In vivo conformation and replication intermediates of circular mitochondrial plasmids in Neurospora and Cryphonectria parasitica.

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.

A 971-bp insertion in the rns gene is associated with mitochondrial hypovirulence in a strain of Cryphonectria parasitica isolated from nature.

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.

Mitochondrial plasmid-like elements in some hypovirulent strains of Cryphonectria parasitica.

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.

Characterization of inhibitor-resistant histone deacetylase activity in plant-pathogenic fungi.

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.