Development of solubilization and purification protocols of the membrane associated alternative oxidase (AOX) from the fungus Moniliophthora perniciosa, the causal agent of witches' broom disease of cocoa

• Grant number: 11/21867-7
• Support Opportunities: Scholarships abroad - Research Internship - Scientific Initiation
• Start date: February 01, 2012
• End date: March 31, 2012
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Gonçalo Amarante Guimarães Pereira
• Grantee: Paula Favoretti Vital Do Prado
• Supervisor: Isabel Moraes
• Host Institution: Instituto de Biologia (IB). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
• Institution abroad: Diamond Light Source, England
• Associated to the scholarship: 11/07928-3 - Structural characterization of the mitochondrial protein alternative oxidase of the fungus Moniliophthora perniciosa , the causal agent of witches' broom disease of cocoa, BP.IC

Abstract

Witches' Broom disease (WBD) of cacao (Theobroma cacao), the raw material for chocolate production, is well-regarded as one of the most important phytopathological problems to afflict the southern hemisphere in recent decades, with devastating consequences to the agro-economy of the affected countries. In Brazil, the WBD started as endemic within the Amazon region, but after an outbreak in 1989, it was introduced into the largest area of cacao production, the state of Bahia. A severe decrease in the production of cacao then followed and, in less than a decade, Brazil shifted from being the second largest cacao exporter to being acacao importer. Genome and transcriptome studies of the fungus, conducted by Dr. Gonçalo’s laboratory during the last 10 years, have provided a better understanding of the fungus-pathogen interaction mechanism. In this context, it was found that the alternative oxidase enzyme (AOX) plays an important role on the metabolism and lifecycle of the pathogen. AOX is a protein attached to the inner mitochondrial membrane that receives electrons directly from reduced ubiquinone and catalyzes the reduction of oxygen to water. AOX is a non-proton motive terminal quinol oxidase that enables cell respiration to continue even in the presence of inhibitors targeting the complexes of the respiratory chain and is the main source of drug-resistance related to respiratory chain inhibition. Our main goal in this project is to express and define a solubilization and purification protocol of the AOX protein of M. perniciosa, aiming at posterior structural studies of this enzyme. In order to accomplish that, we have established a collaboration with Dr. Isabel de Moraes’s group at the world-renowned Membrane Protein Laboratory (MPL), located at the Diamond Light Source in England, due to their vast experience on dealing with membrane proteins. On a long term, the knowledge of AOX-crystallographic structure holds the potential to assist on the development of new fungicide compounds. (AU)