Single-site mutations in the carboxyltransferase domain of plastid acetyl-CoA carboxylase confer resistance to grass-specific herbicides

Abstract

Grass weed populations resistant to aryloxyphenoxypropionate (APP) and cyclohexanedione herbicides that inhibit acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) represent a major problem for sustainable agriculture. We investigated the molecular basis of resistance to ACCase-inhibiting herbicides for nine wild oat (Avena sterilis ssp. ludoviciana Durieu) populations from the northern grain-growing region of Australia. Five amino acid substitutions in plastid ACCase were correlated with herbicide resistance: Ile-1,781-Leu, Trp-1,999-Cys, Trp-2,027-Cys, Ile-2,041-Asn, and Asp-2,078-Gly (numbered according to the Alopecurus myosuroides plastid ACCase). An allele-specific PCR test was designed to determine the prevalence of these five mutations in wild oat populations suspected of harboring ACCase-related resistance with the result that, in most but not all cases, plant resistance was correlated with one (and only one) of the five mutations. We then showed, using a yeast gene-replacement system, that these single-site mutations also confer herbicide resistance to wheat plastid ACCase: Ile-1,781-Leu and Asp-2,078-Gly confer resistance to APPs and cyclohexanediones, Trp-2,027-Cys and Ile-2,041-Asn confer resistance to APPs, and Trp-1,999-Cys confers resistance only to fenoxaprop. These mutations are very likely to confer resistance to any grass weed species under selection imposed by the extensive agricultural use of the herbicides.

Fig. 1.

Amino acid sequence comparisons of the herbicide target site in the CT domain of the plant plastid (pla) and cytosolic (cyt) multidomain ACCase. Mutations associated with resistance are shown with residue numbering following the full-length A. myosuroides plastid ACCase (GenBank accession no. AJ310767) highlighted by dark-gray vertical strips with amino acids found at the corresponding position in yeast shown in the bottom row (underlined with numbering following the yeast ACCase sequence). Amino acid residues implicated in APP binding (5) are highlighted by light-gray vertical strips. Dots indicate identical residues. The composite sequences of the plastid and cytosolic ACCase were derived from sequences available from GenBank in December 2006.

Fig. 2.

Allele-specific PCR tests for ACCase mutations in herbicide-resistant populations of A. sterilis ssp. ludoviciana. L, 1-kb DNA marker; c, no DNA control; S, template DNA from a susceptible plant; R, template DNA from a resistant plant from each of the populations.

Fig. 3.

Response of yeast gene-replacement strains that depends for growth on chimeric wheat ACCase carrying single-site mutations to fenoxaprop-P-ethyl, haloxyfop, and sethoxydim. The control (sensitive) strain (w-t) with wild-type chimeric wheat ACCase was described previously (4).

Fig. 4.

Position of amino acid residues corresponding to the six herbicide-resistance mutations (green) in the 3D structure of the CT domain of yeast ACCase in complex with CoA (blue) and haloxyfop (red) (5, 17). Amino acids shown in yellow were determined to contribute to the haloxyfop-binding site (5). This illustration was prepared by using PyMol (DeLano Scientific, South San Francisco, CA) and coordinates from Protein Data Bank ID codes 1UYS and 1OD2. The numbering follows the yeast ACCase sequence.