Laccase Transcript Research Articles

Cerrena unicolor Laccases, Genes Expression and Regulation of Activity.

A white rot fungus Cerrena unicolor has been identified as an important source of laccase, unfortunately regulation of this enzyme genes expression is poorly understood. Using 1D and 2D PAGE and LC-MS/MS, laccase isoenzymes were investigated in the liquid filtrate of C. unicolor culture. The level of expression of laccase genes was measured using qPCR. The elevated concentrations of copper and manganese in the medium caused greatest change in genes expression and three laccase transcripts were significantly affected after culture temperature was decreased from 28 to 4 °C or increased to 40 °C. The small differences in the PAGE band intensities of individual laccase proteins were also observed, indicating that given compound affect particular laccase’s transcript. Analyses of laccase-specific activity, at all tested conditions, showed the increased activities as compared to the control, suggesting that enzyme is regulated at the post-translational stage. We observed that the aspartic protease purified from C. unicolor, significantly stimulate laccase activity. Moreover, electrochemical analysis of protease-treated laccase sample had 5 times higher redox peaks. The obtained results indicate that laccases released by C. unicolor are regulated at transcriptional, translational, and at the post-translational steps of gene expression helping fungus adapt to the environmental changes.

Sound Vibration-Triggered Epigenetic Modulation Induces Plant Root Immunity Against Ralstonia solanacearum.

Sound vibration (SV) is one of the several environmental stimuli that induce physiological changes in plants including changes in plant immunity. Immune activation is a complicated process involving epigenetic modifications, however, SV-induced epigenetic modifications remain unexplored. Here, we performed an integrative analysis comprising chromatin immunoprecipitation (ChIP) and microRNA sequencing (miRNA-seq) to understand the role of SV-mediated epigenetic modifications in immune activation in Arabidopsis thaliana against the root pathogen Ralstonia solanacearum. Plants exposed to SV (10 kHz) showed abundant H3K27me3 modification in the promoter regions of aliphatic glucosinolate biosynthesis and cytokinin signaling genes, leading to transcriptional changes that promote immunity. Additionally, 10 kHz SV down-regulated miR397b expression, thus activating three target LACCASE transcripts that mediate cell wall reinforcement via lignin accumulation. Taken together, SV triggers epigenetic modification of genes involved in secondary metabolite biosynthesis, defense hormone signaling, and pre-formed defense in A. thaliana, leading to the activation of plant immunity against R. solanacearum.

Substrate-based differential expression analysis reveals control of biomass degrading enzymes in Pycnoporus cinnabarinus

White rot fungi possess a powerful ability to degrade recalcitrant lignin within plant biomass. Pycnoporus cinnabarinus PB 94 accomplishes lignin degradation through the combined activity of laccases, peroxidases, and their supporting enzymes. Assembly of the de novo transcriptome for PB 94 resulted in identification of 45,286 transcripts, including isoforms, and a predicted 27,990 genes. Differential expression analysis revealed how the expression of these ligninolytic enzymes was dictated by the nature of the substrate. Growth on lignin containing poplar and switchgrass resulted in as much as a 285-fold increase in expression of peroxidases (auxiliary activity family AA2) and a 36-fold increase in expression of alcohol oxidases (auxiliary activity family AA3) compared to growth on soluble sugars. These findings suggest that expression of these enzyme classes was controlled by the presence of lignin, or triggered by an aromatic break out product of lignin degradation. Laccases showed little significant regulation under these growth conditions, but one laccase transcript (TR10024|c0_g1_i1), similar to laccase gene lcc2 in Pycnoporus sanguineus, was greatly upregulated during growth on cellobiose, suggesting that this sugar may be a mechanism for enhanced expression. The findings presented here reveal regulation patterns of enzymes critical for the lignin degrading activity of P. cinnabarinus PB 94 providing insight into how the white rot fungus controls lignocellulolytic activity.

Laccase Gene Family in Cerrena sp. HYB07: Sequences, Heterologous Expression and Transcriptional Analysis.

