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 highosmolarity 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 twocomponent signaling system that is different from that of yeast and is responsible for high-osmolarity adaptation and fungicide sensitivity.

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A PCR‐based technique for detection of clavicipitaceous pathogens in rice and related grasses was developed. The target pathogens were Ustilaginoidea virens, which causes rice false smut, and Ephelis japonica, which causes rice udbatta disease and black choke in grasses. To design specific primers, a comparison was made on genetic diversity on the rDNA internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene of U. virens, Ephelis japonica, as well as some other clavicipitaceous fungi. Each fungus was successfully detected by using a specific primer set with high sensitivity. Species‐specific primers designed here were capable of detecting these pathogens in plant tissues. The PCR detection was consistent with conventional histological observation. This nested PCR assay was sensitive and reliable for the detection of U. virens and E. japonica, and thus can be a used to study disease cycles and early prediction of false smut and udbatta‐disease incidence in fields.

All fungi use multiple mitogen-activated protein kinase (MAPK) cascades to respond to external signals to regulate specialized responses. In this study, we cloned and characterized a putative MAPKKK gene ChSte11, orthologous to yeast STE11, of Cochliobolus heterostrophus. DeltaChste11 strains showed defects in conidiation, sexual development, melanization and the formation of appressoria. These mutants were significantly less virulent on corn plants than the wild type. Similar phenotypes were observed in mutants of Chk1-MAPK, a putative downstream protein kinase of ChSte11. These results suggested that ChSte11 regulates various morphological changes and pathogenicity via Chk1 MAPK. Both DeltaChste11 and Deltachk1 strains showed severe sensitivity to oxidative stress, hydrogen peroxide, and heavy metals, cupric or ferric cations. DeltaBmhog1 strains, mutants of the HOG1-type MAPK, did not show sensitivity to these forms of stress. Our results strongly suggested that the Ste11-type MAPKKK regulates not only various morphological changes and pathogenicity, but also adaptations to stress via Chk1-type MAPK in filamentous fungi.

Aspergillus luchuensismut.kawachii(A. kawachii) produces a large amount of citric acid during the process of fermenting shochu, a traditional Japanese distilled spirit. In this study, we characterizedA. kawachiiCtpA and YhmA, which are homologous to the yeastSaccharomyces cerevisiaemitochondrial citrate transporters Ctp1 and Yhm2, respectively. CtpA and YhmA were purified fromA. kawachiiand reconstituted into liposomes. The proteoliposomes exhibited only counterexchange transport activity; CtpA transported citrate using countersubstrates, especiallycis-aconitate and malate, whereas YhmA transported citrate using a wider variety of countersubstrates, including citrate, 2-oxoglutarate, malate,cis-aconitate, and succinate. Disruption ofctpAandyhmAcaused deficient hyphal growth and conidium formation with reduced mycelial weight-normalized citrate production. Because we could not obtain a ΔctpAΔyhmAstrain, we constructed an S-taggedctpA(ctpA-S) conditional expression strain in the ΔyhmAbackground using the Tet-On promoter system. Knockdown ofctpA-Sin ΔyhmAresulted in a severe growth defect on minimal medium with significantly reduced acetyl coenzyme A (acetyl-CoA) and lysine levels, indicating that double disruption ofctpAandyhmAleads to synthetic lethality; however, we subsequently found that the severe growth defect was relieved by addition of acetate or lysine, which could remedy the acetyl-CoA level. Our results indicate that CtpA and YhmA are mitochondrial citrate transporters involved in citric acid production and that transport of citrate from mitochondria to the cytosol plays an important role in acetyl-CoA biogenesis inA. kawachii.IMPORTANCECitrate transport is believed to play a significant role in citrate production by filamentous fungi; however, details of the process remain unclear. This study characterized two citrate transporters fromAspergillus luchuensismut.kawachii. Biochemical and gene disruption analyses showed that CtpA and YhmA are mitochondrial citrate transporters required for normal hyphal growth, conidium formation, cytosolic acetyl-CoA synthesis, and citric acid production. The characteristics of fungal citrate transporters elucidated in this study will help expand our understanding of the citrate production mechanism and facilitate the development and optimization of industrial organic acid fermentation processes.

Copper is an essential trace element that serves as a cofactor for numerous enzymes. In eukaryotes, copper-transporting ATPases deliver copper to various copper-containing proteins in the trans-golgi network. This study identified a copper-transporting ATPase gene BcCcc2 in a fungus pathogenic to plants, Botrytis cinerea. We investigated the biological roles of BcCCC2 by generating null mutants for BcCcc2. Melanization, conidiation and the formation of sclerotia were severely affected in DeltaBcCcc2 mutants. Moreover, a pathogenicity assay using tomato leaves and carnation petals revealed the mutants to be nonpathogenic. Further analysis indicated that they formed fewer appressoria and infection cushions than the wild-type. These structures were aberrant in morphology and in many cases had a significantly reduced ability to penetrate the plant epidermis. An assay also indicated that DeltaBcCcc2 mutants were defective in infection through wounds. BcCCC2 is necessary not only for penetrating a host but also for fungal growth within plant tissues. Our results also imply that B. cinerea requires copper-containing proteins for infection that are inactive in the absence of the copper-transporting ATPase BcCCC2.

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