Transformation of Brassica napus canola cultivars with Arabidopsis thaliana acetohydroxyacid synthase genes and analysis of herbicide resistance

Summary A survey of selected crop species and weeds was conducted to evaluate the inhibition of the enzyme acetohydroxyacid synthase (AHAS) and seedling growth in vitro by the sulfonylurea herbicides chlorsulfuron, DPX A7881, DPX L5300, DPX M6316 and the imidazolinone herbicides AC243,997, AC263,499, AC252,214. Particular attention was given to the Brassica species including canola cultivars and cruciferous weeds such as B. kaber (wild mustard) and Thlaspi arvense (stinkweed). Transgenic lines of B. napus cultivars Westar and Profit, which express the Arabidopsis thaliana wild-type AHAS gene or the mutant gene csr1-1 at levels similar to the resident AHAS genes, were generated and compared. The mutant gene was essential for resistance to the sulfonylurea chlorsulfuron but not to DPX A7881, which appeared to be tolerated by certain Brassica species. Cross-resistance to the imidazolinones did not occur. The level of resistance to chlorsulfuron in transgenic canola greatly exceeded the levels that were toxic to the Brassica species or cruciferous weeds. Direct selection of transgenic lines with chlorsulfuron sprayed at field levels under greenhouse conditions was achieved.     

Herbicide-resistant Acala and Coker cottons transformed with a native gene encoding mutant forms of acetohydroxyacid synthase

Herbicide-resistant transgenic cotton (Gossypium hirsutum L.) plants carrying mutant forms of a native acetohydroxyacid synthase (AHAS) gene have been obtained by Agrobacterium and biolistic transformation. The native gene, A19, was mutated in vitro to create amino acid substitutions at residue 563 or residue 642 of the precursor polypeptide. Transformation with the mutated forms of the A19 gene produced resistance to imidazolinone and sulfonylurea herbicides (563 substitution), or imidazolinones only (642 substitution). The herbicide-resistant phenotype of transformants was also manifested in their in vitro AHAS activity. Seedling explants of both Coker and Acala cotton varieties were transformed with the mutated forms of the A19 gene using Agrobacterium. In these experiments, hundreds of transformation events were obtained with the Coker varieties, while the Acala varieties were transformed with an efficiency about one-tenth that of Coker. Herbicide-resistant Coker and Acala plants were regenerated from a subset of transformation events. Embryonic cell suspension cultures of both Coker and Acala varieties were biolistically transformed at high frequencies using cloned cotton DNA fragments carrying the mutated forms of the A19 gene. In these transformation experiments the mutated A19 gene served as the selectable marker, and the efficiency of selection was comparable to that obtained with the NPT II gene marker of vector Bin 19. Using this method, transgenic Acala plants resistant to imidazolinone herbicides were obtained. Southern blot analyses indicated the presence of two copies of the mutated A19 transgene in one of the biolistically transformed R₀ plants, and a single copy in one of the R₀ plants transformed with Agrobacterium. As expected. progeny seedlings derived from outcrosses involving the R₀ plant transformed with Agrobacterium segregated in a 1:1 ratio with respect to herbicide resistance. The resistant progeny grew normally after irrigation with 175 g/l of the imidazolinone herbicide imazaquin, which is five times the field application rate. In contrast, untransformed sibling plants were severely stunted.Abbreviations AHAS acetohydroxyacid synthase - CaMV cauliflower mosaic virus - ELISA enzyme linked immunosorbent assay - FW fresh weight - GUS -glucuronidase - IC₅₀ herbicide concentration that produces a 50% reduction in the fresh weight growth of cells - NAA -naphthaleneacetic acid - NPT II neomycin phosphotransferase II - MS Murashige and Skoog (1962)     

An acetohydroxy acid synthase mutant reveals a single site involved in multiple herbicide resistance

