Resveratrol acts as a natural profungicide and induces self-intoxication by a specific laccase

Molecular Microbiology (2002) 43(4), 883–894

Resveratrol acts as a natural profungicide and induces self-intoxication by a specific laccase Alexander Schouten, Lia Wagemakers, Francesca L. Stefanato, Rachel M. van der Kaaij and Jan A. L. van Kan* Wageningen University Plant Sciences, Laboratory of Phytopathology, PO Box 8025, 6700 EE Wageningen, The Netherlands. Summary The grapevine (Vitis) secondary metabolite resveratrol is considered a phytoalexin, which protects the plant from Botrytis cinerea infection. Laccase activity displayed by the fungus is assumed to detoxify resveratrol and to facilitate colonization of grape. We initiated a functional molecular genetic analysis of B. cinerea laccases by characterizing laccase genes and evaluating the phenotype of targeted gene replacement mutants. Two different laccase genes from B. cinerea were characterized, Bclcc1 and Bclcc2. Only Bclcc2 was strongly expressed in liquid cultures in the presence of either resveratrol or tannins. This suggested that Bclcc2, but not Bclcc1, plays an active role in the oxidation of both resveratrol and tannins. Gene replacement mutants in the Bclcc1 and Bclcc2 gene were made to perform a functional analysis. Only Bclcc2 replacement mutants were incapable of converting both resveratrol and tannins. When grown on resveratrol, both the wild type and the Bclcc1 replacement mutant showed inhibited growth, whereas Bclcc2 replacement mutants were unaffected. Thus, contrary to the current theory, BcLCC2 does not detoxify resveratrol but, rather, converts it into compounds that are more toxic for the fungus itself. The Bclcc2 gene was expressed during infection of B. cinerea on a resveratrol-producing host plant, but Bclcc2 replacement mutants were as virulent as the wild-type strain on various hosts. The activation of a plant secondary metabolite by a pathogen introduces a new dimension to plant– pathogen interactions and the phytoalexin concept. Introduction Laccases

are

copper-containing

phenol

oxidases

Accepted 14 November, 2001. *For correspondence. E-mail: [email protected]; Tel, (+31) 317 483126; Fax (+31) 317 483412.

© 2002 Blackwell Science Ltd

(p-diphenol:oxygen oxidoreductase, EC 1.10.3.2) belonging to a small group of enzymes known as large blue copper proteins or blue copper oxidases (Thurston,1994). They catalyse the oxidation of phenolic compounds together with the reduction of molecular oxygen into water (Messerschmidt and Huber, 1990) and attract attention because of their industrial applications, such as the bleaching of paper pulp (Akhtar et al., 1992) or the remediation of xenobiotic effluents (Bar and Aust, 1994). Laccases are typically glycoproteins, found in multiple isoforms in plants, fungi and bacteria and have different functions. The substrate specificity differs from one laccase to the other; some convert only one type of compound, whereas others have a wide substrate range (Thurston, 1994). In fungi, laccases are involved in many different processes, e.g. delignification (Lewis and Yamamoto, 1990; Hatakka, 1994), pigmentation (Clutterbuck, 1990), morphogenesis (Leatham and Stahmann, 1981) and the generation of active oxygen species (Guillén et al., 2000). For many species of fungi, two or more laccase genes or laccase isozymes have been characterized (Wahleithner et al., 1996; Jönsson et al., 1997; Muñoz et al., 1997; Scherer and Fischer, 1998; Zhao and Kwan, 1999), some of which have overlapping specificity (Kim et al., 1995). Laccase activity of the plant pathogenic fungus Botrytis cinerea not only converts tannins in grape berries, resulting in a reduced tenability of the wine produced from them, but is also considered to play a role in the degradation of the secondary metabolite resveratrol. Resveratrol (trans-3,5,4¢-trihydroxystilbene) is considered a phytoalexin, which protects the host from fungal infection (Breuil et al., 1999). Grapevine (Vitis vinifera), peanut (Arachis hypogaea) and several other plant species can accumulate resveratrol in leaves to high concentrations (400 mg g–1 fresh weight), and the concentration correlates with the resistance level against B. cinerea (Sbaghi et al., 1995). Transgenic tobacco, tomato, alfalfa and grapevine plants, in which resveratrol synthase genes were introduced, displayed an increased resistance against certain fungi, including B. cinerea (Hain et al., 1993; Thomzik et al., 1997; Hipskind and Paiva, 2000; Coutos-Thévenot et al., 2001). Based on biochemical data, it is generally assumed that B. cinerea must detoxify resveratrol by means of laccase activity in order to colonize grapevine (Pezet et al., 1991; Adrian et al., 1998; Breuil et al., 1999).

884 A. Schouten et al.

Fig. 1. Comparison of the B. cinerea laccases BcLCC1 and BcLCC2 with laccases from Cryphonectria parasitica (CpLAC1, GenBank acc. no. Q03966), Colletotrichum lagenarium (ClLAC1, GenBank acc. no. AB32575), Neurospora crassa (NcLAC1, GenBank acc. no. P06811; and NcLAC2, GenBank acc. no. P10574) and Podospora anserina (PaLAC2, GenBank acc. no. P78722). Black boxes represent amino acids that are identical in all sequences. Open boxes indicate identity in six of the seven aligned protein sequences. The four copper-binding motifs (His-X-His) are underlined, and the two conserved glycosylation signals are indicated by a dotted underline. © 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 883–894

Self-intoxication by a fungal laccase 885 We initiated a functional molecular genetic analysis of B. cinerea laccases by characterizing laccase genes and evaluating the phenotype of targeted gene replacement mutants. Contrary to the current theory, one of the laccase genes that was analysed is induced by resveratrol and encodes an enzyme that converts resveratrol into a fungitoxic compound. The role of fungal genes in the activation of prophytoalexins is discussed.

Results Isolation and characterization of complete laccase genes from B. cinerea

Fig. 2. Expression of Bclcc2 and Bclcc1 mRNA after the addition of potential laccase substrate. Mycelium was pregrown, harvested by filtration and divided equally into flasks with fresh medium. The cultures were supplemented, at t = 0, with 50 mg l–1 resveratrol (dissolved in methanol), 125 mg l–1 tannic acid + 200 mM CuSO4 or, as a control, 0.2% (v/v) methanol, and incubation was continued. Samples were taken at different time points (h.p.i. indicated at the top of the lanes). RNA was extracted and analysed by hybridization using specific probes for Bclcc1, Bclcc2 or B. cinerea 27S ribosomal DNA. The lane numbers below the autoradiograms are referred to in the text.

