1 Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Noda, Chiba, 278-8510, Japan
2 Department of Molecular Biology, Division of Bioscience, Graduate School of Natural Science and Technology, Okayama University, Okayama, 700-8530, Japan
Correspondence
Kengo Sakaguchi
kengo{at}rs.noda.tus.ac.jp
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ABSTRACT |
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The results of Southern analysis of pCcLIM15dsRNA insertions in white-cap transformants of C. cinereus are shown in Supplementary Fig. S1 with the online version of this paper.
Present address: Howard Hughes Medical Institute, Department of Molecular Biology, Massachusetts General Hospital, Department of Genetics, Harvard Medical School, Boston, MA 02114, USA.
Present address: Howard Hughes Medical Institute, Harvard-Partners Center for Genetics and Genomics, Harvard Medical School, Boston, MA 02115, USA.
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INTRODUCTION |
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C. cinereus has been analysed using forward genetics approaches because of its relatively short life cycle and its ease of mutagenesis by transformation of an asexual basidiospore of the haploid mycelium known as the oidium (reviewed by Casselton & Zolan, 2002; Kamada, 2002
; Kues, 2000
). Unfortunately, it has been difficult to repress specific genes through methods such as gene disruption by homologous recombination. Although a low frequency of homologous recombination does seem to occur in C. cinereus (Binninger et al., 1991
), so far, targeted gene disruption by homologous recombination in C. cinereus has not been reported. To get around this problem, we tried gene repression by dsRNA-mediated gene silencing as an alternative reverse-genetics technique in C. cinereus.
In RNA interference, also known as dsRNA-mediated gene silencing, dsRNA is fragmented by the RNase III-like nuclease Dicer into 2125 nt small interfering RNAs (siRNAs; Bernstein et al., 2001). These siRNAs are incorporated into the RNA-induced silencing complex (RISC) to disassemble the target mRNA complementary to the siRNAs in a sequence-specific manner (Hammond et al., 2000
). RNA interference is conserved in various organisms (reviewed by Montgomery, 2004
), including Caenorhabditis elegans (Fire et al., 1998
), plants (Waterhouse et al., 1998
), Drosophila (Misquitta & Paterson, 1999
), mammals (Elbashir et al., 2001
), and also in fungi such as fission yeast (Raponi & Arndt, 2003
), Dictyostelium (Martens et al., 2002
) and the heterobasidiomycete Cryptococcus (Liu et al., 2002
). Because of this broad conservation, we predicted that C. cinereus might have an RNAi-like mechanism. However, RNAi has not been reported so far in homobasidiomycete fungi.
In this study, we select LIM15 as a target of dsRNA-mediated gene silencing. In eukaryotes, there are two types of recA-like recombinase: Rad51 and Lim15/Dmc1. While RAD51 is expressed in both meiotic and somatic cells and functions in the DNA-repair reaction, LIM15/DMC1 expression is restricted to meiotic cells. However, both enzymes are thought to be involved in meiotic recombination, and the functional difference between these enzymes during meiosis is still obscure (reviewed by Masson & West, 2001).
If meiosis is hampered in C. cinereus, the normal black-coloured basidiospore is not produced, and the cap of the mature fruiting body becomes white or grey. Therefore, if we use the meiosis-specific transcript LIM15 as a target of dsRNA-mediated gene silencing, we can evaluate the effects of LIM15 dsRNA by judging the phenotype of the mushroom cap colour without affecting growth and morphogenesis. Here we show that transformation of a LIM15/DMC1 dsRNA expression construct (LIM15dsRNA) into C. cinereus resulted in paucity of LIM15 transcripts and abnormal homologous chromosome synapsis during meiosis. The applications of dsRNA-mediated gene silencing in C. cinereus research are discussed.
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METHODS |
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Construction of pCcLIM15dsRNA.
The expression vector pCcCEX (C. cinereus constitutive expression), which has the constitutive C. cinereus -tubulin promoter, the C. cinereus
-tubulin terminator and a multi-cloning site in the intervening space between the promoter and terminator, was made as follows. A 393 bp fragment of the
-tubulin promoter (Cummings et al., 1999
) was cloned into the pCRII vector (Invitrogen) at HindIII and EcoRI sites. Then, the resulting plasmid was digested with EcoRI and NotI, and a multi-cloning site containing EcoRI, SacII, XhoI, KpnI, XbaI and NotI sites was ligated into the digested site. The 427 bp fragment of the
-tubulin terminator sequence was cloned into the NotI and ApaI sites of the plasmid to generate pCcCEX.
The LIM15 dsRNA expression construct in pCcCEX (pCcLIM15dsRNA) was made as follows. The antisense strand which corresponds to 7501 bp in LIM15 cDNA was cloned into the XhoI and KpnI sites of pCcCEX. Next, the sense strand which corresponds to 101750 bp in LIM15 cDNA was cloned into the XbaI and NotI sites of the resulting plasmid to generate pCcLIM15dsRNA.
Transformations.