Acetohydroxy acid synthase (AHAS) is an essential enzyme for many organisms as it catalyzes the first step in the biosynthesis of the branched-chain amino acids valine, isoleucine, and leucine. The enzyme is under allosteric control by these amino acids. It is also inhibited by several classes of herbicides, such as the sulfonylureas, imidazolinones and triazolopyrimidines, that are believed to bind to a relic quinone-binding site. In this study, a mutant allele of AHAS3 responsible for sulfonylurea resistance in a Brassica napus cell line was isolated. Sequence analyses predicted a single amino acid change (557 TrpLeu) within a conserved region of AHAS. Expression in transgenic plants conferred strong resistance to the three classes of herbicides, revealing a single site essential for the binding of all the herbicide classes. The mutation did not appear to affect feedback inhibition by the branched-chain amino acids in plants.     

Herbicide resistance due to amplification of a mutant acetohydroxyacid synthase gene

Summary 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.     

Genetic characterization of the acetohydroxyacid synthase (AHAS) gene responsible for resistance to imidazolinone in chickpea (Cicer arietinum L.)

Key message

A point mutation in the AHAS1 gene leading to resistance to imidazolinone in chickpea was identified. The resistance is inherited as a single gene. A KASP marker targeting the mutation was developed.

Abstract

Weed control in chickpea (Cicer arietinum L.) is challenging due to poor crop competition ability and limited herbicide options. A chickpea genotype with resistance to imidazolinone (IMI) herbicides has been identified, but the genetic inheritance and the mechanism were unknown. In many plant species, resistance to IMI is caused by point mutation(s) in the acetohydroxyacid synthase (AHAS) gene resulting in an amino acid substitution preventing herbicide attachment to the molecule. The main objective of this research was to characterize the resistance to IMI herbicides in chickpea. Two homologous AHAS genes namely AHAS1 and AHAS2 sharing 80 % amino acid sequence similarity were identified in the chickpea genome. Cluster analysis indicated independent grouping of AHAS1 and AHAS2 across legume species. A point mutation in the AHAS1 gene at C675 to T675 resulting in an amino acid substitution from Ala205 to Val205 confers the resistance to IMI in chickpea. A KASP marker targeting the point mutation was developed and effectively predicted the response to IMI herbicides in a recombinant inbred (RI) population of chickpea. The RI population was used in molecular mapping where the major locus for the reaction to IMI herbicide was mapped to chromosome 5. Segregation analysis across an F₂ population and RI population demonstrated that the resistance is inherited as a single gene in a semi-dominant fashion. The simple genetic inheritance and the availability of KASP marker generated in this study would speed up development of chickpea varieties with resistance to IMI herbicides.     

Selection and molecular characterization of imidazolinone resistant mutation-derived lines of Tritordeum HT621

Imidazolinone herbicides resistant varieties, induced by mutations at the AHAS gene (acetohydroxyacid synthase), have been developed in many crops. Hexaploid tritordeum (Tritordeum Asch. & Graebn.) is the amphiploid derived from the cross between Hordeum chilense (H^chH^ch) and durum wheat Triticum turgidum L. (Thell) (AABB). Tritordeums have the potential to become a new crop with high added-value for food or feed. Mutagenesis with EMS was conducted to obtain imidazolinone resistant lines derived of the tritordeum HT621. Eleven M₃ plants were selected after imidazolinone treatment and five descendants of two of these lines (HT621-M3R1-3 and HT621-M3R10-1) were analyzed at the molecular level. Partial sequences of the three homologous AHAS loci in genomes A, B, and H^ch were obtained as well as those of HT621. A partial sequence of the AHAS gene in Hordeum chilense is first described in this work, and the designation ahasL-H ^ch 1 is proposed. A single Ser-Asn₆₂₇ substitution at the AHAS locus in the B genome is responsible of resistance in both lines. We propose the name AhasL-B2 for this resistance allele. This is the first report of the selection of imidazolinone resistant lines of tritordeum and the molecular characterization of the mutation conferring this resistance.     

