Molecular characterization of strobilurin resistance mechanisms in Moniliophthora perniciosa, the causal agent of the devastating witche's broom disease of cacao

Author(s)

Paula Favoretti Vital do Prado

• Document type: Master's Dissertation
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2016-02-23

Examining board members

Gonçalo Amarante Guimarães Pereira; Celso Eduardo Benedetti; Antonio Vargas de Oliveira Figueira

• Advisor: Daniela Paula de Toledo Thomazella; Gonçalo Amarante Guimarães Pereira

Abstract

Theobroma cacao, the plant that provides the raw material for chocolate production, is one of the most important perennial crops in the world. However, the chocolate tree is seriously affected by severe fungal diseases. The witches' broom disease (WBD) is well regarded as one of the major phytopathological problems that afflict cacao crops in Americas, with devastating consequences to the agro-economy of the affected countries. In Brazil, the damages caused by WBD have stimulated initiatives to control this phytopathogen, which included the application of strobilurin fungicides. Strobilurins are one of the most used classes of fungicides in the global agriculture, these molecules inhibit mitochondrial respiration, compromising ATP generation and leading to oxidative stress. This strategy, however, was not effective against this pathogen. Until now, the molecular mechanisms underpinning the M. perniciosa tolerance to the strobilurins have been little explored. In this context, the present work used the RNA-seq technique to identify differentially expressed genes that might play a role in drug resistance in vitro. In response to Azoxystrobin (a commercial strobilurin) treatment, M. perniciosa transcriptome reveals the induction of many enzymes of the gluconeogenesis pathway, glyoxylate cycle, fatty acid catabolism and amino acid degradation, suggesting an intense metabolism remodeling that probably compensates ATP depletion and allows M. perniciosa survival. On the other hand, enzymes related to cell cycle (mitosis), ribossome biogenesis and sterol metabolism are repressed, which is in agreement with the compromised mycelial growth rate. In addition, the fungus expresses a wide range of genes that mitigates the toxic effects of the inhibition of the main mitochondrial respiratory chain, comprising genes related to oxidative stress protection (e.g. glutathione s-transferases and fungal peroxidases), stress resistance (e.g. heat shock proteins) and detoxification (e.g. cythocromes P450). This RNA-seq analysis also revealed members from different families of transporters, including ABC transporters and MFS (major facilitator superfamily) transporters, classically related to multidrug resistance events. Also, a set of "No Hit" proteins, which have extremely low similarity to any other known protein outside the Moniliophthora genus, were highly upregulated by the fungicide. In parallel experiments, a natural mutant of M. perniciosa with increased strobilurin tolerance phenotype was recently identified in our laboratory. This mutant represents an important tool for elucidating additional mechanisms that leads to resistance and its initial characterization was conducted, revealing extensive transcriptomic changes. This work provides the first steps towards the understanding of the molecular basis of strobilurin resistance in this pathogen, which is a fundamental approach to achieve a higher efficiency in the use of this important class of drugs. At long term, we aim to determine a set of potential molecular targets for inhibition, providing valuable information for the development of effective anti-fungal molecules with the potential to be used as a strategy to control witches' broom disease and possibly other destructive fungal diseases around the world (AU)

• FAPESP's process: 13/05979-5 - Molecular characterization of mechanisms of strobilurin resistance in Moniliophthora perniciosa, the causal agent of witches' broom disease of cacao
• Grantee: Paula Favoretti Vital Do Prado
• Support Opportunities: Scholarships in Brazil - Master