Herbicide resistance due to amplification of a mutant acetohydroxyacid synthase gene

Christian T. Harms, Susan L. Armour, Joseph J. DiMaio, Laura A. Middlesteadt, David Murray, David V. Negrotto, Hope Thompson-Taylor, Kristianna Weymann, Alice L. Montoya, Raymond D. Shillito, George C. Jen

Research output: Contribution to journalArticle

58 Citations (Scopus)

Abstract

We have selected a tobacco cell line, SU-27D5, that is highly resistant to sulfonylurea and imidazolinone herbicides. This line was developed by selection first on a lethal concentration of cinosulfuron and then on increasing concentrations of primisulfuron, both sulfonylurea herbicides. SU-27D5 was tested against five sulfonylureas and one imidazolinone herbicide and was shown, in every case, to be two to three orders of magnitude more resistant than wild-type cells. The acetohydroxyacid synthase (AHAS) of SU-27D5 was 50- to 780-fold less sensitive than that of wild-type cells to herbicide inhibition. The specific activity of AHAS in the SU-27D5 cell lysate was 6 to 7 times greater than that in wild-type cells. Using Southern analysis, we showed that cell line SU-27D5 had amplified its SuRB AHAS gene about 20-fold while maintaining a normal diploid complement of the SuRA AHAS gene. Genomic clones of both AHAS genes were isolated and used to transform wild-type tobacco protoplasts. SuRB clones gave rise to herbicide-resistant transformants, whereas SuRA clones did not. DNA sequencing showed that all SuRB clones contained a point mutation at nucleotide 588 that converted amino acid 196 of AHAS from proline to serine. In contrast, no mutations were found in the SuRA clones. The stability of SuRB gene amplification was variable in the absence of selection. In one experiment, the withdrawal of selection reduced the copy number of the amplified SuRB gene to the normal level within 30 days. In another experiment, amplification remained stable after extended cultivation on herbicide-free medium. This is the first report of amplification of a mutant herbicide target gene that resulted in broad and strong herbicide resistance.

Original languageEnglish (US)
Pages (from-to)427-435
Number of pages9
JournalMGG Molecular & General Genetics
Volume233
Issue number3
DOIs
StatePublished - Jun 1992
Externally publishedYes

Fingerprint

Herbicide Resistance
Herbicides
Clone Cells
Genes
Tobacco
Cell Line
Protoplasts
Gene Amplification
Diploidy
DNA Sequence Analysis
Point Mutation
Proline
Serine
Nucleotides
Amino Acids
Mutation

Keywords

  • Acetohydroxyacid synthase
  • Gene amplification
  • Herbicide resistance
  • Mutant gene
  • Nicotiana tabacum

ASJC Scopus subject areas

  • Genetics

Cite this

Harms, C. T., Armour, S. L., DiMaio, J. J., Middlesteadt, L. A., Murray, D., Negrotto, D. V., ... Jen, G. C. (1992). Herbicide resistance due to amplification of a mutant acetohydroxyacid synthase gene. MGG Molecular & General Genetics, 233(3), 427-435. https://doi.org/10.1007/BF00265440

Herbicide resistance due to amplification of a mutant acetohydroxyacid synthase gene. / Harms, Christian T.; Armour, Susan L.; DiMaio, Joseph J.; Middlesteadt, Laura A.; Murray, David; Negrotto, David V.; Thompson-Taylor, Hope; Weymann, Kristianna; Montoya, Alice L.; Shillito, Raymond D.; Jen, George C.

In: MGG Molecular & General Genetics, Vol. 233, No. 3, 06.1992, p. 427-435.

Research output: Contribution to journalArticle

Harms, CT, Armour, SL, DiMaio, JJ, Middlesteadt, LA, Murray, D, Negrotto, DV, Thompson-Taylor, H, Weymann, K, Montoya, AL, Shillito, RD & Jen, GC 1992, 'Herbicide resistance due to amplification of a mutant acetohydroxyacid synthase gene', MGG Molecular & General Genetics, vol. 233, no. 3, pp. 427-435. https://doi.org/10.1007/BF00265440
Harms CT, Armour SL, DiMaio JJ, Middlesteadt LA, Murray D, Negrotto DV et al. Herbicide resistance due to amplification of a mutant acetohydroxyacid synthase gene. MGG Molecular & General Genetics. 1992 Jun;233(3):427-435. https://doi.org/10.1007/BF00265440
Harms, Christian T. ; Armour, Susan L. ; DiMaio, Joseph J. ; Middlesteadt, Laura A. ; Murray, David ; Negrotto, David V. ; Thompson-Taylor, Hope ; Weymann, Kristianna ; Montoya, Alice L. ; Shillito, Raymond D. ; Jen, George C. / Herbicide resistance due to amplification of a mutant acetohydroxyacid synthase gene. In: MGG Molecular & General Genetics. 1992 ; Vol. 233, No. 3. pp. 427-435.
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abstract = "We have selected a tobacco cell line, SU-27D5, that is highly resistant to sulfonylurea and imidazolinone herbicides. This line was developed by selection first on a lethal concentration of cinosulfuron and then on increasing concentrations of primisulfuron, both sulfonylurea herbicides. SU-27D5 was tested against five sulfonylureas and one imidazolinone herbicide and was shown, in every case, to be two to three orders of magnitude more resistant than wild-type cells. The acetohydroxyacid synthase (AHAS) of SU-27D5 was 50- to 780-fold less sensitive than that of wild-type cells to herbicide inhibition. The specific activity of AHAS in the SU-27D5 cell lysate was 6 to 7 times greater than that in wild-type cells. Using Southern analysis, we showed that cell line SU-27D5 had amplified its SuRB AHAS gene about 20-fold while maintaining a normal diploid complement of the SuRA AHAS gene. Genomic clones of both AHAS genes were isolated and used to transform wild-type tobacco protoplasts. SuRB clones gave rise to herbicide-resistant transformants, whereas SuRA clones did not. DNA sequencing showed that all SuRB clones contained a point mutation at nucleotide 588 that converted amino acid 196 of AHAS from proline to serine. In contrast, no mutations were found in the SuRA clones. The stability of SuRB gene amplification was variable in the absence of selection. In one experiment, the withdrawal of selection reduced the copy number of the amplified SuRB gene to the normal level within 30 days. In another experiment, amplification remained stable after extended cultivation on herbicide-free medium. This is the first report of amplification of a mutant herbicide target gene that resulted in broad and strong herbicide resistance.",
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AU - DiMaio, Joseph J.

AU - Middlesteadt, Laura A.

AU - Murray, David

AU - Negrotto, David V.

AU - Thompson-Taylor, Hope

AU - Weymann, Kristianna

AU - Montoya, Alice L.

AU - Shillito, Raymond D.

AU - Jen, George C.

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