Mutations in corn (Zea mays L.) conferring resistance to imidazolinone herbicides

Summary Three corn (Zea mays L.) lines resistant to imidazolinone herbicides were developed by in vitro selection and plant regeneration. For all three lines, resistance is inherited as a single semidominant allele. The resistance alleles from resistant lines XA17, XI12, and QJ22 have been crossed into the inbred line B73, and in each case homozygotes are tolerant of commercial use rates of imidazolinone herbicides. All resistant selections have herbicide-resistant forms of acetohydroxyacid synthase (AHAS), the known site of action of imidazolinone herbicides. The herbicide-resistant phenotypes displayed at the whole plant level correlate directly with herbicide insensitivity of the AHAS activities of the selections. The AHAS activities from all three selections have normal feedback regulation by valine and leucine, and plants containing the mutations display a normal phenotype.     

Molecular basis of sulfonylurea herbicide inhibition of acetohydroxyacid synthase

Acetohydroxyacid synthase (AHAS) (acetolactate synthase, EC ) catalyzes the first step in branched-chain amino acid biosynthesis and is the target for sulfonylurea and imidazolinone herbicides. These compounds are potent and selective inhibitors, but their binding site on AHAS has not been elucidated. Here we report the 2.8 A resolution crystal structure of yeast AHAS in complex with a sulfonylurea herbicide, chlorimuron ethyl. The inhibitor, which has a K(i) of 3.3 nm, blocks access to the active site and contacts multiple residues where mutation results in herbicide resistance. The structure provides a starting point for the rational design of further herbicidal compounds.     

Directed excision of a transgene from the plant genome

Summary The effectiveness of loxP-Cre directed excision of a transgene was examined using phenotypic and molecular analyses. Two methods of combining the elements of this system, re-transformation and cross pollination, were found to produce different degrees of excision in the resulting plants. Two linked traits, -glucuronidase (GUS) and a gene encoding sulfonylurea-resistant acetolactate synthase (ALS^r), were integrated into the genome of tobacco and Arabidopsis. The ALS^r gene, bounded by loxP sites, was used as the selectable marker for transformation. The directed loss of the ALS^T gene through Cre-mediated excision was demonstrated by the loss of resistance to sulfonylurea herbicides and by Southern blot analysis. The -glucuronidase gene remained active. The excision efficiency varied in F₁ progeny of different lox and Cre parents and was correlated with the Cre parent. Many of the lox × Cre F₁ progeny were chimeric and some F₂ progeny retained resistance to sulfonylureas. Re-transformation of lox/ALS/lox/GUS tobacco plants with cre led to much higher efficiency of excision. Lines of tobacco transformants carrying the GUS gene but producing only sulfonylurea-sensitive progeny were obtained using both approaches for introducing cre. Similarly, Arabidopsis lines with GUS activity but no sulfonylurea resistance were generated using cross pollinations.     

Acetohydroxyacid synthase mutations conferring resistance to imidazolinone or sulfonylurea herbicides in sunflower

Wild biotypes of cultivated sunflower (Helianthus annuus L.) are weeds in corn (Zea mays L.), soybean (Glycine max L.), and other crops in North America, and are commonly controlled by applying acetohydroxyacid synthase (AHAS)-inhibiting herbicides. Biotypes resistant to two classes of AHAS-inhibiting herbicides—imidazolinones (IMIs) or sulfonylureas (SUs)—have been discovered in wild sunflower populations (ANN-PUR and ANN-KAN) treated with imazethapyr or chlorsulfuron, respectively. The goals of the present study were to isolate AHAS genes from sunflower, identify mutations in AHAS genes conferring herbicide resistance in ANN-PUR and ANN-KAN, and develop tools for marker-assisted selection (MAS) of herbicide resistance genes in sunflower. Three AHAS genes (AHAS1, AHAS2, and AHAS3) were identified, cloned, and sequenced from herbicide-resistant (mutant) and -susceptible (wild type) genotypes. We identified 48 single-nucleotide polymorphisms (SNPs) in AHAS1, a single six-base pair insertion-deletion in AHAS2, and a single SNP in AHAS3. No DNA polymorphisms were found in AHAS2 among elite inbred lines. AHAS1 from imazethapyr-resistant inbreds harbored a C-to-T mutation in codon 205 (Arabidopsis thaliana codon nomenclature), conferring resistance to IMI herbicides, whereas AHAS1 from chlorsulfuron-resistant inbreds harbored a C-to-T mutation in codon 197, conferring resistance to SU herbicides. SNP and single-strand conformational polymorphism markers for AHAS1, AHAS2, and AHAS3 were developed and genetically mapped. AHAS1, AHAS2, and AHAS3 mapped to linkage groups 2 (AHAS3), 6 (AHAS2), and 9 (AHAS1). The C/T SNP in codon 205 of AHAS1 cosegregated with a partially dominant gene for resistance to IMI herbicides in two mutant × wild-type populations. The molecular breeding tools described herein create the basis for rapidly identifying new mutations in AHAS and performing MAS for herbicide resistance genes in sunflower.     

