Glyphosate selected amplification of the 5-enolpyruvylshikimate-3-phosphate synthase gene in cultured carrot cells

Summary CAR and C1, two carrot (Daucus carota L.) suspension cultures of different genotypes, were subjected to stepwise selection for tolerance to the herbicide glyphosate [(N-phosphonomethyl)glycine]. The specific activity of the target enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), as well as the mRNA level and copy number of the structural gene increased with each glyphosate selection step. Therefore, the tolerance to glyphosate is due to stepwise amplification of the EPSPS genes. During the amplification process, DNA rearrangement did not occur within the EPSPS gene of the CAR cell line but did occur during the selection step from 28 to 35 mM glyphosate for the C1 cell line, as determined by Southern hybridization of selected cell DNA following EcoRI restriction endonuclease digestion. Two cell lines derived from a previously selected glyphosate-tolerant cell line (PR), which also had undergone EPSPS gene amplification but have been maintained in glyphosate-free medium for 2 and 5 years, have lost 36 and 100% of the increased EPSPS activity, respectively. Southern blot analysis of these lines confirms that the amplified DNA is relatively stable in the absence of selection. These studies demonstrate that stepwise selection for glyphosate resistance reproducibly produces stepwise amplification of the EPSPS genes. The relative stability of this amplification indicates that the amplified genes are not extrachromosomal.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DTT dithiothreitol - EPSPS 5-enolpyruvylshikimate-3-phosphate synthase - I₅₀ 50% inhibitory concentration - Kb Kilobase (pairs) - PEP phosphoenolpyruvate - PMSF phenylmethylsulfonyl fluoride - PVPP polyvinylpolypyrrolidone - S-3-P shikimate-3-phosphate     

Glyphosate selection of gene amplification in suspension cultures of 3 plant species

Stepwise selection was carried out with increasing glyphosate concentrations to produce suspension cultures of Medicago sativa L. (alfalfa), Glycine max L. (Merr.) (soybean) and Nicotiana tabacum L. (tobacco) (two lines) that were at least 100-fold more resistant than the original culture as measured by the I₅₀. The selection process required from 8 to 11 transfers to fresh medium over a total period from 161 to 312 days. The alfalfa and soybean lines contained 62- and 21-fold higher activity levels of the glyphosate target enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), respectively. The tobacco lines had EPSPS enzyme activity levels more than 800-times higher than the original cultures. The EPSPS gene copy number and mRNA were increased in all of the lines as measured by southern and northern hybridization, respectively. Thus, as has been found before with most glyphosate-resistant suspension cultures, the resistance is caused by high EPSPS enzyme activity due to EPSPS gene amplification. Alfalfa and soybean EPSPS gene amplification and the very high EPSPS enzyme activity increases found in the tobacco cultures have not been reported before. These studies show that EPSPS gene amplification can occur in many plant species to confer glyphosate tolerance.     

Expression of glyphosate resistance in carrot somatic hybrid cells through the transfer of an amplified 5-enolpyruvylshikimic acid-3-phosphate synthase gene

Summary A Daucus carota cell line selected as resistant to N-(phosphonomethyl)-glycine (glyphosate) was found to have increased levels of 5-enolpyruvylshikimic acid-3-phosphate synthase (EPSPS) activity of 5.5 times over wild-type carrot and an EPSPS protein level increase of 8.7 times as confirmed by Western hybridization analysis. Southern blot hybridization using a petunia EPSPS probe showed increases in the number of copies of EPSPS genes in the glyphosate-resistant line which correlated with the higher levels of the EPSPS enzyme. The mechanism of resistance to glyphosate is therefore due to amplification of the EPSPS gene. To examine the stability of the amplified genes, cloned lines selected as doubly resistant to Dl-5-methyltryptophan (5MT) and azetidine-2-carboxylate (A2C) were fused with the amplified EPSPS glyphosate-resistant cell line. Somatic hybrids expressed resistances to 5MT in a semidominant fashion while A2C and glyphosate resistance was expressed as dominant, or semi-dominant traits, in a line-specific manner. The hybrid lines possessed additive chromosome numbers of the parental lines used and no double minute chromosomes were observed. The glyphosate-resistant parental line and most somatic hybrids retained the amplified levels of EPSPS in the absence of selection pressure over a 3-year period.     

