Effect of the anilinopyrimidine fungicide pyrimethanil on the cystathionine β-lyase of Botrytis cinerea

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, P-lyase (CBL). More recent works carried on with strains of Botrytis cinerea highly resistant to APs (Ani(R1)) 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 I 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 (Ani(S)) and 10 strains Ani(R1). We showed that aminoethoxyvinylglycine at 2.5 muM strongly inhibited, in a competitive manner, a crude extract of the CBL isolated from a strain Ani(S) or from a strain Ani(R1). 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 Ani(S) and the strain Ani(R1). 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 I gene. (C) 2003 Elsevier Inc. All rights reserved.