Laccases are a class of multi-copper oxidases with industrial potential. In this study, eight laccases (Lac1–8) from Cerrena sp. strain HYB07, a white-rot fungus with high laccase yields, were analyzed. The laccases showed moderate identities to each other as well as with other fungal laccases and were predicted to have high redox potentials except for Lac6. Selected laccase isozymes were heterologously expressed in the yeast Pichia pastoris, and different enzymatic properties were observed. Transcription of the eight laccase genes was differentially regulated during submerged and solid state fermentation, as shown by quantitative real-time polymerase chain reaction and validated reference genes. During 6-day submerged fermentation, Lac7 and 2 were successively the predominantly expressed laccase gene, accounting for over 95% of all laccase transcripts. Interestingly, accompanying Lac7 downregulation, Lac2 transcription was drastically upregulated on days 3 and 5 to 9958-fold of the level on day 1. Consistent with high mRNA abundance, Lac2 and 7, but not other laccases, were identified in the fermentation broth by LC-MS/MS. In solid state fermentation, less dramatic differences in transcript abundance were observed, and Lac3, 7 and 8 were more highly expressed than other laccase genes. Elucidating the properties and expression profiles of the laccase gene family will facilitate understanding, production and commercialization of the fungal strain and its laccases.

Impact of agricultural management on bacterial laccase-encoding genes with possible implications for soil carbon storage in semi-arid Mediterranean olive farming.

Background: In this work, we aimed to gain insights into the contribution of soil bacteria to carbon sequestration in Mediterranean habitats. In particular, we aimed to use bacterial laccase-encoding genes as molecular markers for soil organic C cycling. Using rainfed olive farming as an experimental model, we determined the stability and accumulation levels of humic substances and applied these data to bacterial laccase-encoding gene expression and diversity in soils under four different agricultural management systems (bare soils under tillage/no tillage and vegetation cover under chemical/mechanical management).Materials and Methods: Humic C (> 104 Da) was subjected to isoelectric focusing. The GC-MS method was used to analyze aromatic hydrocarbons. Real-Time PCR quantification and denaturing gradient gel electrophoresis (DGGE) for functional bacterial laccase-like multicopper oxidase (LMCO)-encoding genes and transcripts were also carried out.Results: Soils under spontaneous vegetation, eliminated in springtime using mechanical methods for more than 30 years, showed the highest humic acid levels as well as the largest bacterial population rich in laccase genes and transcripts. The structure of the bacterial community based on LMCO genes also pointed to phylogenetic differences between these soils due to the impact of different management systems. Soils where herbicides were used to eliminate spontaneous vegetation once a year and those where pre-emergence herbicides resulted in bare soils clustered together for DNA-based DGGE analysis, which indicated a certain amount of microbial selection due to the application of herbicides. When LMCO-encoding gene expression was studied, soils where cover vegetation was managed either with herbicides or with mechanical methods showed less than 10% similarity, suggesting that the type of weed management strategy used can impact weed community composition and consequently laccase substrates derived from vegetation decay.Conclusions: We suggest that the low humic acid content retrieved in the herbicide-treated soils was mainly related to the type (due to vegetal cover specialization) and smaller quantity (due to lower vegetal biomass levels) of phenolic substrates for laccase enzymes involved in humification processes. We also found that spontaneous vegetal cover managed using mechanical methods could be the best option for achieving C stabilization in rainfed Mediterranean agroecosystems.

The Trametes hirsuta 072 laccase multigene family: Genes identification and transcriptional analysis under copper ions induction.

Laccases, blue copper-containing oxidases, ≿ an play an important role in a variety of natural processes. The majority of fungal laccases are encoded by multigene families that express closely related proteins with distinct functions. Currently, only the properties of major gene products of the fungal laccase families have been described. Our study is focused on identification and characterization of laccase genes, which are transcribed in basidiomycete Trametes hirsuta 072, an efficient lignin degrader, in a liquid medium, both without and with induction of laccase transcription by copper ions. We carried out production of cDNA libraries from total fungal RNA, followed by suppression subtractive hybridization and mirror orientation selection procedures, and then used Next Generation Sequencing to identify low abundance and differentially expressed laccase transcripts. This approach resulted in description of five laccase genes of the fungal family, which, according to the phylogenetic analysis, belong to distinct clusters within the Trametes genus. Further analysis established similarity of physical, chemical, and catalytic properties between laccases inside each cluster. Structural modeling suggested importance of the sequence differences in the clusters for laccase substrate specificity and catalytic efficiency. The implications of the laccase variations for the fungal physiology are discussed.

