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Effect of the anilinopyrimidine fungicide pyrimethanil on the cystathionine β-lyase of Botrytis cinerea
PESTICIDE Biochemistry & Physiology
Pesticide Biochemistry and Physiology 77 (2003) 54–65 www.elsevier.com/locate/ypest
Effect of the anilinopyrimidine fungicide pyrimethanil on the cystathionine b-lyase of Botrytis cinerea Rene Fritz,* Catherine Lanen, Florence Chapeland-Leclerc, and Pierre Leroux Institut National de la Recherche Agronomique, Unit e de Phytopharmacie et des M ediateurs Chimiques, 78026 Versailles Cedex, France Received 22 April 2003; accepted 24 July 2003
Abstract Previous studies, carried out in our laboratory, upon the mode of action of anilinopyrimidines (APs) suggested that these fungicides inhibit the biosynthesis of methionine and that the primary target could be the cystathionine b-lyase (CBL). More recent works carried on with strains of Botrytis cinerea highly resistant to APs (AniR1 ) suggested that one major gene (Ani 1) confers this phenotype and that this gene segreged independently of the gene of resistance to dicarboximides. This confirmed the fact that the target site of APs must be different from that of dicarboximides and suggested that the mechanism of resistance to APs could be due to a mutation in Ani 1 gene. In this report, we tried to find if the CBL of B. cinerea could be the target site of the APs, pyrimethanil and cyprodinil. To do this, first we searched for an inhibitory effect of APs on the enzyme activity of the B. cinerea CBL and second, we compared the nucleotide sequences of the B. cinerea CBL gene, we called metC, of 3 strains sensitive to APs (AniS ) and 10 strains AniR1 . We showed that aminoethoxyvinylglycine at 2.5 lM strongly inhibited, in a competitive manner, a crude extract of the CBL isolated from a strain AniS or from a strain AniR1 . On the other hand, APs, at 0.1 mM, slightly inhibited the CBL isolated from B. cinerea. This inhibitory effect which was uncompetitive was not different between the strain AniS and the strain AniR1 . We sequenced and analysed the metC gene (Accession No.: AF211176). Based on the cDNA sequence, we found that this gene spans 1377 bp and is interrupted by one intron. We found consensus sequences for the putative CAAT-box, TATA-box, transcription-site, polyadenylation, and splicing signals. The metC gene codes for a polypeptide chain of 459 amino-acids and has a predicted molecular weight of 49,087 Da. The analysis of the sequence polymorphism in 13 strains did not allow us to discriminate between the sensitive strains and the resistant ones. So, it is assumed that the CBL of B. cinerea does not seem to be the primary target site of APs and probably does not correspond to the Ani 1 gene. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Anilinopyrimidines; Botrytis cinerea; Fungicide resistance; Cystathionine b-lyase; Mode of action; Enzyme activity; Sequence polymorphism; metC gene
1. Introduction * Corresponding author. Fax: +33-1-30-83-31-19. E-mail address: [email protected] (R. Fritz).
Pyrimethanil, cyprodinil, and mepanipyrim are broad-spectrum fungicides belonging to the
0048-3575/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0048-3575(03)00094-4
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chemical class of anilinopyrimidines (APs).1 They are highly effective against Botrytis cinerea Pers. ex Fr. (anamorph of Botryotinia fuckeliana (de Bary) Whetz.), the causal agent of grey mould, and show no cross-resistance to any current commercially available botryticides [1–3]. This suggests that APs have a different mode of action. Tested in vitro on Botrytis spp., pyrimethanil does not show any effect on respiration, lipid peroxidation, osmotic stability or on biosynthesis of protein, RNA, DNA, chitin or ergosterol [1]. In Botrytis fabae and B. cinerea, pyrimethanil has been reported to inhibit the secretion of cell wall degrading enzymes required for infection at concentrations lower than those needed to inhibit growth [4,5]. This has also been reported for mepanipyrim [6,7]. This mode of action has been suggested to be the primary one for pyrimethanil [5]. Nevertheless, it has been shown that pyrimethanil and the other APs are effective on B. cinerea growth at low concentrations in culture media lacking amino-acids. Addition of several different amino-acids to the culture media reduced the growth inhibitory activity of pyrimethanil. Among these, methionine did not reverse the activity of pyrimethanil completely but exhibited the highest reversal activity suggesting a direct effect of pyrimethanil on the methionine biosynthesis pathway in B. cinerea [8,9]. We showed that when mycelium of B. cinerea was treated with low concentrations of pyrimethanil the total amount of free aminoacids increased. Qualitative variations were also induced and particularly methionine decreased while cystathionine accumulated [10]. These data indicate that pyrimethanil inhibits the biosynthesis of methionine and suggest that the primary target could be the cystathionine b-lyase (CBL). More recently, works conducted with strains of B. cinerea highly resistant to APs (AniR1 phenotype) suggested that one major gene (Ani 1) confers this phenotype and that this gene segregated independently of the gene of resistance to dicarboximides [11–13]. Therefore, the target site of APs must be different from that of dicarboximides and the 1
Abbreviations used: APs, anilinopyrimidines; CBL, cystathionine b-lyase, AVG, L -a-2-aminoethoxyvinylglycine; PLP, pyridoxal 50 -phosphate; CGS, cystathionine c-synthase.
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mechanism of resistance to APs could be due to a mutation in Ani 1 gene. In this report, we try to demonstrate that APs inhibit the enzyme activity of B. cinerea CBL. We present the nucleotide sequence of the gene encoding this enzyme which we called metC and compare the nucleotide sequences of the metC gene of strains sensitive to APs (AniS phenotype) and strains of AniR1 phenotype. The results are used to discuss about the mode of action of APs.
2. Materials and methods 2.1. Chemicals Technical grade fungicides were provided as follows: pyrimethanil was from AgrEvo (UK), while cyprodinil was from Novartis (Germany). These compounds were dissolved in ethanol whose final concentration was never more than 0.5%. Yeast nitrogen base without amino-acids was from Difco laboratories, Detroit, USA. L -a-2-Aminoethoxyvinylglycine (AVG) and other chemicals were from Sigma–Aldrich, France. Taq DNA polymerase was from Qiagen, USA. 2.2. Fungi The inhibitory effect of the fungicides on the enzyme activity was studied using the strain L of AniS phenotype [9,10] and the strain 4 271 17 a, of AniR1 phenotype [12,13]. The comparison between the different CBL nucleotide sequences was performed on the above strains as well as on two other AniS strains, T4 and SAS56 and nine other AniR1 strains, 1851, 1856, 1864, 7 170 21 2, 8 159 22 d, 8 184 23 a, 8 245 23 a, 99 22 2b 2, and 99 175 22 b. The main characteristics of these strains are presented in Table 1. All the strains isolated from berries collected in vineyards are of transposa type [14]. SAS56, a reference strain for the mating type provided by Faretra from the University of Bari, Italy, contains only the transposable element boty [14]. The strain T4, of transposa type, and whose cDNA library was submitted to NCBI GenBank database, was provided by Yves Brygoo from INRA of Versailles, France [14]. The description
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Table 1 Main characteristics of isolates of B. cinerea used to compare CBL nucleotide sequences or CBL enzyme activity (in bold) Strain
Year
Origin
Phenotypea
L T4 SAS56 1851 1856 1864 4 271 17a 7 170 21 2 8 159 22d 8 184 23a 8 245 23a 9 22 2b 2 9 175 22b
1980 1991
Bordeaux, grapes Eyrague, tomato Reference strain from Faretra Bordeaux, grapes, noble rot Bordeaux, grapes, noble rot Bordeaux, grapes, noble rot Armagnac, grapes Loire Valley, grapes Champagne, grapes Champagne, grapes Champagne, grapes Loire Valley, grapes Loire Valley, grapes
AniS , BenR1 , ImiS AniS , BenS , ImiS AniS , BenS , ImiS AniR1 , BenS , ImiS AniR1 , BenS , ImiS AniR1 , BenS , ImiS AniR1 , BenS , ImiR1 AniR1 , BenS , ImiS AniR1 , BenR1 , ImiS AniR1 , BenS , ImiS AniR1 , BenR1 , ImiR1 AniR1 , BenS , ImiS AniR1 , BenR2 , ImiR1
—
1999 1999 1999 1994 1997 1998 1998 1998 1999 1999
a AniS , sensitive to anilinopyrimidines; BenS , sensitive to benzimidazoles; ImiS , sensitive to dicarboximides; AniR1 , highly resistant to anilinopyrimidines; AniR1 , highly resistant to anilinopyrimidines but to a lesser extent; BenR1 , resistant to benzimidazoles and sensitive to phenylcarbamates; BenR2 , resistant to benzimidazoles and phenylcarbamates; and ImiR1 , resistant to dicarboximides and sensitive to phenylpyrroles (Leroux et al. [15]).
of the different phenotypes has been previously reported by Leroux et al. [15]. All the strains were maintained and sporulated in the dark on a medium containing: 20 g malt, 5 g yeast extract, and 12.5 g agar, for 1 L. Liquid cultures, in exponential growth phase, were prepared by inoculating 7 105 macroconidia ml1 in the medium previously described [16] (150 ml in 300 ml Erlenmeyer flasks) but without yeast extract because of the possible reversal activity of methionine [10]. To set up the best conditions in the production of an enzyme sensitive to APs, the yeast extract was replaced by 2 g L1 of yeast nitrogen base without amino-acids or by 2 g L1 of asparagine [17]. This latter medium was made of: 10 g glucose, 1.5 g K2 HPO4 , 2 g KH2 PO4 , 1 g (NH4 )2 SO4 , 0.5 g MgSO4 7H2 O, and 2 g asparagine, for 1 L. Cultures were shaken (orbital shaker at 150 rev min1 ) for 18 h at 23 °C. To obtain more mycelium, each preliminary culture was filtered through a 100 lm gauze, washed with fresh medium, and resuspended in 1200 ml of the same fresh medium to prepare two cultures of 600 ml each contained in 1 L Erlenmeyer flask. These cultures were incubated for 24 h again under the previous conditions. Finally, mycelial pellets were harvested, filtered through a 100 lm gauze, and washed by cold UHP water (Vivendi Water STI, France).
