5.5.19
MOLECULAR GENETICS OF CARBOXIN RESISTANCE IN MYCOSPHAERELLA GRAMINICOLA

W SKINNERl, A BAILEYl, A RENWICK2, S GURR3, J KEONl and J HARGREAVESl

lIACR-Long Ashton Research Station, University of Bristol, Long Ashton, Bristol BS18 9AF, UK; 2Zeneca Agrochemicals, Jealott's Hill Research Station, Bracknell, Berkshire RG12 6EY, UK; 3Department of Plant Sciences, South Parks Road, University of Oxford, Oxford OX1 3RB, UK.

Background and objective
The fungicide carboxin (Cbx) is a respiratory toxin that prevents the oxidation of succinate by inhibiting the tricarboxylic acid cycle enzyme, succinate dehydrogenase (Sdh). This enzyme is composed of two subunits, a flavoprotein and an iron-sulphur protein (1p), which together with two membrane-anchoring proteins make up succinate-quinone oxidoreductase. Cbx is thought to act by preventing the transfer of electrons from succinate to ubiquinone through inhibiting the reoxidation of the high-potential S3 centre of the lp subunit. Furthermore, Cbx resistance in Ustilago maydis is known to be determined by a single amino acid residing in the Cys-rich cluster which ligates the S3 centre [1]. Our aim was to isolate the gene encoding the lp subunit from the wheat pathogen Mycosphaerella graminicola and to determine the nature of a mutation determining Cbx resistance in this pathogen.

Results and conclusions
The M. graminicoia lp subunit gene:
The gene encoding the lp subunit of Sdh from M. graminicola has been isolated and sequenced. This was done by amplifying a fragment of the gene using degenerate PCR primers and by using this amplified DNA product as a probe to recover the complete gene from a lambda genomic library. The gene, which was interrupted by two introns, encoded a peptide which showed homology to lp subunits from a wide range of other organisms. In particular, three Cys-rich clusters associated with the iron-sulphur centres were well conserved. Furthermore, the aminoterminal of the deduced protein was rich in basic and in hydroxylated amino acids; a characteristic of cleavable presequences of nuclear DNA encoded proteins that are imported into mitochondria.
A single amino acid substitution determines resistance to Cbx:
Seven mutant strains which grew on media supplemented with normally inhibitory concentrations of Cbx were recovered following UV mutagenesis. Comparison of partial sequences of the lp subunit alleles from these strains with that of the wild-type revealed that in each of the mutants a single point mutation had occurred at codon 267, which encodes His in the third Cys-rich duster that binds the S3 centre. In six of the mutants, a C-T nucleotide mutation led to the replacement of the His residue with Tyr, while in the seventh mutant an A-T mutation resulted in the substitution of Leu, a mutation identical to that found in Cbx-resistant mutants of U. maydis [2].

References
l. Broomfield PLJ, Hargreaves JA, 1992. Current Genetics 22, 117-121.
2. Keon JPR, Broomfield PLJ, White GA, Hargreaves JA, 1994. Biochemical Society Transactions 22, 234-237.