Author for correspondence: John W. G. Cairney Tel: +61 29685 9903 Fax: +61 29685 9915 Email: email@example.com
• Ectomycorrhizal (ECM) fungi were screened for laccase-like genes by polymerase chain reaction (PCR) using primers for white rot fungal laccase genes, and expression of the genes was examined by reverse transcriptase polymerase chain reaction (RT-PCR) for Piloderma byssinum in axenic culture under different nutrient conditions.
• Laccase-like genes were present in Rhizopogon roseolus along with several Russulales and Atheliaceae taxa, and showed strong nucleotide sequence similarity to laccase genes in white rot fungi. Multiple laccase-like genes were only identified in Piloderma spp.
• Laccase-like genes were expressed in Piloderma spp., with transcript levels some six times higher under high nitrogen conditions in P. byssinum than when nitrogen availability was lower.
• The potential roles of laccases in nutrient mobilization and/or differentiation of multihyphal ECM fungal structures are discussed.
Extant ectomycorrhizal (ECM) fungi form a taxonomically diverse group that has arisen through convergent evolution from multiple saprotrophic lineages (Hibbett et al., 1997; Bruns et al., 1998; Kretzer & Bruns, 1999). These fungi are known to facilitate host tree access to inorganic, and often organic, forms of N and P, and are regarded as important components of nutrient cycles in many forest habitats (Smith & Read, 1997). While it is widely perceived that ECM fungi have diverged substantially from ancestral saprotrophs in their abilities to degrade plant cell wall polymers, the fact that some ECM fungi appear to have undergone evolutionary reversal to saprotrophy suggests that many may retain genes for saprotrophic enzyme systems (Hibbett et al., 2000). This is supported by evidence that some ECM fungi can partly degrade certain components of plant cell walls, an ability that may provide access to mineral nutrients complexed with cell wall components such as lignins or their phenolic derivatives (Cairney & Burke, 1994; Leake & Read, 1997).
Laccase (EC 220.127.116.11), which catalyses reduction of O2 to H2O using a range of phenolic compounds as hydrogen donor, is also thought to contribute to degradation of lignin by some saprotrophic fungi (Hatakka, 1994; Thurston, 1994). Laccase has also been implicated in a number of other processes, including morphogenesis of multihyphal structures such as rhizomorphs and basidiomes (Thurston, 1994; Burke & Cairney, 2002). In the context of ECM fungi, laccase activities might thus play roles, not only in nutrient mobilization, but also in the development of multihyphal components of the symbiosis, such as rhizomorphic extramatrical mycelium or the hyphal mantle. Some laccase isozymes, at least, can contribute to more than one process; for example, a single gene encodes an isozyme that is involved in both lignin degradation and basidiome pigmentation in Pycnoporus cinnabarinus (Jacq.) P. Karst. (Eggert et al., 1996b). Multiple laccase isozymes are, however, produced by some fungi (Yaver et al., 1996; Mansur et al., 1997) and may variously contribute to decomposition and developmental processes under different conditions (Temp et al., 1999).
There have been numerous reports of laccase production by ECM fungi but, because of substrate overlap with other polyphenol oxidases, many of these activities may reflect activities of the latter rather than laccase per se (reviewed by Burke & Cairney, 2002). There is, however, convincing evidence of extracellular laccase production by the ECM basidiomycete Thelephora terrestris (Ehrh.) Fr. (Kanunfre & Zancan, 1998). Given the difficulties associated with separating laccase from other phenol-oxidizing activities – see Burke & Cairney (2002) for discussion – molecular approaches using, for example, polymerase chain reaction (PCR) amplification of laccase-specific sequences (D’Souza et al., 1996; Zhao & Kwan, 1999), offer an accurate and rapid means of determining the presence potential laccase-producing abilities of ECM fungi. We have used primers developed for studying laccase genes in saprotrophic basidiomycetes to screen a broad taxonomic range of ECM basidiomycetes for laccase-like genes. Here, we report the results of that work, along with investigation of expression of laccase-like genes in P. byssinum under different axenic culture conditions.
Materials and Methods
Fungal material and DNA extraction
Single isolates or basidiomes of 47 ECM basidiomycetes (Table 1) were screened for the presence of laccase genes. With the exception of P. byssinum (P. Karst.) Jülich, Piloderma fallax (Lib.) Stalp., Pisolithus albus (M.C. Cooke & G.E. Massee) M.J. Priest, nom. prov., Pisolithus marmoratus (M.J. Berkeley) M.J. Priest, nom. prov. and Tylospora fibrillosa (Burt) Donk, which were maintained as mycelial cultures on Modified Melin Norkans (MMN) agar medium (Marx & Bryan, 1975), all fungi were obtained as dried basidiome material. DNA was extracted from dried basidiome material using the method of Cubero et al. (1999) and from cultured mycelia using the modified cetyltrimethylammonium bromide (CTAB) method of Gardes & Bruns (1993).