Validation of Reference Genes Aiming Accurate Normalization of qRT-PCR Data in Dendrocalamus latiflorus Munro

Background Dendrocalamus latiflorus Munro distributes widely in subtropical areas and plays vital roles as valuable natural resources. The transcriptome sequencing for D. latiflorus Munro has been performed and numerous genes especially those predicted to be unique to D. latiflorus Munro were revealed. qRT-PCR has become a feasible approach to uncover gene expression profiling, and the accuracy and reliability of the results obtained depends upon the proper selection of stable reference genes for accurate normalization. Therefore, a set of suitable internal controls should be validated for D. latiflorus Munro.ResultsIn this report, twelve candidate reference genes were selected and the assessment of gene expression stability was performed in ten tissue samples and four leaf samples from seedlings and anther-regenerated plants of different ploidy. The PCR amplification efficiency was estimated, and the candidate genes were ranked according to their expression stability using three software packages: geNorm, NormFinder and Bestkeeper. GAPDH and EF1α were characterized to be the most stable genes among different tissues or in all the sample pools, while CYP showed low expression stability. RPL3 had the optimal performance among four leaf samples. The application of verified reference genes was illustrated by analyzing ferritin and laccase expression profiles among different experimental sets. The analysis revealed the biological variation in ferritin and laccase transcript expression among the tissues studied and the individual plants.ConclusionsgeNorm, NormFinder, and BestKeeper analyses recommended different suitable reference gene(s) for normalization according to the experimental sets. GAPDH and EF1α had the highest expression stability across different tissues and RPL3 for the other sample set. This study emphasizes the importance of validating superior reference genes for qRT-PCR analysis to accurately normalize gene expression of D. latiflorus Munro.

Changes in xylem tissue and laccase transcript abundance associated with posture recovery in Chamaecyparis obtusa saplings growing on an incline.

Lignin is a major component of plant cell walls and is synthesised through oxidative polymerisation of monolignols. The transcription level of laccase, an enzyme implicated in monolignol polymerisation, is higher in the tissue forming compression wood than in normal wood. Compression wood, which is a special xylem tissue that develops to reorient inclined stems, also has a higher lignin content than normal wood. In the present study, Chamaecyparis obtusa Endl. saplings were grown on an incline and the following variables were tracked for 10 weeks: posture recovery of the saplings; development of xylem tissue on the lower side of inclined stems; and the transcription level of laccase. The posture of saplings approached vertical after 8 weeks, the development of compression wood reached a peak around 6 weeks and laccase transcription was the highest after 4 weeks. These results suggest a sequence of righting mechanisms. Inclination stimulates an increase in the abundance of laccase transcript and this increase encourages the formation of compression wood. The accumulation of compression wood then causes the stem to bend upward.

Genome expression profile analysis reveals important transcripts in maize roots responding to the stress of heavy metal Pb

Lead (Pb) has become one of the most abundant heavy metal pollutants of the environment. With its large biomass, maize could be an important object for studying the phytoremediation of Pb-contaminated soil. In our previous research, we screened 19 inbred lines of maize for Pb concentration, and line 178 was identified to be a hyperaccumulator for Pb in both the roots and aboveground parts. To identify important genes and metabolic pathways related to Pb accumulation and tolerance, line 178 was underwent genome expression profile under Pb stress and a control (CK). A total of approximately 11 million cDNA tags were sequenced and 4 665 539 and 4 936 038 clean tags were obtained from the libraries of the test and CK, respectively. In comparison to CK, 2379 and 1832 genes were identified up- or downregulated, respectively, more than fivefolds under Pb stress. Interestingly, all the genes were related to cellular processes and signaling, information storage and processing or metabolism functions. Particularly, the genes involved in posttranslational modification, protein turnover and chaperones; signal transduction, carbohydrate transport and metabolism; and lipid transport and metabolism significantly changed under the treatment. In addition, seven pathways including ribosome, photosynthesis, and carbon fixation were affected significantly, with 118, 12, 34, 21, 18, 72 and 43 differentially expressed genes involved. The significant upregulation of the ribosome pathway may reveal an important secret for Pb tolerance of line 178. And the sharp increase of laccase transcripts and metal ion transporters were suggested to account in part for Pb hyperaccumulation in the line.