2.3. Enzyme preparation The mycelium was washed and lyophilized. The freeze-dried mycelium (1.5 g) was homogenized for 3 3 s in a mini-mincer (Seb, France) until a mycelial-powder was obtained. The mycelial-powder was added to 15 ml of a 0.1 M phosphate buffer, pH 7.3, containing 1 mM Na2 -ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol (DTT), and 0.2 mM pyridoxal 50 -phosphate (PLP), cooled to 4 °C. The suspension was homogenized in a Potter tube of 30 ml in less than 30 s (20-fold up and down). The Potter tube was washed by an equal volume of the same buffer. The total homogenate (cell-free extract and wash) was centrifuged at about 15,000g and 4 °C for 45 min. The resulting supernatant was then acidified to pH 6.5 with 4 M acetic acid and then quickly heated up to 55 °C in a water bath, during 4 min, while stirring with a glass rod. The supernatant was immediately cooled in an ice bath under stirring with the same glass rod and submitted to a second centrifugation at about 32,000g and 4 °C for 45 min. The new supernatant was recovered and its pH was balanced to 7.3 with a 20% ammonia solution. This supernatant was subjected to a 35–45% ammonium sulfate fractionation. The two steps of the fractionation were done in a beaker placed in an
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ice bath, upon a magnetic stirrer. A slow rate of stirring must be used to avoid foaming and ammonium sulfate was added in small batches, being dissolved separately during 15 min. During stirring, the pH value was maintained at 7.3 with the precedent acetic acid solution. After the first step of fractionation (35%), the protein suspension was decanted to a centrifugal tube and left to stand for 1 h, in an ice bath, to ensure equilibrium. To remove the precipitated protein, the tube was centrifuged at about 20,000g and 4 °C for 30 min. The supernatant was subjected to the second step of fractionation (45%) using the same precautions and conditions as the precedent, except that the resultant protein suspension was kept in a refrigerator during the night. Finally, the precipitated protein was recovered by centrifugation at about 20,000g and 4 °C for 30 min. The protein pellets were suspended in 4 ml of the precedent buffer without the protectors (EDTA and DTT) but with PLP (PLP buffer). The protein solution obtained was filtered through a glass fibre filter (GF/A from Whatman) and then through filter series of hydrophilic membrane Durapore (Millipore) with three different pore sizes: 0.45, 0.22, and finally 0.1 lm. After each filtration, the filter was washed by 3 0.5 ml of PLP buffer. The filtrates were pooled in a Centricon plus-20 device with a Biomax membrane with a molecular weight cutoff of 50 kDa (Millipore) and centrifuged at 4000g and 4 °C for 95 min. The protein retained on the Centricon membrane was washed by 10 ml of PLP buffer and centrifuged for 1 h. The protein retained (2 ml) was recovered by centrifuging the membrane of the Centricon upside down. This enzyme preparation was glycerol added up to 20% and kept frozen at )20 °C in aliquots of 300 ll each until application. 2.4. Essay of CBL activity CBL catalyses the following reaction: cystathionine þ H2 O ! pyruvate þ homocysteine þ NH3 Enzyme activity was assayed according to the methods modified from Guggenheim [18] and Gi-
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ovanelli [19], whereby pyruvate production was coupled to NADH oxidation via a lactate dehydrogenase (LDH) pyruvate þ NADH $ lactate þ NAD: The resultant decrease in absorption was followed with a thermostated (30 °C) Uvikon 933 spectrophotometer from Kontron Instruments at 340 nm. The assay was carried out in cuvettes of 1-cm light path in 0.1 M Tris–HCl buffer at pH 8.3 containing 2–7 mM L -cystathionine, 0.3 lmol NADH, and 30 lg L -LDH from pig heart provided by Boehringer Mannheim, Germany (300 U mg1 at 25 °C) and 0.1 lmol PLP. The reference cuvette contained identical components except that enzyme was replaced by Tris–HCl buffer. The reaction was started by adding substrate and the optical density at 340 nm versus a buffer blank was recorded continuously. For each substrate concentration, the reaction speed (disappearance of NADH according to time measured at 340 nm which is correlated, via pyruvate formation, to the consumption of cystathionine) was calculated. Each speed value used in the double-reciprocal plot representation resulted in the kinetic analysis of a set of different absorption measures, which was at least linear from 2 to 15 min. Protein concentration was determined using the method of Lowry modified by Hartree [20] with bovine serum albumin fraction V (Sigma) as protein standard. As AVG, an analogue of L -cystathionine, is known to be a potent inhibitor of CBL [21], we tested the reliability of our crude extract by looking at the activity of AVG toward our crude extract. We found that, at 2.5 lM, AVG solubilized in Tris–HCL buffer always showed a strong inhibitory effect. 2.5. Preparation of DNA, RNA, and cDNA from B. cinerea DNA was extracted from lyophilized mycelium as described by Chapeland [13] and conserved at )20 °C. RNA was extracted with the ‘‘RNeasy Plant Mini Kit’’ (Qiagen, USA) according to the protocol described for filamentous fungi by the supplier.
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The synthesis of cDNA from RNA was carried out with the ‘‘first-strand cDNA Synthesis’’ kit (Clontech, USA) according to supplierÕs instructions.
TTACAATGGAGCTGCTGCTGC-30 as 30 -primer. The RT-PCR was carried out as described by Chapeland [13]. 2.8. DNA sequencing
2.6. PCR and genome walker libraries for finding the DNA sequence of cystathionine b-lyase All primers were synthesized by Sigma-Genosys (UK). The PCR amplification of a first fragment of the gene encoding cystathionine b-lyase in B. cinerea was performed with degenerate primers designed to amplify well-conserved regions of genes encoding well-identified CBL of two microorganisms: the CBL gene (Accession No.: P53101) from Saccharomyces cerevisiae [22] and the metG gene (Accession No.: U28383) from Aspergillus nidulans [23]. The 50 -primer was: 50 -TAYGAYTAYACICGITC NGGNAA-30 (Y ¼ C/T; I ¼Inosine, A/G; N ¼ A/ G/T/C). The 30 -primer was 50 -RTCRTCIACRTCY TCDAT-30 (R ¼ A/G; D ¼ A/G/T). The PCR was performed, in a Gene Amp PCR system 2400 (Perkin–Elmer), as described by Chapeland [13]. To obtain the complete sequence of the B. cinerea CBL gene, we used the ‘‘Universal GenomeWalker’’ kit (Clontech, USA). This kit allowed us to find unknown genomic DNA sequences adjacent to the known precedent sequence. From this sequence, four gene-specific primers synthesized by SigmaGenosys (UK) were designed for DNA walking [13]. Two 30 -primers were designed for walking upstream, one for the primary PCR: 50 -GGGGTT GCATAGCATAGGAGAAAGCAT-30 , the other for the nested PCR: 50 -CGCGAGATGTCGTTCC AG-30 . Two 50 -primers were designed for walking downstream, one for the primary PCR: 50 -ATGCT TTCTCCTATGCTATGCAACCCC-30 , the other for the nested PCR: 50 -GGCGATGTTGCTCTCT CTGA G-30 . The GenomeWalker ‘‘libraries’’ were constructed and the PCRs were done according to supplierÕs instructions. 2.7. RT-PCR Two gene-specific primers were designed for RT-PCR from the complete sequence of the CBL of B. cinerea: 50 -CCAATATGTCTTCACCATC AGGCTCC-30 as 50 -primer, and 50 -CATTCG
PCR and RT-PCR fragments were purified with the ‘‘QIAquick PCR’’ kit (Qiagen, USA). All sequences were determined at the Euro Sequences Genes Services (ESGS, France). 2.9. Analysis of the sequences Consensus multiple alignment for nucleotide and amino-acid sequences was performed with CLUSTAL W program [24] available on the Infobiogen web site (http://www.infobiogen.fr). The similarity searches in databases were performed with the Basic Local Alignment Search Tool (BLAST) programs [25] available on the NCBI web site (http://www.ncbi.nlm.nih.gov).
3. Results and discussion 3.1. Properties of CBL from B. cinerea Our preparation method implies that crude CBL from B. cinerea is relatively resistant to heat up to 55 °C. Preparation of crude CBL must be done at an acidic or neutal pH (6.5–7.3) and need the presence of PLP to avoid any degradation of the enzyme activity. The strongest activity was found in the 35–45% ammonium sulfate fraction. We did not test a wide range of temperatures but the best results using LDH were obtained at 30 °C. The activity of crude CBL required PLP and was best in Tris–HCl buffer at pH 8.3. But at this pH the enzyme activity could not be preserved more than 1 day. This was probably due to a degradation process. Our crude CBL was active toward L -djenkolate but better results were obtained toward L -cystathionine. The activity of crude CBL increased when the cystathionine concentration raised (Fig. 1). The apparent Km value, found in the double reciprocal plot, (Fig. 2) was 1.6 mM and the Vmax value was 17.4 nmol min1 mg protein1 .
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Fig. 1. Activity of crude CBL of B. cinerea versus cystathionine concentrations and inhibitory effect of aminoethoxyvinylglycine (AVG), pyrimethanil, and cyprodinil.
