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.
Highlights
Dinitroaniline herbicides have been widely used for selective grass weed control in many crops
Calli, relative to the transformed WT calli (Figures 4, 5). This clearly demonstrates that the Arg-243-Met/Lys mutations endow resistance to dinitroaniline herbicides, with the Arg-243-Lys mutant being more resistant than the Arg-243-Met mutant (Figures 3–5)
In the first binding model, the trifluralin binding site on the α-tubulin surface consists of amino acid residues Arg2, Gly-131, Leu-132, Gln-133, Asn-253, Glu-254, and Thr-257 (Figures 6, 7)
Summary
Dinitroaniline herbicides have been widely used for selective grass weed control in many crops. Trifluralin is a soil-applied, pre-emergence dinitroaniline herbicide long used in Australia, where innovations in seeding machinery have enabled trifluralin to be safely used at a relatively high rate (>960 g ha−1) in no-till/conservation farming systems. In these systems, trifluralin controls germinating grass weed seeds in wheat and other crops. Dinitroaniline herbicides (e.g., trifluralin, pendimethalin and ethalfluralin) bind to tubulin and interrupt the polymerisation of microtubules, arresting cell division and elongation, and resulting in plant death. The precise tubulin binding sites for the dinitroanilines have not been determined, molecular structural modeling, together with analysis of tubulin resistance mutations in plants and protozoa (see below), has demonstrated that dinitroanilines most likely interact with α-tubulin to disrupt microtubule polymerisation (Blume et al, 2003; Morrissette et al, 2004; Nyporko and Blume, 2008, 2014; Nyporko et al, 2009), β-tubulin may be a target (Blume et al, 1998; Nyporko and Blume, 2008)
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