Molecular Characterization of True and Ectopic Gene Targeting Events at the Acetolactate Synthase Gene in Arabidopsis (original) (raw)
Related papers
Molecular and General Genetics, 1988
A gene encoding acetolactate synthase was cloned from a chlorsulfuron-resistant mutant of Arabidopsis. The DNA sequence of the mutant gene differed from that of the wild type by a single base pair substitution. When introduced into tobacco by Ti plasmid-mediated transformation the gene conferred a high level of herbicide resistance. These results suggest that the cloned gene may confer agronomically useful levels of herbicide resistnace in other crop species, and that it may be useful as a selectable marker for plant transformation experiments.
Plant Molecular Biology, 2007
Acetolactate synthase (ALS) is the first common enzyme in the biosynthetic pathway of branched-chain amino acids. Mutations of specific amino acids in ALS have been known to confer resistance to ALS-inhibiting herbicides such as sulfonylureas and pyrimidinyl carboxy (PC) herbicides. However, mutations conferring exclusive resistance to PC have not yet been reported to date. We selected PC resistant rice calli, which were derived from anther culture, using one of the PCs, bispyribac-sodium (BS), as a selection agent. Two lines of BS-resistant plants carrying a novel mutation, the 95th Glycine to Alanine (G95A), in ALS were obtained. In vitro ALS activity assay indicated that the recombinant protein of G95A-mutated ALS (ALS-G95A) conferred highly specific resistance to PC herbicides. In order to determine if the ALS-G95A gene could be used as a selection marker for rice transformation, the ALS-G95A gene was connected to ubiquitin promoter and introduced into rice. PC resistant plants containing integrated ALS-G95A gene were obtained after selection with BS as a selection agent. In conclusion, novel G95A mutated ALS gene confers highly specific resistant to PC-herbicides and can be used as a selection marker.
PLANT PHYSIOLOGY, 1990
Genes encoding wild type acetolactate synthase (ALS) and a sulfonylurea herbicide-resistant form of the enzyme, isolated from Arabidopsis thaliana, were expressed in transgenic Nkotiana tabacum plants under the control of their native promoters or of the highly active cauliflower mosaic virus 35S promoter. Expression of the wild type coding region from the 35S promoter resulted in a small, threefold increase in sulfonylurea tolerance above the levels measured in tissue expressing the nafive wild type gene. A much larger, 300-fold increase in herbicide tolerance was conferred by the mutant gene encoding a herbicide-resistant ALS. An additional 10-fold increase in tolerance was attained by expressing this coding region from the 35S promoter. The increase in both wild type and mutant gene expression directed by the 35S promoter resulted in over 25-fold higher levels of ALS messenger RNA in some transformants as compared with those expressing the native genes. However, ALS specific activity increased at most twofold, indicating that the amount of functional enzyme and messenger RNA are not correlated.
Current Science, 2008
We report in this study, the successful deployment of a double mutant acetolactate synthase gene (ALS dm , containing Pro 197 to Ser and Ser 653 to Asn substitutions) as an efficient in vitro selection marker for the development of transgenic plants in Brassica juncea (oilseed mustard). The ALS enzyme is inhibited by two categories of herbicides, sulfonylureas (e.g. chlorsulfuron) and imidazolinones (e.g. imazethapyr), while the mutant forms are resistant to the same. Three different selection agents (kanamycin, chlorsulfuron and imazethapyr) were tested for in vitro selection efficiency in two B. juncea cultivars, RLM198 and Varuna. For both the cultivars, higher transformation frequencies were obtained using chlorsulfuron (3.870.6% and 4.670.9% for RLM198 and Varuna, respectively) and imazethapyr (10.270.7% for RLM198 and 7.871.2% for Varuna) as compared to that obtained on kanamycin (3.170.2% and 2.870.5% for RLM198 and Varuna, respectively). Additionally, transformation frequencies were higher on imazethapyr than on chlorsulfuron for both the cultivars indicating that imidazolinones are better selective agents than sulfonylureas for the selection of mustard transgenics.
Plant Science, 2011
We investigated the diversity of mechanisms conferring resistance to herbicides inhibiting acetolactate synthase (ALS) in corn poppy (Papaver rhoeas L.) and the processes underlying the selection for resistance. Six mutant ALS alleles, Arg 197 , His 197 , Leu 197 , Ser 197 , Thr 197 and Leu 574 were identified in five Italian populations. Different alleles were found in a same population or a same plant. Comparison of individual plant phenotype (herbicide sensitivity) and genotype (amino-acid substitution(s) at codon 197) showed that all mutant ALS alleles conferred dominant resistance to the field rate of the sulfonylurea tribenuron and moderate or no resistance to the field rate of the triazolopyrimidine florasulam. Depending on the allele, dominant or partially dominant resistance to the field rate of the imidazolinone imazamox was observed. Putative non-target-site resistance mechanisms were also likely present in the populations investigated. The derived Cleaved Amplified Polymorphic Sequence assays targeting ALS codons crucial for herbicide sensitivity developed in this work will facilitate the detection of resistance due to mutant ALS alleles. Nucleotide variation around codon 197 indicated that mutant ALS alleles evolved by multiple, independent appearances. Resistance to ALS inhibitors in P. rhoeas clearly evolved by redundant evolution of a set of mutant ALS alleles and likely of non-target-site mechanisms.
