A codon deletion confers resistance to herbicides inhibiting protoporphyrinogen oxidase

Abstract

Herbicides that act by inhibiting protoporphyrinogen oxidase (PPO) are widely used to control weeds in a variety of crops. The first weed to evolve resistance to PPO-inhibiting herbicides was Amaranthus tuberculatus, a problematic weed in the midwestern United States that previously had evolved multiple resistances to herbicides inhibiting two other target sites. Evaluation of a PPO-inhibitor-resistant A. tuberculatus biotype revealed that resistance was a (incompletely) dominant trait conferred by a single, nuclear gene. Three genes predicted to encode PPO were identified in A. tuberculatus. One gene from the resistant biotype, designated PPX2L, contained a codon deletion that was shown to confer resistance by complementation of a hemG mutant strain of Escherichia coli grown in the presence and absence of the PPO inhibitor lactofen. PPX2L is predicted to encode both plastid- and mitochondria-targeted PPO isoforms, allowing a mutation in a single gene to confer resistance to two herbicide target sites. Unique aspects of the resistance mechanism include an amino acid deletion, rather than a substitution, and the dual-targeting nature of the gene, which may explain why resistance to PPO inhibitors has been rare.

Fig. 1.

Dose–response curves of different A. tuberculatus lines to the PPO inhibitor lactofen. Lactofen was foliar-applied to greenhouse-grown plants from the S (filled diamonds), R (open circles), F₁(R) (open triangles), or F₁(S) (filled triangles) A. tuberculatus lines. Data were collected 15 days after treatment. Vertical bars represent ± SEM (n = 12).

Fig. 2.

Southern blot of A. tuberculatus gDNA probed with a fragment of PPX2L. DNA was isolated from plants that were derived from the S or R biotype and digested with EcoRI or HindIII.

Fig. 3.

PCR-based molecular marker analysis of PPX1 or PPX2L alleles. A. tuberculatus plants used in the study were derived from F₁ hybrids backcrossed to the S parent (BC_S). Markers were used to determine whether the F₁-derived pollen carried an S or R parental allele. BC_S plants were treated with lactofen at 110 g ai·ha⁻¹ plus 1% (vol/vol) COC and harvested 15 days after treatment. Vertical bars represent ± SEM (PPX1, n = 42 or 40 for S or R parental alleles, respectively; PPX2L, n = 39 or 49 for S or R parental alleles, respectively).

Fig. 4.

Selected amino acid residues of N. tabacum PPO2 in proximity to the herbicide-binding site. A. tuberculatus plants resistant to PPO inhibitors are missing a glycine residue equivalent to G178 of N. tabacum. This amino acid deletion is predicted to hinder PPO inhibitor binding. Amino acid residues: D, aspartic acid; G, glycine; C, cysteine; and T, threonine. PPO-inhibiting herbicide: Flz, fluazolate.

Fig. 5.

PPO expression in a hemG mutant strain of E. coli. E. coli were grown on LB medium alone or supplemented with hematin (20 μg·ml⁻¹) or lactofen (100 nM). E. coli isolates were as follows: C1 and C2, nontransformed controls; S1 and S2, transformed with a vector encoding A. tuberculatus-derived PPO2L with glycine at position 210; and R1 and R2, transformed with a vector encoding identical PPO2L with the exception of a deletion of glycine at position 210.