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.