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

Abstract

We describe efficient polyethylene glycol (PEG)-mediated and Agrobacterium-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 of C. passeckerianus. The PEG-mediated transformation of C. passeckerianus protoplasts was successful and generated hygromycinresistant transformants more efficiently than either phleomycin or carboxin resistance. Agrobacterium-mediated transformation with plasmid pBGgHg containing hph gene under the control of the Agaricus bisporus gpdII promoter 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 the Agrobacterium-mediated system within the genome. C. passeckerianus actin and tubulin promoters were amplified from genomic DNA and proved successful in driving green fluorescent protein and DsRed expression in C. 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 using gfp as a target gene easily scored in C. passeckerianus. Upon transformation of gfp antisense 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 in C. passeckerianus. The molecular tools developed in this study should facilitate studies aimed at gene isolation or characterization in this pharmaceutically important species.

Agrobacterium tumefaciens-mediated transformation of the violet root-rot fungus, Helicobasidium mompa, and the effect of activated carbon

“…Error bars indicate standard deviation studies have reported that multiple integrations predominate in T-DNA transfer to filamentous fungi (Micosh et al 2001). However, similar results to those obtained in our analysis have been reported in Clitopilus passeckerianus (Kilaru et al 2009) and Venturia inaequalis (Fitzgerald et al 2003), where a majority of transformants contained a single copy of T-DNA in the genome. Therefore, the genetic manipulation of H. mompa may be possible because A. tumefaciens transformation resulted in singlecopy integration.…”

Enhancement of Ganoderic Acid Accumulation by Overexpression of an N-Terminally Truncated 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Gene in the Basidiomycete Ganoderma lucidum

“…A. tumefaciens-mediated transformation of G. lucidum protoplasts with plasmid pCAMBIA1301 also failed to achieve hygromycin resistance transformants (data not shown), possibly because the constructs containing the heterologous gene may not be functional in G. lucidum. Previous studies reported that the hygromycin resistance gene and phleomycin resistance gene cannot work in the basidiomycetes Schizophyllum commune and Clitopilus passeckerianus unless the appropriate N terminus and appropriate introns were present within the coding sequences (14,30). In the present study, a homologous selection marker gene cbx r was developed and used to transform basidiomycete by ATMT to establish a stable transformation system for G. lucidum.…”

“…The availability of practical transformation systems is a limiting factor for the genetic improvement of basidiomycetous mushrooms for biotechnological applications (11,12,14). Basidiomycete mushrooms generally exhibit higher resistance to genetic transformation compared to ascomycetous fungi (37).…”

Agrobacterium tumefaciens -Mediated Transformation for Investigation of Somatic Recombination in the Fungal Pathogen Armillaria mellea

“…eGFP was not expressed in transformants, likely due to the absence of an intron from the 5Ј end of eGFP in pBGgHg, which has been shown to bring about eGFP expression in other basidiomycetes (10,26). The methods and results are presented only for hph.…”