Omics integration for the investigation of Moniliophthora perniciosa resistance mechanisms against one alternative oxidase-targeting fungicide

The cacao tree (Theobroma cacao) is originated from the Amazon basin and provides the raw material for the production of several products, especially chocolate. However, the Witche¿s Broom Disease of cocoa (WDB), caused by the basidiomycete fungus "Moniliophthora perniciosa", is the major threat for cocoa production in Latin America. M. perniciosa reached Brazilian farms in the state of Bahia ¿ then the largest cacao producing region ¿ in the late 1980¿s and caused major economic and environmental upheaval. This scenario stimulated studies aimed at better understanding the molecular basis for "M. perniciosa" pathogenesis. Genome and transcriptome analyzes of M. perniciosa identified that the mitochondrial enzyme Alternative Oxidase (MpAOX) is an essential factor for pathogen survival and virulence, especially during the early stages of infection. However, known AOX inhibitors are not suitable for use in the field, making AOX a desirable target for the development of novel fungicides. We have recently identified a new class of AOX inhibitors wich were effective in vitro against M. perniciosa AOX and other relevant fungal phytopathogens in Brazilian agricultural scenario, such as Sclerotinia sclerotiorum and Venturia pirina. However, "M. perniciosa" mycelia were resistant to such inhibitors in clear contrast to M. perniciosa spores which were sensitive to AOX inhibition. Therefore, the present study aimed to uncover "M. perniciosa" mycelial resistance mechanisms by investigating metabolic changes after treatment with the AOX inhibitor N-(3-Bromophenyl)-3-fluorobenzamide (3FH). The identification of genes and metabolic pathways involved in fungicide resistance, as well as the identification of any chemical changes suffered by the inhibitor were achieved by RNA-seq, NMR and LC/MS techniques. This information will be relevant for the development of novel crop protection agents which are effective throughout the entire life cycle of "M. perniciosa". At the same time, experiments were carried out to genetically manipulate the model yeast Pichia pastoris for the introduction of MpAOX, aiming at the development of a robust experimental model for the screening and characterization of AOX inhibitors (AU)