Table 1. Details of ectomycorrhizal fungi screened for laccase-like genes
Appropriately sized polymerase chain reaction products were obtained using lac2 and lac3 primers; –, indicates that no products were obtained using lac2 and lac3 primers.
Polymerase chain reaction amplification of laccase-like gene fragments
PCR was conducted using three primer pairs (here designated lac1, lac2 and lac3) that were developed to amplify laccase-like gene fragments from saprotrophic basidiomycetes (D’Souza et al., 1996; Zhao & Kwan, 1999). Primer sequences have been published previously as follows: lac1u/d = lelacU1523 and Lelac1L2129 (Zhao & Kwan, 1999), lac2u/d = lelac2U1 and Lelac2L710 (Zhao & Kwan, 1999), lac3u/d = degenerate primers I and II (D’Souza et al., 1996). Amplifications were performed in 50 µl reaction volumes containing c. 50 ng genomic DNA, 25 pmol of the relevant primer pair, 50 mm KCl, 10 mm Tris-HCl, 0.1% Triton X-100, 2.5 mm MgCl2, 200 mm each of dATP, dCTP, dGTP and dTTP, and 2 units of Taq DNA polymerase (Promega, Madison, WI, USA). All amplifications were performed in a PTC-100 Thermal Cycler (MJ Research, Watertown, MA, USA). For lac1 and lac2, the PCR amplification program was: 1 cycle of 94°C for 4 min then 30 cycles of 94°C for 1 min 58°C for 1 min, and 72°C for 1 min, followed by 72°C for 10 min (Zhao & Kwan, 1999). For lac3 the PCR amplification program was 35 cycles of 94°C for 1 min, 54°C for 1 min, and 72°C for 1 min, followed by 72°C for 10 min (D’Souza et al., 1996). A negative control, containing no fungal DNA was included in each PCR reaction run to test for the presence of contaminating DNA. Amplification products were electrophoresed in 3% (w : v) agarose gels, stained with ethidium bromide under UV light.
DNA sequencing and sequence analysis
In order to confirm the identity of the bands obtained in the PCR amplifications, DNA sequences were obtained for all products from each primer pair. Before sequencing, each PCR product was purified using the Wizard PCR Purification System (Promega) according to the manufacturers instructions. Purified PCR products were cloned with the pGEM-T easy vector system (Promega) and two clones for each isolate sequenced. Each strand was sequenced using pGEMf and pGEMr primers on an ABI 377 automatic sequencer (Applied Biosystems, Inc., Foster City, CA, USA). Final sequences were compared with the GenBank and EMBL nucleotide databases using the fasta 3.0 program (Pearson & Lipman, 1988) and aligned with sequences having close identity using the pileup program (within EGCG extensions to the Wisconsin package, Version 8.1.0; Rice, 1996). All were submitted to the GenBank nucleotide database (see Table 2 for accession codes).
Table 2. Closest matches from fasta searches between DNA sequences of isozyme-specific polymerase chain reaction fragments amplified from various ectomycorrhizal fungi and sequences from the GenBank nucleotide database
Influence of culture conditions on expression of laccase-like genes
To investigate expression of laccase-like genes, P. fallax was grown in MMN liquid medium and P. byssinum was grown in liquid media containing different combinations of carbon and nitrogen as follows: high nitrogen/high carbon (HN-HC), high nitrogen/low carbon (HN-LC) and low nitrogen/high carbon (LN-HC). The basal medium was a modified version of MMN containing (l−1) 10 mm (HN) or 1.2 mm (LN) (NH4)2HPO4, 111 mm (HC) or 5.5 mm (LC) glucose, 140 mg MgSO4·7H2O, 50 mg CaCl2, 25 mg NaCl, 3 mg ZnSO4, 0.133 mg thiamine and 12.5 mg ferric ethylenediaminetetraacetic acid (EDTA). Media were adjusted to pH 5–5.5 before the addition of the ferric EDTA and autoclaving. Discs of inoculum (5 mm diameter) were cut from the leading edge of actively growing colonies from 2- to 3 wk-old MMN agar plates and four discs of fungus were inoculated into 5 cm diameter Petri dishes containing 11 ml liquid medium. Cultures were incubated at 23°C in the dark for 10 or 20 d before mRNA extraction.
mRNA isolation, cDNA synthesis and reverse transcriptase PCR
Mycelia were harvested, blotted briefly on filter paper to remove excess medium, frozen in liquid nitrogen and ground to a powder with a chilled mortar and pestle. mRNA isolation was carried using the Dynalbeads mRNA DIRECT Kit (Dynal, Oslo, Norway) according to the manufacturers instructions, and residual contaminating DNA was digested with amplification grade DNase I (Invitrogen, Groningen, The Netherlands). cDNA was synthesised from mRNA according to the manufacturers protocol with SUPERSCRIPT First-Strand Synthesis System for reverse transcriptase (RT)-PCR (Invitrogen). For RT-PCR amplification, a 2-µl aliquot from each RT reaction mixture was used as template for PCR using the conditions outlined above. Primers for a gene encoding glyceraldehyde-3-phosphate dehydrogenase were used as a positive control for cDNA amplification, as described by Franken et al. (1997).