Fig. 2. Double-reciprocal plot of the data shown in Fig. 2, about activity of crude CBL of B. cinerea, and inhibitory effect of aminoethoxyvinylglycine (AVG), pyrimethanil, and cyprodinil.
Sierotzki et al. [26], working on a CBL crude extract from B. cinerea homogenized with a French press and tested in a potassium phosphate buffer at
pH 7.5 and at 30 °C, obtained a comparable Km value but their Vmax value was about 8-fold lower. We found that AVG did not have any effect on the
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LDH coupling reaction. At 2.5 lM, AVG strongly inhibited our crude CBL, but this effect decreased as the cystathionine concentration increased (Fig. 1): it was probably due to a competition between the substrate, cystathionine, and the inhibitor, AVG. Therefore, in accordance with kinetic models, AVG showed an inhibition of competitive type with an apparent Km value of 10.2 mM and a Vmax value of 17.1 nmol min1 mg protein1 (Figs. 1 and 2). As expected, assuming a simple Michaelis–Menten mechanism, the apparent Km value strongly increased while the Vmax value tended to stay constant. With our crude CBL, at 2.5 lM AVG did not cause a time-dependent inactivation of B. cinerea CBL. All the CBL enzyme properties presented above were shared by the AP sensitive strain L and the AP resistant strain 4 271 17a.
more polar than pyrimethanil. After 30 min, the overall recovery of [14 C]pyrimethanil was identical in the 2 strains tested (97%), but after 6 h this recovery was slightly better in the strain L than in the strain 4 271 17a, respectively, 96 and 86% [13]. It was assumed that these results cannot account for the resistance because the difference in the recovery of pyrimethanil did not take place earlier enough. But the occurrence of metabolites, even in small amount, might support the hypothesis of an activation. However, these metabolites separated by thin-layer chromatography were not fungitoxic toward conidia of the strain L sprayed on the thinlayer [13].
3.2. Effect of pyrimethanil and cyprodinil on CBL of B. cinerea
The complete nucleotide (nt) sequence of the CBL (metC gene) and 50 -flanking region of the strain L sensitive to anilinopyrimide fungicides (2921 bp) was submitted to the NCBI GenBank database under Accession No.: AF211176. The translation initiation codon for the metC gene was assigned to the ATG at nt 1135. The sequence around this presumed translation start site (50 CAATATGTC-30 ) fits well the consensus sequence for the translation initiation site in Neurospora crassa (50 -CAM‘‘T3’’ATGGC-30 ) [27]. The metC gene coding region spans 1377 bp and is interrupted only by one intron, unlike the metG gene of A. nidulans which is interrupted by two introns [23]. The position of this small intron (49 bp) was determined through a comparison of cDNA and genomic metC sequences. Nucleotide sequences at all exon–intron junctions conform to an established filamentous fungi splice-site consensus sequence: 50 -donor site g^ GTAAGT and 30 -acceptor site CCAG^ g [28]. Similarly, internal lariat sequence agrees with a filamentous fungi lariat consensus: TGCTAAC [28]. As described in our submission to the NCBI GenBank (Accession No.: AF211176), we found several putative consensus sequences, which match well with those reported in N. crassa [29]: TCATCATC for transcription site, ATTATAA for TATA-box, CAAAT for CAATbox, and AATAAA for polyadenylation site signal. Translation terminates at the TAA stop codon
With the two strains tested, the AP sensitive strain L and the AP resistant strain 4 271 17a, the activity of our crude CBL was not affected by pyrimethanil and cyprodinil at concentrations below 0.1 mM. But at 0.1 mM we observed a significant inhibitory effect of these two APs. With pyrimethanil and cyprodinil, we obtained an uncompetitive inhibition with an apparent Km value of 1.0 mM and a Vmax value of 10.6 nmol min1 mg protein1 (Fig. 2). At 0.1 mM, we found, with both fungicides, an equal level of inhibition of Vmax of 39% (Fig. 1). In their experimental conditions, Sierotzki et al. [26] reported that at 0.1 mM cyprodinil the enzyme activity of CBL of B. cinerea was not affected whatever the concentrations of substrate or PLP. These results did not take account for a possible mechanism of activation of anilinopyrimidines. In previous studies, [14 C]pyrimethanil was not metabolized after a 6 h incubation in a medium having contained the strain L or the strain 4 271 17a [12,13]. In the presence of these 2 strains, only 1% of the radioactivity brought by 100 lM [14 C]pyrimethanil was recovered in the mycelial pellet. This very small fungicidal uptake did not make easy the interpretation of the results. After 6 h, the 2 strains studied produced, in small amount, 4 metabolites
3.3. Nucleotide-sequence analysis of the CBL of the sensitive strain L
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(nt 2561). The metC gene of B. cinerea codes for a polypeptide chain of 459 amino-acids and has a predicted molecular weight of 49,087 Da. This is in agreement with the monomer molecular weight estimated for various CBL: 48,280 Da for A. nidulans [23], 53,000 Da for Spinacia oleracea L. [30], and 50,372 Da for Arabidopsis thaliana [31]. Using the BLAST program for protein sequences [25], several putative conserved domains were detected. Only one conserved domain showed a credible score. This domain is 384 residues long and belongs to the protein family, PF01053, named cysteine/methionine metabolism PLP-dependent enzyme. This family includes enzymes involved in cysteine and methionine metabolism as cystathionine c-lyases, cystathionine c-synthases (CGS), cystathionine b-lyases (CBL), and homocysteine synthases. In the metC gene of B. cinerea, this conserved protein-domain starts at position 52 (ATEL. . .) and ends at position 422 (. . .SRAL) as shown in CLUSTAL W alignment [24] between the protein sequence of the CBL gene of B. cinerea and those of 2 other filamentous fungi: N. crassa and A. nidulans (Fig. 3). So, B. cinerea CBL belongs to the c-family of pyridoxal 50 -phosphatedependent enzymes described by Alexander et al. [32]. The conserved Lys-243 residue (K) in the B. cinerea enzyme, aligned with Lys-237 in N. crassa and Lys-230 in A. nidulans (Fig. 3), seems to be homologous to Lys-210 in Escherichia coli CBL [33], Lys-198 in E. coli CGS [33], and Lys-278 in A. thaliana CBL [31,34], which were identified as being covalently bound to the PLP cofactor [35]. Two other conserved residues in B. cinerea CBL, Glu-187 (E) and Asp-218 (D), respectively, aligned, on the one hand, with Glu-181 and Asp212 in N. crassa and, on the other hand, with Glu174 and Asp-205 in A. nidulans (Fig. 3), seem to be homologous to Glu-154 and Asp-185 of E. coli CBL [33] and to Glu-224 and Asp-253 in A. thaliana CBL [31,34]. These three residues are conserved in the whole c-family [23] and participate in PLP-binding [33]. The analysis of identities between the B. cinerea CBL sequence and those of the closest organisms found in the NCBI GenBank database reveals that they are better with the filamentous fungi N. crassa (80%) and A. nidulans (68%) than with S. cerevisiae (41%) or plant CBL
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(41–42%). B. cinerea CBL shows the better identity with those of N. crassa. This fact is in accordance with the similarities found between the consensus sequences of these two fungi. 3.4. Comparison between the CBL gene sequences from sensitive and resistant strains The sequence polymorphism has been studied in the coding region (including the intron) and in the 50 -flanking region of B. cinerea metC gene for the AniS strains, L, T4, and SAS56 and for the AniR1 strains: 1851, 1856, 1864, 4 271 17 a, 7 170 21 2, 8 159 22 d, 8 184 23 a, 8 245 23 a, 9 22 2b 2, and 9 175 22 b (Table 1). The results are shown in Table 2. In the coding region, we found 13 single-nucleotide substitutions. Among them, 11 are transition-mutations at nt 1200 1561, 1578, 1594, 1680, 1740, 1815, 1971, 2038, 2145, and 2244. The others are transversion-mutations at nt 1959 and 2416. Only one of the 13 substitutions is in the intron at nt 2416 (1282 from ATG). The others are in the coding region. Among those substitutions, 11 are silent-mutations and one, at nt 904 from ATG, is a missense-mutation which leads to an amino-acid difference: a threonine (T, ACA) at position 302 (Fig. 3) is replaced by an alanine (A, GCA). This substitution, found in 2 strains of AniR1 phenotype, 8 245 23 a and 9 22 2b 2 is without any effect on the growth of these strains. Although situated in the conserved domain (PF01053), this aminoacid substitution, at position 302 in B. cinerea CBL, should be without any effect on the efficiency of the resulting enzyme because this amino-acid position corresponds to an alanine in N. crassa CBL, at position 296, and in A. nidulans CBL, at position 289 (Fig. 3). So, the substitution of a threonine by an alanine at position 302 seems not to play a major role. As reported by Sienko et al. [23] in A. nidulans CBL gene, we observed that the silent-mutations are distributed along the gene while the missense-mutation is found in the Cterminal part of the gene which is less determining. Above all, we are not able to find any correlation between the observed polymorphisms and the phenotype of the strains used (Tables 1 and 2). In the promoter region (50 -flanking region of 1.134 kb), we found 12 single-nucleotide substitu-
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Fig. 3. Alignment of B. cinerea (BCMETC; AF211176), N. crassa (NCMET2; AF401237) and A. nidulans (ANMETG; U28383) cystathionine b-lyase sequences using Clustal W program [24]. Identical residues are marked by asterisks and similar ones by two dots, when codons differ in one base, or one dot when codons differ in two bases. Residues (in bold) involved in PLP binding are indicated by arrows. Conserved domain found by BLASTP program [25] is indicated by the dotted line between arrows.