A novel selection system for potato transformation using a mutated AHAS gene

Acetohydroxyacid synthase (AHAS) is the target enzyme for a number of herbicides. A S653N mutation in the AHAS gene results in an increased tolerance to imidazolinone herbicides. We have investigated the use of the mutated gene as selection gene for potato transformation. This resulted in a transformation system with a very high transformation frequency and low rate of escapes. The mutated AHAS gene was introduced into transformed potato together with a -glucuronidase (GUS) gene. Selection on 0.5 M Imazamox yielded GUS expression in 93–100% of regenerated shoots. Furthermore the mutated AHAS gene was used as selection gene for production of high-amylopectin potato lines. The high transformation frequency was verified and potato lines with the desirable starch quality were obtained.Abbreviations ABA Abscisic acid - AHAS Acetohydroxyacid synthase - BAP 6-Benzylaminopurine - 2,4-D 2, 4-Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - GBSS Granule bound starch synthase - GUS -Glucuronidase - MS medium Murashige and Skoog medium - NAA -Naphthaleneacetic acid - nos Nopaline synthase - OCS Octopine synthase - PCR Polymerase chain reaction - X-gluc 5-Bromo-4-chloro-3-indolyl-beta-d-glucuronic acid - YEB Yeast extract brothCommunicated by R. Schmidt     

Expression,Characterization, and Site-Directed Mutation of a Multiple Herbicide-Resistant Acetohydroxyacid Synthase (rAHAS) from <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. Lm10

A multiple herbicide-resistant acetohydroxyacid synthase (rAHAS) gene was cloned from Pseudomonas sp. Lm10. Sequence analysis showed that the rAHAS regulatory subunit was identical to that of Pseudomonas putida KT2440 (sensitive AHAS, sAHAS), whereas six different sites [H134→N (rAHAS→sAHAS), A135→P, S136→T, I210→V, F264→Y, and S486→W] were found in the catalytic subunit. The rAHAS and sAHAS were over expressed, purified and characterized. rAHAS showed higher resistance to four kinds of AHAS-inhibitor herbicides than sAHAS. The resistance factor of rAHAS was 56.0-fold, 12.6-fold, 6.5-fold, and 9.2-fold as compared with sAHAS when metsulfuron-methyl, imazethapyr, flumetsulam, and pyriminobac-methyl used as inhibitor, respectively. The specific activity of rAHAS was lower than that of sAHAS and the K _m value of rAHAS for pyruvate was approximately onefold higher than the corresponding value for sAHAS. Data from site-directed mutagenesis demonstrated that alteration at A135, F264, and S486 resulted in resistance reduction, while the mutation at H134, S136, and I210 has little effect on the resistance. A135 was mainly responsible for resistance to imidazolinone; F264 conferred resistance to sulfonylurea and triazolopyrimidine sulfonamide; and S486 showed multiple herbicides resistance to the four herbicides.     