Expression and stability of amplified genes encoding 5-enolpyruvylshikimate-3-phosphate synthase in glyphosate-tolerant tobacco cells

Two distinct cDNAs for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) were obtained from a glyphosate-tolerant tobacco cell line. The cDNAs were 89% identical and the predicted sequences of the mature proteins were greater than 83% identical with EPSPS proteins from other plants. Tobacco EPSPS proteins were more similar to those from tomato and petunia than Arabidopsis. One cDNA clone, EPSPS-1, represented a gene that was amplified in glyphosate-tolerant cells, while the gene for EPSPS-2 was unaltered in these cells. Consequently, EPSPS-1 mRNA was more abundant in tolerant than unselected cells, whereas EPSPS-2 mRNA was at relatively constant levels in these cell lines. Exposure of unselected cells and tobacco leaves to glyphosate produced a transient increase in EPSPS mRNA. However, glyphosate-tolerant cells containing amplified copies of EPSPS genes did not show a similar response following exposure to glyphosate. A significant proportion of the EPSPS gene amplification was maintained when tolerant cells were grown in the absence of glyphosate for eight months. Plants regenerated from these cells also contained amplified EPSPS genes.     

Structure of the amplified 5-enolpyruvylshikimate-3-phosphate synthase gene in glyphosate-resistant carrot cells

The structure of amplified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) DNA of carrot suspension-cultured cell lines selected for glyphosate resistance was analyzed to determine the mechanism of gene amplification in this plant system. Southern hybridization of the amplified DNA digested with several restriction enzymes probed with a petunia EPSPS cDNA clone showed that there were differences in fragment sizes in the amplified DNA from one highly resistant cell line in comparison with the parental line. Cloning of the EPSPS gene and 5 flanking sequences was carried out and two different DNA structures were revealed. A 13 kb clone contained only one copy of the EPSPS gene while a 16 kb clone contained an inverted duplication of the gene. Southern blot analysis with a carrot DNA probe showed that only the uninverted repeated DNA structure was present in all of the cell lines during the selection process and the inverted repeat (IR) was present only in highly amplified DNA. The two structures were present in about equal amounts in the highly amplified line, TC 35G, where the EPSPS gene was amplified about 25-fold. The presence of the inverted repeat (IR) was further verified by resistance to S1 nuclease hydrolysis after denaturation and rapid renaturation, showing foldback DNA with the IR length being 9.5 kb. The junction was also sequenced. Mapping of the clones showed that the size of the amplified carrot EPSPS gene itself is about 3.5 kb. This is the first report of an IR in amplified DNA of a target enzyme gene in selected plant cells.     

Selection of a Nicotiana plumbaginifolia universal hybridizer and its use in intergeneric somatic hybrid formation

Summary A Nicotiana plumbaginifolia cell strain carrying a positive (dominant) trait, resistance to azetidine-2-carboxylate (A2C), was selected in strain NX1 which lacked nitrate reductase activity (a negative or recessive trait). This universal hybridizer strain, denoted NXA^r, was fused with dextran to a Daucus carota strain, PR, which carried glyphosate (GLP) resistance. A large number of hybrids were selected in a medium with NO ₃ ^- as the sole nitrogen source and A2C as inhibitor, conditions which prevent the growth of both parents. When the selected colonies were then tested for GLP resistance, 93% carried this trait. In addition the hybrid nature was indicated by additive chromosome numbers, both A2C and GLP resistance in suspension cultures, intermediate nitrate reductase activity and the presence of banding patterns for three isozymes which match those of the parents. Southern hybridization analysis using an enolpyruvylshikimic acid-3-phosphate synthase (EPSPS) probe, pMON 6145, also showed the presence of the gene from both parents in the hybrid strains based on restriction length polymorphisms. The PR strain contains increased levels of EPSPS which confers GLP^r due to gene amplification. Since the universal hybridizer can be used as a fusion partner with any wild-type line many protoplast fusion studies can be carried out easily.Abbreviations A2C azetidine-2-carboxylate - 2,4-D 2,4-dichlorophenoxyacetic acid - EPSPS 5-enolpyruvylshikimic acid-3-phosphate synthase - GLP glyphosate - HAT hypoxanthine, aminopterin, glycine and thymidine medium - IDH isocitrate dehydrogenase - MDH malate dehydrogenase - 5MT 5-methyltryptophan - NBT nitroblue tetrazolium - PGI phosphoglucoisomerase - SDS sodium dodecylsulfate     

Stability and expression of amplified EPSPS genes in glyphosate resistant tobacco cells and plantlets