Plants
Chickpea (Cicer arietinum L.) is an important crop in crop-rotation management in Israel. Imidazolinone herbicides have a wide spectrum of weed control, but chickpea plants are sensitive to acetohydroxyacid synthase (AHAS; also known as acetolactate synthase [ALS]) inhibitors. Using the chemical mutagen ethyl methanesulfonate (EMS), we developed a chickpea line (M2033) that is resistant to imidazolinone herbicides. A point mutation was detected in one of the two genes encoding the AHAS catalytic subunit of M2033. The transition of threonine to isoleucine at position 192 (203 according to Arabidopsis) conferred resistance of M2033 to imidazolinones, but not to other groups of AHAS inhibitors. The role of this substitution in the resistance of line M2033 was proven by genetic transformation of tobacco plants. This resistance showed a single-gene semidominant inheritance pattern. Conclusion: A novel mutation, T192I (T203I according to Arabidopsis), providing resistance to IMI herbicide...
Pest Management Science
BACKGROUND: Herbicide resistant weeds are a serious problem worldwide. Recently, two populations of Amaranthus palmeri with suspected cross-resistance to ALS-inhibiting herbicides (R1 and R2) were found by farmers in two locations from Argentina (Vicuña Mackenna and Totoras, respectively). We conducted studies to confirm and elucidate the mechanism of resistance. RESULTS: We performed in vivo dose-response assays, and confirmed both populations had a strong resistance to chlorimuron-ethyl, diclosulam and imazethapyr when compared to a susceptible population (S). In vitro ALS activity inhibition tests only indicated a considerable resistance to imazethapyr and chlorimuron-ethyl, indicating that other non-target mechanisms could be involved in diclosulam resistance. Subsequently, molecular analysis of als nucleotide sequences revealed three single base-pair mutations conferring substitutions in amino acids previously associated with resistance to ALS inhibitors, A122, W574, and S653 CONCLUSION: This is the first report of als resistant alleles in Amaranthus palmeri from Argentina. The data support the involvement of a target-site mechanism of resistance to ALS inhibiting herbicides.
Molecular Basis of Imidazolinone Herbicide Resistance in Arabidopsis thaliana var Columbia
Plant Physiology, 1991
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 ALScoding sequence. This would result in Ser to Asn substitution at ' This work was supported in part by research assistantship to K.S. provided by the Louisiana State University College of Agriculture and Dr. John B. Baker and by grants from Louisiana Education Quality Support Funds (1987-90)-RD-A-6 to NM.
The Plant Journal, 2002
Precise modi®cation by gene targeting (GT) provides an important tool for studies of gene function in vivo. Although routine with many organisms, only isolated examples of GT events have been reported for¯owering plants. These were at low frequencies precluding reliable estimation of targeting ef®ciency and evaluation of GT mechanisms. Here we present an unambiguous and straightforward system for detection of GT events in Arabidopsis using an endogenous nuclear gene encoding protoporphyrinogen oxidase (PPO), involved in chlorophyll and heme syntheses. Inhibition of PPO by the herbicide Butafenacil results in rapid plant death. However, the combination of two particular mutations renders PPO highly resistant to Butafenacil. We exploited this feature for selection of GT events by introducing the mutations into the PPO gene by homologous recombination. We have estimated the basal GT frequency to be 2.4 Q 10 ±3 . Approximately one-third of events were true GT (TGT) leading to the anticipated modi®cation of the chromosomal PPO copy. The remaining events could be classi®ed as ectopic GT (EGT) arising by modi®cation of vector DNA by the chromosomal template and its random integration into the Arabidopsis genome. Thus the TGT frequency in our experimental setup is 0.72 Q 10 ±3 . In view of the high ef®ciency of Arabidopsis transformation, GT experiments of a reasonable size followed by a PCR screen for GT events should also allow for modi®cation of non-selectable targets. Moreover, the system presented here should contribute signi®cantly to future improvement of GT technology in plants.
Molecular Basis of Imidazolinone Herbicide Resistance in Arabidopsis thaliana var Columbia
Plant Physiology, 1991
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 ALScoding sequence. This would result in Ser to Asn substitution at ' This work was supported in part by research assistantship to K.S. provided by the Louisiana State University College of Agriculture and Dr. John B. Baker and by grants from Louisiana Education Quality Support Funds (1987-90)-RD-A-6 to NM.