A survey on optimization of Agrobacterium-mediated genetic transformation (AtMT) of Colletotrichum gloeosporioides

The fungus Colletotrichum gloeosporioides is the causative agent of anthracnose disease of many tropical, subtropical and temperate fruits, and a microbial source of the anticancer drug, Taxol. Here, we introduce an optimized Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for genetic manipulation of this fungus using hph and gfp-tagged hph genes as selection markers. Results showed that falcate spores can be easily used instead of protoplasts for transformation. Several experimental parameters were shown to affect transformation efficiencies, among which the length of co-cultivation, the ratio of fungal conidia to bacterium during co-cultivation, the kind of membrane during co-cultivation, and the kind of fungal growth medium during transformants selection, showed the highest influences on ATMT frequencies. Results indicated that the optimal ATMT of C. gloeosporioides was achived after 3 days of co-cultivation, at 107 per ml fungal conidia, via the use of Fabriano 808 filter paper and Czapek''s culture medium. Successive subculturing of transformants on selective and non-selective media demonstrated the stable expression of transgens, and subsequent PCR based analyses of transformants revealed the presence (100%) of transferred genes. Flourescence microscopy analyses showed a punctuate pattern for localization of an expressed Gfp-tagged Hph protein inside fungal hyphae. The optimized ATMT protocol generated mutants that showed different phenotypes based on their vegetation and pigmentation. This suggests the possible applicability of this technique for functional genetics studies in C. gloeosporioides, through insertional mutagenesis.