Two different laccase genes, including flanking regions, were isolated from a l EMBL3 genomic library of B. cinerea. The first, Bclcc1 (GenBank accession number AF243854), is homologous to the partial B. cinerea laccase gene fragment (GenBank accession number U20192) that was used for screening. The 1871nucleotide-long sequence contains three putative introns and encodes a protein of 561 amino acids. An algorithm for signal peptidase prediction indicates that the first 20 amino acids serve as a putative signal sequence for secretion (Nielsen et al., 1997). The second, Bclcc2 (GenBank accession number AF243855), is significantly different from Bclcc1 and spans 2042 nucleotides. It also contains three introns, which were confirmed by cDNA analysis, and encodes a protein of 581 amino acids. The first 21 amino acids serve as a putative signal sequence for secretion. At the amino acid level, BcLCC2 shows a high homology with the predicted BcLCC1 (amino acid sequence identity of 52.8%) and with laccases from Cryphonectria parasitica, Colletotrichum lagenarium, Neurospora crassa and Podospora anserina (Fig. 1). The four His-X-His copper binding site motifs are conserved, and the areas surrounding these motifs are highly homologous. In addition, two signals for N-linked glycosylation (Asn-X-Ser/Thr) are conserved.

Bclcc2 was expressed in wild-type B. cinerea immediately after the addition of methanol in the control culture. This may result from a general stress response to methanol, as the accumulation of transcripts decreased relatively quickly again (Fig. 2, lanes 2 and 3). In contrast, Bclcc2 transcripts accumulated to considerably higher levels between 30 min and 11 h after adding resveratrol (Fig. 2, lanes 6–10). The addition of tannic acid, dissolved in water, also resulted in the rapid accumulation of Bclcc2 transcripts, especially after 1 h (Fig. 2, lane 14). During the incubation, the liquid cultures containing either resveratrol or tannic acid gradually turned brown, indicating that the substrate was being converted (Hoos and Blaich, 1990). This conversion seemed to have been completed within 24 h, as no Bclcc2 transcripts could be detected 24 h after induction (Fig. 2, lanes 11 and 16). The rapid Bclcc2 induction and the complete absence of any detectable Bclcc1 expression suggest that Bclcc2, but not Bclcc1, plays an active role in the oxidation of both resveratrol and tannins.

Bclcc2 expression is induced by tannic acid and resveratrol

Bclcc2-deficient mutants are incapable of converting both tannic acid and resveratrol

To determine whether or not expression of Bclcc1 and Bclcc2 was inducible by potential substrates, mycelium of wild-type B. cinerea was grown in liquid cultures and supplemented with resveratrol, tannic acid or methanol, which is the solvent of resveratrol and served as a control. The mycelium was harvested at various time points after induction. Total RNA was isolated and analysed by hybridization. None of these growing conditions resulted in the accumulation of detectable levels of Bclcc1 transcripts (Fig. 2).

The role of Bclcc1 and Bclcc2 in tannin and resveratrol conversion was analysed further by generating B. cinerea gene replacement mutants. Each laccase gene was individually mutated by targeted integration of a hygromycin selection marker through homologous recombination (Fig. 3A and B). Transformants carrying the proper mutation in either the Bclcc1 or the Bclcc2 gene were selected by Southern blot analysis (Fig. 3C and D). Therefore, genomic DNA was isolated from mycelium of several independent Bclcc1 and Bclcc2 transformants and

© 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 883–894

886 A. Schouten et al.

Fig. 3. Organization of the Bclcc1 (A) and Bclcc2 (B) loci before and after disruption by integration of a hygromycin selection marker through homologous recombination. The translation start (arrow) and stop codons (asterisk), the introns (grey boxes) and restriction sites are indicated. Dotted boxes represent the hygromycin selection marker (Hygr). C. Southern analysis of individual Bclcc1 transformants. Genomic DNA was digested with EcoRI and hybridized with a partial B. cinerea Bclcc1 gene fragment (GenBank accession number U20192). The arrows indicate the positions of the 2.3 kb EcoRI wild-type fragment and the 4.6 kb EcoRI replacement fragment that are indicative of proper recombination. The lane numbers representing independent transformants with the proper gene replacement are underlined (lanes 1, 3 and 6). D. Southern analysis of individual Bclcc2 transformants. Genomic DNA was digested with BspHI and hybridized with Bclcc2 cDNA. The arrows indicate the positions of the 2.0 kb BspHI wild-type fragment and the 3.7 kb BspHI replacement fragment that are indicative of proper recombination. The lane numbers representing independent transformants with the proper gene replacement are underlined (lanes 4 and 6–11).

digested with EcoRI and BspHI respectively. The blot containing EcoRI-digested DNA from the Bclcc1 transformants was hybridized with a partial Bclcc1 gene fragment. Three individual Bclcc1 transformants with the proper replacement were identified (Fig. 3A and C, lanes 1, 3 and 6), in which the 2.3 kb wild-type EcoRI fragment (Fig. 3A and C, lane 11) is replaced by a 4.6 kb fragment. As a consequence, part of the Bclcc1 gene is replaced by the hygromycin selection marker. The blot containing BspHI-digested DNA from the Bclcc2 transformants was hybridized with the Bclcc2 cDNA probe. In the event of a proper homologous recombination, the 2.0 kb wild-type BspHI fragment (Fig. 3B and D, lane 12) is replaced by a 3.7 kb fragment, in which part

of the Bclcc2 gene has been replaced by the selection marker. Seven independent Bclcc2 transformants were identified that carried the desired recombination. One of them contained an additional ectopic integration (Fig. 3D, lane 5), whereas the remaining six were clean Bclcc2 replacement mutants (Fig. 3D, lanes 4 and 6–11). Kerssies (1990) reported that B. cinerea wild-type strains are capable of converting tannic acid, added to agar medium, by a laccase-like enzyme into a brown pigment (Fig. 4). All three independent Bclcc1 replacement mutants remained, like the wild-type B05.10, capable of converting tannic acid, whereas all six independent Bclcc2 replacement mutants had lost the capability to convert tannic acid (Fig. 4). © 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 883–894

Self-intoxication by a fungal laccase 887

Fig. 4. Plate assay to visualize the tannin-converting ability of wildtype B. cinerea (w.t.), two independent Bclcc2 replacement mutants (DBclcc2, A and B) and one Bclcc1 replacement mutant (DBclcc1). Tannin conversion results in a brown precipitate underneath and around the growing mycelium.

