The development of effective strategies to control witches' broom disease in cacao (WBD) is clearly required in view of the current status of cocoa production in the Americas. The use of strobilurins, one of the most commonly used fungicides in agriculture, has already been employed, but with no success. The Witches' Broom Genome Project allowed the identification of possible mechanisms of strobilurin resistance in the fungus Moniliophthora perniciosa, the causative agent of WBD. A gene encoding an alternative oxidase enzyme (Mp-aox) was identified, and genetics and biochemical assays indicated that MpAOX is an important mechanism of strobilurin resistance in this fungus as well as a potential target to control the disease. Recently, transcriptomic analysis demonstrated that a gene encoding an efflux transporter (Mpflr1) is among the most highly expressed genes of M. perniciosa in response to strobilurin, suggesting the involvement of Mpflr1 in fungicide resistance. Based on that, the present project aims to perform functional assays with both proteins (MpAOX and Mpflr1), using the yeast Saccharomyces cerevisiae as expression system. In the case of MpAOX, an established target, the expression in yeast will allow the realization of high throughput screenings with the aim of identifying new inhibitory molecules that are stable enough to be used in the field. Remarkably, we recently identified a natural mutant of M. perniciosa, which has a phenotype of increased strobilurin resistance. In this context, the project also proposes an initial characterization of this mutant in order to identify the possible genetic alterations associated with this phenotype. Overall, with this project, we expect to provide the basis for the development of new strategies to control witches' broom disease, which also have the potential of being used in the control of other fungal pathogens that employ similar strategies of fungicide resistance.
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