Functional characterization of a chitinase-like protein of Moniliophthora perniciosa with a potential role in fungal pathogenicity during the development of the witches' broom disease of cacao

Abstract

Cacao (Theobroma cacao) is a perennial crop of remarkable economic importance worldwide, being responsible for the generation of a considerable portion of national currency. The productivity of cacao farming in Brazil was high and provided great benefits to the country until the end of the 80s, when the witches' broom disease, caused by the basidiomycete Moniliophthora perniciosa, was introduced in cacao plantations of the main producing region of Brazil, southern Bahia. As a consequence, the local cocoa agribusiness was destroyed, driving Brazil from an exporter to an importer of cocoa and causing drastic social and environmental problems in the region. Traditional strategies to control the disease proved to be ineffective. Therefore, the understanding of this pathology became essential for the development of more appropriate programs to combat the disease. Important advances in this direction have been obtained with the Witches' Broom Genome Project. Additionally, in 2010, it was launched the Witches' broom Disease Transcriptome Atlas and the first inspection of this database led to the identification of a fungal chitinase gene (Mp-Chi) that is highly and exclusively expressed during M. perniciosa interaction with cacao. Strikingly, the encoded enzyme has an amino acid substitution in its catalytic site that probably leads to the lack of chitinolytic activity. Based on these two observations - the Mp-Chi expression pattern and the potential absence of enzymatic activity - it is plausible that this protein comprehends a protective mechanism of M. perniciosa against chitinases produced by the plant. This protection would be achieved by ligation of Mp-Chi to the fungal cell wall, sterically blocking the action of plant hydrolytic enzymes. Therefore, this project aims to verify if the chitinase Mp-Chi plays such role in the pathology of cacao caused by M. perniciosa. To this purpose, the Mp-Chi will be cloned and heterologously expressed, and the protein activity will be evaluated in order to confirm the absence of chitinolytic activity. Tests to prove the ability of Mp-Chi to bind chitin will also be performed. The functional characterization of this chitinase in the witches' broom disease represents an important step in the understanding of the evasion strategies of M. perniciosa against plant defenses, as well as it comprises a relevant novelty in modern plant pathology.(AU)