Diverse mechanisms associated with cyhalofop-butyl resistance in Chinese sprangletop ( Leptochloa chinensis (L.) Nees): Characterization of target-site mutations and metabolic resistance-related genes in two resistant populations

Resistance of Chinese sprangletop ( (L.) Nees) to the herbicide cyhalofop-butyl has recently become a severe problem in rice cultivation. However, the molecular mechanisms of target-site resistance (TSR) in cyhalofop-butyl resistant as well as the underlying non-target-site resistance (NTSR) have not yet been well-characterized. This study aimed to investigate cyhalofop-butyl resistance mechanisms using one susceptible population (LC-S) and two resistant populations (LC-1701 and LC-1704) of We analyzed two gene copies encoding the entire carboxyltransferase (CT) domain of chloroplastic acetyl-CoA carboxylase (ACCase) from each population. Two non-synonymous substitutions were detected in the resistant populations (Trp -Cys in the 1 of LC-1701 and Leu -Phe in the 2 of LC-1704), which were absent in LC-S. As Trp -Cys confers resistance to ACCase-inhibiting herbicides, the potential relationship between the novel Leu -Phe mutation and cyhalofop-butyl resistance in LC-1704 was further explored by single-nucleotide polymorphism (SNP) detection. Metabolic inhibition assays indicated that cytochrome P450 monooxygenases (P450s) and glutathione -transferases (GSTs) contributed to cyhalofop-butyl resistance in specific resistant populations. RNA sequencing showed that the P450 genes , , , , and and the GST genes , , and were upregulated in at least one resistant population, which indicated their putative roles in cyhalofop-butyl resistance of . Correlation analyses revealed that the constitutive or inducible expression patterns of , , , and in were strongly associated with the resistant phenotype. For this reason, attention should be directed towards these genes to elucidate metabolic resistance to cyhalofop-butyl in . The findings of this study improve the understanding of mechanisms responsible for resistance to ACCase-inhibiting herbicides in grass-weed species at the molecular level, thus aiding in the development of weed management strategies that delay the emergence of resistance to this class of pest control products.