Development of a CRISPR/Cas9-based methodology for genome engineering in the fungus Moniliophthora perniciosa, the causative agent of 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 and witches' broom disease transcriptome atlas, which have contributed for identification and functional analysis of multiple fungal pathogenicity-related factors. However, functional studies have been severely hampered by the lack of tools for genome engineering in Moniliophthora perniciosa, this being the main bottleneck in the current study of witches' broom. With this in mind, this project aims to establish a procedure for production of knockout strains of the fungus using the CRISPR - Cas9 system. For this, an inactivation cassette targeted to Mp-aox gene (which encode an alternative oxidase) will be prepared and used in the transformation of M. perniciosa. This gene has been well characterized by our group and tools for phenotypic screening in vitro are well established, allowing the validation of knockout strains in a simple and robust way. Once established, the procedure of genome edition in M. perniciosa may be used for the production of defective strains in numerous other genes of interest, providing a powerful tool for the study of factors involved in the pathogenicity of M. perniciosa. (AU)