A naturally occurring point mutation confers broad range tolerance to herbicides that target acetolactate synthase

Acetolactate synthase (ALS) inhibitors are among the most commonly used herbicides. They fall into four distinct families of compounds: sulfonylureas, imidazolinones, triazolopyrimidine sulfonanilides, and pyrimidinyl oxybenzoates. We have investigated the molecular basis of imidazolinone tolerance of two field isolates of cocklebur (Xanthium sp.) from Mississippi and Missouri. In both cases, tolerance was conferred by a form of ALS that was less sensitive to inhibitors than the wild type. The insensitivity pattern of the Mississippi isolate was similar to that of a commercial mutant of corn generated in the laboratory: ICI 8532 IT. Sequencing revealed that the same residue (Ala57-->Thr) was mutated in both Mississippi cocklebur and ICI 8532 IT corn. ALS from the Missouri isolate was highly insensitive to all the ALS herbicide families, similar in this respect to another commercial corn mutant: Pioneer 3180 IR corn. Sequencing of ALS from both plants revealed a common mutation that changed Trp552 to Leu. The sensitive cocklebur ALS cDNA, fused with a glutathione S-transferase, was functionally expressed in Escherichia coli. The recombinant protein had enzymatic properties similar to those of the plant enzyme. All the possible point mutations affecting Trp552 were investigated by site-directed mutagenesis. Only the Trp-->Leu mutation yielded an active enzyme. This mutation conferred a dramatically reduced sensitivity toward representatives of all four chemical families, demonstrating its role in herbicide tolerance. This study indicates that mutations conferring herbicide tolerance, obtained in an artificial environment, also occur in nature, where the selection pressure is much lower. Thus, this study validates the use of laboratory models to predict mutations that may develop in natural populations.