Transformations of C. cinereus strain AmutBmut protoplasts were performed exactly as described elsewhere (Binninger et al., 1987). For the co-transformation of pCcLIM15dsRNA with pPAB1-2, we used total of 4 µg intact plasmids of pCcLIM15dsRNA and pPAB1-2 in a molar ratio of 2 : 1. As a control, the strain AmutBmut was co-transformed with pCcCEX and pPAB1-2, or transformed with pPAB1-2 alone. The transformed cells were spread onto minimal medium (Binninger et al., 1987
). After incubation for 1 week at 28 °C, transformants were selected.
Genetic technique for mating to make heterozygotes.
The inocula (1x1 mm) of strain 5337 (Murata et al., 1998) on MY agar plates were removed 2 days later, followed by the inoculation of white-cap transformants to the place from which 5337 had been removed. After two more days, mated dikaryons were isolated from the marginal region (Makino & Kamada, 2004
).
Basidiospore production and viability.
The number of basidiospores produced per milligram of cap tissue was determined using the procedure described by Ramesh & Zolan (1995). The viability of basidiospores was established using the spotted-drop method described in Ramesh & Zolan (1995)
, with minor modifications. To inoculate the MY medium, the basidiospores were suspended in PBS and spotted onto the medium, which had been directly solidified on the glass slide. After overnight incubation at 37 °C, the germinated basidiospores were counted with an Olympus BH2 microscope. For each strain tested, basidiospores from three caps were analysed; the mean and standard deviation are reported.
Electron microscopy.
Spreads of C. cinereus chromosomes were prepared and stained with silver nitrate, as described elsewhere (Pukkila & Lu, 1985), and grids were viewed with a JEOL-1010 electron microscope.
Other methods.
Southern and Northern analyses were performed as described previously (Namekawa et al., 2003b; Nara et al., 1999
). For the Northern analysis, we used the C. cinereus homologue of the glyceraldehyde-3-phosphate dehydrogenase gene (G3PDH) as a loading control. The blots were stripped and sequentially reprobed for LIM15, RAD51 and G3PDH, in that order. For the DAPI staining of meiotic cells, a small piece of meiotic tissue was picked up by forceps and squashed immediately with DAPI in PBS and viewed with an Olympus BH2 microscope.
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RESULTS |
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In C. cinereus, normal meiosis results in a black cap on the fruiting body (Fig. 2A). Meiotic defects cause the fruiting body to have a pale or white cap due to the absence of healthy black basidiospores. This feature makes it easy to detect meiotic defects by the cap colour of the fruiting body after mutagenesis. To investigate the effect of LIM15dsRNA, we co-transformed pCcLIM15dsRNA and pPAB1-2 (Granado et al., 1997
), which contains the PAB synthetase gene, into the oidia. We obtained 10 white-cap transformants out of a total of 44 PAB+ transformants (Table 1
). These white-cap transformants specifically correspond to the transformation of the pCcLIM15dsRNA. No white-cap mutants were obtained when we used pPAB1-2 alone or pPAB1-2 and pCcCEX (Table 1
). Fig. 2(B)
shows an example of a white-cap transformant (lineage no. 2; line #2). All 10 white-cap transformants appeared completely normal with regard to mitotic mycelial growth and fruiting-body formation. We fruited the transformants more than 30 times, then scored for cap colour. The cap colour from white-cap transformants was always white, and these phenotypes were stable during passaging and after silica-gel stocks.
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We examined whether the phenotype survived sporulation. We obtained spores from transformant #2, from which mycelium was cultured, and 100 % of spores (n=7) displayed a white cap. This result indicated that the phenotype was heritable.
Meiotic phenotype of LIM15 repression strain
To investigate the meiotic defect in white-cap transformants, we examined meiotic chromosome behaviour using DAPI, and compared meiotic chromosomes from line #2 with chromosomes from AmutBmut. We defined the time of karyogamy (K) to be the time at which 5 % of cells undergo nuclear fusion. In the wild-type, nuclear fusion continues for some hours after karyogamy, with almost all nuclei being fused by K+4. Subsequently, meiotic prophase I can be observed in single nuclei until K+7. Meiotic prophase I is followed by meiosis I and meiosis II, and results in tetrads at K+12.
Consistent with previous reports (Celerin et al., 2000), we found that AmutBmut followed this standard progression. We observed one broad nucleus per cell at meiotic prophase I (K+4; Fig. 6A
), followed by the first and second divisions of meiosis at K+9 (Fig. 6D
). Tetrad and beetling sterigmata formation, which will become the basidiospore after nuclear migration, was also observed in normal basidiospore development (Fig. 6G
). AmutBmutx5337 also showed normal progression of meiosis (data not shown). In contrast, although line #2 shows one broad nucleus per cell with meiotic prophase I proceeding normally (Fig. 6B
), the majority of cells of line #2 could not divide into two nuclei, and meiosis did not proceed beyond meiosis I. Discrete chromosomes were observed in the nucleus in K+9, indicative of abnormal meiosis (41·4 %, n=175; Fig. 6E, J
). Following this discrete stage, we observed these cells to contain a condensed nucleus (52·6 %, n=175 at K+9; 43·2 %, n=81 at K+12; Fig. 6E, H and J
), and they eventually became anucleate cells (33·3 %, n=81 at K+12; Fig. 6H, J
). This suggested that line #2 arrested at meiotic metaphase I. Chromatin condensation and anucleate cells are common features in all meiotic mutants of C. cinereus (Lu et al., 2003
). Also, in #2x5337, cells showed discrete chromosomes at K+9 (55·2 %, n=134; Fig. 6F, J
). By K+12, we observed cells containing a condensed nucleus (Fig. 6I
) and anucleate cells (indicated by the arrow in Fig. 6I
). In line #2 and in #2x5337, we rarely observed normal progression of meiosis and sporulation. These phenotypes are common to the other white-cap transformants that show normal progression around karyogamy.
