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Gene amplification of 5-enol-pyruvylshikimate-3-phosphate synthase in glyphosate-resistant Kochia scoparia

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Abstract

Main conclusion

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.

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Abbreviations

ALS:

Acetolactate synthase

EPSPS:

5-enol-pyruvylshikimate-3-phosphate synthase

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Acknowledgments

The authors thank Dr. Phil Stahlman, Dr. Randall Currie, and Dr. Mike Moechnig for providing kochia seed samples; and Dr. Doug Sammons and Monsanto Co. for providing the EPSPS antibody.

Conflict of interest

This research was financially supported by the Colorado Wheat Administrative Committee and Monsanto Company.

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Authors and Affiliations

  1. Department of Bioagricultural Science and Pest Management, Colorado State University, 1177 Campus Delivery, Fort Collins, CO, 80523, USA

    Andrew T. Wiersma, Todd A. Gaines, Darci Giacomini, Jan E. Leach & Philip Westra

  2. School of Agriculture, Food and Wine, University of Adelaide, Adelaide, South Australia, Australia

    Christopher Preston

  3. Department of Plant Biology, Michigan State University, East Lansing, MI, USA

    John P. Hamilton & C. Robin Buell

Authors
  1. Andrew T. Wiersma
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  2. Todd A. Gaines
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  5. Darci Giacomini
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  7. Jan E. Leach
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  8. Philip Westra
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Corresponding author

Correspondence to Todd A. Gaines.

Electronic supplementary material

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425_2014_2197_MOESM1_ESM.tif

Online Resource 1 Top panel: Geographical distribution of glyphosate-susceptible and -resistant K. scoparia populations in western Kansas. Susceptible (white circles) and resistant (black circles) K. scoparia population sample locations. Middle panel: Glyphosate-resistant K. scoparia field streak pattern. Glyphosate-resistant K. scoparia persist after glyphosate field treatment. Bottom panel: Representative glyphosate-susceptible and -resistant K. scoparia at 3 weeks after treatment. Glyphosate-susceptible (left) K. scoparia could be clearly differentiated from -resistant (right) individuals based on whole plant response to glyphosate treatment (TIFF 1238 kb)

425_2014_2197_MOESM2_ESM.png

Online Resource 2 EPSPS genomic copy number was measured in all individuals used for the transcriptomics study. Susceptible individuals had the expected EPSPS copy number, and all resistant individuals had increased EPSPS copy number (PNG 51 kb)

425_2014_2197_MOESM3_ESM.tif

Online Resource 3 Alignments of Illumina reads to A, the gDNA sequence of EPSPS in K. scoparia, and B, the assembled EPSPS transcript sequence. Glyphosate-susceptible, GS; glyphosate-resistant, GR. The alignments are evidence for no retained introns or exon rearrangements. R read depth was much greater than S read depth (see scale, indicative of higher expression in R); coverage pattern was nearly identical between R and S read alignments; and higher read coverage was observed in the center of the transcript. Alternatively spliced alleles would be expected to appear as reads aligned to segments of the genomic sequence other than the exons of the expected gene model (such as introns); no reads aligned outside of the expected exons (A). No noticeable gaps or drops in read coverage occur across the EPSPS transcript sequence (B), which would be expected if any exons were not transcribed (TIFF 217 kb)

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Wiersma, A.T., Gaines, T.A., Preston, C. et al. Gene amplification of 5-enol-pyruvylshikimate-3-phosphate synthase in glyphosate-resistant Kochia scoparia . Planta 241, 463–474 (2015). https://doi.org/10.1007/s00425-014-2197-9

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