Molecular bases for sensitivity to tubulin-binding herbicides in green foxtail

Abstract

We investigated the molecular bases for resistance to several classes of herbicides that bind tubulins in green foxtail (Setaria viridis L. Beauv.). We identified two alpha- and two beta-tubulin genes in green foxtail. Sequence comparison between resistant and sensitive plants revealed two mutations, a leucine-to-phenylalanine change at position 136 and a threonine-to-isoleucine change at position 239, in the gene encoding alpha2-tubulin. Association of mutation at position 239 with herbicide resistance was demonstrated using near-isogenic lines derived from interspecific pairings between green foxtail and foxtail millet (Setaria italica L. Beauv.), and herbicide sensitivity bioassays combined with allele-specific PCR-mediated genotyping. Association of mutation at position 136 with herbicide resistance was demonstrated using herbicide sensitivity bioassays combined with allele-specific PCR-mediated genotyping. Both mutations were associated with recessive cross resistance to dinitroanilines and benzoic acids, no change in sensitivity to benzamides, and hypersensitivity to carbamates. Using three-dimensional modeling, we found that the two mutations are adjacent and located into a region involved in tubulin dimer-dimer contact. Comparison of three-dimensional alpha-tubulin models for organisms with contrasted sensitivity to tubulin-binding herbicides enabled us to propose that residue 253 and the vicinity of the side chain of residue 251 are critical determinants for the differences in herbicide sensitivity observed between organisms, and that positions 16, 24, 136, 239, 252, and 268 are involved in modulating sensitivity to these herbicides.

Figure 1.

Phylogenetic tree of foxtail α- and β-tubulin sequences, including introns. The tree was constructed using the maximum parsimony method as implemented in the Mega2.1 software (www.megasoftware.net).

Figure 2.

Ribbon diagram view of the surface of the region involved in dimer-dimer tubulin contact in three-dimensional models for α-tubulins. Sequences used for modeling are wild type, Leu-136, Thr-239 foxtail α2-tubulin (A); mutant, Leu-136, Ile-239 foxtail α2-tubulin (B); mutant, Phe-136, Thr-239 foxtail α2-tubulin (C); T. gondii α-tubulin (GenBank/EMBL/DDBJ accession no. M20024; D); carrot α-tubulin (GenBank/EMBL/DDBJ accession no. AY007250; E); C. reinhardtii α1-tubulin (GenBank/EMBL/DDBJ accession no. M11447; F); L. major α-tubulin (GenBank/EMBL/DDBJ accession no. AL359683; G); and rat α-tubulin (GenBank/EMBL/DDBJ accession no. V01227; H). On the figure, α-helixes are pink, β-sheets are blue, and the areas with no particular structure are orange. Atom colors are green, C; red, O; blue, N; yellow, S. Side chains of residues not discussed in the text and hydrogen atoms are not shown for clarity. Amino acid positions are labeled at the base of the corresponding side chain. Amino acid positions containing side chains different from those in wild type, Leu-136, Thr-239 foxtail α2-tubulin are in yellow boxes, and the resulting structural differences are circled.