Novel α-Tubulin Mutations Conferring Resistance to Dinitroaniline Herbicides in Lolium rigidum

Abstract

The dinitroaniline herbicides (particularly trifluralin) have been globally used in many crops for selective grass weed control. Consequently, trifluralin resistance has been documented in several important crop weed species and has recently reached a level of concern in Australian Lolium rigidum populations. Here, we report novel mutations in the L. rigidum α-tubulin gene which confer resistance to trifluralin and other dinitroaniline herbicides. Nucleotide mutations at the highly conserved codon Arg-243 resulted in amino acid substitutions of Met or Lys. Rice calli transformed with the mutant 243-Met or 243-Lys α-tubulin genes were 4- to 8-fold more resistant to trifluralin and other dinitroaniline herbicides (e.g., ethalfluralin and pendimethalin) compared to calli transformed with the wild type α-tubulin gene from L. rigidum. Comprehensive modeling of molecular docking predicts that Arg-243 is close to the trifluralin binding site on the α-tubulin surface and that replacement of Arg-243 by Met/Lys-243 results in a spatial shift of the trifluralin binding domain, reduction of trifluralin-tubulin contacts, and unfavorable interactions. The major effect of these substitutions is a significant rise of free interaction energy between α-tubulin and trifluralin, as well as between trifluralin and its whole molecular environment. These results demonstrate that the Arg-243 residue in α-tubulin is a determinant for trifluralin sensitivity, and the novel Arg-243-Met/Lys mutations may confer trifluralin resistance in L. rigidum.

Keywords: Lolium rigidum; dinitroanilines; mutation; trifluralin resistance; α-tubulin.

FIGURE 1

Vector construct for co-expression of α- and β-tubulin genes in rice callus. HPT, hygromycin phosphotransferase; N, Nopaline synthase terminator; Ubi, Ubiquitin promotor; HA, Hemagglutinin epitope tag; c-myc, epitope tag; Kan, kanamycin.

FIGURE 2

Western blot analysis of expression of WT, and mutant 243-Met and 243-Lys α-tubulin variants in transgenic rice seedlings in comparison to an untransformed control, probed by antibody against fused HA (hemagglutinin) epitope tag. HSP (Heat shock protein 70) expression was used as a protein loading control.

FIGURE 3

Growth of rice calli transformed with the WT, mutant 243-Met or 243-Lys α-tubulin genes in medium containing (A) 0, (B) 0.5, (C) 2.0, and (D) 4.0 mg L^-1 trifluralin.

FIGURE 4

Growth of rice calli transformed with the WT, mutant 243-Met or 243-Lys α-tubulin genes in medium containing (A) 2.0, (B) 4.0, (C) 6.0, and (D) 8.0 mg L^-1 ethalfluralin.

FIGURE 5

Growth of rice calli transformed with the WT, mutant 243-Met or 243-Lys α-tubulin genes in medium containing (A) 2.0, (B) 4.0, (C) 6.0, and (D) 10 mg L^-1 pendimethalin.

FIGURE 6

Ribbon diagram view of predicted trifluralin-binding amino acids relative to the position of amino acid Arg-243 in Lolium rigidum α-tubulin. Positions of binding site amino acids and Arg-243 (arrowed) are colored by burgundy. Trifluralin atoms colored by standard color scheme for atom representation.

FIGURE 7

Spatial structure of contact interface between trifluralin and wild type (WT) α-tubulin. Protein contact surface is colored by H-bond donor/acceptor distribution, binding site amino acids represented by sticks, and intermolecular contacts indicated by dotted lines.

FIGURE 8

Spatial arrangement of trifluralin-binding amino acids in WT and the Arg-243-Met mutant α-tubulin isoform. Residues of WT and mutant isoforms are colored by green and violet, respectively.

FIGURE 9

Spatial arrangement of trifluralin-binding amino acids in WT and the Arg-243-Lys mutant α-tubulin isoforms. Residues of WT and mutant isoforms are colored by green and orange, respectively.

FIGURE 10

Spatial structure of contact interface between trifluralin and the Arg-243-Met (A), and between trifluralin and the Arg-243-Lys (B) mutant α-tubulins. Protein contact surface is colored by H-bond donor/acceptor distribution, binding site amino acids represented by sticks, and intermolecular contacts indicated by dotted lines.

FIGURE 11

2D diagram of intermolecular interactions (shown by dotted lines) between trifluralin and the Arg-243-Met (A), and between trifluralin and the Arg-243-Lys (B) mutant α-tubulins.

FIGURE 12

Helical growth of the 243-Met mutant (right side) as compared to the WT (left side).