Establishing Molecular Tools for Genetic Manipulation of the Pleuromutilin-Producing FungusClitopilus passeckerianus

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Abstract
We describe efficient polyethylene glycol (PEG)-mediated andAgrobacterium-mediated transformation systems for a pharmaceutically important basidiomycete fungus,Clitopilus passeckerianus, which produces pleuromutilin, a diterpene antibiotic. Three dominant selectable marker systems based on hygromycin, phleomycin, and carboxin selection were used to study the feasibility of PEG-mediated transformation ofC. passeckerianus. The PEG-mediated transformation ofC. passeckerianusprotoplasts was successful and generated hygromycin-resistant transformants more efficiently than either phleomycin or carboxin resistance.Agrobacterium-mediated transformation with plasmid pBGgHg containinghphgene under the control of theAgaricus bisporus gpdIIpromoter led to hygromycin-resistant colonies and was successful when homogenized mycelium and fruiting body gill tissue were used as starting material. Southern blot analysis of transformants revealed the apparently random integration of the transforming DNA to be predominantly multiple copies for the PEG-mediated system and a single copy for theAgrobacterium-mediated system within the genome.C. passeckerianusactin and tubulin promoters were amplified from genomic DNA and proved successful in driving green fluorescent protein and DsRed expression inC. passeckerianus, but only when constructs contained a 5′ intron, demonstrating that the presence of an intron is prerequisite for efficient transgene expression. The feasibility of RNA interference-mediated gene silencing was investigated usinggfpas a target gene easily scored inC. passeckerianus. Upon transformation ofgfpantisense constructs into a highly fluorescent strain, transformants were recovered that exhibited either reduced or undetectable fluorescence. This was confirmed by Northern blotting showing depletion of the target mRNA levels. This demonstrated that gene silencing is a suitable tool for modulating gene expression inC. passeckerianus. The molecular tools developed in this study should facilitate studies aimed at gene isolation or characterization in this pharmaceutically important species.

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