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Table 2 Sequence polymorphism in the 50 -flanking region and in the coding region of the metC gene of B. cinerea Nta
Change in relation to the sensitive strain L
Strains concernedb
30 81 96 186 198 375 700 718 719 745 748 1033 1200, 1561, 1578, 1594, 1680, 1740, 1815, 1959, 1971, 2038, 2145, 2244, 2416,
T!C G!A G!A G!A T!C A!G A!T G!A T!C G!A G!A C!G G!A T!C T!C C!T C!T T!C C!T T!G G!A A!G T!C C!T A!T
T4, 8 159 22d, 9 175 22b, 1851 T4, 8 159 22d, 9 175 22b, 1851 T4, 8 159 22d, 9 175 22b, 1851 T4, 8 159 22d, 9 175 22b, 1851 T4, 8 159 22d, 9 175 22b, 1851 T4, 8 159 22d, 9 175 22b, 1851 T4, 8 159 22d, 9 175 22b, 1851 T4, 9 175 22b All strains except, L, 4 271 17a and 1856 All strains except, L, 4 271 17a and 1856 All strains except, L and 4 271 17a SAS56, 7 170 21 2, 8 184 23a, 8 245 23a, 9 22 2b 2, 1864 All strains except, L and 4 271 17a SAS56, 7 170 21 2 T4, SAS56, 7 170 21 2, 9 175 22b, 1851 T4, SAS56, 7 170 21 2, 9 175 22b, 1851 SAS56, 7 170 21 2 SAS56, 7 170 21 2 8 159 22d, 8 184 23a, 8 245 23a, 9 22 2b 2, 1864 8 159 22d, 8 184 23a, 8 245 23a, 9 22 2b 2, 1864 T4, 9 175 22b, 1851 8 245 23a, 9 22 2b 2 8 159 22d, 8 184 23a, 8 245 23a, 9 22 2b 2, 1864 8 159 22d, 8 184 23a, 8 245 23a, 9 22 2b 2, 1864 T4, SAS56, 7 170 21 2, 9 175 22b, 1851
66 427 444 460 546 606 681 825 837 904c 1011 1110 1282d
a
Nucleotide positions (see Accession No.: AF211176); those counted from ATG are in bold. Strains sensitive to anilinopyrimidines are in bold. c Mutation resulting in an amino-acid substitution (see text). d Mutation in the intron. b
tions. Among them, 10 are transition-mutations at nt 30, 81, 96, 186, 198, 375, 718, 719, 745, and 748. The others are transversion-mutations at nt 700 and 1033. None of the observed mutation occur in the putative CAAT-box or TATA-box and none of them correlate with AniR1 phenotypes and/or AniS phenotypes. Our results about the sequence polymorphism presented in Table 2 as those reported by Giraud et al. [14] show that a wide sequence polymorphism is encountered in B. cinerea. However, the sequence polymorphism uncountered in the CBL gene of B. cinerea seems to be less important than those reported in the CBL gene of A. nidulans [23] where, between two unrelated strains, the authors revealed up to 37 single-nucleotide differences of which 30 were in the coding region.
4. Conclusion We show that APs, at 0.1 mM, inhibit the CBL isolated from B. cinerea while Sierotzki et al. [26], in their experimental conditions, did not find any effect of cyprodinil at the same concentration. Nevertheless, this inhibition, compared to those of AVG, is too low to consider the CBL of B. cinerea as the primary target site of APs. However, as we mentioned above, if the APs might be activated we were not able to look for any activity of APs on our crude CBL. Moreover, the sequence polymorphism encountered in the metC gene of B. cinerea, in the coding region or upstream in the promoter region, does not discriminate between AP-sensitive strains and AP-resistant ones even if we found in two cases an amino-acid change in the enzyme. Similarly, Sierotzki et al. [26], analysing
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R. Fritz et al. / Pesticide Biochemistry and Physiology 77 (2003) 54–65
the eight ascospore progeny isolates of a cross between an AP-sensitive strain and an AP-resistant one, found point mutations but without amino-acid change in the enzyme. However, the fact that APs could interfere with the expression of the metC gene cannot be ruled out. To answer this question, we tried to study the expression the metC gene by Northern blotting but we were unable to highlight any transcription product of this gene (not shown). This failure was probably due to the very weak level of expression of metC gene. Structure similarities exist between APs and the experimental anilide SC-0858 whose fungitoxicity is also reversed by methionine and additionally by cystathionine [36]. CBL, and above all, cystathionine c-synthase (CGS) were proposed to be putative target sites for SC-0858 [36]. Moreover, Belfaiza et al. [37] suggested that CBL and CGS enzymes, because of their homology, originate from a common ancestral gene. For these reasons, it could be proposed that APs inhibit CGS. However, this hypothesis could be rejected because of the absence of any cross-resistance between APs and SC-0858 [26,38]. Finally, the question is to know why a treatment by APs leads to an increase of L -cystathionine in B. cinerea [10]. To answer this question, we must determine the exact nature of Ani 1 gene [12,13], which confers resistance toward APs. For this purpose, we plan to transform a sensitive strain with genomic bank of an AniR1 strain.
References [1] G.L. Neumann, E.H. Winter, J.E. Pittis, Pyrimethanil: a new fungicide, Brighton Crop Prot. Conf. Pests Dis. 1 (1992) 395. [2] U.J. Heye, J. Speich, H. Siegle, A. Steinemann, B. Forster, G. Knauf-Beiter, J. Herzog, A. Hubele, CGA 219417: a novel broad-spectrum fungicide, Crop Prot. 13 (1994) 541. [3] S. Maeno, I. Miura, K. Masuda, T. Nagata, Mepanipyrim (KIF-3535), a new pyrimidine fungicide, Brighton Crop Prot. Conf. Pests Dis. 2 (1990) 415. [4] A. Daniels, J.A. Lucas, Mode of action of the anilinopyrimidine fungicide pyrimethanil. 1. In vivo activity against Botrytis fabae on broad bean (vicia faba) leaves, Pestic. Sci. 45 (1995) 33.
[5] R.J. Milling, C.J. Richardson, Mode of action of the anilinopyrimidine fungicidepyrimethanil. 2. Effects on enzyme secretion in Botrytis cinerea, Pestic. Sci. 45 (1995) 43. [6] I. Miura, T. Kamakura, S. Maeno, S. Hayashi, I. Yamaguchi, Inhibition of enzyme secretion in plant pathogens by mepanipyrim, a novel fungicide, Pestic. Biochem. Physiol. 48 (1994) 222. [7] I. Miura, T. Kamakura, S. Maeno, T. Nagata, S. Hayashi, I. Yamaguchi, Effect of mepanipyrim on uptake of various substrates and macromolecular biosyntheses in Botrytis cinerea, J. Pestic. Sci. 19 (1994) 103. [8] P. Masner, P. Muster, J. Schmid, Possible methionine biosynthesis inhibition by pyrimidinamine fungicides, Pestic. Sci. 42 (1994) 163. [9] P. Leroux, Effect of pH, amino acids and various organic compounds on the fungitoxicity of pyrimethanil, glufosinate, captafol, cymoxanil and fenpiclonil in Botrytis cinerea, Agronomie 14 (1994) 541. [10] R. Fritz, C. Lanen, V. Colas, P. Leroux, Inhibition of methionine biosynthesis in Botrytis cinerea by the anilinopyrimidine fungicide pyrimethanil, Pestic. Sci. 49 (1997) 40. [11] U.W. Hilber, M. Hilber-Bodmer, Genetic basis and monitoring of resistance of Botryotinia fuckeliana to anilinopyrimidines, Plant Dis. 82 (1998) 496. [12] F. Chapeland, R. Fritz, C. Lanen, M. Gredt, P. Leroux, Inheritance and mechanism of resistance to anilinopyrimidine fungicides in Botrytis cinerea (Botryotinia fuckeliana), Pestic. Biochem. Physiol. 64 (1999) 85. [13] F. Chapeland, Etude de la resistance aux anilinopyrimidines et a dÕautres fongicides chez le champignon phytopathogene Botrytis cinerea, PhD thesis, University of Paris VI, France, 2000. [14] T. Giraud, D. Fortini, C. Levis, P. Leroux, Y. Brygoo, RFLP markers show genetic recombination in Botryotinia fuckeliana (Botrytis cinerea) and transposable elements reveal two sympatric species, Mol. Biol. Evol. 14 (1997) 1177. [15] P. Leroux, F. Chapeland, D. Desbrosses, M. Gredt, Patterns of cross-resistance to fungicides in Botryotinia fuckeliana (Botrytis cinerea) isolated from French vineyards, Crop Prot. 18 (1999) 687. [16] R. Fritz, P. Leroux, M. Gredt, Mecanisme de lÕaction fongitoxique de la promidione (26019 RP ou glycophene), de la vinchlozoline et du dicloran sur Botrytis cinerea Pers, Phytopathol. Z. 90 (1977) 152. [17] B. Forster, U. Heye, C. Pillonel, T. Staub, Methods to determine the sensitivity to cyprodinil, a new broadspectrum fungicide, in Botrytis cinerea, in: H. Lyr, P.E. Russel, T. Staub (Eds.), Modern Fungicides and Antifungal Compounds, Intercept, Andover, 1996, pp. 365–376. [18] S. Guggenheim, b-Cystathionase (Salmonella), in: S.P. Colowick, N.O. Kaplan (Eds.), Methods in Enzymology, vol. XIIB, Academic Press, New York, 1971, pp. 439– 442. [19] J. Giovanelli, Cystathionine b-lyase from spinach, in: S.P. Colowick, N.O. Kaplan (Eds.), Methods in Enzymology, vol. 143, Academic Press, New York, 1987, pp. 443–449.
