RNA-Mediated Gene Silencing in Monokaryons and Dikaryons of Schizophyllum commune

Disruption of genes by homologous recombination occurs at alow frequency in the basidiomycete Schizophyllum commune. Forinstance, the SC3 and SC15 genes were inactivated at frequenciesof 1 and 5%, respectively. As an alternative to disruption,we used gene silencing through the introduction of a hairpinconstruct. The SC15 gene, which encodes an abundantly secretedstructural protein, was silenced at a frequency of 80% in monokaryonsof S. commune after introduction of a hairpin construct of thegene. Silencing also occurred in dikaryons in which one of thepartners was not a silenced strain. The silencing mechanismresembles RNAi in other filamentous fungi and is a powerfultool for the functional analysis of genes expressed in monokaryonsor dikaryons.

INTRODUCTION

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Introduction

RNA interference (RNAi) can be used for the functional analysisof genes. In this process double-stranded RNA (dsRNA) moleculesinduce the degradation of transcripts that are homologous insequence. RNAi can result from the direct administration ofdsRNA to cells or by inducing dsRNA formation through the expressionof complementary sequences (10). Sequences similar to the dcl-2,qde-2, and qde-3 genes of Neurospora crassa, which are requiredfor RNAi in N. crassa, are present in a partial genomic sequenceof the homobasidiomycete Schizophyllum commune that represents $~$ 40% of the single-copy DNA (GenBank DQ275629, DQ275628, andDQ275630). dcl-2 (GenBank NCU06766.1) is an ortholog of Dicer(3), which encodes an endonuclease that processes dsRNA in smallinterfering RNAs (siRNAs) (1). qde-2 (GenBank AF217760) encodesa component of the RNA-induced silencing complex (RISC), whichis guided by a siRNA to mRNA with a homologous sequence (2).Finally, qde-3 (GenBank AF205407) encodes a putative RecQ DNAhelicase (4). The presence of sequences in S. commune homologousto dcl-2, qde-2, and qde-3 suggests that the proteins requiredfor RNAi are present and that this mechanism of gene silencingmight be functional in this basidiomycete.

Our objective here was to determine whether a mechanism similarto RNAi is operating in S. commune. To this end, a hairpin constructof SC15 was made. This gene encodes an abundantly secreted 17-kDaprotein of S. commune, which is involved in aerial hyphae formationand attachment in the absence of the SC3 protein (9). This isthe first report of RNA-mediated gene silencing in a filamentoushomobasidiomycete.

MATERIALS AND METHODS

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Materials and Methods

Strains and growth conditions.
The isogenic Schizophyllum commune strains 4-39 (MATA41 MATB41,CBS 341.81), 4-40 (MATA43 MATB43, CBS 340.81), and FL1 (MATA43MATB43) were used. The latter strain contains the SC15::GFPfusion construct pSC15gfp (see DNA constructs). Strains weregrown at 25°C in the light or at 30°C in the dark onminimal medium (6) solidified with 1.5% agar. For microscopy,strains were grown in a thin layer of medium between a coverglass and a porous polycarbonate (PC) membrane (diameter, 76mm; pore size, 0.1 µm; Osmonics; GE Water Technologies,Trevose, PA).

DNA constructs.
A fragment of SC15 (GenBank AJ007503) cDNA encompassing theregion between the start of the 5' untranslated region and the93rd codon was amplified by PCR with primers sc15kpnfw (5'-GGTACCAGTCGAACCCACACCGACTACC-3')and sc15kpnrv (5'-GGTACCTGAGCTCCTCAATGCC-GTCGTTGG-3'), whichboth introduce KpnI recognition sites. The fragment was clonedin inverse orientation in the KpnI site just after the stopcodon of genomic SC15 in plasmid pSC15gspz (Fig. 1). The resultinghairpin construct, pSC15hp, encodes a SC15 mRNA with a stemof 334 nucleotides and with a loop of 333 bp. Plasmid pSC15gspzconsists of a pUC20 backbone containing a phleomycin resistancecassette (11) and a 4.1-kb SalI genomic fragment encompassingthe SC15 gene. pSC15gfp is a derivative of pSC15gspz in whicha green fluorescent protein (GFP) cDNA (GenBank AF188479) hasbeen cloned in frame with the SC15 coding region (A. Vinck andL. G. Lugones, in preparation).

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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).

Transformation procedure.
S. commune was transformed essentially as previously described(11), except that protoplast generation was done in 1 M MgSO₄containing 1 mg of lysing enzymes ml^–1 from Trichodermaharzianum (Applied Plant Research, Horst, The Netherlands).A total of 5 to 10 µg of DNA was added to 3 x 10⁷ protoplastsin 100 µl of 1 M sorbitol. Transformants were selectedon minimal medium containing 25 µg of phleomycin (Cayla,Toulouse, France) ml^–1.

RNA and protein analysis.
RNA was isolated from mycelium that had been ground in liquidnitrogen using TRIzol reagent (Invitrogen, Carlsbad, CA). Electrophoresisand hybridization of RNA were performed as previously described(5), except that the filters were washed at 65°C. For analysisof SC3 and SC15 secretion, colonies were grown for 3 to 4 dayson the surface of a PC membrane overlaying solidified minimalmedium. The membrane supporting the colonies was transferredto a fresh plate topped with a polyvinylidene difluoride (PVDF)membrane. SC15 and SC3 secretion was monitored by immunodetectionafter incubation of the colonies on PVDF membrane for 2 and17 h, respectively. The SC3 and SC15 antisera (9, 17) were diluted10,000- and 5,000-fold, respectively. Goat anti-rabbit-conjugatedalkaline phosphatase was used as a second antibody with BCIP(5-bromo-4-chloro-3-indolylphosphate) and nitroblue tetrazoliumas substrates (7).

