Evolution of a Double Amino Acid Substitution in the 5-Enolpyruvylshikimate-3-Phosphate Synthase in Eleusine indica Conferring High-Level Glyphosate Resistance

Glyphosate is the most important and widely used herbicide in world agriculture. Intensive glyphosate selection has resulted in the widespread evolution of glyphosate-resistant weed populations, threatening the sustainability of this valuable once-in-a-century agrochemical. Field-evolved glyphosate resistance due to known resistance mechanisms is generally low to modest. Here, working with a highly glyphosate-resistant Eleusine indica population, we identified a double amino acid substitution (T102I + P106S TIPS]) in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene in glyphosate-resistant individuals. This TIPS mutation recreates the biotechnology-engineered commercial first generation glyphosate-tolerant EPSPS in corn (Zea mays) and now in other crops. In E. indica, the naturally evolved TIPS mutants are highly (more than 180-fold) resistant to glyphosate compared with the wild type and more resistant (more than 32-fold) than the previously known P106S mutants. The E. indica TIPS EPSPS showed very high-level (2,647-fold) in vitro resistance to glyphosate relative to the wild type and is more resistant (600-fold) than the P106S variant. The evolution of the TIPS mutation in crop fields under glyphosate selection is likely a sequential event, with the P106S mutation being selected first and fixed, followed by the T102I mutation to create the highly resistant TIPS EPSPS. The sequential evolution of the TIPS mutation endowing high-level glyphosate resistance is an important mechanism by which plants adapt to intense herbicide selection and a dramatic example of evolution in action.Modern herbicides make major contributions to global food production by easily removing weeds while maintaining sustainable soil conservation practices. However, persistent herbicide selection of huge weed numbers across vast areas has resulted in the widespread evolution of herbicide-resistant weed populations. Worldwide, there are currently more than 449 unique cases of herbicide resistance, with about 11 new cases reported annually, on average (Heap, 2015). Target site resistance due to target gene mutation is one of the major mechanisms enabling resistance evolution (Gressel, 2002; Powles and Yu, 2010).The most important and globally used herbicide in crop fields is glyphosate (Duke and Powles, 2008). Glyphosate disrupts the shikimate pathway by specifically inhibiting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; Steinrücken and Amrhein, 1980). Glyphosate resistance was initially considered to be unlikely to evolve in nature based on the facts that intentional selection for glyphosate tolerance using whole plants and cell/tissue culture was unsuccessful, and laboratory-generated highly resistant EPSPS mutants displayed undesirable enzyme kinetics (Bradshaw et al., 1997; for review, see Pline-Srnic, 2006). This seemed to be true, as resistance was not found during the first 15 years of glyphosate use, primarily as a nonselective herbicide. However, unprecedented intensive glyphosate use for controlling large numbers of weeds over massive areas, especially after the introduction of glyphosate-resistant transgenic crops, imposed high selection pressure on weeds, resulting in the evolution of glyphosate resistance in populations of 32 weed species (Heap, 2015). Since the first identification of a resistance-endowing EPSPS point mutation, P106S, in a glyphosate-resistant Eleusine indica population (Baerson et al., 2002), several other resistance-endowing single-amino acid substitutions at P106 (P106T, P106A, and P106L) have been reported in glyphosate-resistant weeds (e.g. Ng et al., 2004; Yu et al., 2007; Kaundun et al., 2011; for review, see Sammons and Gaines, 2014). These single-codon EPSPS resistance mutations only endow low-level glyphosate resistance (2- to 3-fold the recommended rates). This is not surprising, because glyphosate is a competitive inhibitor of the second substrate, phosphoenolpyruvate (PEP; Boocock and Coggins, 1983), and is considered a transition state mimic of the catalyzed reaction course (Schönbrunn et al., 2001). Indeed, highly glyphosate-resistant EPSPS variants (e.g. mutants at G101 or T102) have greatly increased K_m values (decreased affinity) for PEP when expressed in Escherichia coli (Eschenburg et al., 2002; Funke et al., 2009; for review, see Sammons and Gaines, 2014). In contrast, P106 substitutions confer weak glyphosate resistance but preserve adequate EPSPS functionality (Healy-Fried et al., 2007; for review, see Sammons and Gaines, 2014). Aside from P106 EPSPS gene mutations, there are other glyphosate resistance mechanisms, including EPSPS gene amplification and nontarget-site reduced glyphosate translocation/nontarget-site increased vacuole sequestration (Lorraine-Colwill et al., 2002; Gaines et al., 2010; Ge et al., 2010; for review, see Powles and Preston, 2006; Shaner, 2009; Powles and Yu, 2010; Sammons and Gaines, 2014). Generally, each of these mechanisms endows moderate-level (4- to 8-fold the recommended rates) glyphosate resistance.Low-level glyphosate resistance due to the EPSPS P106 mutations was reported in Malaysian E. indica (Baerson et al., 2002; Ng et al., 2004). Recently, we reported a highly (more than 10-fold the recommended rates) glyphosate-resistant Malaysian E. indica population (Jalaludin et al., 2015). This paper investigates the high-level glyphosate resistance in this population, and is, to our knowledge, the first to reveal the sequential evolution of a double amino acid substitution in EPSPS.