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DISCUSSION |
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We confirmed that the introduction of LIM15dsRNA correlates with specific reduction of the LIM15 transcript. However, all white-cap transformants had multiple insertions of intact and non-intact constructs (Table 2). We cannot completely exclude the possibility of mutagenesis by random insertion of plasmids into the C. cinereus genome. In C. cinereus, transformation is associated with random cutting of DNA and random insertion into the genome (Binninger et al., 1991
). We think that this feature is a shortcoming of C. cinereus in terms of evaluating the effect of transformation. To exclude the integration of non-intact constructs, further technical modification of the transformation procedure will be required.
In spite of these caveats, we argue that the effects of our LIM15dsRNA are specific, for four reasons. First, as shown in Table 1, repression of LIM15 occurred specifically when we transformed with pCcLIM15dsRNA. Second, paucity of LIM15 mRNA occurred without disturbing the endogenous LIM15 locus. A previous report has indicated that the frequency of genomic insertion at homologous sites by transformation is approximately 5 % in C. cinereus (Binninger et al., 1991
). However, we did not detect any insertion at the homologous site of LIM15 and at the
-tubulin gene locus in any of our transformants. Third, the trans effect of the construct in the heterozygote might be associated with the action of dsRNA on the other normal allele. This phenotype is different from that of recessive meiotic mutants which show no meiotic phenotype in the heterozygous state. Fourth, the meiosis-specific phenotype in these transformants supports the specific action of LIM15dsRNA. Abnormal homologous synapsis is a typical feature of LIM15/DMC1 mutants in eukaryotic species (reviewed by Masson & West, 2001
).
From these observations, we speculate that an RNAi pathway is likely to exist in C. cinereus. The proteins required for the RNAi pathway are known to be conserved in various eukaryotic organisms. Using the C. cinereus genomic database (http://www.broad.mit.edu/annotation/fungi/coprinus_cinereus/), we found a Dicer homologue in C. cinereus. Similarly, the C. cinereus genomic database also has a sequence which has homology to the Zwill/ARGONAUTE/Piwi family that has been implicated in the RNAi pathway in Caen. elegans (RDE-1; Tabara et al., 1999) and Neurospora (QDE-2; Catalanotto et al., 2000
). Further analysis of dsRNA-mediated gene silencing in C. cinereus will shed light on the detailed mechanism of RNAi in C. cinereus and in other homobasidiomycete fungi.
Interestingly, although the LIM15 repression strain (line #2) showed a defect in SC formation, a significant amount of basidiospore production and basidiospore viability was still observed. One possible explanation of these results is that Rad51 compensates for the loss of Lim15 function due to redundant function of Rad51 and Lim15. However, we support another explanation: incomplete repression of LIM15 permits a significant amount of sporulation. We could detect faint signals of LIM15 by Northern analysis. This low-level expression might result in occasional normal progression of meiosis and sporulation. These results may indicate that gene silencing by LIM15dsRNA is incomplete.
We propose several further applications of dsRNA-mediated gene silencing in C. cinereus. First, dsRNA-mediated gene silencing can produce partial repression of a gene of interest. This may be useful for the study of essential genes, since incomplete silencing might rescue the lethality and display an intermediate phenotype. Second, dsRNA-mediated gene silencing can be regulated spatially and temporally by dsRNA expression using a specific promoter. For example, we have previously studied DNA-replication-related factors, such as DNA polymerase and PCNA, during meiosis (Namekawa et al., 2003b
; Hamada et al., 2002
). Using a meiosis-specific promoter to express the dsRNA of these cDNAs, it will be possible to investigate the function of these genes during meiotic prophase I separately from pre-meiotic S phase or mitotic S phase. Third, we can simultaneously induce repression of multiple genes by introducing multiple dsRNAs. Lastly, future experiments can take advantage of the trans effect of the construct specifically to study whether heterozygoticity is sufficient to induce the phenotype. It may be easy to induce the repression of multiple genes at the dikaryonic stage by making a heterozygote of different dsRNA expression monokaryons.
In these ways, this study opens up doors for the use of the reverse-genetics approach in C. cinereus. The C. cinereus genome project was published in 2003. dsRNA-mediated gene silencing will be an important tool for C. cinereus research in the post-genomic era.
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ACKNOWLEDGEMENTS |
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Received 20 May 2005;
revised 15 August 2005;
accepted 18 August 2005.
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