No abstract

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.

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.

No abstract

A metallo-oxidase gene from a phytopathogenic filamentous fungus, Cochliobolus heterostrophus was cloned. Structural prediction of ChMco1 indicated that this protein lacks a transmembrane helix and is soluble, whereas other known fungal metallo-oxidases including Saccharomyces cerevisiae FET3 are localized to the cell membrane. The results of searches in fungal genomic databases and phylogenetic analysis of fungal metallooxidases revealed that ChMco1 and its allies are distinct homologues of Fet3 and unique to filamentous ascomycetous species including C. heterostrophus. We performed a functional analysis of ChMCO1 by generating null mutants for the ChMco1 gene. The ChMco1 null (DChMco1) mutants clearly had reduced melanization, although they showed normal growth and conidiation. Results also show that DChMco1 mutants lost laccase activity. These results suggest that ChMCO1 is a novel class of metallo-oxidase that is necessary for laccase activity and melanization.

No abstract

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 DBcHpb1 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 DBcHpb1 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.

A melanin biosynthetic gene, Sal1, in the southern corn leaf blight fungus Cochliobolus heterostrophus, was identified by degenerate and inverse PCR. Structural analysis revealed that the open reading frame is 555 bp in length, interrupted by one intron. Melanin deficiency of the sal1 mutant strain, which accumulates the intermediate scytalone, was successfully complemented by introducing the gene fragment. These results suggest that Sal1 is a scytalone dehydratase gene of C. heterostrophus.