RNA-mediated gene silencing in monokaryons and dikaryons of Schizophyllum commune

Abstract

Disruption of genes by homologous recombination occurs at a low frequency in the basidiomycete Schizophyllum commune. For instance, the SC3 and SC15 genes were inactivated at frequencies of 1 and 5%, respectively. As an alternative to disruption, we used gene silencing through the introduction of a hairpin construct. The SC15 gene, which encodes an abundantly secreted structural protein, was silenced at a frequency of 80% in monokaryons of S. commune after introduction of a hairpin construct of the gene. Silencing also occurred in dikaryons in which one of the partners was not a silenced strain. The silencing mechanism resembles RNAi in other filamentous fungi and is a powerful tool for the functional analysis of genes expressed in monokaryons or dikaryons.

FIG. 1.

Silencing construct pSC15hp and resulting hairpin mRNA. The SC15 promoter and terminator (white boxes), 5′ untranslated region (black box), and coding sequence (cross-hatched box) are indicated, as well as the SC15 introns (I1, I2, and I3).

FIG. 2.

Secretion of SC15 is severely affected in strains transformed with the SC15 hairpin construct. (A and B) A PVDF membrane was placed underneath colonies grown on top of a PC membrane. This was followed by immunodetection for SC15 (C) and SC3 (D), respectively. The colonies on each plate are arranged in the following order: wild-type strain 4-40 (top, left), and recombinant strains S1 (top, right), S3 (bottom, left), and S5 (bottom, right).

FIG. 3.

SC15 mRNA level is severely reduced in strains transformed with the SC15 hairpin construct. (A) Accumulation of SC15 mRNA in 4-day-old colonies of the wild-type strain 4-40 (lane 1) and transformed strains S1 (lane 2), S3 (lane 3), and S5 (lane 4). (B) Methylene blue staining of 18S rRNA (loading control).

FIG. 4.

Bright-field (A, B, and C) and fluorescence (D, E, and F) images of 4-day-old colonies of wild-type strains 4-39 × 4-40 (A and D) and strains 4-39 × FL1 (B and E) and S1 × FL1 (C and F).

FIG. 5.

The expression level of SC15 and SC15::GFP is reduced in dikaryons that express the SC15 hairpin construct in one of the parental nuclei. (A) Hybridization of an SC15 probe with RNA from 4-day-old colonies of crosses of the wild-type strains 4-39 × 4-40 (lane 1), 4-39 × FL1 (lane 2), and FL1 crossed with, respectively, transformants S1 (lane 3), S3 (lane 4), and S5 (lane 5). SC15 mRNA is indicated by a filled arrowhead, and SC15::GFP is indicated by an open arrowhead. (B) Same as in panel A but probed with GFP. (C) Methylene blue staining of 18S rRNA (loading control).