Transcriptional analysis of Pleurotus ostreatus laccase genes

Fungal laccases (p-diphenol:oxygen oxidoreductase; EC 1.10.3.2) are multi-copper-containing oxidases that catalyse the oxidation of a great variety of phenolic compounds and aromatic amines through simultaneous reduction of molecular oxygen to water. Fungi generally produce several laccase isoenzymes encoded by complex multi-gene families. The Pleurotus ostreatus genome encodes 11 putative laccase coding genes, and only six different laccase isoenzymes have been isolated and characterised so far. Laccase expression was found to be regulated by culture conditions and developmental stages even if the redundancy of these genes still raises the question about their respective functions in vivo. In this context, laccase transcript profiling analysis has been used to unravel the physiological role played by the different isoforms produced by P. ostreatus. Even if reported results depict a complex picture of the transcriptional responses exhibited by the analysed laccase genes, they were allowed to speculate on the isoform role in vivo. Among the produced laccases, LACC10 (POXC) seems to play a major role during vegetative growth, since its transcription is downregulated when the fungus starts the fructification process. Furthermore, a new tessera has been added to the puzzling mosaic of the heterodimeric laccase LACC2 (POXA3). LACC2 small subunit seems to play an additional physiological role during fructification, beside that of LACC2 complex activation/stabilisation.

Phenol-oxidizing laccases from the termite gut

cDNAs encoding two gut laccase isoforms ( RfLacA and RfLacB) were sequenced from the termite Reticulitermes flavipes. Phylogenetic analyses comparing translated R. flavipes laccases to 67 others from prokaryotes and eukaryotes indicate that the R. flavipes laccases are evolutionarily unique. Alignments with crystallography-verified laccases confirmed that peptide motifs involved in metal binding are 100% conserved in both isoforms. Laccase transcripts and phenoloxidase activity were most abundant in symbiont-free salivary gland and foregut tissue, verifying that the genes and activities are host-derived. Using a baculovirus-insect expression system, the two isoforms were functionally expressed with histidine tags and purified to near homogeneity. ICP-MS (inductively coupled plasma – mass spectrometry) analysis of RfLacA identified bound metals consisting mainly of copper (∼4 copper molecules per laccase protein molecule and ∼3 per histidine tag) with lesser amounts of calcium, manganese and zinc. Both recombinant enzyme preparations showed strong activity towards the lignin monomer sinapinic acid and four other phenolic substrates. By contrast, both isoforms displayed much lower or no activity against four melanin precursors, suggesting that neither isoform is involved in integument formation. Modification of lignin alkali by the recombinant RfLacA preparation was also observed. These findings provide evidence that R. flavipes gut laccases are evolutionarily distinct, host-derived, produced in the salivary gland, secreted into the foregut, bind copper, and play a role in lignocellulose digestion. These findings contribute to a better understanding of termite digestion and gut physiology, and will assist future translational studies that examine the contributions of individual termite enzymes in lignocellulose digestion.

Hydroquinone and H2O2differentially affect the ultrastructure and expression of ligninolytic genes in the basidiomyceteCeriporiopsis subvermispora

The biodegradation of lignin is a highly oxidative process in which various oxidases and peroxidases play a major role. During lignin decay, the generation of aromatic compounds and reactive oxygen species leads to oxidative stress. In this work, the effect of the oxidative compounds H(2)O(2) and hydroquinone in the ligninolytic fungus Ceriporiopsis subvermispora was studied, both at the ultrastructural and at the transcriptional level. Transmission electron microscopy revealed the presence of microvesicles and extensive cytoplasm degeneration after incubation with hydroquinone, but not with H(2)O(2). Studies of the intracellular redox state of the fungus showed that hydroquinone causes a transient decrease in the reduced glutathione/oxidized glutathione (GSH/GSSG) ratio and an increase in the glutathione-S-transferase mRNA levels. These results suggest that hydroquinone produces oxidative stress in this microorganism. On the other hand, it was observed that hydroquinone, but not H(2)O(2), affects Mn-dependent peroxide and laccase transcripts levels. We propose that the mechanism by which the fungus reacts against oxidative stress contributes to its selectivity toward lignin during wood decay.

