Mechanism of metamifop inhibition of the carboxyltransferase domain of acetyl-coenzyme A carboxylase in Echinochloa crus-galli

Acetyl-coenzyme A carboxylase (ACCase) plays crucial roles in fatty acid metabolism and is an attractive target for herbicide discovery. Metamifop is a novel ACCase-inhibiting herbicide that can be applied to control sensitive weeds in paddy fields. In this study, the effects of metamifop on the chloroplasts, ACCase activity and carboxyltransferase (CT) domain gene expression in Echinochloa crus-galli were investigated. The results showed that metamifop interacted with the CT domain of ACCase in E. crus-galli. The three-dimensional structure of the CT domain of E. crus-galli ACCase in complex with metamifop was examined by homology modelling, molecular docking and molecular dynamics (MD) simulations. Metamifop has a different mechanism of inhibiting the CT domain compared with other ACCase inhibitors as it interacted with a different region in the active site of the CT domain. The protonation of nitrogen in the oxazole ring of metamifop plays a crucial role in the interaction between metamifop and the CT domain. The binding mode of metamifop provides a foundation for elucidating the molecular mechanism of target resistance and cross-resistance among ACCase herbicides, and for designing and optimizing ACCase inhibitors.

. Ramachandran plot of CT domain of ACCase in Echinochloa crus-galli. The quality of the homology model of the barnyard grass CT domain was good, with most of the residues (91.5%) in the most favored region and 0.2% in the disallowed region.

Methods
Sequencing of chloroplastic ACCase CT domain. Total RNA was isolated from leaves of Echinochloa crus-galli using an RNAprep Pure Plant Kit (Tiangen Biotech Co., Ltd., Beijing, China). For RT-PCR, first-strand cDNA was reverse transcribed from the total RNA using the RevertAid RT Kit (Thermo Scientific). Based on the barnyard grass ACCase encoding sequence (GenBank accession HQ395758), a pair of primers (CT4889:5' AAACGCTGCTCTGCTAGGAA 3', CT7342: 5' CTTGCAAACT GTCACTCGGC 3') was designed for PCR amplification of the DNA fragment of the entire CT domain of barnyardgrass chloroplastic ACCase. Each amplification reaction was performed in a total volume of 50 µl with 37.75µl distilled deionized (dd) H2O, 1 µl of cDNA, 1 µl of each primer (10 µM), 5 µl of 10X Ex Taq Buffer, 4 µl of dNTP Mixture, and 0.25 µl of TaKaRa Ex Taq DNA Polymerase (250 U/µl). The RT-PCR procedure was as follows: an initial denaturation step at 95°C for 5 min, followed by 35 cycles of denaturation for 30 s at 95°C, annealing for 1 min at 57°C, extension for 2 min at 72°C, and a final elongation step at 72°C for 10 min. The amplification product obtained from at least 6 independent PCR reactions was purified and then sequenced on both strands using gene-specific primers. The qPCR amplification conditions included an initial denaturation at 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. After amplification, a melting curve analysis from 65°C to 95°C was verified to produce a specific amplified product.
A pair of primers (CT4F 5' TTGCCTCTGTTCATCCTT 3', CT4R 5' TCAGCATAACACTCAATTCG 3') for the qPCR amplification of the accD gene was designed, and the primers used for β-actin were as described by Zhibo H. et al. 18 .
Relative changes in gene expression were calculated by the 2 −△△Ct method 33 .
ACCase activity assay. When plants grew to the 2-to 3-leaf stage, the shoots (3 g per sample) were harvested at the base, quickly frozen in liquid nitrogen, and stored at -80°C. The next day, these materials were ground in liquid nitrogen and homogenized in 15 mL of extraction buffer (100 mM Tris-HCl, pH 8.0, 1 mM EDTA, 10% glycerol, 2 mM isoascorbic acid, 0.5% PVP-40, 0.5% PVPP, 20 mM DTT and 1 mM PMSF).
The homogenate was then filtered through four layers of muslin and maintained on ice until centrifuged (27 000 g for 15 min at 4°C) to remove cell debris. The pellet was discarded, and the supernatant was adjusted using 10% ammonium sulphate and stirred for 30 min. The supernatant was transferred to a new tube and centrifuged at 27 000 g for 30 min. The pellet was discarded. The supernatant was adjusted with 40% ammonium sulphate and stirred for 30 min. The supernatant was centrifuged at 27 000 g for 30 min. The pellet containing the protein extract was resuspended in 2 mL of elution buffer and desalted on a Sephadex G-25 column equilibrated with elution buffer (50 mM Tricine-KOH, pH 8.0, 2.5 mM MgCl2, 50 mM KCl and 1 mM DTT) 34 . The protein extracts were frozen at -80°C and assayed the next day. The total protein contents of the ACCase isolations were determined as described by Bradford 35 .
The malonyl-CoA concentration in each sample was determined with an injection volume of 10.0 µl using an HPLC system (Agilent 1200, USA) to record the absorbance peak area at 260 nm. A Thermo Scientific Hypersil ODS-2 (C18) HPLC column (4.6 mm i.d. ×250 mm, 5.0 µm particle size) was used to separate the substances. The