Resveratrol has a UV absorption spectrum with a peak at 302 nm that is absent in oxidation products. This property can be used in a spectrophotometric enzyme assay (Pezet et al., 1991). The wild-type and mutant strains were grown in liquid cultures in the presence or absence of tannins or resveratrol, and the culture filtrate was analysed 19 h after induction. Figure 6 shows that culture filtrates obtained from the B. cinerea wild-type strain and the Bclcc1 replacement mutants converted resveratrol only when the culture was induced with either tannins or resveratrol (Fig. 6A and B). Culture filtrates from Bclcc2 replacement mutants did not convert resveratrol in any of the treatments (Fig. 6C). In all, disruption of the Bclcc2 gene leads to the loss of both tannin and resveratrol-converting ability. Bclcc1 and

The resveratrol conversion activity was determined both in a plate assay (Fig. 5) and spectrophotometrically (Fig. 6). Resveratrol degradation was visualized on agar plates containing 200 mg ml–1 resveratrol (Fig. 5). At this concentration, resveratrol is only partly dissolved, and particles can be observed. When such plates were inoculated with wild-type B. cinerea or Bclcc1 replacement mutants, particles were dissolved 1–2 mm in front of the advancing mycelium, forming a yellow halo in the agar around the colony as a result of the accumulation of oxidation products (Hoos and Blaich, 1990). This oxidation was not observed for any of the six independent Bclcc2 replacement mutants. The resveratrol particles were unaffected and remained present under the growing mycelium (Fig. 5).

Fig. 5. Plate assay to visualize resveratrol conversion by B. cinerea. Agar plates contain 200 mg ml–1 resveratrol. Precipitates of resveratrol in the agar are dissolved by the advancing wild-type B. cinerea mycelium (on the left) and converted to yellow compounds. White arrows indicate the border of the halo resulting from resveratrol conversion. The mycelium itself stains yellow. Resveratrol conversion cannot be observed for the Bclcc2 replacement mutant (on the right). © 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 883–894

Fig. 6. Evaluation of resveratrol conversion by spectrophotometric analysis. Mycelium from wild-type B. cinerea (A) and Bclcc1 (B) and Bclcc2 (C) replacement mutants was grown in liquid in the presence of resveratrol (open diamonds), tannins (open circles) or methanol (control, open triangles) for 19 h. The culture filtrate was collected and incubated with fresh resveratrol. The OD302 of the reaction mixture was monitored over time. A decrease in OD302 is indicative of resveratrol conversion.

888 A. Schouten et al. additional laccase-like genes in the B. cinerea genome do not functionally complement for the absence of the Bclcc2 gene under the circumstances tested. Characterization of resveratrol conversion products The samples used for spectrophotometric analysis (Fig. 6) were analysed by high-performance liquid chromatography (HPLC) after 2 h incubation with resveratrol. Culture filtrates of the uninduced wild-type, Bclcc1 replacement mutants, as well as the induced and uninduced Bclcc2 replacement mutants showed identical HPLC profiles containing a single peak with a retention time of ª 19.6 min (Fig. 7A). The retention time and the UV spectrum of the peak were identical to that of pure trans-resveratrol (see Fig. 7C, spectrum A). In all samples, the amount of resveratrol (calculated from the peak area) was also very similar when compared with the control assay of resveratrol to which no culture filtrate was added. Samples of culture filtrates from the induced wild type or Bclcc1 replacement mutants, incubated for 2 h with resveratrol, showed peaks with retention times of ª 19.9 and 23.6 min respectively (Fig. 7B). UV spectrum analysis confirmed that the first peak represented transresveratrol, albeit at an ª 150-fold lower concentration than in the control, thus confirming the conversion of resveratrol as shown in Fig. 6. The second peak showed a different spectrum (Fig. 7C, spectrum B) when compared with trans-resveratrol (Fig. 7C, spectrum A). Spectrum B is in full agreement with the spectrum of the resveratrol dimer e-viniferin reported by Langcake and Pryce (1977a) and Jeandet et al. (1997). In vitro growth of Bclcc2 replacement mutants is not affected by resveratrol To study the effect of resveratrol and its oxidation products on growth, agar plugs containing mycelium of the wild type and Bclcc1 and Bclcc2 replacement mutants were placed on the same agar plate containing 200 mg ml–1 resveratrol (Fig. 8). This is a physiologically relevant concentration, as grapevine leaves can accumulate resveratrol up to 400 mg g–1 fresh weight, depending on the cultivar (Sbaghi et al., 1995). The Bclcc2 mutant is hardly affected by the presence of resveratrol (Fig. 8A and B). The mycelium grew almost as rapidly as on the control plate lacking resveratrol; it had a white colour and formed no yellow halo in the agar. In contrast, both the wild type and the Bclcc1 mutant showed inhibited growth (Fig. 8A and B). Growth of the Bclcc2 replacement mutant was severely inhibited as soon as the colony reached the halo produced by the wild type or the Bclcc1 replacement mutant colony (Fig. 8C). This result opposes the hypothesis that laccase activity detoxifies resveratrol (Pezet

Fig. 7. HPLC analysis and UV spectra of resveratrol conversion products. Mycelium of wild-type B. cinerea and Bclcc2 replacement mutants were grown in liquid in the presence of resveratrol for 19 h. The culture filtrate was collected and incubated with resveratrol for 2 h. The reaction mixture was separated by HPLC on a reversephase column. A. Incubation with culture filtrate obtained from the Bclcc2 replacement mutants yields a peak at 19.6 min, with a similar retention time and an identical spectrum to pure resveratrol (C, spectrum A). B. Incubation with culture filtrate obtained from wild-type B. cinerea yields two peaks at 19.9 and 23.6 min respectively. The first peak, identified by its UV spectrum as resveratrol, was present in ª 150-fold lower amounts than in (A) (note the different scales). The UV spectrum of peak B was different from that of peak A. C. Comparison of UV spectra of peaks A and B. The maximum peaks in spectrum A have shifted to higher wavelengths. The spectrum of peak B matches that of e-viniferin reported by Langcake and Pryce (1977a) and Jeandet et al. (1997). © 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 883–894

Self-intoxication by a fungal laccase 889

Fig. 9. Accumulation of Bclcc2 mRNA in peanut leaves at different time points (h.p.i., indicated at the top of the lanes) after spray inoculation with B. cinerea conidia. RNA was extracted and analysed by hybridization using specific probes for Bclcc1 (not shown), Bclcc2 (top) or a plant ribosomal DNA probe (bottom), the latter serving as a measure of total RNA loading.