Molecular Basis of Imidazolinone Herbicide Resistance in Arabidopsis thaliana var Columbia

Acetolactate synthase (ALS), the first enzyme in the biosynthetic pathway of leucine, isoleucine, and valine, is inhibited by imidazolinone herbicides. To understand the molecular basis of imidazolinone resistance, we isolated the ALS gene from an imazapyr-resistant mutant GH90 of Arabidopsis thaliana. DNA sequence analysis of the mutant ALS gene demonstrated a single-point mutation from G to A at nucleotide 1958 of the ALS-coding sequence. This would result in Ser to Asn substitution at residue 653 near the carboxyl terminal of the matured ALS. The mutant ALS gene was introduced into tobacco using Agrobacterium-mediated transformation. Imidazolinone-resistant growth of transformed calli and leaves of transgenic plants was 100-fold greater than that of nontransformed control plants. The relative levels of imidazolinone-resistant ALS activity correlated with the amount of herbicide-resistant growth in the leaves of transgenic plants. Southern hybridization analysis confirmed the existence of transferred ALS gene in the transformant showing high imazapyr resistance. The results demonstrate that the mutant ALS gene confers resistance to imidazolinone herbicides. This is the first report, to our knowledge, of the molecular basis of imidazolinone resistance in plants.     

Structure and mechanism of inhibition of plant acetohydroxyacid synthase

Plants and microorganisms synthesize valine, leucine and isoleucine via a common pathway in which the first reaction is catalysed by acetohydroxyacid synthase (AHAS, EC 2.2.1.6). This enzyme is of substantial importance because it is the target of several herbicides, including all members of the popular sulfonylurea and imidazolinone families. However, the emergence of resistant weeds due to mutations that interfere with the inhibition of AHAS is now a worldwide problem. Here we summarize recent ideas on the way in which these herbicides inhibit the enzyme, based on the 3D structure of Arabidopsis thaliana AHAS. This structure also reveals important clues for understanding how various mutations can lead to herbicide resistance.     

Two consecutive aspartic acid residues conferring herbicide resistance in tobacco acetohydroxy acid synthase

Acetohydroxy acid synthase (AHAS) catalyzes the first common step in the biosynthesis pathway of the branch chain amino acids in plants and microorganisms. A great deal of interest has been focused on AHAS since it was identified as the target of several classes of potent herbicides. In an effort to produce a mutant usable in the development of an herbicide-resistant transgenic plant, two consecutive aspartic acid residues, which are very likely positioned next to the enzyme-bound herbicide sulfonylurea as the homologous residues in AHAS from yeast, were selected for this study. Four single-point mutants and two double mutants were constructed, and designated D374A, D374E, D375A, D375E, D374A/D375A, and D374E/D375E. All mutants were active, but the D374A mutant exhibited substrate inhibition at high concentrations. The D374E mutant also evidenced a profound reduction with regard to catalytic efficiency. The mutation of D375A increased the K(m) value for pyruvate nearly 10-fold. In contrast, the D375E mutant reduced this value by more than 3-fold. The double mutants exhibited synergistic reduction in catalytic efficiencies. All mutants constructed in this study proved to be strongly resistant to the herbicide sulfonylurea Londax. The double mutants and the mutants with the D375 residue were also strongly cross-resistant to the herbicide triazolopyrimidine TP. However, only the D374A mutant proved to be strongly resistant to imidazolinone Cadre. The data presented here indicate that the two residues, D374 and D375, are located at a common binding site for the herbicides sulfonylurea and triazolopyrimidine. D375E may be a valuable mutant for the development of herbicide-resistant transgenic plants.     

