Development of a transformation method for the nematophagous fungus Dactylellina cionopaga

Abstract

Dactylellina cionopaga is a trapping fungus that produces adhesive columns and a two-dimensional network. The factors that influence protoplast preparation and regeneration of D. cionopaga were analyzed, and poly ethylene glycol (PEG)-CaCl₂- or Agrobacterium tumefaciens-mediated transformation was conducted to develop a transformation system for the fungus and provide a tool for studying the function of nematode infection-related genes. The results indicate that between 4.175±1.025×10⁶ and 3.08±1.4×10⁷, protoplasts/ml were obtained under optimized conditions and that the protoplasts could be regenerated on potato dextrose agar (PDA), RA and IM regeneration media. D. cionopaga transformation using PEG-CaCl2 or A. tumefaciens displayed 4.2 to 11 resistant colonies/μg DNA using 10⁶ protoplasts and 180-270 resistant colonies using 10⁶ conidia. Molecular analysis and microscopy of randomly selected transformants showed that the target genes were integrated into the genome of D. cionopaga and that green fluorescence could be detected in transformants containing pK2-BarGFP, which carried a glufosinate ammonium resistance gene and the enhanced green fluorescence protein gene. The methods used in this study for protoplast preparation and convenient Agrobacterium-mediated transformation of D. cionopaga represent useful tools for genetic research on this nematophagous fungus. This is the first report on protoplast generation and transformation of D. cionopaga.

Key words: Nematophagous fungi, Dactylellina cionopaga, Agrobacterium tumefaciens-mediated transformation, PEG-CaCl2-mediated transformation, protoplast preparation and regeneration.