Abstract Detail

Mycological Section

Auxier, Benjamin [1], Dee, Jaclyn [2], Berbee, Mary [3].

Challenges and progress towards developing a selection system for genetic transformation in Phylum Chytridiomycota.

Techniques for transformation, allowing the insertion of foreign genetic elements into a genome, are a vital part of molecular biology. Routine transformation of species in Ascomycota and Basidiomycota has led to revolutionary discoveries about the function of genes involved in cell cycle regulation and plant pathogenesis. Owing to their early divergence from other fungi, genes in Chytridiomycota are of interest to evolutionary biologists, plant and animal pathologists, and biotechnologists. Unfortunately, reliable transformation has yet to be achieved for any fungus in the core chytrid clade, Chytridiomycota. Developing transformation methods for a core chytrid would allow us to test the function of genes that may control processes such as thallus morphogenesis and pathogenesis. For example, we could determine the roles of unicellular chytrid homologs of genes used by multicellular fungi to form septa and elongate hyphae. Also, a transformation protocol would be useful for researching Batrachochytrium dendrobatidis, a cause of global frog population decline. With transformation techniques, we could knock out putative B. dendrobatidis pathogenesis genes such as extracellular proteases to rigorously test their importance in amphibian infection. We are developing a method for transforming the chytrid fungus Chytriomyces hyalinus based on resistance to the antibiotic hygromycin B. We first analyzed concentrations of hygromycin needed to eliminate growth of C. hyalinus in the absence of the transforming DNA. We then engineered DNA constructs to express the hph gene that confers hygromycin B resistance. We codon-optimized the hph gene using expressed sequence tag (EST) data from B. dendrobatidis and put the gene under the control of promoter and terminator regions endogenous to B. dendrobatidis. To introduce the hph constructs into C. hyalinus, we are using biolistic transformation. One initial result from biolistic transformation was promising; an isolate survived selection and with PCR we detected a band corresponding to the expected size of the transforming hph construct. However, we were unable to confirm the long-term persistence of the insertion or its integration into the genome of C. hyalinus. We are continuing transformation experiments with C. hyalinus at different stages of development and with alternative conditions of transformation. Establishing the conditions for efficient, stable transformation is challenging due the evolutionary divergence of the species and due to the number of variables that may be important in success. Challenging though it is, we are convinced that establishing chytrid transformation protocols will lead to rapid progress in the fields of fungal ecology, evolution and pathology.

1 - University of British Columbia, Botany, 6270 University Boulevard, Vancouver, BC, V6T 1Z4, CA
2 - University of British Columbia, Botany, 6270 University Blvd., Vancouver, BC, V6T-1Z4, Canada
3 - University of British Columbia, Department of Botany, 6270 University Blvd, Vancouver, BC, V6T1Z4, CA

Keywords:
transformation
chytrid
hygromycin
Chytridiomycota.

Presentation Type: Poster:Posters for Sections
Session: P
Location: Hall D/The Shaw Conference Centre
Date: Monday, July 27th, 2015
Time: 5:30 PM
Number: PMY063
Abstract ID:565
Candidate for Awards:MSA Best Poster Presentation Award by an Undergraduate Student