Cell selection and inheritance of imidazolinone resistance in sugarbeet (Beta vulgaris)

 Sugarbeets are sensitive to imidazolinone herbicide residues applied to rotational crops. Two imidazolinone-resistance (IMI-R) sugarbeet traits were developed by somatic cell selection to overcome rotation restrictions for sugarbeets where imidazolinones have been applied. Sir-13 is an IMI-R/SU-S (sulfonylurea-sensitive) variant selected from an imidazolinone-sensitive (IMI-S) sugarbeet clone, REL-1. A second variant, 93R30B, resistant to imidazolinone as well as to sulfonylurea herbicides (IMI-R/SU-R), was selected from a plant homozygous for a previously described sulfonylurea-specific resistance trait, Sur (IMI-S/SU-R). The IMI-R alleles (Sir-13 and 93R30B) were found to be corresponding allelic variants at the same ALS locus and both were tightly associated with the Sur allele. Each resistant allele is dominant to the sensitive wild-type allele; however, incomplete dominance is shown among resistance alleles. Diploid sugarbeet contains a single ALS gene copy, limiting the ability to stack these resistance traits in the same plant by traditional breeding. Received: 1 May 1997 / Accepted: 30 June 1997     

乙酰羟酸合成酶同工酶基因ilvBN、ilvGM和ilvIH的表达及对除草剂抗性的比较

乙酰羟酸合成酶(AHAS)是磺酰脲类和咪唑啉酮类等AHAS抑制剂类除草剂的作用靶标。获得抗此类除草剂的AHAS突变基因资源具有非常重要的理论和应用价值。本研究从抗甲磺隆菌株Klebsiella sp.HR11和甲磺隆敏感菌株Klebsiella pneumoniae MGH 78578中分别克隆到AHAS三种同工酶基因ilvBN、ilvGM和ilvIH。抗性菌株和敏感菌株AHAS三种同工酶基因在氨基酸水平上差异位点主要集中在ilvBN和ilvGM的大亚基上。将2株菌的ilvBN、ilvGM和ilvIH分别构建到表达载体pET29a(+)中,在Escherichia coli BL21(DE3)中进行表达,测得只有含菌株HR11 ilvBN和ilvGM的转化子细胞破碎液AHAS对各类AHAS抑制剂类除草剂具有较强的抗性,而含菌株HR11 ilvIH和菌株MGH78578 ilvBN、ilvGM和ilvIH的转化子细胞破碎液AHAS对各类AHAS抑制剂类除草剂敏感。     

Molecular and biochemical characterization of an induced mutation conferring imidazolinone resistance in sunflower

A partially dominant nuclear gene conferring resistance to the imidazolinone herbicides was previously identified in the cultivated sunflower (Helianthus annuus L.) line CLHA-Plus developed by seed mutagenesis. The objective of this study was to characterize this resistant gene at the phenotypic, biochemical and molecular levels. CLHA-Plus showed a complete susceptibility to sulfonylureas (metsulfuron, tribenuron and chlorsulfuron) but, on the other hand, it showed a complete resistance to imidazolinones (imazamox, imazapyr and imazapic) at two rates of herbicide application. This pattern was in close association with the AHAS-inhibition kinetics of protein extracts of CLHA-Plus challenged with different doses of imazamox and chlorsulfuron. Nucleotide and deduced amino acid sequence comparisons between resistant and susceptible lines indicated that the imidazolinone-resistant AHAS of CLHA-Plus has a threonine codon (ACG) at position 122 (relative to the Arabidopsis thaliana AHAS sequence), whereas the herbicide-susceptible enzyme from BTK47 has an alanine residue (GCG) at this position. Since the resistance genes to AHAS-inhibiting herbicides so far characterized in sunflower code for the catalytic (large) subunit of AHAS, we propose to redesignate the wild type allele as ahasl1 and the incomplete dominant resistant alleles as Ahasl1-1 (previously Imr1 or Ar _pur ), Ahasl1-2 (previously Ar _kan ) and Ahasl1-3 (for the allele present in CLHA-Plus). The higher tolerance level to imidazolinones and the lack of cross-resistance to other AHAS-inhibiting herbicides of Ahasl1-3 indicate that this induced mutation can be used to develop commercial hybrids with superior levels of tolerance and, at the same time, to assist weed management where control of weedy common sunflower is necessary.