Aimone Porri, Quincy D Law, Charles M Geddes, Joseph Ikley, Samuel Willingham, Philipp Johnen, Ingo Meiners, Sama Al-Sammarraie, Kim Crommar, Pieter B F Ouwerkerk, Michael Betz, Brian M Jenks, Heike Heiser, Fabienne Baumann, Frank Braendle, Jens Lerchl
PPO-inhibiting herbicides are widely used to manage weeds in different cropping systems, yet resistance evolution threatens their long-term efficacy. Here, we investigated the molecular basis of resistance to PPO-inhibiting herbicides in Bassia scoparia biotypes collected from four locations in North Dakota, USA. Greenhouse dose-response assays revealed high levels of resistance to saflufenacil and carfentrazone-ethyl, while fomesafen retained full efficacy across all biotypes. Resistant plants did not show increased copy number or elevated expression of PPX1 or PPX2. Sequencing of survivor plants revealed conserved PPO2 protein sequences, but consistent target-site substitutions at position F454 in PPO1, including F454I (Phenylalanine, to Isoleucine) F454L (Phenylalanine, to Leucine), and F454V (Phenylalanine, to Valine). In vitro enzyme assays demonstrated that these substitutions impair PPO1 enzyme sensitivity to saflufenacil and carfentrazone, but not to fomesafen. Ectopic expression of B. scoparia PPX1 encoding F454I, L and V variants in Arabidopsis thaliana conferred tolerance to saflufenacil and carfentrazone-ethyl, but not to fomesafen, supporting greenhouse and in vitro results. Molecular modeling indicated that the conformational flexibility and interaction profile of fomesafen enables it to maintain binding to mutated PPO1 enzyme variants, in contrast to the more rigid structures of saflufenacil and carfentrazone. A yeast-based complementation system further confirmed that F454 substitutions decrease herbicide sensitivity. In addition, developmental profiling showed distinct expression patterns of PPX1 and PPX2 during early growth stages in B. scoparia and Amaranthus palmeri, highlighting isoform-specific roles. Together, these findings represent the first reported PPO1 target-site amino acid substitutions in a broadleaf weed species as a key mechanism of resistance and highlight that fomesafen is effective to control resistant B. scoparia populations.