Competitive RT-PCR reactions were conducted using P. byssinum cDNA from the different media, as described by Soden & Dobson (2001), whereby a series of plasmids (of known concentration) containing the gDNA sequence for the lac2 or lac3 fragments were spiked into the PCR reactions containing constant amounts of cDNA. The presence of introns (see Fig. 2) within the gDNA competitive templates allowed distinction from the smaller cDNA PCR products when run on agarose gels. Images of ethidium bromide-stained gels were analysed by densitometry based on average density using quantity one software 4.1.1 (Bio-Rad, Hercules, CA, USA). The transcript concentrations were calculated by determining concentrations at which the competitor gDNA and cDNA targets were equal, as described by Soden & Dobson, 2001). Competitive RT-PCR reactions were performed using cDNA from three replicate cultures on each harvest day and for each treatment and mean data compared by t-tests using Minitab software (Minitab Inc., State College, PA, USA, 1997).
Amplification using the lac1 primers failed to yield products for any of the fungi screened (data not shown). Using the lac2 primers, products (631–645 bp) were obtained from genomic DNA for only Lactarius sp. 2, Rhizopogon luteolus Fr., P. byssinum and P. fallax (Table 1, Fig. 1). Comparison of the ECM sequences with sequences available in the GenBank nucleotide database revealed closest matches with sequences for laccase genes from decomposer basidiomycetes, with sequence similarities of 55–61% (over 441–624 bp) (Table 2). Amplification products (198–207 bp) were obtained from genomic DNA using the lac3 primers for all Russulales taxa screened (except Lactarius sp. 2) along with the Atheliaceae taxa P. byssinum, P. fallax and T. fibrillosa (Table 1). These products had 63–78% identity over 50–153 bp with sequences for decomposer basidiomycete laccase genes (Table 2). Amino acid sequences deduced from P. byssinum lac2 and lac3 cDNA fragments had 70% and 63% identity, respectively, with amino acid sequences for Trametes villosa (Fr.) Kreisel (AAB47734) and Pleurotus sajor-caju (Fr.) Singer (AAG27434) laccase sequence, but only 56% and 32%, respectively, with those for a Agaricus bisporus (AAA17035) laccase sequence (data not shown).
The RT-PCR yielded appropriately sized products (478 and 144 bp for lac2 and lac3, respectively) for P. fallax in MMN (data not shown) and P. byssinum in all media (Fig. 1), indicating that both genes were expressed by the two fungi. Sequencing of cDNA products of RT-PCR reactions confirmed their identity as lac2 and lac3 fragments, and comparison of genomic and cDNA sequences confirmed the presence of three introns (52–56 bp) in the lac2 fragment and a single 58 bp intron in the lac3 fragment. All intron splice junctions conformed to the GT–AG rule (Fig. 2).
Under some conditions lac2 and lac3 transcript levels in P. byssinum were significantly higher on day 10 than on day 20; however, for other treatments there were no significant differences (P < 0.05) (Table 3). On day 10 there were clear differences between transcript concentrations in the three treatments, with HN-HC being significantly higher than LN-HC (P < 0.05) for both lac2 and lac3 (Table 3). Regardless of the treatment, lac3 transcript was present at a significantly higher concentration than lac2 transcript on day 10 (P < 0.05). While transcript concentrations were also highest for lac3 in the HN-HC treatment on day 20, the transcript concentration for HN-LC was significantly higher (P < 0.05) than for HNHC or LN-HC for lac2 and did not differ significantly (P < 0.05) between HN-LC and LN-HC for lac3 on this day (Table 3).
