title: Gene amplification of 5-enol-pyruvylshikimate-3-phosphate synthase in glyphosate-resistant Kochia scoparia.
abstract: Field-evolved resistance to the herbicide glyphosate is due to amplification of one of two EPSPS alleles, increasing transcription and protein with no splice variants or effects on other pathway genes. The widely used herbicide glyphosate inhibits the shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Globally, the intensive use of glyphosate for weed control has selected for glyphosate resistance in 31 weed species. Populations of suspected glyphosate-resistant Kochia scoparia were collected from fields located in the US central Great Plains. Glyphosate dose response verified glyphosate resistance in nine populations. The mechanism of resistance to glyphosate was investigated using targeted sequencing, quantitative PCR, immunoblotting, and whole transcriptome de novo sequencing to characterize the sequence and expression of EPSPS. Sequence analysis showed no mutation of the EPSPS Pro106 codon in glyphosate-resistant K. scoparia, whereas EPSPS genomic copy number and transcript abundance were elevated three- to ten-fold in resistant individuals relative to susceptible individuals. Glyphosate-resistant individuals with increased relative EPSPS copy numbers had consistently lower shikimate accumulation in leaf disks treated with 100 μM glyphosate and EPSPS protein levels were higher in glyphosate-resistant individuals with increased gene copy number compared to glyphosate-susceptible individuals. RNA sequence analysis revealed seven nucleotide positions with two different expressed alleles in glyphosate-susceptible reads. However, one nucleotide at the seven positions was predominant in glyphosate-resistant sequences, suggesting that only one of two EPSPS alleles was amplified in glyphosate-resistant individuals. No alternatively spliced EPSPS transcripts were detected. Expression of five other genes in the chorismate pathway was unaffected in glyphosate-resistant individuals with increased EPSPS expression. These results indicate increased EPSPS expression is a mechanism for glyphosate resistance in these K. scoparia populations.
authors.affiliation: Department of Bioagricultural Science and Pest Management, Colorado State University, 1177 Campus Delivery, Fort Collins, CO, 80523, USA.
authors.forename: Andrew T
authors.initials: AT
authors.lastname: Wiersma
authors.forename: Todd A
authors.initials: TA
authors.lastname: Gaines
authors.forename: Christopher
authors.initials: C
authors.lastname: Preston
authors.forename: John P
authors.initials: JP
authors.lastname: Hamilton
authors.forename: Darci
authors.initials: D
authors.lastname: Giacomini
authors.forename: C
authors.initials: C
authors.lastname: Robin Buell
authors.forename: Jan E
authors.initials: JE
authors.lastname: Leach
authors.forename: Philip
authors.initials: P
authors.lastname: Westra
mesh_terms: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase / Q000235:genetics / Q000378:metabolism*; D027467:Bassia scoparia / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D005784:Gene Amplification / Q000235:genetics / Q000502:physiology*; D018506:Gene Expression Regulation, Plant; D005998:Glycine / Q000031:analogs & derivatives* / Q000494:pharmacology; D053209:Herbicide Resistance / Q000235:genetics; D006540:Herbicides / Q000494:pharmacology*; D000097797:Glyphosate
chemical_list: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D005998:Glycine; D006540:Herbicides; D000097797:Glyphosate