Transcription Factor STE12α Has Distinct Roles in Morphogenesis, Virulence, and Ecological Fitness of the Primary Pathogenic YeastCryptococcus gattii

Cryptococcus gattii is a primary pathogenic yeast, increasingly important in public health, but factors responsible for its host predilection and geographical distribution remain largely unknown. We have characterized C. gattii STE12alpha to probe its role in biology and pathogenesis because this transcription factor has been linked to virulence in many human and plant pathogenic fungi. A full-length STE12alpha gene was cloned by colony hybridization and sequenced using primer walk and 3' rapid amplification of cDNA ends strategies, and a ste12alpha delta gene knockout mutant was created by URA5 insertion at the homologous site. A semiquantitative analysis revealed delayed and poor mating in ste12alpha delta mutant; this defect was not reversed by exogenous cyclic AMP. C. gattii parent and mutant strains showed robust haploid fruiting. Among putative virulence factors tested, the laccase transcript and enzymatic activity were down regulated in the ste12alpha delta mutant, with diminished production of melanin. However, capsule, superoxide dismutase, phospholipase, and urease were unaffected. Similarly, Ste12 deficiency did not cause any auxotrophy, assimilation defects, or sensitivity to a large panel of chemicals and antifungals. The ste12alpha delta mutant was markedly attenuated in virulence in both BALB/c and A/Jcr mice models of meningoencephalitis, and it also exhibited significant in vivo growth reduction and was highly susceptible to in vitro killing by human neutrophils (polymorphonuclear leukocytes). In tests designed to simulate the C. gattii natural habitat, the ste12alpha delta mutant was poorly pigmented on wood agar prepared from two tree species and showed poor survival and multiplication in wood blocks. Thus, STE12alpha plays distinct roles in C. gattii morphogenesis, virulence, and ecological fitness.

A molecular method to evaluate basidiomycete laccase gene expression in forest soils

Fungal laccases, exo-enzymes lacking high substrate specificity, play a central role in cycling of soil organic matter. In a precedent work based on a PCR technique on soil DNA extracts, we showed that the highest diversity of laccase genes occurred in the most organic horizon (i.e., O h) of a brown forest soil. In the present article, we develop a method of RNA extraction, RT-PCR, and semi-quantitative PCR to analyze the expression of Basidiomycete laccase genes. We performed a very first assessment of the methodological approach on five cores of the O h horizon of the same brown forest soil. The level of laccase transcripts was heterogeneous amongst the soil cores. Two samples gave strong expression levels, two showed very faint ones, and the last replicate presented no detectable laccase transcripts. A control with a transcript analysis of the actin gene, which is constitutively expressed in fungi, allowed to rule out that the differences in the transcript level of laccases were due to experimental failures or inhibiting substances in the RNA extracts. A cladistic analysis showed that most laccase transcript sequences detected were grouped in two clades closely related to the ectomycorrhizal fungus Xerocomus chrysenteron. Comparison between DNA laccase sequences found in our previous study and RNA laccase sequence profiles found here showed that less than 30% of the laccases genes detected in a soil core were expressed. This preliminary study demonstrates the potential of RT-PCR for gene expression profiling in forest soils. The number of analyzed samples cannot allow us to draw definitive ecological conclusions, but there are some indications that differences between rhizospheric and bulk soil samples (polyphenol abundances, microbial densities, etc.) might be a potential explanation for the variable laccase expression observed.

Tannic acid induces transcription of laccase gene cglcc1 in the white-rot fungus Coriolopsis gallica.

Laccase, a phenoloxidase enzyme secreted by white-rot fungi, has a significant role in the degradation of lignin and environmental pollutants. Coriolopsis gallica is a ligninolytic basidiomycete that produces high levels of this extracellular enzyme. A laccase gene cglcc1 from this fungus has been cloned and sequenced. The capacity of C. gallica to efficiently degrade polyphenols has been successfully applied in our laboratory to the biotreatment and decolorization of several industrial wastewaters. This study focused on the effect of tannic acid, a natural compound widely distributed in plants, on the production of laccase activity by C. gallica. Our results showed an evident increase of extracellular laccase levels when C. gallica was grown in the presence of tannic acid. Concentrations of 50 and 100 microM of this compound increased laccase activity when compared with control samples grown without tannic acid. In addition, we found an increase in laccase transcript levels in C. gallica grown in culture media supplemented with tannic acid. The role of tannic acid was shown to be an inductor of laccase activity in this fungus, due to the enhancement of expression of the laccase gene at the transcriptional level.