R. Fritz et al. / Pesticide Biochemistry and Physiology 77 (2003) 54–65 [20] E.F. Hartree, Determination of protein: a modification of Lowry method that gives a linear photometric response, Anal. Biochem. 48 (1972) 422. [21] M. Droux, S. Ravanel, R. Douce, Methionine biosynthesis in higher plants. II. Purification and characterisation of cystathionine b-lyase from Spinach chloroplasts, Arch. Biochem. Biophys. 316 (1995) 585. [22] M. Coglievina, R. Klima, I. Bertani, D. Delneri, P. Zaccaria, C.V. Bruschi, Sequencing of a 40.5-kb fragment located on the left arm of the chromosome VII from Saccharomyces cerevisiae, Yeast 12 (1997) 149. [23] M. Sienko, A. Paszewski, The metG gene of Aspergillus nidulans encoding cystathionine b-lyase: cloning and analysis, Curr. Genet. 35 (1999) 638. [24] J.D. Thompson, D.G. Higgins, T.J. Gibson, CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice, Nucleic Acids Res. 22 (1994) 4673. [25] S.F. Altschul, T.L. Madden, A.A. Sch€affer, J. Zhang, Z. Zhang, W. Miller, D.J. Lipman, Gapped BLAST and PSIBLAST: a new generation of protein database search programs, Nucleic Acids Res. 25 (1997) 3389. [26] H. Sierotzki, J. Wullschleger, M. Alt, Thierry Bruyere, C. Pillonel, S. Parisi, U. Gisi, Potential mode of resistance to anilino-pyrimidine fungicides in Botrytis cinerea, in: H.-W. Dehne, U. Gisi, K.H. Kuck, P.E. Russell, H. Lyr (Eds.), Modern Fungicides and Antifungal Compounds III, AgroConcept GmbH, Bonn, Verlag Th Mann GmbH & Co. KG, Gelsenkirchen, 2002, pp. 141–148. [27] J.J.P. Bruchez, J. Eberle, V.E.A. Russo, Regulatory sequences involved in the translation Neurospora crassa mRNA: Kozak sequences and stop codons, Fungal Genet. Newsl. 40 (1993) 85. [28] D.J. Ballance, Sequences important for gene expression in filamentous fungi, Yeast 2 (1986) 229. [29] J.J.P. Bruchez, J. Eberle, V.E.A. Russo, Regulatory sequences in the transcription of Neurospora crassa genes: CAAT box, TATA box, introns, poly(A) tail formation sequences, Fungal Genet. Newsl. 40 (1993) 89.
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[30] A.L. Stanton, M. Mazelis, The C-S lyases of higher plants: homogeneous b-cystathionase of Spinach leaves, Arch. Biochem. Biophys. 290 (1991) 46. [31] S. Ravanel, M.-L. Ruffet, R. Douce, Cloning of an Arabidopsis thaliana cDNA encoding cystathionine b-lyase by functional complementation in Escherichia coli, Plant Mol. Biol. 29 (1995) 875. [32] F.W. Alexander, E. Sandmeier, P.K. Mehta, P. Christen, Evolutionary relationships among pyridoxal-50 -phosphatedependent enzymes. Regio-specific a; b and c families, Eur. J. Biochem. 219 (1994) 953. [33] T. Clausen, R. Huber, B. Laber, H.-D. Pohlenz, A. Messerschmidt, Crystal structure of the pyridoxal-50 -phosphate dependent cystathionine b-lyase from Escherichia , J. Mol. Biol. 262 (1996) 202. coli at 1.83 A [34] U. Breitinger, T. Clausen, S. Ehlert, R. Huber, B. Laber, F. Schmidt, E. Pohl, A. Messerschmidt, The three-dimensional strusture of cystathionine b-lyase from Arabidopsis and its substrate specificity, Plant Physiol. 126 (2001) 631. [35] A. Martel, C. Bouthier de la Tour, F. Le Goffic, Pyridoxal50 -phosphate binding of Escherichia coli beta-cystathionase and cystathionine gamma synthase: comparison of their sequences, Biochem. Biophys. Res. Commun. 147 (1987) 565. [36] B.C. Baldwin, K.H. Brownell, S.B. Rees, T.E. Wiggins, R. Furter, P. Masner, and P. Zobrist, The mode of action of the anilide fungicide SC-0858, 8th IUPAC International Congress on Pesticide Chemistry, Washington, USA, poster, 1994, p. 423. [37] J. Belfaiza, C. Parsot, A. Martel, C. Bouthier de la Tour, D. Margarita, G.N. Cohen, I. Saint-Girons, Evolution in biosynthetic pathways: two enzymes catalysing consecutive steps in methionine biosynthesis originate from a common ancestor and possess a similar regulatory region, Proc. Natl. Acad. Sci. USA 83 (1986) 867. [38] P. Leroux, R. Fritz, D. Debieu, C. Albertini, C. Lanen, J. Bach, M. Gredt, F. Chapeland, Mechanisms of resistance to fungicides in field strains of Botrytis cinerea, Pest Manag. Sci. 58 (2002) 876.
Pesticide Biochemistry and Physiology 77 (2003) 54–65 www.elsevier.com/locate/ypest
Effect of the anilinopyrimidine fungicide pyrimethanil on the cystathionine b-lyase of Botrytis cinerea Rene Fritz,* Catherine Lanen, Florence Chapeland-Leclerc, and Pierre Leroux Institut National de la Recherche Agronomique, Unit e de Phytopharmacie et des M ediateurs Chimiques, 78026 Versailles Cedex, France Received 22 April 2003; accepted 24 July 2003
Abstract Previous studies, carried out in our laboratory, upon the mode of action of anilinopyrimidines (APs) suggested that these fungicides inhibit the biosynthesis of methionine and that the primary target could be the cystathionine b-lyase (CBL). More recent works carried on with strains of Botrytis cinerea highly resistant to APs (AniR1 ) suggested that one major gene (Ani 1) confers this phenotype and that this gene segreged independently of the gene of resistance to dicarboximides. This confirmed the fact that the target site of APs must be different from that of dicarboximides and suggested that the mechanism of resistance to APs could be due to a mutation in Ani 1 gene. In this report, we tried to find if the CBL of B. cinerea could be the target site of the APs, pyrimethanil and cyprodinil. To do this, first we searched for an inhibitory effect of APs on the enzyme activity of the B. cinerea CBL and second, we compared the nucleotide sequences of the B. cinerea CBL gene, we called metC, of 3 strains sensitive to APs (AniS ) and 10 strains AniR1 . We showed that aminoethoxyvinylglycine at 2.5 lM strongly inhibited, in a competitive manner, a crude extract of the CBL isolated from a strain AniS or from a strain AniR1 . On the other hand, APs, at 0.1 mM, slightly inhibited the CBL isolated from B. cinerea. This inhibitory effect which was uncompetitive was not different between the strain AniS and the strain AniR1 . We sequenced and analysed the metC gene (Accession No.: AF211176). Based on the cDNA sequence, we found that this gene spans 1377 bp and is interrupted by one intron. We found consensus sequences for the putative CAAT-box, TATA-box, transcription-site, polyadenylation, and splicing signals. The metC gene codes for a polypeptide chain of 459 amino-acids and has a predicted molecular weight of 49,087 Da. The analysis of the sequence polymorphism in 13 strains did not allow us to discriminate between the sensitive strains and the resistant ones. So, it is assumed that the CBL of B. cinerea does not seem to be the primary target site of APs and probably does not correspond to the Ani 1 gene. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Anilinopyrimidines; Botrytis cinerea; Fungicide resistance; Cystathionine b-lyase; Mode of action; Enzyme activity; Sequence polymorphism; metC gene
1. Introduction * Corresponding author. Fax: +33-1-30-83-31-19. E-mail address: [email protected] (R. Fritz).
Pyrimethanil, cyprodinil, and mepanipyrim are broad-spectrum fungicides belonging to the
0048-3575/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0048-3575(03)00094-4
R. Fritz et al. / Pesticide Biochemistry and Physiology 77 (2003) 54–65
chemical class of anilinopyrimidines (APs).1 They are highly effective against Botrytis cinerea Pers. ex Fr. (anamorph of Botryotinia fuckeliana (de Bary) Whetz.), the causal agent of grey mould, and show no cross-resistance to any current commercially available botryticides [1–3]. This suggests that APs have a different mode of action. Tested in vitro on Botrytis spp., pyrimethanil does not show any effect on respiration, lipid peroxidation, osmotic stability or on biosynthesis of protein, RNA, DNA, chitin or ergosterol [1]. In Botrytis fabae and B. cinerea, pyrimethanil has been reported to inhibit the secretion of cell wall degrading enzymes required for infection at concentrations lower than those needed to inhibit growth [4,5]. This has also been reported for mepanipyrim [6,7]. This mode of action has been suggested to be the primary one for pyrimethanil [5]. Nevertheless, it has been shown that pyrimethanil and the other APs are effective on B. cinerea growth at low concentrations in culture media lacking amino-acids. Addition of several different amino-acids to the culture media reduced the growth inhibitory activity of pyrimethanil. Among these, methionine did not reverse the activity of pyrimethanil completely but exhibited the highest reversal activity suggesting a direct effect of pyrimethanil on the methionine biosynthesis pathway in B. cinerea [8,9]. We showed that when mycelium of B. cinerea was treated with low concentrations of pyrimethanil the total amount of free aminoacids increased. Qualitative variations were also induced and particularly methionine decreased while cystathionine accumulated [10]. These data indicate that pyrimethanil inhibits the biosynthesis of methionine and suggest that the primary target could be the cystathionine b-lyase (CBL). More recently, works conducted with strains of B. cinerea highly resistant to APs (AniR1 phenotype) suggested that one major gene (Ani 1) confers this phenotype and that this gene segregated independently of the gene of resistance to dicarboximides [11–13]. Therefore, the target site of APs must be different from that of dicarboximides and the 1
Abbreviations used: APs, anilinopyrimidines; CBL, cystathionine b-lyase, AVG, L -a-2-aminoethoxyvinylglycine; PLP, pyridoxal 50 -phosphate; CGS, cystathionine c-synthase.