Fig. 8. Growth of wild-type and mutant B. cinerea in the presence of resveratrol. Agar plates containing 200 mg ml–1 resveratrol were inoculated with agar plugs containing mycelium of wild-type B. cinerea, two individual Bclcc2 (DBclcc2, A and B) replacement mutants and one Bclcc1 (DBclcc1) replacement mutant. Pictures were taken 4 (A) and 6 (C) days after inoculation. B. A control plate lacking resveratrol at 4 days after inoculation.

et al., 1991; Adrian et al., 1998; Breuil et al., 1999). Apparently, the resveratrol derivatives formed by BcLCC2 activity are more toxic for the fungus than resveratrol itself. Bclcc2 expression during colonization and its role in virulence on peanut and grapevine leaves Resveratrol accumulation can be observed in grapevine leaves around B. cinerea infection sites under UV light on the basis of its blue fluorescence (Langcake and © 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 883–894

McCarthy, 1979). Strong blue fluorescence was observed in both grapevine and peanut leaves infected with B. cinerea at the periphery of the lesion but not inside the colonized tissue (results not shown). In an infection assay on peanut leaves, Bclcc2 transcripts were detected (Fig. 9) from 24 h post infection (h.p.i.) onwards. The transcript levels increased steadily up to 120 h.p.i. At this time point, the peanut leaves were almost entirely colonized by the fungus. Bclcc1 transcripts could not be detected at any time during the infection (results not shown). The presence of resveratrol in infected plant tissue as a substrate for BcLCC2, the induction of Bclcc2 gene expression in planta, combined with the generation of fungitoxic resveratrol oxidation products by BcLCC2 raised the question whether virulence of a Bclcc2deficient mutant might be increased on resveratrolproducing plants. Several independently obtained Bclcc2 replacement mutants did not show any increase in virulence on peanut and grapevine leaves when compared with the wild type (results not shown). Primary lesion formation and the rate of lesion outgrowth remained unaltered. Also, on hosts that do not produce resveratrol (tomato, common bean Phaseolus vulgaris), the virulence of Bclcc2 mutants was unaltered compared with the wild type (results not shown). Discussion Plants can produce a vast array of antimicrobial secondary metabolites, known as phytoalexins. Chemically divergent groups of phytoalexins have been described (Ingham, 1973; Bailey and Mansfield, 1982), and their production is considered to be an important disease resistance mechanism. The detoxification of phytoalexins by fungi is considered as an important countermeasure to deal with this chemical barrier that facilitates the colonization of a host (van den Heuvel, 1976; Tegtmeier and VanEtten, 1982; VanEtten et al., 1989; Osbourn, 1996; Morrissey and Osbourn, 1999). Our results demonstrate for the first time that this straightforward view may not always apply. In fact, a plant pathogen itself can be

890 A. Schouten et al. actively involved in the process leading to the chemical activation of a phytoalexin, which in this case acts as a natural profungicide. It was reported previously that, in grapevine, the phytoalexin resveratrol is converted into more fungitoxic compounds, the viniferins, around a B. cinerea infection site by a poorly understood mechanism (Langcake and Pryce, 1977b; Langcake, 1981). Resveratrol synthesis can also be induced to high levels by UV irradiation or infection with the fungus Plasmopara viticola but, under these circumstances, no accumulation of viniferins could be detected (Langcake and Pryce, 1977b; Dercks and Creasy, 1989). Using a molecular genetic approach, we firmly prove that B. cinerea carries a laccase gene that is induced by resveratrol and is responsible for converting non-toxic resveratrol into fungitoxic compounds, causing self-intoxication. This is in full agreement with the previous findings that the viniferins can only be detected in grapevine tissue infected with B. cinerea. Based on biochemical data, laccase activity of B. cinerea was always considered to be involved in the detoxification of resveratrol by oxidation (Pezet et al., 1991; Adrian et al., 1998; Breuil et al., 1999). However, the reported laccase was isolated from 15-day-old uninduced B. cinerea cultures (Pezet et al., 1991; Adrian et al., 1998; Breuil et al., 1999). These cultures may have contained a different phenol oxidase, not inducible by resveratrol, that is capable of converting resveratrol into another derivative, e.g. the resveratrol trans-dehydromer. It is unclear at this point which resveratrol oxidation product is the active toxic compound against B. cinerea. The major conversion product that we identified by HPLC displays a UV spectrum (Fig. 7C) that matches that of e-viniferin (Langcake and Pryce, 1977a; Jeandet et al., 1997). To confirm that viniferins are indeed the active toxic compounds is, however, complicated. Recent studies show that an additional class of resveratrol derivatives, named restrytisols, can be synthesized by B. cinerea grown in the presence of resveratrol (Cichewiz et al., 2000). Both viniferins and restrytisols encompass a range of isomeric dimers and oligomers that readily undergo auto-oxidation to form a spectrum of products, all potentially varying in toxicity. The different resveratrol derivatives that were reported may very well be generated by different laccases encoded in the B. cinerea genome. It is highly unlikely that Bclcc1 is responsible for this additional laccase activity, as we could never detect its expression, either during colonization of peanut leaves or in liquid cultures. Recently, we partially characterized a third laccase gene, Bclcc3 (GenBank accession no. AY047482), which is different from Bclcc1 and Bclcc2 and is expressed in liquid cultures only at prolonged incubation times and independently of the presence of resveratrol (data not shown). We also

found resveratrol-converting activity in 5-day-old liquid cultures of both wild type and Bclcc2 replacement mutants grown in the absence of resveratrol (data not shown). It remains to be determined by mutational analysis whether Bclcc3 is the gene encoding the laccase responsible for this particular activity. It is conceivable that even more than three laccase genes are present, as reported for another plant pathogenic fungus, Rhizoctonia solani (Wahleithner et al., 1996). The presence of additional functional laccase genes may explain how distinct resveratrol derivatives can be synthesized during different growth conditions and during pathogenesis. It may also explain why the Bclcc2 gene replacement mutants do not display an increased virulence level on leaves of both peanut and grapevine. The principle of induced self-intoxication raises the question why a potentially undesirable gene is retained in the B. cinerea genome. If it would impose a serious disadvantage, new strains would be selected in which the Bclcc2 gene is deleted or its expression repressed. To our knowledge, no such strains have been reported. It may be that, in the long run, the fungus does benefit from the expression of the Bclcc2 gene. In grapevine, it was reported that resveratrol levels decline during ripening of the berries, resulting in an increased susceptibility to B. cinerea infection (Sarig et al., 1997). Infection with B. cinerea often occurs at the flowering stage, giving rise to quiescent infection without apparent damage (Williamson, 1994). The activation of resveratrol synthesis in response to the infection and the subsequent self-intoxication by B. cinerea laccase might keep the pathogen at bay until the seeds in the fruits have ripened and resveratrol levels have dropped. From ripening onwards, protection is no longer necessary, and the plant may even benefit from the rapid fruit tissue decay that facilitates seed dispersal. The pathogen in turn benefits from the easy digestibility of ripe fruit tissue as opposed to unripe tissue. Fungistasis by means of resveratrol oxidation products might therefore be of mutual benefit during the development of the fruit. Increased accumulation of resveratrol in transgenic grapevine plants altered this balance and conferred partial resistance to the host (Coutos-Thévenot et al., 2001). This might impose a selective pressure on the pathogen in favour of Bclcc2-deficient strains. The principle of metabolic activation of toxicant precursors is common knowledge in toxicology (Sultatos, 1994; Takahashi et al., 1999) and is used for designing anti-tumour drugs (Graham et al., 1991), fungicides (de Waard and van Nistelrooy, 1980) and insecticides (Hedley et al., 1998). We present the first example of substrateinduced self-intoxication in nature, in which the expression of the gene that is necessary for self-intoxication is induced by the non-toxic precursor. Similar processes may occur in other plant–pathogen interactions, possibly © 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 883–894

