A functional analysis of mitochondrial respiratory chain cytochrome bc1 complex in Gaeumannomyces tritici by RNA silencing as a possible target of carabrone

Gaeumannomyces tritici, an ascomycete soilborne fungus, causes a devastating root disease in wheat. Carabrone, a botanical bicyclic sesquiterpenic lactone, is a promising fungicidal agent that can effectively control G. tritici. However, the mechanism of action of carabrone against G. tritici remains largely unclear. Here, we used immunogold for subcellular localization of carabrone and the results showed that carabrone is subcellularly localized in the mitochondria of G. tritici. We then explored the functional analysis of genes GtCytc₁, GtCytb, and GtIsp of the mitochondrial respiratory chain cytochrome bc₁ complex in G. tritici by RNA silencing as a possible target of carabrone. The results showed that the silenced mutant ∆GtIsp is less sensitive to carabrone compared to ∆GtCytc₁ and ∆GtCytb. Compared with the control, the activities of complex III in all the strains, except ∆GtIsp and carabrone-resistant isolate 24-HN-1, were significantly decreased following treatment with carabrone at EC₂₀ and EC₈₀ in vitro (40%–50% and 70%–80%, respectively). The activities of mitochondrial respiratory chain complex III and the mitochondrial respiration oxygen consumption rates in all the strains, except ∆GtIsp and 24-HN-1, were higher with respect to the control when treated with carabrone at EC₂₀ in vivo. The rates of mitochondrial respiration of all strains, except ∆GtIsp, were significantly inhibited following treatment with carabrone at EC₈₀ (ranging from 57% to 81%). This study reveals that the targeting of the iron–sulphur protein encoded by GtIsp is highly sensitive to carabrone and provides a direction for the research of carabrone's target.