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The in vivo conformation and replication intermediates of fungal circular mitochondrial plasmids and plasmid-like mitochondrial element (plMEs) were analyzed by two-dimensional gel electrophoresis and electron microscopy. Plasmids with circular restriction maps exist predominantly as circular molecules and were found to replicate by rolling circle mechanisms. However, the reverse transcriptase-encoding Mauriceville plasmid of Neurospora crassa was observed to replicate by two possible mechanisms: one that is consistent with a reverse transcriptase-mediated process and a second one might involve rolling circle DNA replication. Like the mtDNA-derived plasmid-like elements of N. crassa (Hausner et al. 2006a, b), a plasmid-like element of Cryphonectria parasitica (plME-C9), which consists predominantly of a 1.4 kb nucleotide sequence different from mitochondrial DNA, also was found to replicate by a rolling circle mechanism. Although the techniques used in this study were not suited for the establishment of the in vivo conformation and mode of replication of the mtDNAs of Neurospora or Cryphonectria, we surmise that the rolling circle mechanism might be the predominant mode of DNA replication in fungal mitochondria., (Copyright © 2012 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

In the chestnut blight fungus Cryphonectria parasitica, cytoplasmically transmissible hypovirulence phenotypes are elicited by debilitating mitochondrial DNA (mtDNA) mutations. In virus-free hypovirulent strains of C. parasitica from nature, the presence of a mitochondrial DNA element, named InC9, has been reported to cause similar disease syndromes. We have detected an additional mitochondrial element, termed plME-C9 (plasmid-like mitochondrial element C9) in some of the strains rendered hypovirulent by InC9. This element is a 1.4-kb DNA that exists in the mitochondria as monomeric and multimeric circular forms. Only a short 127-bp sequence of the plME-C9 DNA is derived from a region of the C. parasitica mtDNA that contains a reverse transcriptase-like open reading frame. The accumulation of the plME-C9 DNA in the mitochondria appears to adversely affect the growth of the fungus on synthetic medium. However, the presence plME-C9 in different strains did not correlate with the manifestation of the hypovirulence phenotype, indicating that it is not the primary reason for the prevalence of attenuated C. parasitica strains in the Kellogg Forest in Michigan., (Copyright © 2011 Elsevier Inc. All rights reserved.)

In the chestnut-blight fungus Cryphonectria parasitica, cytoplasmically transmissible hypovirulence phenotypes frequently are elicited by double-stranded RNA (dsRNA) virus infections. However, some strains manifest cytoplasmically transmissible hypovirulence traits without containing any mycovirus. In this study, we describe an altered form of mtDNA that is associated with hypovirulence and senescence in a virus-free strain of C. parasitica, KFC9, which was obtained from nature and has an elevated level of cyanide-resistant respiration. In this strain, a 971-bp DNA element, named InC9, has been inserted into the first exon of the mitochondrial small-subunit ribosomal RNA (rns) gene. Sequence analysis indicates that InC9 is a type A1 group II intron that lacks a maturase-encoding ORF. RT-PCR analyses showed that the InC9 sequence is spliced inefficiently from the rRNA precursor. The KFC9 strain had very low amounts of mitochondrial ribosomes relative to virulent strains, thus most likely is deficient in mitochondrial protein synthesis and lacks at least some of the components of the cyanide-sensitive, cytochrome-mediated respiratory pathway. The attenuated-virulence trait and the splicing-defective intron are transferred asexually and concordantly by hyphal contact from hypovirulent donor strains to virulent recipients, confirming that InC9 causes hypovirulence., (Copyright © 2011 Elsevier Inc. All rights reserved.)

HC-toxin, a cyclic peptide made by the filamentous fungus Cochliobolus carbonum, is an inhibitor of histone deacetylase (HDAC) from many organisms. It was shown earlier that the HDAC activity in crude extracts of C. carbonum is relatively insensitive to HC-toxin as well as to the chemically unrelated HDAC inhibitors trichostatin and D85, whereas the HDAC activity of Aspergillus nidulans is sensitive (G. Brosch et al., Biochemistry 40:12855-12863, 2001). Here we report that HC-toxin-resistant HDAC activity was present in other, but not all, plant-pathogenic Cochliobolus species but not in any of the saprophytic species tested. The HDAC activities of the fungi Alternaria brassicicola and Diheterospora chlamydosporia, which also make HDAC inhibitors, were resistant. The HDAC activities of all C. carbonum isolates tested, except one non-toxin-producing isolate, were resistant. In a cross between a sensitive isolate and a resistant isolate, resistance genetically cosegregated with HC-toxin production. When fractionated by anion-exchange chromatography, extracts of resistant and sensitive isolates and species had two peaks of HDAC activity, one that was fully HC-toxin resistant and a second that was larger and sensitive. The first peak was consistently smaller in extracts of sensitive fungi than in resistant fungi, but the difference appeared to be insufficiently large to explain the differential sensitivities of the crude extracts. Differences in mRNA expression levels of the four known HDAC genes of C. carbonum did not account for the observed differences in HDAC activity profiles. When mixed together, resistant extracts protected extracts of sensitive C. carbonum but did not protect other sensitive Cochlibolus species or Neurospora crassa. Production of this extrinsic protection factor was dependent on TOXE, the transcription factor that regulates the HC-toxin biosynthetic genes. The results suggest that C. carbonum has multiple mechanisms of self-protection against HC-toxin.

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.

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.

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.

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.