An inhibitor-resistant histone deacetylase in the plant pathogenic fungus Cochliobolus carbonum.

We have partially purified and characterized histone deacetylases of the plant pathogenic fungus Cochliobolus carbonum. Depending on growth conditions, this fungus produces HC-toxin, a specific histone deacetylase inhibitor. Purified enzymes were analyzed by immunoblotting, by immunoprecipitation, and for toxin sensitivity. The results demonstrate the existence of at least two distinct histone deacetylase activities. A high molecular weight complex (430,000) is sensitive to HC-toxin and trichostatin A and shows immunoreactivity with an antibody against Cochliobolus HDC2, an enzyme homologous to yeast RPD3. The second activity, a 60,000 molecular weight protein, which is resistant even to high concentrations of well-known deacetylase inhibitors, such as HC-toxin and trichostatin A, is not recognized by antibodies against Cochliobolus HDC1 (homologous to yeast HOS2) or HDC2 and represents a different and/or modified histone deacetylase which is enzymatically active in its monomeric form. This enzyme activity is not present in the related filamentous fungus Aspergillus nidulans. Furthermore, in vivo treatment of Cochliobolus mycelia with trichostatin A and analysis of HDACs during the transition from non-toxin-producing to toxin-producing stages support an HC-toxin-dependent enzyme activity profile.

A gene related to yeast HOS2 histone deacetylase affects extracellular depolymerase expression and virulence in a plant pathogenic fungus.

A gene, HDC1, related to the Saccharomyces cerevisiae histone deacetylase (HDAC) gene HOS2, was isolated from the filamentous fungus Cochliobolus carbonum, a pathogen of maize that makes the HDAC inhibitor HC-toxin. Engineered mutants of HDC1 had smaller and less septate conidia and exhibited an approximately 50% reduction in total HDAC activity. Mutants were strongly reduced in virulence as a result of reduced penetration efficiency. Growth of hdc1 mutants in vitro was normal on glucose, slightly decreased on sucrose, and reduced by 30 to 73% on other simple and complex carbohydrates. Extracellular depolymerase activities and expression of the corresponding genes were downregulated in hdc1 mutant strains. Except for altered conidial morphology, the phenotypes of hdc1 mutants were similar to those of C. carbonum strains mutated in ccSNF1 encoding a protein kinase necessary for expression of glucose-repressed genes. These results show that HDC1 has multiple functions in a filamentous fungus and is required for full virulence of C. carbonum on maize.

A circular mitochondrial plasmid incites hypovirulence in some strains of Cryphonectria parasitica.

In the chestnut-blight fungus Cryphonectria parasitica, a plasmid, pCRY1, occurs in the mitochondria of several strains isolated at various locations in the northeastern United States and Canada. The monomer of this plasmid is a 4.2-kb circular double-stranded DNA that has no detectable sequence homology with the 160-kb mitochondrial DNA of Ep155, a standard virulent laboratory strain of C. parasitica. The circular nature and oligomeric characteristics of the plasmid were deduced from the heterogeneous size of plasmid DNA molecules as detected by one- and two-dimensional gel-electrophoresis, the nature and alignment of restriction fragments, and the lack of detectable termini in the nucleotide sequence. The cytoplasmic location of the plasmid was deduced from its co-purification with mitochondria, uniparental (maternal) transmission in sexual crosses, dissociation from the nuclei of the donor strain during its horizontal transfer between vegetatively compatible strains through hyphal anastomoses, and mitochondrial codon usage (UGA = Try). The pCRY1 plasmid contains a long open reading frame that is transcribed and potentially encodes a unique 1214 amino-acid, B-family DNA polymerase similar to those encoded by the LaBelle and Fiji circular mitochondrial plasmids of Neurospora. In this subgroup of proteins, the DTD motif characteristic of B-family DNA polymerases is replaced by TTD. Amino-acid motifs related to those that are characteristic of the 3'-->5' exonuclease domains of B-family DNA polymerases have been located in the amino-terminal portion of the proteins. A comparison of isogenic plasmid-free and plasmid-containing cultures indicates that pCRY1 is an infectious agent that effects a reduction in the pathogenicity of some, but not all, strains of C. parasitica.

Dynamics of asexual transmission of a mitochondrial plasmid in Cryphonectria parasitica.

In the chestnut blight fungus Cryphonectria parasitica, as in most fungi, little is known about the efficiency of the asexual transmission of optional mitochondrial plasmids, vertically through conidia, and horizontally through hyphal anastomoses. In this paper, we show that pCRY1, a circular mitochondrial plasmid, is transmitted vertically with 100%-efficiency through conidia. Moreover, the plasmid is transmitted horizontally through hyphal contact from donor strains to vegetatively compatible and most incompatible strains. An allelic difference between the donor and recipient strain, at only one of the five nuclear incompatibility genes that were tested strongly inhibited, but did not absolutely prevent, the transfer of pCRY1 through hyphal fusions. In contrast, allelic differences in any one or several of the other four heterokaryon-compatibility loci suppressed the transmission of the plasmid only partially or not at all. The plasmid was also transmitted among incompatible strains by protoplast fusion without the concomitant transfer of mitochondrial DNA (mtDNA). A comparison of plasmid-bearing with plasmid-free isogenic strains revealed that pCRY1 significantly diminishes the pathogenic potency of some strains of the fungus, but does not affect the virulence of others. Collectively, the observations indicate that the introduction of deleterious mitochondrial genetic elements into natural populations may be a means for managing fungal pathogens.

Transmissible mitochondrial hypovirulence in a natural population of Cryphonectria parasitica.

A cytoplasmically transmissible hypovirulence syndrome has been identified in virus-free strains of the chestnut blight fungus Cryphonectria parasitica isolated from healing cankers on American chestnut trees in southwestern Michigan. The syndrome is associated with symptoms of fungal senescence, including a progressive decline in the growth potential and abundance of conidia, and elevated levels of respiration through the cyanide-insensitive alternative oxidase pathway. Conidia from senescing mycelia exhibited varying degrees of senescence ranging from normal growth to death soon after germination. Cytoplasmic transmission of hypovirulence between mycelia occurred by hyphal contact and coincided with the transfer of a specific restriction fragment length polymorphism from the mitochondrial DNA (mtDNA) of the donor strains into the mtDNA of virulent recipients. The transmission of the senescence phenotype was observed not only among vegetatively compatible strains but also among incompatible strains. Hypovirulence was present in isolates from the same location with different nuclear genotypes as identified by DNA fingerprinting. This study confirms that mitochondrial hypovirulence can occur spontaneously and spread within a natural population of a phytopathogenic fungus.