Summary The stability and expression of amplified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) genes was examined in glyphosate resistant tobacco cells grown in glyphosate-free medium, and in plantlets regenerated from resistant cells. Amplified DNA was maintained in resistant cells grown in the absence of glyphosate for three years. Amplified EPSPS genes were retained in regenerated plantlets at levels comparable to those observed in the resistant cells, and EPSPS mRNA was overexpressed (compared to unselected plantlets). However, glyphosate resistance in cell lines grown in glyphosate-free medium declined 7-fold, and in regenerated plantlets approximately 20-fold, compared to resistant cells maintained under glyphosate selection. In plantlets, reduced resistance correlated with lower levels of EPSPS mRNA. Plantlets regenerated from resistant cells exhibited morphological variation, and had an approximate doubling of their nuclear genome size.     

Expression of a bacterial <Emphasis Type="Italic">aroA</Emphasis> mutant, <Emphasis Type="Italic">aroA-M1</Emphasis>, encoding 5-enolpyruvylshikimate-3-phosphate synthase for the production of glyphosate-resistant tobacco plants

Glyphosate is a non-selective broad-spectrum herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants. We have previously reported a strategy for engineering glyphosate-resistant class I EPSPS based on staggered-PCR technology. Selected mutant enzymes exhibited high K_i[glyphosate] and low K_m[PEP] values compared to the parental enzymes from Escherichia coli (EcaroA) and Salmonella typhimurium (StaroA). One mutant, aroA-M1, was further engineered with a tobacco chloroplast leader sequence, and then placed in the binary vector pCAMBIA1300 for Agrobacterium-mediated gene transfer to tobacco (Nicotiana tabacum cv. Xanthi). Transgenic plants with increased resistance to glyphosate were generated.     

Simultaneous substitution of Gly96 to Ala and Ala183 to Thr in 5-enolpyruvylshikimate-3-phosphate synthase gene of <Emphasis Type="Italic">E. coli</Emphasis> (k12) and transformation of rapeseed (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) in order to make tolerance to glyphosate

Glyphosate is a non-selective broad-spectrum herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). This is a key enzyme in the aromatic amino acid biosynthesis pathway of microorganisms and plants. The manipulation of bacterial EPSPS gene in order to reduce its affinity for glyphosate, followed by its transfer to plants is one of the most effective approaches for the production of glyphosate-tolerant plants. In this study, we chose to focus on amino acid residues glycine96 and alanine183 of the E. coli (k12) EPSPS enzyme. These two amino acids are important residues for glyphosate binding. We used site directed mutagenesis (SDM) to induce point mutations in the E. coli EPSPS gene, in order to convert glycine96 to alanine (Gly96Ala) and alanine183 to threonine (Ala183Thr). After confirming the mutation by sequencing, the altered EPSPS gene was transferred to rapeseed (Brassica napus L.) via Agrobacterium-mediated transformation. The transformed explants were screened in shoot induction medium containing 25 mg L⁻¹ kanamycin. Glyphosate tolerance was assayed in putative transgenic plants. Statistical analysis of data showed that there was a significant difference between the transgenic and control plants. It was observed that transgenic plants were resistant to glyphosate at a concentration of 10 mM whereas the non-transformed control plants were unable to survive 1 mM glyphosate. The presence and copy numbers of the transgene were confirmed with PCR and Southern blotting analysis, respectively.     

Distinct non-target site mechanisms endow resistance to glyphosate,ACCase and ALS-inhibiting herbicides in multiple herbicide-resistant <Emphasis Type="Italic">Lolium rigidum</Emphasis>

This study investigates mechanisms of multiple resistance to glyphosate, acetyl-coenzyme A carboxylase (ACCase) and acetolactate synthase (ALS)-inhibiting herbicides in two Lolium rigidum populations from Australia. When treated with glyphosate, susceptible (S) plants accumulated 4- to 6-fold more shikimic acid than resistant (R) plants. The resistant plants did not have the known glyphosate resistance endowing mutation of 5-enolpyruvylshikimate-3 phosphate synthase (EPSPS) at Pro-106, nor was there over-expression of EPSPS in either of the R populations. However, [¹⁴C]-glyphosate translocation experiments showed that the R plants in both populations have altered glyphosate translocation patterns compared to the S plants. The R plants showed much less glyphosate translocation to untreated young leaves, but more to the treated leaf tip, than did the S plants. Sequencing of the carboxyl transferase domain of the plastidic ACCase gene revealed no resistance endowing amino acid substitutions in the two R populations, and the ALS in vitro inhibition assay demonstrated herbicide-sensitive ALS in the ALS R population (WALR70). By using the cytochrome P450 inhibitor malathion and amitrole with ALS and ACCase herbicides, respectively, we showed that malathion reverses chlorsulfuron resistance and amitrole reverses diclofop resistance in the R population examined. Therefore, we conclude that multiple glyphosate, ACCase and ALS herbicide resistance in the two R populations is due to the presence of distinct non-target site based resistance mechanisms for each herbicide. Glyphosate resistance is due to reduced rates of glyphosate translocation, and resistance to ACCase and ALS herbicides is likely due to enhanced herbicide metabolism involving different cytochrome P450 enzymes.     

