A single amino-acid substitution in the iron-sulphur protein subunit of succinate dehydrogenase determines resistance to carboxin in Mycosphaerella graminicola.
A gene encoding the iron-sulphur protein (Ip) subunit of succinate dehydrogenase (Sdh, EC 1.3.99.1) from Mycosphaerella graminicola (Septoria tritici) has been cloned andsequenced. The deduced amino-acid sequence exhibited a high degree of homology to Ip subunits of Sdh from other organisms; three cysteine-rich clusters associated with the iron-sulphur centres involved in electron transport were particularly conserved. Expression studies using a synthetic green fluorescent protein (SGFP) expression vector demonstrated that the cloned DNA also contained a functional promoter region and confirmed that the deduced initiation codon could act as a translational start site. Mutants resistant to the fungicide carboxin (Cbx), a known inhibitor of Sdh, were found to contain a single amino-acid substitution in the third cysteine-rich domain of the Ip protein. These mutations resulted in the conversion of a highly conserved His residue, located in a region of the protein associated with the [3Fe-4 S] high-potential non-heme iron sulphur-redox (S3) centre, to either Tyr or Leu. AnIp gene containing the His -> Tyr mutation was constructed and shown to confer Cbx resistance following co-transformation into the Cbx-sensitive wild-type strain. This confirmed that the mutation identified by sequence analysis was responsible for determining Cbx resistance.
Skinner
W
W
Department of Cell Biology, IACR-Long Ashton Research Station, University of Bristol, Long Ashton, Bristol BS18 9AF, UK.
Bailey
A
A
Renwick
A
A
Keon
J
J
Gurr
S
S
Hargreaves
J
J
D000483:Alleles; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001203:Ascomycota / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D002261:Carboxin / Q000494:pharmacology*; D004252:DNA Mutational Analysis; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004352:Drug Resistance, Microbial / Q000235:genetics; D005659:Fungicides, Industrial / Q000494:pharmacology*; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal / Q000235:genetics; D007506:Iron-Sulfur Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D013385:Succinate Dehydrogenase / Q000235:genetics*
D004271:DNA, Fungal; D005659:Fungicides, Industrial; D007506:Iron-Sulfur Proteins; D002261:Carboxin; D013385:Succinate Dehydrogenase