Kosuke Izumitsu

The MAPKK kinase ChSte11 regulates sexual/asexual development, melanization, pathogenicity, and adaptation to oxidative stress in Cochliobolus heterostrophus

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. ΔChste11 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 ΔChste11 and Δchk1 strains showed severe sensitivity to oxidative stress, hydrogen peroxide, and heavy metals, cupric or ferric cations. ΔBmhog1 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.

A copper-transporting ATPase BcCCC2 is necessary for pathogenicity of Botrytis cinerea.

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 ∆BcCcc2 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 ∆BcCcc2 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.

Phylogenetic analysis of heavy-metal ATPases in fungi and characterization of the copper-transporting ATPase of Cochliobolus heterostrophus

We performed a phylogenetic analysis of heavy-metal ATPases (HMAs) in fungi and found that HMAs can be divided into three groups, A, B, and C. Group A is predicted to deliver copper ions to copper-containing proteins, while Groups B and C are thought to function as cell-membrane copper-efflux pumps. Furthermore, Groups B and C consist of fungal-specific HMAs, while Group A consists of fungal orthologues that have been well conserved in eukaryotes. We also cloned and characterized a Group A-type HMA gene (i.e., ChCcc2) of the filamentous plant pathogen, Cochliobolus heterostrophus. Mutation of ChCcc2 severely affected growth, pigmentation, conidiation, and colonial morphology. Activity of the copper-containing protein, laccase, was also lost in ChCcc2 mutants, suggesting that ChCCC2 plays an important role in growth and morphology by activating various copper-containing proteins in C. heterostrophus.

Dic2 and Dic3 loci confer osmotic adaptation and fungicidal sensitivity independent of the HOG pathway in Cochliobolus heterostrophus

Previously, we identified three gene loci, Dic1, Dic2, and Dic3, that confer high-osmolarity adaptation and dicarboximide/phenylpyrrole fungicide sensitivity in Cochliobolus heterostrophus. Dic1 encoded a group III histidine kinase, but the other genes were not characterized. In the present study, we revealed that both Dic2 and Dic3 are involved in the Skn7 pathway. Dic2 encoded an Skn7-type response regulator, ChSkn7. Strain N4502 contained D359N in the response regulator domain of ChSkn7. Strain E4503 contained a deletion of 50 amino acids in the DNA-binding domain. Strain N4507 was a null mutant of the ChSkn7 gene. All of the dic2 mutant strains showed similar levels of sensitivity to high osmolarity and similar levels of resistance to fungicides. These results strongly suggested that both the DNA-binding domain and response regulator domain are essential for Skn7 function in osmotic adaptation and fungicide sensitivity. A western blot analysis revealed that Dic3 is not involved in the regulation of Hog1-type MAPKs. The Chssk1/dic3 double mutant strains clearly showed greater resistance to fungicides than the single mutant strains. An additive effect was also observed in the high-osmolarity experiments. On the other hand, the dic3/dic2 double mutant strains did not show higher levels of resistance to fungicides and greater sensitivity to KCl than the single mutant strains. These results strongly suggested that the dic3 locus confer high-osmolarity adaptation and fungicide sensitivity independently from Ssk1-Hog1 pathway, but not the Skn7 pathway. Moreover, the dic3 strain and all dic2 strains showed similar levels of sensitivity to high-osmolarity stress and similar levels of resistance to fungicides, suggesting Dic3 to have an essential role in the Skn7 pathway. Our results provide new insight into the functions of the Skn7 pathway in filamentous fungi.

Class I hydrophobin BcHpb1 is important for adhesion but not for later infection of Botrytis cinerea

Hydrophobins are small secreted proteins unique to filamentous fungi. In this study, we cloned and characterized the class I hydrophobin gene BcHpb1 in the necrotrophic pathogen Botrytis cinerea. The BcHpb1 protein consisted of 117 amino acids. Similar to class I hydrophobins from other fungi, BcHpb1 contains eight conserved cysteine residues. The hydropathy plot of the BcHpb1 amino acid sequence was characteristic of a class I hydrophobin. These results indicated that the BcHpb1 gene encodes a class I hydrophobin. Vegetative growth of ΔBcHpb1 strains, null mutants of BcHpb1, was similar to that of the wild-type strain as were the conidiophores, conidia, appressoria and virulence on host plants. However, adherence of ΔBcHpb1 strains to hydrophobic surfaces was greatly reduced, implying that BcHpb1 is important for the hydrophobicity of conidia and that BcHpb1 may be required to adhere to plant surfaces under certain environmental conditions.

Characterization of the autophagy-related gene BmATG8 in Bipolaris maydis

Autophagy is involved in cellular development and the maintenance of viability under nutrient deprivation in a wide range of eukaryotes. A filamentous ascomycete Bipolaris maydis, responsible for southern corn leaf blight, is also studied as a model fungus for sexual reproduction in filamentous ascomycetes that form filiform ascospores. In order to clarify the roles of autophagy in various stages of the life cycle of B. maydis, we constructed null mutants of BmATG8, an orthologue of the Saccharomyces cerevisiae autophagy gene ATG8 in B. maydis. Deletion of BmATG8 impaired localization of cytosolic components to the vacuole under nitrogen starvation, suggesting that autophagy was deficient in the null mutants. Additionally, fluorescent microscopic observations on a eGFP-fused BmATG8 expressing strain showed that BmATG8 is associated with autophagy-related structures. In vegetative growth, ΔBmATG8 strains showed a reduction in conidiation and aerial mycelial growth. Interestingly, the mutant conidia indicated loss of the germination rate under starvation conditions and affected longevity. However, germinated mutant conidia were still capable of infecting the host plant via appressoria. In sexual reproduction, ascospores with ΔBmATG8 genetic background were aborted. Our results revealed that autophagy plays a crucial role in the function of conidia, not in host infection via appressoria in B. maydis. In addition, conservation of the importance of autophagy in ascospore development is suggested among ascomycetes including species that form bitunicate ascus.