Recovery of transgenic plants by pollen-mediated transformation in <Emphasis Type="Italic">Brassica juncea</Emphasis>

The aroA-M1 encoding the mutant of 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS) was introduced into the Brassica juncea genome by sonication-assisted, pollen-mediated transformation. The plasmid DNA and collected pollen grains were mixed in 0.3 mol/L sucrose solution and treated with mild ultrasonication. The treated pollen was then pollinated onto the oilseed stigmas after the stamens were removed artificially. Putative transgenic plants were obtained by screening germinating seeds on a medium containing glyphosate. Southern blot analysis of glyphosate-resistant plants indicated that the aroA-M1 gene had been integrated into the oilseed genome. Western blot analysis further confirmed that the EPSPS coded by aroA-M1 gene was expressed in transgenic plants. The transgenic plants exhibited increased resistance to glyphosate compared to untransformed plants. Some of those transgenic plants had considerably high resistance to glyphosate. The genetic analysis of T₁ progeny further confirmed that the inheritance of the introduced genes followed the Mendelian rules. The results indicated that foreign genes can be transferred by pollen-mediated transformation combined with mild ultrasonication.     

Plastid-expressed 5-enolpyruvylshikimate-3-phosphate synthase genes provide high level glyphosate tolerance in tobacco

Plastid transformation (transplastomic) technology has several potential advantages for biotechnological applications including the use of unmodified prokaryotic genes for engineering, potential high-level gene expression and gene containment due to maternal inheritance in most crop plants. However, the efficacy of a plastid-encoded trait may change depending on plastid number and tissue type. We report a feasibility study in tobacco plastids to achieve high-level herbicide resistance in both vegetative tissues and reproductive organs. We chose to test glyphosate resistance via over-expression in plastids of tolerant forms of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Immunological, enzymatic and whole-plant assays were used to prove the efficacy of three different prokaryotic (Achromobacter, Agrobacterium and Bacillus) EPSPS genes. Using the Agrobacterium strain CP4 EPSPS as a model we identified translational control sequences that direct a 10,000-fold range of protein accumulation (to >10% total soluble protein in leaves). Plastid-expressed EPSPS could provide very high levels of glyphosate resistance, although levels of resistance in vegetative and reproductive tissues differed depending on EPSPS accumulation levels, and correlated to the plastid abundance in these tissues. Paradoxically, higher levels of plastid-expressed EPSPS protein accumulation were apparently required for efficacy than from a similar nuclear-encoded gene. Nevertheless, the demonstration of high-level glyphosate tolerance in vegetative and reproductive organs using transplastomic technology provides a necessary step for transfer of this technology to other crop species.     

A single residue mutation of 5-enoylpyruvylshikimate-3-phosphate synthase in Pseudomonas stutzeri enhances resistance to the herbicide glyphosate

A novel class II 5-enoylpyruvylshikimate-3-phosphate synthase (EPSPS) was identified from Pseudomonas stutzeri A1501 by complementation of an Escherichia coli auxotrophic aroA mutant. The single amino acid substitution of serine (Ser) for asparagine (Asn)-130 of the A1501 EPSPS enhanced resistance to 200 mM glyphosate. The mutated EPSPS had a 2.5-fold increase for IC(50) [glyphosate] value, a 2-fold increase for K (i) [glyphosate] value, but a K (m) [PEP] value similar to that of wild type. The effect of the single residue mutation on glyphosate resistance was also analyzed using a computer-based three-dimensional model.     

