Genetic manipulation of Fonsecaea pedrosoi using particles bombardment and Agrobacterium mediated transformation

Publication date: Available online 2 January 2018 Source:Microbiological Research Author(s): Camille Silva Florencio, Fabiana Alves Silva Brandão, Marcus de Mello Teixeira, Anamélia Lorenzetti Bocca, Maria Sueli S. Felipe, Vânia Aparecida Vicente, Larissa Fernandes Fonsecaea pedrosoi, a melanized fungal pathogen that causes Chromoblastomycosis, a human disease with a worldwide distribution. Biolistic is a widely used technique for direct delivery of genetic material into intact cells by particles bombardment. Another well-established transformation method is Agrobacterium-mediated transformation (ATMT), which involves the transfer of a T-DNA from the bacterium to the target cells. In F. pedrosoi there are no reports of established protocols for genetic transformation, which require optimization of physical and biological parameters. In this work, intact conidia of F. pedrosoi were particle bombarded and subjected to ATMT. In addition, we proposed Hygromycin B, Nourseothricin and Neomycin as dominant selective markers for F. pedrosoi and vectors were constructed. We tested two parameters for biolistic: the distance of the particles to the target cells and time of cells recovery in nonselective medium. The biolistic efficiency was 37 transformants/μg of pFpHYG, and 45 transformants/μg of pAN7.1. Transformants expressing GFP were successfully obtained by biolistic. A co-culture ratio of 10: 1 (bacterium: conidia) and co-incubation time of 72 hours yielded the lar...
Source: Microbiological Research - Category: Infectious Diseases Source Type: research