Self-intoxication by a fungal laccase 891 involving entirely different chemical classes of plant defence compounds and different enzymes secreted by the pathogen. It is conceivable that plants have adapted to exploit pathogen-derived enzymes to defend themselves. The non-toxic metabolite resveratrol is less prone to auto-oxidation and, therefore, more stable than its dimers, the viniferins, that readily undergo subsequent rounds of oxidative polymerization. Thus, resveratrol provides a reservoir of a defence compound precursor that is only activated when the plant is challenged by pathogens with the appropriate laccase activity (such as B. cinerea). Experimental procedures Fungal strains and growth conditions Botrytis cinerea wild-type haploid strain B05.10 (Büttner et al., 1994), derived from strain SAS56 (van der VlugtBergmans et al., 1993), was used for analysis, for transformation and as a wild-type control strain for comparison. Conidia of B. cinerea were stored in glycerol stocks. To grow mycelium or isolate conidia, malt extract agar plates (Difco) were inoculated with conidia and incubated at 20∞C. Plates, which were completely covered with mycelium, were placed under near-UV light for 16 h to induce sporulation. Conidia were harvested from the sporulating plates 7–14 days later using 5 ml of sterile water containing 0.05% (v/v) Tween 80. The suspension was filtered through glass wool, washed once by 5¢ centrifugation at 114 g and resuspended in sterile water. Filter-sterilized Gamborg’s B5 medium including vitamins (Duchefa), supplemented with 10 mM sucrose and 10 mM NaH2PO4, was inoculated with 5 ¥ 108 conidia l–1. After 2–4 h of preincubation with occasional shaking, the germinating conidia were incubated for 56 h at 20∞C in a rotary shaker at 180 r.p.m. The mycelium was harvested by filtration over Miracloth and transferred to 100 ml of fresh B5 medium, supplemented with 10 mM sucrose, 10 mM NaH2PO4 and 50 mg ml–1 resveratrol (Sigma; 25 mg ml–1 stock solution in methanol), 200 mM CuSO4 and 125 mg l–1 tannic acid (Sigma) or, as a control, 0.2% (v/v) methanol. Depending on the experiment, incubation was continued for various time intervals at 20∞C in a rotary shaker at 180 r.p.m. The culture filtrate was separated from the mycelium by filtration over Miracloth, concentrated six times by ultrafiltration over an Amicon PM30 filter and stored at 4∞C. Wild-type B. cinerea and transformants were tested for their ability to convert either tannic acid or resveratrol. Therefore, plates were made containing autoclaved water agar (15 g l–1), cooled to 50∞C, supplemented with filter-sterilized Gamborg’s B5 medium including vitamins, 10 mM sucrose and 10 mM NaH2PO4. Resveratrol (100 mg ml–1 dissolved in methanol) was added to a final concentration of 200 mg ml–1, and the plates were poured immediately. For tannic acid plates, a 25 g l–1 filter-sterilized tannic acid solution (Sigma) in water was added to a final concentration of 2.5 g l–1, and the plates were poured immediately. The plates were inoculated with a 2 ml droplet containing 106 conidia ml–1. © 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 883–894

Isolation of laccase genes A genomic library of B. cinerea strain SAS56 in lambda EMBL3 (van der Vlugt-Bergmans et al., 1997) was screened for the presence of laccase genes. As a probe, we used a 1250 bp fragment (GenBank accession no. U20192), kindly provided by Dr R. C. Staples. The individual hybridizing phages were isolated, and phage DNA was isolated. From these phages, hybridizing restriction fragments were subcloned in the pBluescript SK+ phagemid (Stratagene) and subsequently sequenced.

Genomic DNA isolation and Southern analysis Mycelium from a liquid culture was harvested by filtration over Miracloth (Calbiochem) and freeze dried. The dried mycelium was homogenized in liquid nitrogen by placing a pestle in the tube while vortexing. A sample of 3 ml of TES [100 mM TrisHCl, pH 8.0, 10 mM EDTA and 2% (w/v) SDS] and 60 ml of proteinase K (20 mg ml–1) were added, and the suspension was incubated for 1 h at 60∞C. Subsequently, 840 ml of 5 M NaCl and 130 ml of 10% (w/v) N-cetyl-N,N,N-trimethylammonium bromide (CTAB) was added, and incubation was continued for 20 min at 65∞C. Then, the suspension was extracted by adding 4.2 ml of chloroform–isoamyl alcohol (24:1) followed by vortexing shortly, incubation for 30 min on ice and centrifugation for 5 min at 18 000 g. The aqueous top phase was transferred, and 1350 ml of 7.5 M NH4Ac was added, incubated on ice for 1 h and centrifuged for 15 min at 18 000 g. To precipitate the DNA, 0.7 volumes of isopropanol was added. The DNA was transferred from the liquid using a glass rod, washed in 70% (v/v) ethanol and dried. The genomic DNA was dissolved in 1 ml of TE (10 mM Tris-HCl, pH 7.5, and 0.1 mM EDTA) containing 2.5 units of RNase A, incubated for 30 min at 50∞C and precipitated with ethanol. The pellet was dissolved in 200 ml of TE. Genomic DNA (1mg) was digested to completion with 100 units of the desired restriction enzyme in a total volume of 100 ml. DNA fragments were separated on a 0.8% (w/v) agarose gel and subsequently blotted using the protocol for alkali blotting on Hybond-N+ membrane (Amersham). A capillary blot was set up according to the method of Sambrook et al. (1989) using 0.4 M NaOH as blotting solution. After DNA transfer, the membrane was rinsed in 2 ¥ SSC (0.3 M NaCl and 0.03 M sodium citrate, pH 7) and dried. DNA was cross-linked to the membrane by UV treatment (312 nm, 0.6 J cm–2). The Random Primers DNA labelling system (Life Technologies) was used to obtain radioactively labelled probes. The necessary DNA fragment (20 ng) was labelled according to the manufacturer’s instructions. Labelled DNA fragments were purified on Sephadex G50. Hybridization was performed according to the method of Church and Gilbert (1984). Autoradiograms were made using Kodak X-OMAT AR film.

