Establishing an efficient protoplast isolation and PEG-mediated transformation system for functional genomic studies in Colletotrichum lindemuthianum

Abstract

Colletotrichum lindemuthianum is a significant threat to global bean production. The increasing availability of genomic data has led to a substantial accumulation of knowledge regarding the pathogenicity determinants in C. lindemuthianum, particularly focusing on effector proteins. A highly efficient transformation system is essential to effectively characterize these effectors and elucidate the molecular mechanisms behind C. lindemuthianum pathogenesis. The Agrobacterium-mediated transformation method has proven ineffective for site-directed mutagenesis in C. lindemuthianum. Additionally, the enzymes previously used for protoplast isolation are no longer accessible. We developed a highly efficient method for producing protoplasts using a new set of enzymes from the virulent C. lindemuthianum race 2047. The study involves generating and regenerating viable protoplasts from conidial and mycelial cultures. We treated the conidia of race 2047 with various combinations of enzymes and osmotic agents. Our results showed that a combination of 20mg/ml of Driselase and 1M D-sorbitol yielded the highest number of viable protoplasts. The treatment of 3g mycelial with 1.2g VinoTaste Pro in 30ml of buffer (0.6M (NH₄)₂S0₄, 50mM maleic acid, pH 5.5), yielded 5.8 ± 0.54 × 10⁷protoplasts/g of mycelium, with 85% viability. Isolated protoplasts were transformed with the pBV367 vector and regenerated onto a TB3 medium. The transformants selected for G418 antibiotic resistance colonies were monitored for mRFP1 expression. The protocol achieved a transformation efficiency ranging from 56 to 110 transformants/μg of plasmid DNA. The phenotypic bioassay of ten randomly selected transformants depicted no significant difference between the wild-type race 2047 and the transformants. Additionally, the mRFP1 tagging allowed for accurate fluorescence microscopy visualization of the infection process. This robust transformation method provides a solid foundation for functional genomic studies, including gene knockout and site-directed mutagenesis in C. lindemuthianum.