Targeted Gene Silencing in the Model Mushroom Coprinopsis cinerea ( Coprinus cinereus ) by Expression of Homologous Hairpin RNAs

Abstract

The ink cap Coprinopsis cinerea is a model organism for studying fruiting body (mushroom) formation in homobasidiomycetes. Mutant screens and expression studies have implicated a number of genes in this developmental process. Functional analysis of these genes, however, is hampered by the lack of reliable reverse genetics tools for C. cinerea. Here, we report the applicability of gene targeting by RNA silencing for this organism. Efficient silencing of both an introduced GFP expression cassette and the endogenous cgl1 and cgl2 isogenes was achieved by expression of homologous hairpin RNAs. In latter case, silencing was the result of a hairpin construct containing solely cgl2 sequences, demonstrating the possibility of simultaneous silencing of whole gene families by a single construct. Expression of the hairpin RNAs reduced the mRNA levels of the target genes by at least 90%, as determined by quantitative real-time PCR. The reduced mRNA levels were accompanied by cytosine methylation of transcribed and nontranscribed DNA at both silencing and target loci in the case of constitutive high-level expression of the hairpin RNA but not in the case of transient expression. These results suggest the presence of both posttranscriptional and transcriptional gene silencing mechanisms in C. cinerea and demonstrate the applicability of targeted gene silencing as a powerful reverse genetics approach in this organism.The ink cap Coprinopsis cinerea is a model organism for studying fruiting body (mushroom) formation in homobasidiomycetes (reviewed in references 32 and 35). Mutant screens and expression studies have implicated a number of genes in this developmental process. An example for the latter case are the cgl1 and cgl2 isogenes, which code for two isogalectins that are highly induced during fruiting body formation (5). In addition, orthologues of genes involved in fruiting body formation in other fungi are revealed by the sequence of the C. cinerea genome (http://www.broad.mit.edu/annotation/fungi /coprinus_cinereus) and its annotation, which is in progress (http://fungal.genome.duke.edu/cgi-bin/gbrowse/ccin/). Functional analysis of C. cinerea genes, however, is hampered by the lack of reliable tools for gene targeting. Although homologous recombination seems to occur in C. cinerea (3), targeted gene knockouts appear difficult to achieve, possibly due to very efficient nonhomologous DNA end joining, as has been shown for the filamentous ascomycete Neurospora crassa (47).Recently, RNA-induced gene silencing (RNA silencing) has been emerging as a powerful tool for gene targeting in fungi, plants, and animals (reviewed in references 7, 11, and 13). This strategy exploits an endogenous gene regulatory mechanism of eukaryotic cells in which regulatory double-stranded RNAs (dsRNAs) interfere with homologous mRNA either by triggering its degradation or inhibiting its transcription or translation (see references 1 and 42 for recent reviews and specific references therein). For gene targeting, dsRNA homologous to the target gene...

Oligonucleotide sequences forming short self-complimentary hairpins can expedite the down-regulation of Coprinopsis cinerea genes

“…Coprinopsis cinerea is a model homobasidiomycete used in many studies such as sexual development and meiosis, and also to exploit tools for functional genomics in filamentous fungi (Heneghan et al, 2007;Namekawa et al, 2005;Wälti et al, 2006).…”

Efficient cloning system for construction of gene silencing vectors in Aspergillus niger

“…On the one hand, rearrangements may affect copy number determinations (even using Southern blotting techniques, which frequently miss smaller variations), and on the other hand, agarose gel analysis of amplified segments of genomic DNA is insufficient to provide reliable quantification. Nevertheless, combined analysis of these two methods may be used: Fr example, copy numbers of pXLNRir do not correlate with a higher degree of silencing efficiency, a situation similar to others reported (Nakayashiki et al 2005;Nicolas et al 2003;Tanguay et al 2006;Fitzgerald et al 2004;Henry et al 2007;Yamada et al 2007;Walti et al 2006). Apart from genome instability and the occurrence of rearrangements, other factors may be responsible for a lower silencing efficiency of integrated hairpin-expression units.…”

Wood production, wood technology, and biotechnological impacts

“…They are however not essential as recently shown by gene inactivation in a self-compatible homokaryon through the posttranscriptional acting RNAi technique that leads to degradation of gene transcripts (Wälti et al 2006). Establishing this technique in C. cinerea in various laboratories (Namekawa et al , Heneghan et al 2006, Wälti et al 2006) is a great achievement for analysis of those genes for which mutants are not available. The gene for the LIM15/DCM1 recombinase has been inactivated in a self-compatible homokaryon with the result of an abnormal chromosomal pairing in meiosis, a highly reduced basidiospore number and viability (Namekawa et al 2005).…”

“…The proteins have been suggested to contribute to hyphal aggregation (Boulianne et al 2000). They are however not essential as recently shown by gene inactivation in a self-compatible homokaryon through the posttranscriptional acting RNAi technique that leads to degradation of gene transcripts (Wälti et al 2006). Establishing this technique in C. cinerea in various laboratories (Namekawa et al , Heneghan et al 2006, Wälti et al 2006) is a great achievement for analysis of those genes for which mutants are not available.…”

“…It will be a major effort to individually inactive the various hydrophobin and laccase genes in C. cinerea to test their specific functions in the fruiting bodies. The simultaneous silencing of the two galectin genes cgl1 and cgl2 by the RNAi technique (Wälti et al 2006) gives hope that this could also become the case at least for the closer related hydrophobin genes, respectively laccase genes.…”