Herbicidal inhibitors of amino acid biosynthesis and herbicide-tolerant crops

Summary. Acetohydroxyacid synthase (AHAS) inhibitors interfere with branched-chain amino acid biosynthesis by inhibiting AHAS. Glyphosate affects aromatic amino acid biosynthesis by inhibiting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Glufosinate inhibits glutamine synthetase and blocks biosynthesis of glutamine. AHAS gene variants that confer tolerance to AHAS inhibitors have been discovered in plants through selection or mutagenesis. Imidazolinone-tolerant crops have been commercialized based on these AHAS gene variants. A modified maize EPSPS gene and CP4-EPSPS gene from Agrobacterium sp. have been used to transform plants for target-based tolerance to glyphosate. A gox gene isolated from Ochrobactrum anthropi has also been employed to encode glyphosate oxidoreductase to detoxify glyphosate in plants. Glyphosate-tolerant crops with EPSPS transgene alone or both EPSPS and gox transgenes have been commercialized. Similarly, bar and pat genes isolated from Streptomyces hygroscopicus and S. viridochromogenes, respectively, have been inserted into plants to encode phosphinothricin N-acetyltransferase to detoxify glufosinate. Glufosinate-tolerant crops have been commercialized using one of these two transgenes.     

Glyphosate,paraquat and ACCase multiple herbicide resistance evolved in a <Emphasis Type="Italic">Lolium rigidum</Emphasis> biotype

Glyphosate is the world’s most widely used herbicide. A potential substitute for glyphosate in some use patterns is the herbicide paraquat. Following many years of successful use, neither glyphosate nor paraquat could control a biotype of the widespread annual ryegrass (Lolium rigidum), and here the world’s first case of multiple resistance to glyphosate and paraquat is confirmed. Dose–response experiments established that the glyphosate rate causing 50% mortality (LD₅₀) for the resistant (R) biotype is 14 times greater than for the susceptible (S) biotype. Similarly, the paraquat LD₅₀ for the R biotype is 32 times greater than for the S biotype. Thus, based on the LD₅₀ R/S ratio, this R biotype of L. rigidum is 14-fold resistant to glyphosate and 32-fold resistant to paraquat. This R biotype also has evolved resistance to the acetyl-coenzyme A carboxylase (ACCase) inhibiting herbicides. The mechanism of paraquat resistance in this biotype was determined as restricted paraquat translocation. Resistance to ACCase-inhibiting herbicides was determined as due to an insensitive ACCase. Two mechanisms endowing glyphosate resistance were established: firstly, a point mutation in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene, resulting in an amino acid substitution of proline to alanine at position 106; secondly, reduced glyphosate translocation was found in this R biotype, indicating a co-occurrence of two distinct glyphosate resistance mechanisms within the R population. In total, this R biotype displays at least four co-existing resistance mechanisms, endowing multiple resistance to glyphosate, paraquat and ACCase herbicides. This alarming case in the history of herbicide resistance evolution represents a serious challenge for the sustainable use of the precious agrochemical resources such as glyphosate and paraquat.     

植物莽草酸途径EPSPS蛋白的分子进化和基因结构分析

EPSPS既是植物、微生物和真菌等生物芳香族氨基酸生物合成途径——莽草酸途径中的关键酶,也是除草剂草甘膦的靶标酶。EPSPS的克隆能为草甘膦抗性转基因作物的研发提供候选基因。该研究运用比较基因组学方法,通过对41种不同植物的43条EPSPS蛋白序列进行进化分析,取得主要结果如下:(1)不同植物EPSPS蛋白的相似性很高,且具有相同的结构域、保守基序和保守位点,但是其叶绿体转运肽序列差异显著;(2)系统发育分析表明,EPSPS基因按照双子叶植物纲和单子叶植物纲分为2个大的分支,各个小的分支又按照植物的种属亲缘关系进行分支和聚类;(3)基因结构分析表明,植物EPSPS基因基本都含有8个外显子和7个内含子,且所对应外显子的长度相当,而内含子的长度差异很大,说明在植物基因组进化过程中造成EPSPS基因结构差异的主要因素是内含子的改变。研究结果将为揭示植物EPSPS蛋白的结构功能提供参考。     

5-Enolpyruvylshikimate-3-phosphate synthase from Staphylococcus aureus is insensitive to glyphosate

The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes the penultimate step of the shikimate pathway, and is the target of the broad-spectrum herbicide glyphosate. Kinetic analysis of the cloned EPSPS from Staphylococcus aureus revealed that this enzyme exerts a high tolerance to glyphosate, while maintaining a high affinity for its substrate phosphoenolpyruvate. Enzymatic activity is markedly influenced by monovalent cations such as potassium or ammonium, which is due to an increase in catalytic turnover. However, insensitivity to glyphosate appears to be independent from the presence of cations. Therefore, we propose that the Staphylococcus aureus EPSPS should be classified as a class II EPSPS. This research illustrates a critical mechanism of glyphosate resistance naturally occurring in certain pathogenic bacteria.     