55
mechanism of resistance to APs could be due to a mutation in Ani 1 gene. In this report, we try to demonstrate that APs inhibit the enzyme activity of B. cinerea CBL. We present the nucleotide sequence of the gene encoding this enzyme which we called metC and compare the nucleotide sequences of the metC gene of strains sensitive to APs (AniS phenotype) and strains of AniR1 phenotype. The results are used to discuss about the mode of action of APs.
2. Materials and methods 2.1. Chemicals Technical grade fungicides were provided as follows: pyrimethanil was from AgrEvo (UK), while cyprodinil was from Novartis (Germany). These compounds were dissolved in ethanol whose final concentration was never more than 0.5%. Yeast nitrogen base without amino-acids was from Difco laboratories, Detroit, USA. L -a-2-Aminoethoxyvinylglycine (AVG) and other chemicals were from Sigma–Aldrich, France. Taq DNA polymerase was from Qiagen, USA. 2.2. Fungi The inhibitory effect of the fungicides on the enzyme activity was studied using the strain L of AniS phenotype [9,10] and the strain 4 271 17 a, of AniR1 phenotype [12,13]. The comparison between the different CBL nucleotide sequences was performed on the above strains as well as on two other AniS strains, T4 and SAS56 and nine other AniR1 strains, 1851, 1856, 1864, 7 170 21 2, 8 159 22 d, 8 184 23 a, 8 245 23 a, 99 22 2b 2, and 99 175 22 b. The main characteristics of these strains are presented in Table 1. All the strains isolated from berries collected in vineyards are of transposa type [14]. SAS56, a reference strain for the mating type provided by Faretra from the University of Bari, Italy, contains only the transposable element boty [14]. The strain T4, of transposa type, and whose cDNA library was submitted to NCBI GenBank database, was provided by Yves Brygoo from INRA of Versailles, France [14]. The description
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Table 1 Main characteristics of isolates of B. cinerea used to compare CBL nucleotide sequences or CBL enzyme activity (in bold) Strain
Year
Origin
Phenotypea
L T4 SAS56 1851 1856 1864 4 271 17a 7 170 21 2 8 159 22d 8 184 23a 8 245 23a 9 22 2b 2 9 175 22b
1980 1991
Bordeaux, grapes Eyrague, tomato Reference strain from Faretra Bordeaux, grapes, noble rot Bordeaux, grapes, noble rot Bordeaux, grapes, noble rot Armagnac, grapes Loire Valley, grapes Champagne, grapes Champagne, grapes Champagne, grapes Loire Valley, grapes Loire Valley, grapes
AniS , BenR1 , ImiS AniS , BenS , ImiS AniS , BenS , ImiS AniR1 , BenS , ImiS AniR1 , BenS , ImiS AniR1 , BenS , ImiS AniR1 , BenS , ImiR1 AniR1 , BenS , ImiS AniR1 , BenR1 , ImiS AniR1 , BenS , ImiS AniR1 , BenR1 , ImiR1 AniR1 , BenS , ImiS AniR1 , BenR2 , ImiR1
—
1999 1999 1999 1994 1997 1998 1998 1998 1999 1999
a AniS , sensitive to anilinopyrimidines; BenS , sensitive to benzimidazoles; ImiS , sensitive to dicarboximides; AniR1 , highly resistant to anilinopyrimidines; AniR1 , highly resistant to anilinopyrimidines but to a lesser extent; BenR1 , resistant to benzimidazoles and sensitive to phenylcarbamates; BenR2 , resistant to benzimidazoles and phenylcarbamates; and ImiR1 , resistant to dicarboximides and sensitive to phenylpyrroles (Leroux et al. [15]).
of the different phenotypes has been previously reported by Leroux et al. [15]. All the strains were maintained and sporulated in the dark on a medium containing: 20 g malt, 5 g yeast extract, and 12.5 g agar, for 1 L. Liquid cultures, in exponential growth phase, were prepared by inoculating 7 105 macroconidia ml1 in the medium previously described [16] (150 ml in 300 ml Erlenmeyer flasks) but without yeast extract because of the possible reversal activity of methionine [10]. To set up the best conditions in the production of an enzyme sensitive to APs, the yeast extract was replaced by 2 g L1 of yeast nitrogen base without amino-acids or by 2 g L1 of asparagine [17]. This latter medium was made of: 10 g glucose, 1.5 g K2 HPO4 , 2 g KH2 PO4 , 1 g (NH4 )2 SO4 , 0.5 g MgSO4 7H2 O, and 2 g asparagine, for 1 L. Cultures were shaken (orbital shaker at 150 rev min1 ) for 18 h at 23 °C. To obtain more mycelium, each preliminary culture was filtered through a 100 lm gauze, washed with fresh medium, and resuspended in 1200 ml of the same fresh medium to prepare two cultures of 600 ml each contained in 1 L Erlenmeyer flask. These cultures were incubated for 24 h again under the previous conditions. Finally, mycelial pellets were harvested, filtered through a 100 lm gauze, and washed by cold UHP water (Vivendi Water STI, France).
2.3. Enzyme preparation The mycelium was washed and lyophilized. The freeze-dried mycelium (1.5 g) was homogenized for 3 3 s in a mini-mincer (Seb, France) until a mycelial-powder was obtained. The mycelial-powder was added to 15 ml of a 0.1 M phosphate buffer, pH 7.3, containing 1 mM Na2 -ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol (DTT), and 0.2 mM pyridoxal 50 -phosphate (PLP), cooled to 4 °C. The suspension was homogenized in a Potter tube of 30 ml in less than 30 s (20-fold up and down). The Potter tube was washed by an equal volume of the same buffer. The total homogenate (cell-free extract and wash) was centrifuged at about 15,000g and 4 °C for 45 min. The resulting supernatant was then acidified to pH 6.5 with 4 M acetic acid and then quickly heated up to 55 °C in a water bath, during 4 min, while stirring with a glass rod. The supernatant was immediately cooled in an ice bath under stirring with the same glass rod and submitted to a second centrifugation at about 32,000g and 4 °C for 45 min. The new supernatant was recovered and its pH was balanced to 7.3 with a 20% ammonia solution. This supernatant was subjected to a 35–45% ammonium sulfate fractionation. The two steps of the fractionation were done in a beaker placed in an
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ice bath, upon a magnetic stirrer. A slow rate of stirring must be used to avoid foaming and ammonium sulfate was added in small batches, being dissolved separately during 15 min. During stirring, the pH value was maintained at 7.3 with the precedent acetic acid solution. After the first step of fractionation (35%), the protein suspension was decanted to a centrifugal tube and left to stand for 1 h, in an ice bath, to ensure equilibrium. To remove the precipitated protein, the tube was centrifuged at about 20,000g and 4 °C for 30 min. The supernatant was subjected to the second step of fractionation (45%) using the same precautions and conditions as the precedent, except that the resultant protein suspension was kept in a refrigerator during the night. Finally, the precipitated protein was recovered by centrifugation at about 20,000g and 4 °C for 30 min. The protein pellets were suspended in 4 ml of the precedent buffer without the protectors (EDTA and DTT) but with PLP (PLP buffer). The protein solution obtained was filtered through a glass fibre filter (GF/A from Whatman) and then through filter series of hydrophilic membrane Durapore (Millipore) with three different pore sizes: 0.45, 0.22, and finally 0.1 lm. After each filtration, the filter was washed by 3 0.5 ml of PLP buffer. The filtrates were pooled in a Centricon plus-20 device with a Biomax membrane with a molecular weight cutoff of 50 kDa (Millipore) and centrifuged at 4000g and 4 °C for 95 min. The protein retained on the Centricon membrane was washed by 10 ml of PLP buffer and centrifuged for 1 h. The protein retained (2 ml) was recovered by centrifuging the membrane of the Centricon upside down. This enzyme preparation was glycerol added up to 20% and kept frozen at )20 °C in aliquots of 300 ll each until application. 2.4. Essay of CBL activity CBL catalyses the following reaction: cystathionine þ H2 O ! pyruvate þ homocysteine þ NH3 Enzyme activity was assayed according to the methods modified from Guggenheim [18] and Gi-
57
ovanelli [19], whereby pyruvate production was coupled to NADH oxidation via a lactate dehydrogenase (LDH) pyruvate þ NADH $ lactate þ NAD: The resultant decrease in absorption was followed with a thermostated (30 °C) Uvikon 933 spectrophotometer from Kontron Instruments at 340 nm. The assay was carried out in cuvettes of 1-cm light path in 0.1 M Tris–HCl buffer at pH 8.3 containing 2–7 mM L -cystathionine, 0.3 lmol NADH, and 30 lg L -LDH from pig heart provided by Boehringer Mannheim, Germany (300 U mg1 at 25 °C) and 0.1 lmol PLP. The reference cuvette contained identical components except that enzyme was replaced by Tris–HCl buffer. The reaction was started by adding substrate and the optical density at 340 nm versus a buffer blank was recorded continuously. For each substrate concentration, the reaction speed (disappearance of NADH according to time measured at 340 nm which is correlated, via pyruvate formation, to the consumption of cystathionine) was calculated. Each speed value used in the double-reciprocal plot representation resulted in the kinetic analysis of a set of different absorption measures, which was at least linear from 2 to 15 min. Protein concentration was determined using the method of Lowry modified by Hartree [20] with bovine serum albumin fraction V (Sigma) as protein standard. As AVG, an analogue of L -cystathionine, is known to be a potent inhibitor of CBL [21], we tested the reliability of our crude extract by looking at the activity of AVG toward our crude extract. We found that, at 2.5 lM, AVG solubilized in Tris–HCL buffer always showed a strong inhibitory effect. 2.5. Preparation of DNA, RNA, and cDNA from B. cinerea DNA was extracted from lyophilized mycelium as described by Chapeland [13] and conserved at )20 °C. RNA was extracted with the ‘‘RNeasy Plant Mini Kit’’ (Qiagen, USA) according to the protocol described for filamentous fungi by the supplier.