Transcriptional regulation of laccase and cellulase genes during growth and fruiting of Lentinula edodes on supplemented sawdust

Transcription of laccase and cellulase genes of Lentinula edodes was examined during growth and development under different temperature and moisture levels on a sawdust-based substrate. RNA was extracted from samples of mycelium and fruit bodies at various stages of development and gene expression was determined by competitive RT-PCR. The level of laccase transcripts was maximal during the mycelial growth stage, and then declined rapidly at the fruiting stage. In contrast, the cellulase transcript level peaked at the veil–break stage during fruit body development. Gene expressions for laccase and cellulase were triggered by microclimatic changes, specifically lower temperature and osmotic pressure.

Transcriptional regulation of laccase and cellulase genes during growth and fruiting ofLentinula edodeson supplemented sawdust

Transcription of laccase and cellulase genes of Lentinula edodes was examined during growth and development under different temperature and moisture levels on a sawdust-based substrate. RNA was extracted from samples of mycelium and fruit bodies at various stages of development and gene expression was determined by competitive RT-PCR. The level of laccase transcripts was maximal during the mycelial growth stage, and then declined rapidly at the fruiting stage. In contrast, the cellulase transcript level peaked at the veil-break stage during fruit body development. Gene expressions for laccase and cellulase were triggered by microclimatic changes, specifically lower temperature and osmotic pressure.

Molecular Cloning and Expression of Eight Laccase cDNAs in Loblolly Pine (Pinus taeda)*

Eight cDNA clones for laccase (Lacl-Lac8) were ilated and characterized for analysis of the role of laccases in xylem formation by molecular and genetic approaches. Five of them (Lacl-5) were isolated from cDNA libraries derived from differentiating xylem through a cDNA sequencing project in loblolly pine xylem (Allona et a/. 1998) and other three (Lac6-8) were isolated using a probe from a tobacco blue copper oxidase. Analysis of the cDNAs revealed that the proteins predicted had N-terminal signal sequences, 8-21 glycosylation sites, and four copper binding sites. The putative mature laccases range between 59.2 and 61.7 kD. The predicted isoelectric points vary between 7.3 and 9.9. Phylogenetic analysis shows these laccase cDNAs form three clusters in the cationic plant laccase group. Northern analysis indicated that all eight of the laccase transcripts are most abundant in differentiating xylem of the six tissues and organs tested. The Lac1 transcript is also detectable in immature pollen cones, and the Lac 7 transcript is detectable in several organs tested. The predominance of transcript abundance in differentiating xylem suggests that laccases play an important role in pine xylem development, consistent with previous hypotheses that laccases are involved in lignin synthesis in differentiating xylem.

Transcriptional regulation of laccase and cellulase genes in the mycelium of Agaricus bisporus during fruit body development on a solid substrate

The cultivated mushroom Agaricus bisporus was grown and fruited in wheat straw compost. RNA was extracted from samples of mycelium growing in compost during colonization and fruit body development. Laccase gene (combined lcc1 and lcc2) expression and cel3 gene (cellobiohydrolase) expression were determined by Northern blot analysis and by competitive RT-PCR. The level of laccase transcripts was highest in colonized compost (prior to the onset of fruiting), declined during the period of fruit body enlargement and increased again after harvesting and during the second flush of fruit body production. No transcripts of the cel3 gene were detectable in colonized or pre-fruiting cultures, but the level became detectable and rose to a maximum at the veil-break stage of fruiting, declined after harvesting and rose again during the second flush. Gene expression for laccase and cellobiohydrolase correlated with the known changes of laccase and cellulase enzyme activities during the fruiting life cycle.

Regulation of Laccase Gene Transcription in Trametes versicolor

The expression of laccase in the white rot fungus Trametes versicolor is regulated at the level of gene transcription by copper and nitrogen. We used reverse transcription-PCR to demonstrate that as the concentration of copper or nitrogen in fungal cultures was increased, an increase in laccase activity, corresponding to increased laccase gene transcription levels, was observed. In addition, we demonstrated that the amounts of laccase mRNA and laccase activity in 10-day-old cultures were a direct function of the concentration of either 1-hydroxybenzotriazole, a previously described laccase substrate, or 2,5-xylidine, a well-known laccase inducer, in the medium. No induction was observed after the addition of two aromatic acids, ferulic acid and veratric acid, which have been shown to induce laccase production in other white rot fungi. When either copper, 2,5-xylidine, or both compounds were added to cultures grown in the absence of copper, increased laccase transcript levels were detected within 15 min. Corresponding increases in laccase activity were observed after 24-h incubation only when copper was present.