Fluorescence microscopy.
GFP fluorescence was monitored with an Axioskop 2 Plus microscope(Zeiss, Jena, Germany) equipped with an HBO 100-W mercury lampand a Photometrics Cool SNAP camera (1,392 x 1,024 pixels) usingstandard fluorescein isothiocyanate filters.

RESULTS

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Results

The homokaryotic wild-type S. commune strain 4-39 was transformedwith pSC15hp. This construct encodes an SC15 hairpin mRNA witha stem of 334 nucleotides and a loop of 333 bp. Of 34 transformants,27 produced less SC15 protein than did the 4-39 wild-type strain.The intensity of the immunolocalization signal of the otherseven colonies was similar to that of the wild type. Of thetransformants showing no secretion of SC15, three (S1, S3, andS5) were randomly selected for further study. These three strainsand the wild-type strain were grown on minimal medium on a porousPC membrane. Immunodetection on PVDF membranes that had beenplaced underneath the colonies in between the medium and thePC membrane revealed that, in contrast to SC15, secretion ofSC3 was unaffected (Fig. 2). This shows that the reduction inSC15 secretion was not due to a thn mutation. (The thn mutationoccurs frequently spontaneously and has a pleiotropic phenotypethat includes downregulation of SC3 and SC15 and a reductionin the formation of aerial hyphae [16].) The low amount of SC15secreted by transformants S1, S3, and S5 was accompanied bygreatly reduced accumulation of SC15 mRNA (Fig. 3).

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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).

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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).

The silencing of SC15 was not related to the introduction ofextra copies of the gene rather than due to the hairpin mRNA,since when S. commune strain 4-39 was transformed with pSC15gspzcontaining SC15 there was no change in SC15 secretion. Silencingalso was not due to cytosine methylation, since when coloniesof transformants S1, S3, and S5 were grown on solid minimalmedium containing 10 µM 5-azacytidine (AZC), there wasno change in the amount of detectable SC15 protein. (AZC inhibitsDNA-methylation by incorporating into the DNA in place of cytidineand/or by direct inhibition of the activity of methylases [8].)

Transformants S1, S3, and S5 were crossed with FL1, which expressesthe endogenous copy of SC15 and a translational fusion of SC15and GFP. GFP fluorescence in the dikaryons was significantlyreduced relative to a dikaryon composed of nuclei from FL1 and4-39 (Fig. 4). Fluorescence intensity corresponded with mRNAlevels of the SC15::GFP fusion, as shown by hybridization withprobes against SC15 and GFP (Fig. 5). SC15 mRNA was not detectedin dikaryons that expressed pSC15hp in only one of the parentalnuclei (Fig. 5). Accumulation of SC15::GFP mRNA was also reduced.Thus, the hairpin RNA not only leads to degradation of SC15mRNA originating from the parental nucleus but also of comparablemRNA produced by the partner nucleus in the dikaryotic cell.

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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 x 4-40 (A and D) and strains 4-39 x FL1 (B and E) and S1 x FL1 (C and F).

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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 x 4-40 (lane 1), 4-39 x 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).

DISCUSSION

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Discussion

The introduction of extra copies of the SC3 hydrophobin geneinto S. commune resulted in silencing of both the introducedand endogenous SC3 genes in 90% of the transformants (13). Inthis case, the silencing was triggered by SC3 mRNA through acytosine methylation mechanism, and the gene in the wild-typenucleus of the dikaryon was not silenced (12). SC15 was notsilenced by the introduction of multiple copies of the gene.These results distinguish silencing of SC3 (13) and the silencingof SC15 we describe here and suggest that two different silencingmechanisms are operative in S. commune.

We anticipate that the RNA-based silencing mechanism describedhere also operates in other homobasidiomycetes, e.g., the commerciallyimportant species Agaricus bisporus and Pleurotus ostreatus.Orthologs of the N. crassa genes dcl-2, qde-2, and qde-3 arealso present in the genomic sequences of Coprinus cinereus (http://www.broad.mit.edu/cgi-in/annotation/fungi/coprinus_cinereus/blast_page.cgi)and Phanerochaete chrysosporium (http://genome.jgi-psf.org/Phchr1/Phchr1.home.html),which is consistent with this hypothesis. In the homobasidiomycetes,RNA-based gene silencing is a powerful alternative to gene inactivationby homologous recombination, which occurs at low frequency inthis group of fungi. For instance, gene inactivation of SC3,SC4, and SC15 in S. commune occurs at a frequency of 1 to 5%(9, 14, 15). More importantly, functional analysis of genesthat are active in the heterokaryotic phase, e.g., in mushroomformation and sporulation, requires inactivation of the genein both nuclei of the dikaryon. RNA-based gene silencing occursat a much higher frequency than homologous recombination andenables silencing of functional genes in both nuclei of a dikaryonby the expression of a hairpin construct in only one of thenuclei.

ACKNOWLEDGMENTS

We thank Ischa Lamot for technical support.

FOOTNOTES

* Corresponding author. Mailing address: Microbiology, Institute of Biomembranes, University of Utrecht, Padualaan 8, NL-3584 CH Utrecht, The Netherlands. Phone: (31) 30 2533016. Fax: (31) 30 2513655. E-mail: l.g.lugones{at}bio.uu.nl.

REFERENCES

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