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. 2006 Apr;5(4):732-44.
doi: 10.1128/EC.5.4.732-744.2006.

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

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Free PMC article

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

Martin A Wälti et al. Eukaryot Cell. .
Free PMC article

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.

Figures

FIG. 1.
FIG. 1.
Schematic representation of DNA constructs used in the present study. Promoter (p), terminator (t), and coding (c) regions of indicated genes are represented as boxes, and introns (i) as triangles. Bold arrows indicate the direction of transcription of the respective DNA element at the original locus, and the fine arrows indicate the transcriptional start sites of the constructs. The length of the longer DNA fragments is given in kilobases. Hatched boxes and block arrows below the boxes indicate hybridization probes used for Southern blot analyses and primers used for genomic PCR analyses, respectively. Restriction sites used for Southern blot analyses are indicated by thick (HindIII) or thin lines (HpaII/MspI).
FIG. 2.
FIG. 2.
Molecular analysis of GFP silencing in C. cinerea strain KK7. Analysis is shown for the strain without (lane 16) and with the GFP-expression construct 373 (lane 15), as well as for individual transformants of the latter transformant with either a mock plasmid carrying only the marker gene (pPAB1.2; lanes 11 to 14) or the GFP-silencing construct 367 (lanes 1 to 10). (A) Analysis of GFP protein levels in WCEs. A total of 3 μg of WCE prepared from the individual strains or transformants was analyzed by immunoblotting using a specific anti-GFP antiserum (see the text for details). (B) Analysis of DNA integration by genomic PCR and Southern blotting. Genomic DNA was prepared from the individual strains or transformants and used as a template for two PCRs indicative for the integration of either the GFP silencing construct (PCR A, primers A1 and AB2) or the GFP expression construct (PCR B, primers B1 and AB2) in the genome. A 100-bp DNA ladder (Fermentas International, Inc., Burlington, Ontario, Canada) was used as a size standard (m). The bottom panel shows a Southern blot analysis of the same genomic DNAs with HindIII as restriction enzyme and a GFP hybridization probe (see Materials and Methods for details). DNA-Molecular-Weight-Marker VII (Roche) was used as a size standard (m). The positions of the expected GFP and GFPhp DNA fragments are indicated. Plasmids 373 (lane 17) and 367 (lane 18) were included as controls. (C) GFP fluorescence microscopy of silenced and nonsilenced KK7 transformants. A silenced and a nonsilenced transformant in comparison to the appropriate control strains or transformants (the numbering in the brackets refers to panels A and B) were examined by GFP fluorescence and phase-contrast microscopy (PC) as described in Materials and Methods. Bar, 20 μm.
FIG. 3.
FIG. 3.
Determination of GFP silencing efficiency using quantitative real-time PCR. Relative GFP mRNA levels were determined for the same representative series of transformants as in Fig. 2 (numbering refers to Fig. 2A and B). Values (above the histogram bars) are given as the percentage of a mock transformant. The analysis was performed as described in Materials and Methods. The error bars represent the standard deviations of three different measurements of the same cDNA.
FIG. 4.
FIG. 4.
Molecular analysis of cgl2 silencing in C. cinerea strain AmutBmut. Individual transformants with cgl2-silencing construct 359 (lanes 1 to 21) were analyzed and compared to mock transformants carrying the vector control 336 (lanes C1 to C6). (A) Analysis of DNA integration by Southern blotting. Genomic DNA of the individual transformants was analyzed by using the restriction enzyme HindIII and a cgl2 hybridization probe (see Materials and Methods for details). The positions of the endogenous cgl2 and the introduced cgl2hp fragments are indicated. A HindIII digest of plasmid 359 served as a positive control. DNA-Molecular-Weight-Marker VII (Roche) was used as size standard (m). (B) Analysis of CGL1/2 and CGL3 protein levels in WCEs prepared from fruiting mycelium and primordia of the individual transformants (see Materials and Methods for details). A total of 5 μg of WCE from the indicated sample of the indicated transformants (numbering refers to panel A) was analyzed by immunoblotting with specific antisera against CGL1/2 and related protein CGL3 as described in Materials and Methods. In case of the anti-CGL1/2 blots, the HRP-coupled secondary antibody was detected either by using ECL (fruiting mycelium) or by using less sensitive chloronaphthol development (primordia). The positions of the individual proteins are indicated.
FIG. 5.
FIG. 5.
DNA methylation analysis in GFP and cgl2 silencing. Individual transformants of C. cinerea strains KK7 silenced for GFP (A and B) and AmutBmut silenced for cgl2 (C) were analyzed for methylation of their genomic DNA in comparison to corresponding mock transformants and original strains. DNA-Molecular-Weight-Marker VII (Roche) was used as a size standard (m). (A) DNA methylation analysis in GFP silencing. Oidial DNA of the indicated KK7 transformants and original strains (numbering refers to Fig. 2) was analyzed by Southern blotting with the indicated pairs of isoschizomeric restriction endonucleases, HpaII/MspI (H/Ms) or Sau3AI/MboI (S/Mb), and hybridization probes for the indicated genes (see Materials and Methods for details). (B) Cytosine methylation of GFP target and silencing locus. GFP target and silencing locus of indicated KK7 transformants (numbering refers to Fig. 2) were isolated as HindIII fragments as described in Materials and Methods and analyzed for cytosine methylation by Southern blotting using restriction endonucleases HpaII (H) and MspI (Ms) and a GFP hybridization probe. (C) Cytosine methylation in cgl2 silencing during fruiting body formation. Genomic DNA from different tissues (primordia, premeiotic oidia of mycelium before fruiting, and postmeiotic oidia of mycelium derived from two independent basidiospores [-1, -2] of respective fruiting bodies) of individual transformants (numbering refers to Fig. 4) was analyzed for cytosine methylation by Southern blotting using the restriction endonucleases HpaII (H) and MspI (Ms) and a cgl2 hybridization probe.
FIG. 5.
FIG. 5.
DNA methylation analysis in GFP and cgl2 silencing. Individual transformants of C. cinerea strains KK7 silenced for GFP (A and B) and AmutBmut silenced for cgl2 (C) were analyzed for methylation of their genomic DNA in comparison to corresponding mock transformants and original strains. DNA-Molecular-Weight-Marker VII (Roche) was used as a size standard (m). (A) DNA methylation analysis in GFP silencing. Oidial DNA of the indicated KK7 transformants and original strains (numbering refers to Fig. 2) was analyzed by Southern blotting with the indicated pairs of isoschizomeric restriction endonucleases, HpaII/MspI (H/Ms) or Sau3AI/MboI (S/Mb), and hybridization probes for the indicated genes (see Materials and Methods for details). (B) Cytosine methylation of GFP target and silencing locus. GFP target and silencing locus of indicated KK7 transformants (numbering refers to Fig. 2) were isolated as HindIII fragments as described in Materials and Methods and analyzed for cytosine methylation by Southern blotting using restriction endonucleases HpaII (H) and MspI (Ms) and a GFP hybridization probe. (C) Cytosine methylation in cgl2 silencing during fruiting body formation. Genomic DNA from different tissues (primordia, premeiotic oidia of mycelium before fruiting, and postmeiotic oidia of mycelium derived from two independent basidiospores [-1, -2] of respective fruiting bodies) of individual transformants (numbering refers to Fig. 4) was analyzed for cytosine methylation by Southern blotting using the restriction endonucleases HpaII (H) and MspI (Ms) and a cgl2 hybridization probe.

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