Electrophoresis, blotting and hybridization of total RNA Isolation of total RNA from infected leaves and mycelium, electrophoresis under denaturing conditions, blotting and hybridization were performed as described previously (Prins et al., 2000).

892 A. Schouten et al. cDNA analysis Mycelium of B. cinerea strain B05.10 was grown in liquid culture in the presence of resveratrol for 30 min. The mycelium was harvested and freeze dried. Total RNA was isolated using TRIzol (Life Technologies) according to the manufacturer’s recommendations. Complete cDNA fragments were obtained by the Superscript One-Step reverse transcriptase–polymerase chain reaction (RT–PCR) system (Life Technologies) according to the manufacturer’s recommended conditions, using 0.01 mg of total RNA and the specific primers Lcc2-cDNA(+1)for (5¢-CATGAAGTATTCTACAGTCTTTACTGCCCTCACTG-3¢) and Lcc2-cDNA(+1870)rev (5¢-CCCTTAGATTCCAGAATCG TCCTCGGCG-3¢). The cDNA was cloned into the vector pCR4-TOPO (Invitrogen) and sequenced.

Construction of the Bclcc1 and Bclcc2 gene replacement vectors The Bclcc1 replacement (pLCC1) vector contained the vector pOHT (van Kan et al., 1997), in which the 1.1 kb SstI–EcoRV fragment and the 1.5 kb HindIII fragment were ligated on either side of the hygromycin resistance cassette. The complete Bclcc2 gene including flanking sequences was located on two SalI fragments, which were each cloned in pBluescript. The Sal-A fragment contained the promoter region and the first 1300 bp of the gene. The Sal-B fragment contained the remaining part of the gene, including the stop codon, followed by the terminator. From the Sal-A fragment, a 1.6 kb HindIII fragment was isolated and cloned in the HindIII site of the transformation vector pOHT, resulting in the vector pLCC2-H. From the vector containing the Sal-B fragment, a 1.6 kb SstI fragment was isolated and cloned in the SstI site of the transformation vector pLCC2-H, resulting in the vector pLCC2-HS.

Generation of Bclcc1 and Bclcc2 replacement mutants Transformation of B. cinerea was performed as described previously (Hamada et al., 1994) with minor modifications (van Kan et al., 1997). To obtain protoplasts for transformation, 100 ml of 1% (w/v) malt extract (Oxoid) was inoculated with 2 ¥ 107 B. cinerea strain B05.10 conidia. After 2 h, the germinating conidia were incubated at 20∞C in a rotary shaker at 180 r.p.m. for 24 h. The mycelium was harvested using a 22.4 mm sieve and incubated in 50 ml of KC solution, containing 0.6 M KCl and 50 mM CaCl2, supplemented with 5 mg ml–1 Novozyme. After protoplasting, the suspension was filtrated over a glass wool filter and subsequently sieved over a 22.4 mm and 10 mm sieve. The protoplasts were washed and resuspended to a final concentration of 108 protoplasts ml–1. For gene replacement, 2 mg of transformation vector (pLCC2-HS or pLCC1) was diluted in 95 ml of KC, and 2 ml of 50 mM spermidine was added. After incubation on ice for 5 min, 100 ml of protoplast suspension was added to the DNA and incubated further on ice for 5 min. Polyethylene glycol (PEG; 100 ml) solution, containing 25% (v/v) PEG 3350 in 10 mM Tris-HCl, pH 7.4, and 50 mM CaCl2, was added, and

the suspension was mixed. After 20 min at room temperature, 500 ml of PEG was added again, and the tubes were left at room temperature for another 10 min. Finally, 200 ml of KC solution was added. The transformation mixture with the transformed protoplasts was mixed with 200 ml of SH agar and dispensed immediately over 20 Petri dishes. SH agar contains 0.6 M saccharose, 5 mM HEPES, pH 6.5, 1.2% (w/v) purified agar and 1 mM NH4(H2)PO4. After 24 h incubation at 20∞C, an equal volume of SH agar containing 50 mg ml–1 hygromycin was added. Individual emerging colonies were transferred to malt agar plates containing 100 mg ml–1 hygromycin for further selection. Growing colonies were transferred to malt agar plates without hygromycin, and sporulation was induced by near-UV treatment. To obtain monospore isolates, conidia were isolated, diluted and plated on malt agar plates containing 100 mg ml–1 hygromycin. Conidia obtained from these plates were isolated and used for further analysis.

Spectrophotometric detection of laccase enzyme activity and HPLC analysis The spectrophotometric assay to detect laccase activity was modified from the method of Pezet et al. (1991). To 1 ml of 0.1 M citrate buffer, pH 5.2, 30 ml of 0.2 mg ml–1 resveratrol (dissolved in methanol) and 50 ml of culture filtrate were added. The OD at 302 nm was measured at various time points. HPLC analysis was used to characterize the products arising from the resveratrol conversion by laccase activity. The analyses were performed on a Waters HPLC system containing a Symmetry C18 reversed phase column (150 mm ¥ 3.9 mm, 5 mm), connected to a system comprising a Waters 600S system controller, a Waters 616 pump unit, a Waters 996 photodiode array detector and a Rheodyne 7725i sample injector. Samples from the spectrophotometric assays (50 ml) were injected onto the HPLC column, which was eluted with a linear gradient of 10–90% acetonitrile in water within 50 min. The flow rate was 0.5 ml min–1. The column was re-equilibrated for 5 min with 10% acetonitrile in water before injection of the next sample. Identification of trans-resveratrol and related compounds was carried out by comparison of the retention time and UV spectrum analysis.

