoxification Gene

ABSTRACT Maize is subject to ear rot caused by toxigenic Aspergillus and Fusarium species, resulting in contamination with aflatoxins, fumonisins, trichothecenes, and zearalenone (ZEN). The trichothecene group and ZEN mycotoxins are produced by the cereal pathogen Fusarium graminearum. A transgenic detoxification system for the elimination of ZEN was previously developed using an egfp::zhd101 gene (gfzhd101), encoding an enhanced green fluorescent protein fused to a ZEN-degrading enzyme. In this study, we produced a transgenic maize line expressing an intact copy of gfzhd101 and examined the feasibility of transgene-mediated detoxification in the kernels. ZEN-degrading activity has been detected in transgenic kernels during seed maturation (for a period of 6 weeks after pollination). The level of detoxification activity was unaltered after an additional storage period of 16 weeks at 6°C. When the seeds were artificially contaminated by immersion in a ZEN solution for 48 h at 28°C, the total amount of the mycotoxin in the transgenic seeds was uniformly reduced to less than 1/10 of that in the wild type. The ZEN in the transgenic maize kernels was also efficiently decontaminated under conditions of lower water activity (aw) and temperature; e.g., 16.9 μg of ZEN was removed per gram of seed within 48 h at an aw of 0.90 at 20°C. F. graminearum infection assays demonstrated an absence of ZEN in the transgenic maize seeds, while the mycotoxin accumulated in wild-type kernels under the same conditions. Transgene-mediated detoxification may offer simple solutions to the problems of mycotoxin contamination in maize.

A screening system for inhibitors of trichothecene biosynthesis: hydroxylation of trichodiene as a target

Fusarium Tri4 encodes a key cytochrome P450 monooxygenase for hydroxylation of trichodiene early in the biosynthesis of trichothecenes. In this study, we established a system for screening for inhibitors of trichothecene biosynthesis using transgenic Saccharomyces cerevisiae expressing Tri4. For easy evaluation of the TRI4 activity, trichodiene-11-one was used as a substrate and the formation of 2α-hydroxytrichodiene-11-one was monitored by HPLC. Using this system, TRI4 proved to be inhibited by various flavones and furanocoumarins. We also found that a catechin-containing commercial beverage product, Catechin Supplement 300 (CS300), inhibited TRI4 activity, at a concentration which did not significantly affect the growth of the transgenic yeast. At an early stage of culture, both flavone and CS300 exhibited a toxin-inhibitory activity against Fusarium graminearum. However, inhibition of trichothecene production was not observed with longer incubation periods at minimum concentrations necessary to inhibit >50% of the TRI4 activity, presumably due to the metabolism by the fungus. The results suggest that this yeast screening system with TRI4 is useful for the rapid identification of lead compounds for the design of trichothecene biosynthesis inhibitors that are resistant to modification by the fungus.

OS-2 Mitogen Activated Protein Kinase Regulates the Clock-Controlled Gene ccg-1 in Neurospora crassa

OS-2 MAP kinase is involved in osmoadaptation in Neurospora crassa. Clock-controlled genes ccg-1, bli-3, and con-10 were induced by osmotic stress in an OS-2 dependent manner. In contrast, osmotic stress did not affect the expression of clock genes frq, wc-1, and wc-2 or of clock-controlled genes ccg-2 and bli-4. These results suggest that OS-2 participates in the regulation of certain circadian-clock output genes.