Volume 19 Part 1 Article 139: Genetic transformation of the model basidiomycete Coprinopsis cinerea

Volume 19 Part 1 Article 139
Year 2016
Title: Genetic transformation of the model basidiomycete Coprinopsis cinerea
Author: B. Dörnte and U. Kües

Abstract:
Coprinopsis cinerea was amongst the first fungi for which a functional transformation system had been established. Transformation made use of a trp1.1,1.6 auxotrophy that can be complimented by the cloned trp1+ wild type gene which randomly integrates into the host genome (Binninger et al., 1987). Transformation in C. cinerea is especially efficient since the fungus produces unicellular haploid oidia in its aerial mycelium which are easily transformed into protoplasts and also easily regenerated (Dörnte and Kües 2012). With time, also the trp3+ and the pab1+ genes have been appointed for complementation of auxotrophies in C. cinerea (Bhattiprolu et al. 1993; Granado et al. 1997). In addition, the bacterial hygromycin and phleomycin resistance genes hph and ble and fungal carboxin-resistance sdi1R genes are available for selection of transformants (Kilaru et al. 2009). Despite of using DNA-mediated transformation in numerous molecular analyses for nearly 30 years, there are still factors limiting its applicability. More selection markers of homologous and heterologous origin are desirable, for example to allow sequential transformations of a strain or as markers in knocking out host genes without any interference at markers´ homologous sites. Therefore, the focus of this study is on the further improvement of the oidia transformation technique of C. cinerea, better vector design, development of new selection markers and optimizing screening techniques for further characterization of transformants. We summarize important factors in generating highly competent protoplasts, present new homologous and heterologous trp1+-based vectors, introduce partly inactivated bifunctional enzymatic markers, visual screening for successful expression of co-transformed genes and a fast microwave-PCR transformant screening method.

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