Analysis of gene expression in planta Conidia of sporulating B. cinerea cultures, wild-type strain B05.10, were harvested, resuspended in Gamborg’s B5 medium (Duchefa), supplemented with 10 mM glucose and 10 mM potassium phosphate, pH 6.0 (106 conidia ml–1), and sprayed onto detached peanut leaves (Arachis hypogaea). The inoculum was air dried, and the compound leaves were incubated with their stem inserted in wet florist’s foam oasis, in closed plastic boxes with a transparent lid to obtain a humidity of 100%. The boxes were placed at 18∞C with a diurnal cycle of 16 h light and 8 h darkness. Leaves were harvested at 0, 16, 24, 36, 48, 72, 96 and 120 h.p.i. and stored at –80∞C until further use. © 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 883–894

Self-intoxication by a fungal laccase 893 Virulence assay The virulence of B. cinerea mutants was compared with that of the wild type as described by Benito et al. (1998) on detached leaves of peanut (A. hypogaea), grapevine (Vitis vinifera), tomato (Lycopersicon esculentum) and common bean (Phaseolus vulgaris) at both 15∞C and 20°C with a 16 h photoperiod. Of the conidial suspension, two rows of two 2 ml droplets were applied on the leaf, wild-type B05.10 on the right side and mutants on the left side. After inoculation (65 h), the diameter of the spreading lesions was measured. Statistical analysis was performed using Student’s t-test (two-tailed distribution, two-sample unequal variance) on each leaflet.

Acknowledgements We thank Professor Richard Staples and Professor Alfred Mayer for providing the partial Bclcc1 gene fragment. We acknowledge Paul Wood (University of Bristol, UK) for very fruitful discussions during the course of the project. Jenny Stewart and Brian Williamson (Scottish Crop Research Institute, Dundee, UK) are acknowledged for evaluating the virulence of gene replacement mutants on bean. Jos Raaijmakers is acknowledged for assistance with the HPLC analysis. This research was supported financially by grants from the European Commission (EU-FAIR PL97-3351) and the ‘Cassa di Risparmio di Cesena’ Foundation (Italy).

References Adrian, M., Rajaei, H., Jeandet, P., Veneau, J., and Bessis, R. (1998) Resveratrol oxidation in Botrytis cinerea conidia. Phytopathology 88: 472–476. Akhtar, M., Attridge, M., Myers, G.C., Blanchette, R.A., and Kirk, T.K. (1992) Biochemical pulping of loblolly pine with different strains of the white rot fungus Ceriporiopsis subvermispora. TAPPI J 75: 105–109. Bailey, J.A., and Mansfield, J.W. (1982) Phytoalexins. Glasgow: Blackie & Son. Bar, D.P., and Aust, S.D. (1994) Mechanisms white rot fungi use to degrade pollutants. Environ Sci Technol 28: 78– 87. Benito, E.P., ten Have, A., van’t Klooster, J.W., and van Kan, J.A.L. (1998) Fungal and plant gene expression during synchronized infection of tomato leaves by Botrytis cinerea. Eur J Plant Pathol 104: 207–220. Breuil, A.-C., Jeandet, P., Adrian, M., Chopin, F., Pirio, N., Meunier, P., and Bessis, R. (1999) Characterization of a pterostilbene dehydrodimer produced by laccase of Botrytis cinerea. Phytopathology 89: 298–302. Büttner, P., Koch, F., Voigt, K., Quidde, T., Risch, S., Blaich, R., et al. (1994) Variations in ploidy among isolates of Botrytis cinerea: implications for genetic and molecular analysis. Curr Genet 25: 445–450. Church, C.M., and Gilbert, W. (1984) Genomic sequencing. Proc Natl Acad Sci USA 81: 1991–1995. Cichewiz, R.H., Kouzi, S.A., and Hamann, M.T. (2000) Dimerization of resveratrol by the grapevine pathogen Botrytis cinerea. J Nat Prod 63: 29–33. © 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 883–894

Clutterbuck, A.J. (1990) The genetics of conidiophore pigmentation in Aspergillus nidulans. J Gen Microbiol 136: 1731–1738. Coutos-Thévenot, P., Poinssot, B., Bonomelli, A., Yean, H., Breda, C., Buffard, D., et al. (2001) In vitro tolerance to Botrytis cinerea of grapevine 41B rootstock in transgenic plants expressing the stilbene synthase Vst1 gene under the control of a pathogen-inducible PR10 promoter. J Exp Bot 52: 901–910. Dercks, W., and Creasy, L.L. (1989) The significance of stilbene phytoalexins in the Plasmopara viticola–grapevine interaction. Physiol Mol Plant Pathol 34: 189–202. Graham, M.A., Riley, R.J., and Kerr, D.J. (1991) Drug metabolism in carcinogenesis and cancer chemotherapy. Pharmacol Ther 51: 275–289. Guillén, F., Muñoz, C., Gómez-Toribio, V., Martínez, A.T., and Martínez, M.J. (2000) Oxygen activation during oxidation of methoxyhydroquinones by laccase from Pleurotus eryngii. Appl Environ Microbiol 66: 170–175. Hain, R., Reif, H.-J., Krause, E., Langebartels, R., Kindl, H., Vornam, B., et al. (1993) Disease resistance results from foreign phytoalexin expression in a novel plant. Nature 361: 153–156. Hamada, W., Reignault, P., Bompeix, G., and Boccara, M. (1994) Transformation of Botrytis cinerea with the hygromycin B resistance gene, hph. Curr Genet 26: 251–255. Hatakka, A. (1994) Lignin-modifying enzymes from selected white-rot fungi: production and role in lignin degradation. FEMS Microbiol Rev 13: 125–135. Hedley, D., Khambay, B.P.S., Hooper, A.M., Thomas, R.D., and Devonshire, A.L. (1998) Proinsecticides effective against insecticide-resistant peach-potato aphid Myzus persicae (Sulzer). Pestic Sci 53: 201–208. van den Heuvel, J. (1976) Sensitivity to, and metabolism of, phaseollin in relation to the pathogenicity of different isolates of Botrytis cinerea to bean (Phaseolus vulgaris). Neth J Plant Pathol 82: 153–160. Hipskind, J.D., and Paiva, N.L. (2000) Constitutive accumulation of a resveratrol-glucoside in transgenic alfalfa increases resistance to Phoma medicaginis. Mol Plant– Microbe Interact 13: 551–562. Hoos, G., and Blaich, R. (1990) Influence of resveratrol on germination of conidia and mycelial growth of Botrytis cinerea and Phomopsis viticola. J Phytopathol 129: 102–110. Ingham, J.L. (1973) Disease resistance in higher plants: The concept of pre-infectional and post-infectional resistance. Phytopathol Z 78: 314–335. Jeandet, P., Breuil, A.C., Adrian, M., Weston, L.A., Debord, S., Meunier, P., et al. (1997) HPLC analysis of grapevine phytoalexins coupling photodiode array detection and fluorometry. Anal Chem 69: 5172–5177. Jönsson, L.J., Saloheimo, M., and Penttillä, M. (1997) Laccase from the white-rot fungus Trametes versicolor: cDNA cloning of lcc1 and expression in Pichia pastoris. Curr Genet 32: 425–430. van Kan, J.A.L., van’t Klooster, J.W., Wagemakers, C.A.M., Dees, D.C.T., and van der Vlugt-Bergmans, C.J.B. (1997) Cutinase A of Botrytis cinerea is expressed, but not essential, during penetration of gerbera and tomato. Mol Plant–Microbe Interact 10: 30–38.