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The synthesis of cDNA from RNA was carried out with the ‘‘first-strand cDNA Synthesis’’ kit (Clontech, USA) according to supplierÕs instructions.
TTACAATGGAGCTGCTGCTGC-30 as 30 -primer. The RT-PCR was carried out as described by Chapeland [13]. 2.8. DNA sequencing
2.6. PCR and genome walker libraries for finding the DNA sequence of cystathionine b-lyase All primers were synthesized by Sigma-Genosys (UK). The PCR amplification of a first fragment of the gene encoding cystathionine b-lyase in B. cinerea was performed with degenerate primers designed to amplify well-conserved regions of genes encoding well-identified CBL of two microorganisms: the CBL gene (Accession No.: P53101) from Saccharomyces cerevisiae [22] and the metG gene (Accession No.: U28383) from Aspergillus nidulans [23]. The 50 -primer was: 50 -TAYGAYTAYACICGITC NGGNAA-30 (Y ¼ C/T; I ¼Inosine, A/G; N ¼ A/ G/T/C). The 30 -primer was 50 -RTCRTCIACRTCY TCDAT-30 (R ¼ A/G; D ¼ A/G/T). The PCR was performed, in a Gene Amp PCR system 2400 (Perkin–Elmer), as described by Chapeland [13]. To obtain the complete sequence of the B. cinerea CBL gene, we used the ‘‘Universal GenomeWalker’’ kit (Clontech, USA). This kit allowed us to find unknown genomic DNA sequences adjacent to the known precedent sequence. From this sequence, four gene-specific primers synthesized by SigmaGenosys (UK) were designed for DNA walking [13]. Two 30 -primers were designed for walking upstream, one for the primary PCR: 50 -GGGGTT GCATAGCATAGGAGAAAGCAT-30 , the other for the nested PCR: 50 -CGCGAGATGTCGTTCC AG-30 . Two 50 -primers were designed for walking downstream, one for the primary PCR: 50 -ATGCT TTCTCCTATGCTATGCAACCCC-30 , the other for the nested PCR: 50 -GGCGATGTTGCTCTCT CTGA G-30 . The GenomeWalker ‘‘libraries’’ were constructed and the PCRs were done according to supplierÕs instructions. 2.7. RT-PCR Two gene-specific primers were designed for RT-PCR from the complete sequence of the CBL of B. cinerea: 50 -CCAATATGTCTTCACCATC AGGCTCC-30 as 50 -primer, and 50 -CATTCG
PCR and RT-PCR fragments were purified with the ‘‘QIAquick PCR’’ kit (Qiagen, USA). All sequences were determined at the Euro Sequences Genes Services (ESGS, France). 2.9. Analysis of the sequences Consensus multiple alignment for nucleotide and amino-acid sequences was performed with CLUSTAL W program [24] available on the Infobiogen web site (http://www.infobiogen.fr). The similarity searches in databases were performed with the Basic Local Alignment Search Tool (BLAST) programs [25] available on the NCBI web site (http://www.ncbi.nlm.nih.gov).
3. Results and discussion 3.1. Properties of CBL from B. cinerea Our preparation method implies that crude CBL from B. cinerea is relatively resistant to heat up to 55 °C. Preparation of crude CBL must be done at an acidic or neutal pH (6.5–7.3) and need the presence of PLP to avoid any degradation of the enzyme activity. The strongest activity was found in the 35–45% ammonium sulfate fraction. We did not test a wide range of temperatures but the best results using LDH were obtained at 30 °C. The activity of crude CBL required PLP and was best in Tris–HCl buffer at pH 8.3. But at this pH the enzyme activity could not be preserved more than 1 day. This was probably due to a degradation process. Our crude CBL was active toward L -djenkolate but better results were obtained toward L -cystathionine. The activity of crude CBL increased when the cystathionine concentration raised (Fig. 1). The apparent Km value, found in the double reciprocal plot, (Fig. 2) was 1.6 mM and the Vmax value was 17.4 nmol min1 mg protein1 .
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Fig. 1. Activity of crude CBL of B. cinerea versus cystathionine concentrations and inhibitory effect of aminoethoxyvinylglycine (AVG), pyrimethanil, and cyprodinil.
Fig. 2. Double-reciprocal plot of the data shown in Fig. 2, about activity of crude CBL of B. cinerea, and inhibitory effect of aminoethoxyvinylglycine (AVG), pyrimethanil, and cyprodinil.
Sierotzki et al. [26], working on a CBL crude extract from B. cinerea homogenized with a French press and tested in a potassium phosphate buffer at
pH 7.5 and at 30 °C, obtained a comparable Km value but their Vmax value was about 8-fold lower. We found that AVG did not have any effect on the
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LDH coupling reaction. At 2.5 lM, AVG strongly inhibited our crude CBL, but this effect decreased as the cystathionine concentration increased (Fig. 1): it was probably due to a competition between the substrate, cystathionine, and the inhibitor, AVG. Therefore, in accordance with kinetic models, AVG showed an inhibition of competitive type with an apparent Km value of 10.2 mM and a Vmax value of 17.1 nmol min1 mg protein1 (Figs. 1 and 2). As expected, assuming a simple Michaelis–Menten mechanism, the apparent Km value strongly increased while the Vmax value tended to stay constant. With our crude CBL, at 2.5 lM AVG did not cause a time-dependent inactivation of B. cinerea CBL. All the CBL enzyme properties presented above were shared by the AP sensitive strain L and the AP resistant strain 4 271 17a.
more polar than pyrimethanil. After 30 min, the overall recovery of [14 C]pyrimethanil was identical in the 2 strains tested (97%), but after 6 h this recovery was slightly better in the strain L than in the strain 4 271 17a, respectively, 96 and 86% [13]. It was assumed that these results cannot account for the resistance because the difference in the recovery of pyrimethanil did not take place earlier enough. But the occurrence of metabolites, even in small amount, might support the hypothesis of an activation. However, these metabolites separated by thin-layer chromatography were not fungitoxic toward conidia of the strain L sprayed on the thinlayer [13].
3.2. Effect of pyrimethanil and cyprodinil on CBL of B. cinerea
The complete nucleotide (nt) sequence of the CBL (metC gene) and 50 -flanking region of the strain L sensitive to anilinopyrimide fungicides (2921 bp) was submitted to the NCBI GenBank database under Accession No.: AF211176. The translation initiation codon for the metC gene was assigned to the ATG at nt 1135. The sequence around this presumed translation start site (50 CAATATGTC-30 ) fits well the consensus sequence for the translation initiation site in Neurospora crassa (50 -CAM‘‘T3’’ATGGC-30 ) [27]. The metC gene coding region spans 1377 bp and is interrupted only by one intron, unlike the metG gene of A. nidulans which is interrupted by two introns [23]. The position of this small intron (49 bp) was determined through a comparison of cDNA and genomic metC sequences. Nucleotide sequences at all exon–intron junctions conform to an established filamentous fungi splice-site consensus sequence: 50 -donor site g^ GTAAGT and 30 -acceptor site CCAG^ g [28]. Similarly, internal lariat sequence agrees with a filamentous fungi lariat consensus: TGCTAAC [28]. As described in our submission to the NCBI GenBank (Accession No.: AF211176), we found several putative consensus sequences, which match well with those reported in N. crassa [29]: TCATCATC for transcription site, ATTATAA for TATA-box, CAAAT for CAATbox, and AATAAA for polyadenylation site signal. Translation terminates at the TAA stop codon
With the two strains tested, the AP sensitive strain L and the AP resistant strain 4 271 17a, the activity of our crude CBL was not affected by pyrimethanil and cyprodinil at concentrations below 0.1 mM. But at 0.1 mM we observed a significant inhibitory effect of these two APs. With pyrimethanil and cyprodinil, we obtained an uncompetitive inhibition with an apparent Km value of 1.0 mM and a Vmax value of 10.6 nmol min1 mg protein1 (Fig. 2). At 0.1 mM, we found, with both fungicides, an equal level of inhibition of Vmax of 39% (Fig. 1). In their experimental conditions, Sierotzki et al. [26] reported that at 0.1 mM cyprodinil the enzyme activity of CBL of B. cinerea was not affected whatever the concentrations of substrate or PLP. These results did not take account for a possible mechanism of activation of anilinopyrimidines. In previous studies, [14 C]pyrimethanil was not metabolized after a 6 h incubation in a medium having contained the strain L or the strain 4 271 17a [12,13]. In the presence of these 2 strains, only 1% of the radioactivity brought by 100 lM [14 C]pyrimethanil was recovered in the mycelial pellet. This very small fungicidal uptake did not make easy the interpretation of the results. After 6 h, the 2 strains studied produced, in small amount, 4 metabolites
3.3. Nucleotide-sequence analysis of the CBL of the sensitive strain L