Table 3. Effect of medium composition on the transcript levels of laccase-like genes of Piloderma byssinum
The data we have presented indicate that several ECM fungal taxa possess laccase-like genes. While Chen et al. (2001) found that sequences for lignin peroxidase-like (LiP-like) genes were widespread in a broad taxonomic range of ECM basidiomycetes, laccase-like gene fragments were amplified from a more restricted group of taxa in the present study. The DNA from all taxa was PCR amplified using the internal transcribed spacer (ITS) primers ITS1 and ITS4 (White et al., 1990) to ensure viability before screening with the lac primers. Internal transcribed spacer products were obtained for all fungi screened (data not shown), allowing us to exclude the possibility that failure to obtain a product with one or more lac primer pair for a taxon simply reflected poor quality DNA template. With the exception of the Boletales taxon R. luteolus (lac2), laccase-like gene fragments were only amplified from the Russulales taxa (Lactarius spp. and Russula spp.) and from the resupinate Atheliaceae taxa (Piloderma spp. and Tylospora spp.). The Piloderma species were notable as being the only taxa from which two laccase-like genes were identified. Similarly, while Chen et al. (2001) found evidence for one or two lignin peroxidase-like genes in many ECM fungal taxa, four such genes were found in P. byssinum and P. fallax and three in T. fibrillosa. Moreover, manganese peroxidase-like genes were only observed in Piloderma and Tylospora spp., along with one other ECM basidiomycete, suggesting that these Atheliaceae taxa may have greater potential for producing ligninolytic enzymes than other ECM fungi (Chen et al., 2001). The presence of multiple laccase-like genes in Piloderma spp. provides further support for this tenet, as does the fact that mycelia of P. fallax and Tylospora spp. are generally associated with soil organic matter and are frequently observed in decaying wood in the field (Goodman & Trofymow, 1998; Erland & Taylor, 1999; Smith et al., 2000).
As would be predicted from their broader specificity, the degenerate primers (lac3) were more successful in identifying laccase-like sequences in ECM fungi than the more specific lac1 and lac2 primer pairs which were based on sequences from only a single saprotrophic taxon (Lentinula edodes) (Zhao & Kwan, 1999). It must be noted, however, that the lac3 primers have previously been shown to amplify laccase sequences from a broad range of saprotrophic basidiomycetes, but failed to yield PCR products from some white rot taxa that are known to produce laccase activities (D’Souza et al., 1996). This may reflect the presence of introns at the primer binding sites in the taxa for which amplification was unsuccessful (D’Souza et al., 1996), and it is possible that some of the ECM taxa in the present study failed to yield a PCR product for similar reasons. We therefore cannot exclude the possibility that laccase-like genes may be more widespread in ECM fungi than our data suggest. While many purported extracellular laccase activities associated with some ECM taxa probably reflect activities of other polyphenol oxidases (Burke & Cairney, 2002), there is convincing evidence for extracellular laccase production by T. terrestris (Kanunfre & Zancan, 1998). Our PCR-based screening, however, failed to identify laccase-like genes in T. terrestris or several other Thelephoraceae taxa. This emphasizes that failure to identify a laccase-like gene in many ECM taxa cannot necessarily be taken to indicate that they lack genes for laccases. Determining relative Km values for purified proteins against a range of appropriate substrates (Kanunfre & Zancan, 1998) may thus identify laccases in some of the taxa for which no laccase-like genes were identified in the present study.
Fungal laccases have been implicated in a number of processes, including lignin degradation, fungal growth, rhizomorph and basidiome morphogenesis, detoxification of phenolic compounds and pathogenesis (Thurston, 1994; Burke & Cairney, 2002). Laccase gene sequences from white rot basidiomycetes generally show a high degree of similarity to each other at the nucleotide level (Soden & Dobson, 2001), but show less similarity to others such as A. bisporus or ascomycetes (D’Souza et al., 1996; Mansur et al., 1998; Temp et al., 1999). It has been proposed that laccase genes that encode for different isozymes may have evolved in different fungi and that these might underpin different functions in different fungi and under different conditions (Temp et al., 1999; Soden & Dobson, 2001). Multiple genes that encode different laccase isozymes have been identified in several decomposer fungi (Yaver et al., 1996; Mansur et al., 1997; Temp et al., 1999; Soden & Dobson, 2001) and our data indicate that multiple laccase-like genes exist in ECM Piloderma spp. There is also strong evidence for differential expression of laccase genes in some decomposer fungal taxa, supporting the hypothesis that different laccase genes may be important under different conditions (Mansur et al., 1998; Zhao & Kwan, 1999; Soden & Dobson, 2001). The widespread occurrence of laccases in white rot basidiomycetes (Smith et al., 1998), their high redox potential (Youn et al., 1995) and a requirement for laccase expression for lignin degradation by some white rot fungi (Eggert et al., 1996b) strongly implicates white rot fungal laccases in lignin degradation processes. Indeed, the greater similarity of gene sequences for white rot laccases to each other than to sequences for laccases from other basidiomycetes has been suggested as indicating functional similarity in white rot laccases (Temp et al., 1999). The fact that the gene fragments from the ECM taxa had greatest similarities to white rot laccase genes may thus suggest a potential role in lignin degradation. Single laccase isozymes may, however, perform more than one function (Eggert et al., 1996a,b). Further work is clearly required to investigate the potential roles of ECM fungal laccase activities in nutrient mobilization and/or development of the various phases of the symbiosis.
This work was supported by an ARC large Grant (A00000685) awarded to JWGC. We thank Dr R. M. Burke for insightful discussions on laccases.