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

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Abstract
The basidiomyceteCoprinus cinereushas 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 theLIM15/DMC1dsRNA expression construct (LIM15dsRNA) resulted in genomic insertion ofLIM15dsRNA and paucity of theLIM15/DMC1transcript. First,LIM15dsRNA was transformed into the homothallic strainAmutBmutto generate a homozygote in which both nuclei had a copy ofLIM15dsRNA. TheLIM15/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 ofLIM15dsRNA. 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 inC. cinereusresearch.