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(nt 2561). The metC gene of B. cinerea codes for a polypeptide chain of 459 amino-acids and has a predicted molecular weight of 49,087 Da. This is in agreement with the monomer molecular weight estimated for various CBL: 48,280 Da for A. nidulans [23], 53,000 Da for Spinacia oleracea L. [30], and 50,372 Da for Arabidopsis thaliana [31]. Using the BLAST program for protein sequences [25], several putative conserved domains were detected. Only one conserved domain showed a credible score. This domain is 384 residues long and belongs to the protein family, PF01053, named cysteine/methionine metabolism PLP-dependent enzyme. This family includes enzymes involved in cysteine and methionine metabolism as cystathionine c-lyases, cystathionine c-synthases (CGS), cystathionine b-lyases (CBL), and homocysteine synthases. In the metC gene of B. cinerea, this conserved protein-domain starts at position 52 (ATEL. . .) and ends at position 422 (. . .SRAL) as shown in CLUSTAL W alignment [24] between the protein sequence of the CBL gene of B. cinerea and those of 2 other filamentous fungi: N. crassa and A. nidulans (Fig. 3). So, B. cinerea CBL belongs to the c-family of pyridoxal 50 -phosphatedependent enzymes described by Alexander et al. [32]. The conserved Lys-243 residue (K) in the B. cinerea enzyme, aligned with Lys-237 in N. crassa and Lys-230 in A. nidulans (Fig. 3), seems to be homologous to Lys-210 in Escherichia coli CBL [33], Lys-198 in E. coli CGS [33], and Lys-278 in A. thaliana CBL [31,34], which were identified as being covalently bound to the PLP cofactor [35]. Two other conserved residues in B. cinerea CBL, Glu-187 (E) and Asp-218 (D), respectively, aligned, on the one hand, with Glu-181 and Asp212 in N. crassa and, on the other hand, with Glu174 and Asp-205 in A. nidulans (Fig. 3), seem to be homologous to Glu-154 and Asp-185 of E. coli CBL [33] and to Glu-224 and Asp-253 in A. thaliana CBL [31,34]. These three residues are conserved in the whole c-family [23] and participate in PLP-binding [33]. The analysis of identities between the B. cinerea CBL sequence and those of the closest organisms found in the NCBI GenBank database reveals that they are better with the filamentous fungi N. crassa (80%) and A. nidulans (68%) than with S. cerevisiae (41%) or plant CBL
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(41–42%). B. cinerea CBL shows the better identity with those of N. crassa. This fact is in accordance with the similarities found between the consensus sequences of these two fungi. 3.4. Comparison between the CBL gene sequences from sensitive and resistant strains The sequence polymorphism has been studied in the coding region (including the intron) and in the 50 -flanking region of B. cinerea metC gene for the AniS strains, L, T4, and SAS56 and for the AniR1 strains: 1851, 1856, 1864, 4 271 17 a, 7 170 21 2, 8 159 22 d, 8 184 23 a, 8 245 23 a, 9 22 2b 2, and 9 175 22 b (Table 1). The results are shown in Table 2. In the coding region, we found 13 single-nucleotide substitutions. Among them, 11 are transition-mutations at nt 1200 1561, 1578, 1594, 1680, 1740, 1815, 1971, 2038, 2145, and 2244. The others are transversion-mutations at nt 1959 and 2416. Only one of the 13 substitutions is in the intron at nt 2416 (1282 from ATG). The others are in the coding region. Among those substitutions, 11 are silent-mutations and one, at nt 904 from ATG, is a missense-mutation which leads to an amino-acid difference: a threonine (T, ACA) at position 302 (Fig. 3) is replaced by an alanine (A, GCA). This substitution, found in 2 strains of AniR1 phenotype, 8 245 23 a and 9 22 2b 2 is without any effect on the growth of these strains. Although situated in the conserved domain (PF01053), this aminoacid substitution, at position 302 in B. cinerea CBL, should be without any effect on the efficiency of the resulting enzyme because this amino-acid position corresponds to an alanine in N. crassa CBL, at position 296, and in A. nidulans CBL, at position 289 (Fig. 3). So, the substitution of a threonine by an alanine at position 302 seems not to play a major role. As reported by Sienko et al. [23] in A. nidulans CBL gene, we observed that the silent-mutations are distributed along the gene while the missense-mutation is found in the Cterminal part of the gene which is less determining. Above all, we are not able to find any correlation between the observed polymorphisms and the phenotype of the strains used (Tables 1 and 2). In the promoter region (50 -flanking region of 1.134 kb), we found 12 single-nucleotide substitu-
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Fig. 3. Alignment of B. cinerea (BCMETC; AF211176), N. crassa (NCMET2; AF401237) and A. nidulans (ANMETG; U28383) cystathionine b-lyase sequences using Clustal W program [24]. Identical residues are marked by asterisks and similar ones by two dots, when codons differ in one base, or one dot when codons differ in two bases. Residues (in bold) involved in PLP binding are indicated by arrows. Conserved domain found by BLASTP program [25] is indicated by the dotted line between arrows.
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Table 2 Sequence polymorphism in the 50 -flanking region and in the coding region of the metC gene of B. cinerea Nta
Change in relation to the sensitive strain L
Strains concernedb
30 81 96 186 198 375 700 718 719 745 748 1033 1200, 1561, 1578, 1594, 1680, 1740, 1815, 1959, 1971, 2038, 2145, 2244, 2416,
T!C G!A G!A G!A T!C A!G A!T G!A T!C G!A G!A C!G G!A T!C T!C C!T C!T T!C C!T T!G G!A A!G T!C C!T A!T
T4, 8 159 22d, 9 175 22b, 1851 T4, 8 159 22d, 9 175 22b, 1851 T4, 8 159 22d, 9 175 22b, 1851 T4, 8 159 22d, 9 175 22b, 1851 T4, 8 159 22d, 9 175 22b, 1851 T4, 8 159 22d, 9 175 22b, 1851 T4, 8 159 22d, 9 175 22b, 1851 T4, 9 175 22b All strains except, L, 4 271 17a and 1856 All strains except, L, 4 271 17a and 1856 All strains except, L and 4 271 17a SAS56, 7 170 21 2, 8 184 23a, 8 245 23a, 9 22 2b 2, 1864 All strains except, L and 4 271 17a SAS56, 7 170 21 2 T4, SAS56, 7 170 21 2, 9 175 22b, 1851 T4, SAS56, 7 170 21 2, 9 175 22b, 1851 SAS56, 7 170 21 2 SAS56, 7 170 21 2 8 159 22d, 8 184 23a, 8 245 23a, 9 22 2b 2, 1864 8 159 22d, 8 184 23a, 8 245 23a, 9 22 2b 2, 1864 T4, 9 175 22b, 1851 8 245 23a, 9 22 2b 2 8 159 22d, 8 184 23a, 8 245 23a, 9 22 2b 2, 1864 8 159 22d, 8 184 23a, 8 245 23a, 9 22 2b 2, 1864 T4, SAS56, 7 170 21 2, 9 175 22b, 1851
66 427 444 460 546 606 681 825 837 904c 1011 1110 1282d
a
Nucleotide positions (see Accession No.: AF211176); those counted from ATG are in bold. Strains sensitive to anilinopyrimidines are in bold. c Mutation resulting in an amino-acid substitution (see text). d Mutation in the intron. b
tions. Among them, 10 are transition-mutations at nt 30, 81, 96, 186, 198, 375, 718, 719, 745, and 748. The others are transversion-mutations at nt 700 and 1033. None of the observed mutation occur in the putative CAAT-box or TATA-box and none of them correlate with AniR1 phenotypes and/or AniS phenotypes. Our results about the sequence polymorphism presented in Table 2 as those reported by Giraud et al. [14] show that a wide sequence polymorphism is encountered in B. cinerea. However, the sequence polymorphism uncountered in the CBL gene of B. cinerea seems to be less important than those reported in the CBL gene of A. nidulans [23] where, between two unrelated strains, the authors revealed up to 37 single-nucleotide differences of which 30 were in the coding region.
4. Conclusion We show that APs, at 0.1 mM, inhibit the CBL isolated from B. cinerea while Sierotzki et al. [26], in their experimental conditions, did not find any effect of cyprodinil at the same concentration. Nevertheless, this inhibition, compared to those of AVG, is too low to consider the CBL of B. cinerea as the primary target site of APs. However, as we mentioned above, if the APs might be activated we were not able to look for any activity of APs on our crude CBL. Moreover, the sequence polymorphism encountered in the metC gene of B. cinerea, in the coding region or upstream in the promoter region, does not discriminate between AP-sensitive strains and AP-resistant ones even if we found in two cases an amino-acid change in the enzyme. Similarly, Sierotzki et al. [26], analysing
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the eight ascospore progeny isolates of a cross between an AP-sensitive strain and an AP-resistant one, found point mutations but without amino-acid change in the enzyme. However, the fact that APs could interfere with the expression of the metC gene cannot be ruled out. To answer this question, we tried to study the expression the metC gene by Northern blotting but we were unable to highlight any transcription product of this gene (not shown). This failure was probably due to the very weak level of expression of metC gene. Structure similarities exist between APs and the experimental anilide SC-0858 whose fungitoxicity is also reversed by methionine and additionally by cystathionine [36]. CBL, and above all, cystathionine c-synthase (CGS) were proposed to be putative target sites for SC-0858 [36]. Moreover, Belfaiza et al. [37] suggested that CBL and CGS enzymes, because of their homology, originate from a common ancestral gene. For these reasons, it could be proposed that APs inhibit CGS. However, this hypothesis could be rejected because of the absence of any cross-resistance between APs and SC-0858 [26,38]. Finally, the question is to know why a treatment by APs leads to an increase of L -cystathionine in B. cinerea [10]. To answer this question, we must determine the exact nature of Ani 1 gene [12,13], which confers resistance toward APs. For this purpose, we plan to transform a sensitive strain with genomic bank of an AniR1 strain.
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