894 A. Schouten et al. Kerssies, A. (1990) A selective medium for Botrytis cinerea to be used in a spore trap. Neth J Plant Pathol 96: 247–250. Kim, D.H., Rigling, D., Zhang, L., and Van Alfen, N.K. (1995) A new extracellular laccase of Cryphonectria parasitica is revealed by deletion of Lac1. Mol Plant–Microbe Interact 8: 259–266. Langcake, P. (1981) Disease resistance of Vitis spp. and the production of the stress metabolites resveratrol, e-viniferin, a-viniferin and pterostilbene. Physiol Plant Pathol 18: 213–226. Langcake, P., and McCarthy, W.V. (1979) The relationship of resveratrol production to infection of grapevine leaves by Botrytis cinerea. Vitis 18: 244–253. Langcake, P., and Pryce, R.J. (1977a) A new class of phytoalexins from grapevines. Experientia 33: 151–152. Langcake, P., and Pryce, R.J. (1977b) The production of resveratrol and the viniferins by grapevines in response to ultraviolet radiation. Phytochemistry 16: 1193–1196. Leatham, G.F., and Stahmann, M.A. (1981) Studies on laccase of Lentinus edodes: specificity, localization and association with the development of fruiting bodies. J Gen Microbiol 125: 147–157. Lewis, N.G., and Yamamoto, E. (1990) Lignin: occurrence, biogenesis and biodegradation. Annu Rev Plant Physiol Plant Mol Biol 41: 455–496. Messerschmidt, A., and Huber, R. (1990) The blue oxidases, ascorbate oxidase, laccase and ceruplasmin. Eur J Biochem 187: 341–352. Morrissey, J.P., and Osbourn, A.E. (1999) Fungal resistance to plant antibiotics as a mechanism to pathogenesis. Microbiol Mol Biol Rev 63: 708–724. Muñoz, C., Guillén, F., Martínez, A.T., and Martínez, M.J. (1997) Laccase isoenzymes of Pleurotus eryngii: Characterization, catalytic properties, and participation in activation of molecular oxygen and Mn2+ oxidation. Appl Environ Microbiol 63: 2166–2174. Nielsen, H., Engelbrecht, J., Brunak, S., and von Heijne, G. (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10: 1–6. Osbourn, A.E. (1996) Preformed antimicrobial compounds and plant defense against fungal attack. Plant Cell 8: 1821–1831. Pezet, R., Pont, V., and Hoang-Van, K. (1991) Evidence for detoxification of pterostilbene and resveratrol by laccaselike stilbene oxidase produced by Botrytis cinerea. Physiol Mol Plant Pathol 39: 441–450. Prins, T.W., Wagemakers, L., Schouten, A., and van Kan, J.A.L. (2000) Cloning and characterization of a glutathione S-transferase homologue from the plant pathogenic fungus Botrytis cinerea. Mol Plant Pathol 1: 169–178. Sambrook, J., Fritsch, E.F., and Maniatis, F. (1989) Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. Sarig, P., Zutkhi, Y., Monjauze, A., Lisker, N., and BenArie,

R. (1997) Phytoalexin elicitation in grape berries and their susceptibility to Rhizopus stolonifer. Physiol Mol Plant Pathol 50: 337–347. Sbaghi, M., Jeandet, P., Faivre, B., Bessis, R., and Fournioux, J.C. (1995) Development of methods using phytoalexin (resveratrol) assessment as a selection criterion to screen grapevine in vitro cultures for resistance to grey mould (Botrytis cinerea). Euphytica 86: 41–47. Scherer, M., and Fischer, R. (1998) Purification and characterization of laccase II of Aspergillus nidulans. Arch Microbiol 170: 78–84. Sultatos, L.G. (1994) Mammalian toxicology of organophosphorus pesticides. J Toxicol Environ Health 43: 271–289. Takahashi, N., Tamura, M., Kajiwara, S., and Ohtaguchi, K. (1999) Genetically engineered herbicide resistance in cyanobacterium Synechococcus sp. by bialaphos resistance gene from Streptomyces hygroscopicus. Biotechnol Lett 21: 751–757. Tegtmeier, K.J., and VanEtten, H.D. (1982) The role of pisatin tolerance and degradation in the virulence of Nectria haematococca on peas: a genetic analysis. Phytopathology 72: 608–612. Thomzik, J.E., Stenzel, K., Stocker, R., Schreier, P.H., Hain, R., and Stahl, D.J. (1997) Synthesis of a grapevine phytoalexin in transgenic tomatoes (Lycopersicon esculentum Mill.) conditions resistance against Phytophthora infestans. Physiol Mol Plant Pathol 51: 265–278. Thurston, C.F. (1994) The structure and function of fungal laccases. Microbiology 140: 19–26. VanEtten, H.D., Mathews, D.E., and Mathews, P.S. (1989) Phytoalexin detoxification: importance for pathogenicity and practical implications. Annu Rev Phytopathol 27: 143–164. van der Vlugt-Bergmans, C.J.B., Brandwagt, B.F., van’t Klooster, J.W., Wagemakers, C.A.M., and van Kan, J.A.L. (1993) Genetic variation and segregation of DNA polymorphisms in Botrytis cinerea. Mycol Res 97: 1193–1200. van der Vlugt-Bergmans, C.J.B., Wagemakers, C.A.M., and Van Kan, J.A.L. (1997) Cloning and expression of the cutinase A gene of Botrytis cinerea. Mol Plant–Microbe Interact 10: 21–29. de Waard, M.A., and van Nistelrooy, J.G.M. (1980) Mechanism of resistance to pyrasophos in Pyricularia oryzae. Neth J Plant Pathol 86: 251–258. Wahleithner, J.A., Xu, F., Brown, K.M., Brown, S.H., Golightly, E.J., Halkier, T., et al. (1996) The identification and characterisation of four laccases of the plant pathogenic fungus Rhizoctonia solani. Curr Genet 29: 395–403. Williamson, B. (1994) Latency and quiescence in survival and success of fungal plant pathogens. In Ecology of Plant Pathogens. Blakeman, J.P., and Williamson, B. (eds). Oxford: CAB International, pp. 187–207. Zhao, J., and Kwan, H.S. (1999) Characterization, molecular cloning, and differential expression analysis of laccase genes from the edible mushroom Lentinula edodes. Appl Envion Microbiol 65: 4908–4913.

© 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 883–894