Genetic Transformation and Mutagenesis of the Entomopathogenic Fungus Paecilomyces fumosoroseus

Abstract

We have developed a DNA-mediated transformation system for the entomopathogenic fungus Paecilomyces fumosoroseus based on resistance to the herbicide glufosinate ammonium. The resistance is provided by the bar gene from Streptomyces hygroscopicus and is under the control of the Aspergillus nidulans trpC promoter and terminator sequences. Frequencies of up to 110 transformants/μg of linearized plasmid DNA were obtained aided by the addition of the nuclease inhibitor, aurintricarboxylic acid (ATA). The transformants were stable for drug resistance for five consecutive vegetative transfers under selective and nonselective conditions. Southern analyses revealed that the transforming DNA integrated into the P. fumosoroseus genome in single and multiple copies. Single-copy integration events were generated with higher efficiency by restriction enzyme-mediated integration (REMI), although rates of transformation were generally not as high as those treatments containing ATA. Two mutant strains altered in sporulation capacity and virulence toward the Russian wheat aphid were recovered using this approach. This transformation-based manipulation of P. fumosoroseus will facilitate insertional mutagenesis and the functional analysis of various genes.