Knockdown of LIM15/DMC1 in the mushroom Coprinus cinereus by double-stranded RNA-mediated gene silencing

Abstract

The basidiomycete Coprinus cinereus has many advantages as a model organism for studying sexual development and meiosis, but it has been difficult to investigate using reverse-genetics methods, such as gene disruption by homologous recombination. Here, gene repression by dsRNA-mediated gene silencing was tried as an alternative method for reverse-genetics studies. It was shown that transformation of the LIM15/DMC1 dsRNA expression construct (LIM15dsRNA) resulted in genomic insertion of LIM15dsRNA and paucity of the LIM15/DMC1 transcript. First, LIM15dsRNA was transformed into the homothallic strain AmutBmut to generate a homozygote in which both nuclei had a copy of LIM15dsRNA. The LIM15/DMC1-repressed strain showed abnormal homologous chromosome synapsis during meiosis. Basidiospore production was reduced to 16 % by the induction of dsRNA. However, approximately 60 % of basidiospores were viable. Next, a heterozygote was generated in which one nucleus had a copy of LIM15dsRNA. The phenotype was similar to that of the homozygote. These results are not only the first demonstration of dsRNA-mediated gene silencing in a member of the homobasidiomycete fungi, to which 90 % of mushroom species belong, but also the first successful use of a reverse-genetics approach in C. cinereus research.

Senescence of the Lentinula edodes Fruiting Body After Harvesting

“…DMC1 is a recombinase that is required for DNA pairing during recombination. Two DMC1-encoding genes were previously identified in C. cinerea, namely Rad51 (Stassen et al, 1997) and LIM15 (Namekawa et al, 2005). Whereas Rad51 is involved in both mitosis and meiosis, LIM15 is specifically involved in meiosis, being expressed during meiosis and disappearing immediately after meiosis (Nara et al, 1999).…”

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

“…In 8 of the pab+ transformants the promoter region could be detected but not the hairpin expression sequences. When transforming with closed circular plasmid, the integration of partial constructs and/or rearrangements are not unusual in C. cinerea (Heneghan et al, 2007;Namekawa et al, 2005;Wälti et al, 2006).…”

“…The study of gene function in filamentous fungi has been hampered by the lack of efficient tools but RNA mediated gene silencing has recently been successfully achieved in these fungi (Heneghan et al, 2007;Namekawa et al, 2005;Wälti et al, 2006;De Jong et al, 2006). Most of these studies have involved laborious and time consuming multiple-steps to clone target sequence in sense and antisense orientations to yield dsRNA forming hairpin constructs that initiate RNAi mediated gene silencing.…”

“…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).…”

Wood production, wood technology, and biotechnological impacts

“…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). Classical knocking-out of genes via exchange by homologous recombination of wildtype genes in the fungal genome by non-functional gene copies with an inserted transformation marker gene has been attempted before in C. cinerea.…”