Meunier, B. | bioRxiv Science

bioRxiv2021

Study of Metyltetraprole, an unusual agrofungicide targeting the Qo-site of cytochrome bc1 complex

The mitochondrial respiratory chain bc1 complex is a proven target of agrofungicides. Most of them are Qo-site antagonists (i.e QoIs), competing with the substrate ubiquinol, and likely share the same binding mode as the widespread Qo-site resistance mutation G143A confers cross-resistance. Metyltetraprole (MTP) presents an exception as studies with phytopathogenic fungi showed that the inhibitor was unaffected by G143A. Here, we used the yeast model to investigate its mode of action. Analysis of bc1 complex mutants supports a Qo-site binding for MTP. However the compound seems distinct to other QoIs, such as azoxystrobin, in various ways, namely; 1) G143A was without effect on MTP, as previously reported. 2) The level of MTP resistance of mutants was higher in bc1 complex activity assays than in growth assays while the opposite was observed with azoxystrobin. 3) Steady-state kinetics used to characterise the mode of action of MTP also revealed differences compared to other QoIs.

biochemistry↗

bioRxiv2021

Directed evolution predicts cytochrome b G37V target site modification as probable adaptive mechanism towards the QiI fungicide fenpicoxamid in Zymoseptoria tritici.

Acquired resistance is a threat for antifungal efficacy in medicine and agriculture. The diversity of possible resistance mechanisms, as well as the highly adaptive traits of pathogens make it difficult to predict evolutionary outcomes of treatments. We used directed evolution as an approach to assess the risk of resistance to the new fungicide fenpicoxamid in the wheat pathogenic fungus Zymoseptoria tritici. Fenpicoxamid inhibits complexIII of the respiratory chain at the ubiquinone reduction site (Qi site) of the mitochondrially encoded cytochrome b, a different site than the widely-used strobilurins which the respiratory complex by binding to the ubiquinol oxidation site (Qo site). We identified the G37V change, within the cytochrome b Qi site, as the most likely resistance mechanism to be selected in Z. tritici. This change triggered high fenpicoxamid resistance and halved the enzymatic activity of cytochrome b, despite no significant penalty for in vitro growth. In addition, we identified a negative cross-resistance between isolates harboring G37V or G143A, a Qo site change previously selected by strobilurins. Moreover, double mutants were less resistant to both QiIs and QoIs compared to single mutants. This work is a proof of concept that experimental evolution can be used to predict adaptation to fungicides, and provides new perspectives for the management of QiIs. Originality-Significance StatementO_LIThe highly adaptive traits of pathogens render evolutionary outcomes of antifungal treatments difficult to predict. C_LIO_LIWe used directed evolution to assess the risk of resistance to the new fungicide fenpicoxamid in the wheat pathogenic fungus Zymoseptoria tritici. C_LIO_LIWe identified a target modification as the most likely resistance mechanism to be selected. C_LIO_LIThis change triggered high fenpicoxamid resistance and halved the activity of the target enzyme despite no significant penalty for in vitro growth. C_LIO_LIThis work supports the use of experimental evolution as a method to predict adaptation to fungicides and provides important information for the management of QiIs. C_LI

microbiology↗