Inhibition profile of trifludimoxazin towards PPO2 target site mutations

Abstract

Background: Target site resistance to herbicides that inhibit protoporphyrinogen IX oxidase (PPO; EC 1.3.3.4) has been described mainly in broadleaf weeds based on mutations in the gene designated protoporphyrinogen oxidase 2 (PPO2) and in one monocot weed species in protoporphyrinogen oxidase 1 (PPO1). To control PPO target site resistant weeds in future it is important to design new PPO-inhibiting herbicides that can control problematic weeds expressing mutant PPO enzymes. In this study, we assessed the efficacy of a new triazinone-type inhibitor, trifludimoxazin, to inhibit PPO2 enzymes carrying target site mutations in comparison with three widely used PPO-inhibiting herbicides.

Results: Mutated Amaranthus spp. PPO2 enzymes were expressed in Escherichia coli, purified and measured biochemically for activity and inhibition kinetics, and used for complementation experiments in an E. coli hemG mutant that lacks the corresponding microbial PPO gene function. In addition, we used ectopic expression in Arabidopsis and structural PPO protein modeling to support the enzyme inhibition study. The generated data strongly suggest that trifludimoxazin is a strong inhibitor both at the enzyme level and in transgenics Arabidopsis ectopically expressing PPO2 target site mutations.

Conclusion: Trifludimoxazin is a potent PPO-inhibiting herbicide that inhibits various PPO2 enzymes carrying target site mutations and could be used as a chemical-based control strategy to mitigate the widespread occurrence of PPO target site resistance as well as weeds that have evolved resistance to other herbicide mode of actions. © 2022 BASF SE and The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Keywords: PPO; efficacy; herbicide; herbicide resistance; mutations; target site; trifludimoxazin.

FIGURE 1

Binding mode of trifludimoxazin and binding pose fomesafen in Amaranthus tuberculatus protoporphyrinogen oxidase 2 (PPO2) wild‐type. The protein crystal structure of PPO2 co‐crystalized with trifludimoxazin is shown in white. Secondary structural elements are shown in cartoon style. A white surface shows the shape of the binding site. Trifludimoxazin is shown in stick representation with carbon in cyan, the binding mode of the co‐factor flavin adenine dinucleotide in gray and the modeled binding pose of fomesafen in magenta. Oxygen is shown in red, nitrogen in blue, sulfur in yellow and fluorine in green. The binding of PPO2 inhibitors is stabilized by pi‐stacking effects of the ring systems (magenta lines). The triazindionthione head group with the sulfur and two methyl groups fits very well in a hydrophobic environment and is covered between Phe420 and the tip of an α‐helical structure. The heterocyclic benzoxazinone exhibits many favoring van der Waals interactions with the β‐sheet elements shaping the binding site of protoporphyrinogen IX oxidase. The hydrophobic amino acids Leu384 and Leu400 interact favorably with the pi‐electron system. The propargyl group anchors in a small hydrophobic pocket, establishing a bond with a carbonyl via the polarized hydrogen. Arg128 can establish a charge‐assisted hydrogen bond upon binding. In particular, the CF2 group, with its multipolar interactions (green lines) to the carbonyl oxygens of Phe381, Gly382 and Val383 protein backbone, but also to Gly398, is key for affinity. Mutations at the position of Phe381 or Val383 would not change the interaction between the fluorine atoms to the backbone because the altered side chains would point away from trifludimoxazin. Any mutation of Gly382, the middle amino acid, generally results in an inactive protein.

FIGURE 2

T1 35S::AMATU PPO2 R128(X) ∆G210 Arabidopsis lines sprayed with saflufenacil and trifludimoxazin. Pictures were taken 7 days after treatment. The five pots contain independent transgenic events (T1 plants, selected with Imazamox by confirming presence of resistance gene AHAS). (a) Untreated wild‐type (WT) Arabidopsis (left) and treatment with the adjuvant DASH (right). (b) WT Arabidopsis treated with 2×, 1× and 0.5× (right to left) saflufenacil (upper) or trifludimoxazin (lower). (c–g) T1 Arabidopsis transgenics treated with 2×, 1× and 0.5× saflufenacil (upper) or trifludimoxazin (lower). 1× trifludimoxazin = 12.5 g ha⁻¹; 1× saflufenacil = 25 g ha⁻¹.

FIGURE 3

Trifludimoxazin binding in Amaranthus tuberculatus protoporphyrinogen oxidase 2 (PPO2) wild‐type with mutation sites. The protein crystal structure of PPO2 together with trifludimoxazin is shown in white. The binding mode of trifludimoxazin in the presence of target site mutations is shown in cyan. The β‐sheet wall (upper), and the α‐helical structure (middle) are depicted white, in cartoon style. The co‐factor flavin adenine dinucleotide is depicted by sticks with gray carbon color. Oxygen is shown in red, nitrogen in blue, sulfur in yellow and fluorine in green. The shape of the wild‐type binding site is highlighted by white hatching. R128G and G398A (cyan stick representation) reshaping the binding site are highlighted by blue hatching.