Glyphosate inhibits the translocation of green fluorescent protein and sucrose from a transgenic tobacco host to<Emphasis Type="Italic"> Cuscuta campestris</Emphasis> Yunk.

The parasitic plant Cuscuta campestris is dependent on its host for water, assimilates and amino acids. It can be controlled by the herbicide glyphosate, which inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), resulting in shikimate accumulation. In this study, C. campestris was parasitic on transgenic tobacco plants expressing green fluorescent protein (GFP) in the phloem. Changes in [¹⁴C]sucrose and GFP accumulation in the parasite were used as indicators of the herbicides effect on translocation between the host and parasite. Host plants were treated with glyphosate 22 days after sowing. Shikimate accumulation in the parasite 1 day after glyphosate treatment (DAGT) confirmed EPSPS inhibition in C. campestris. No damage was visible in the host plants for the first 3 DAGT, while during that same time, a significant reduction in [¹⁴C]sucrose and GFP accumulation was observed in the parasite. Thus, we propose that the parallel reduction in GFP and sucrose accumulation in C. campestris is a result of a glyphosate effect on the parasites ability to withdraw assimilates from the host.Abbreviations CLSM Confocal laser-scanning microscope - DAGT Days after glyphosate treatment - DAS Days after sowing - EPSPS 5-Enolpyruvylshikimate-3-phosphate synthase - GFP Green fluorescent protein     

A Novel 5-Enolpyruvylshikimate-3-Phosphate Synthase from Rahnella aquatilis with Significantly Reduced Glyphosate Sensitivity

The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19) is a key enzyme in the shikimate pathway for the production of aromatic amino acids and chorismate-derived secondary metabolites in plants, fungi, and microorganisms. It is also the target of the broad-spectrum herbicide glyphosate. Natural glyphosate resistance is generally thought to occur within microorganisms in a strong selective pressure condition. Rahnella aquatilis strain GR20, an antagonist against pathogenic agrobacterial strains of grape crown gall, was isolated from the rhizosphere of grape in glyphosate-contaminated vineyards. A novel gene encoding EPSPS was identified from the isolated bacterium by complementation of an Escherichia coli auxotrophic aroA mutant. The EPSPS, named AroA(R.aquatilis), was expressed and purified from E. coli, and key kinetic values were determined. The full-length enzyme exhibited higher tolerance to glyphosate than the E. coli EPSPS (AroA(E.coli)), while retaining high affinity for the substrate phosphoenolpyruvate. Transgenic plants of AroA(R.aquatilis) were also observed to be more resistant to glyphosate at a concentration of 5 mM than that of AroA(E.coli). To probe the sites contributing to increased tolerance to glyphosate, mutant R.aquatilis EPSPS enzymes were produced with the c-strand of subdomain 3 and the f-strand of subdomain 5 (Thr38Lys, Arg40Val, Arg222Gln, Ser224Val, Ile225Val, and Gln226Lys) substituted by the corresponding region of the E. coli EPSPS. The mutant enzyme exhibited greater sensitivity to glyphosate than the wild type R.aquatilis EPSPS with little change of affinity for its first substrate, shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). The effect of the residues on subdomain 5 on glyphosate resistance was more obvious.     

Glyphosate as a selective agent for the production of fertile transgenic maize (Zea mays L.) plants

Efficient and reproducible selection of transgenic cells is an essential component of a good transformation system. In this paper, we describe the development of glyphosate as a selective agent for the recovery of transgenic embryogenic corn callus and the production of plants tolerant to Roundup^® herbicide. Glyphosate, the active ingredient in Roundup^® herbicide inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and thus prevents the synthesis of chorismate-derived aromatic amino acids and secondary metabolites in plants. A maize EPSPS gene has been cloned, mutated to produce a modified enzyme resistant to inhibition by glyphosate, and engineered into a monocot expression vector. In addition, a bacterial gene which degrades glyphosate (glyphosate oxidoreductase, or GOX) was also cloned into a similar expression vector. Stably transformed callus has been reproducibly recovered following introduction of mutant maize EPSPS and GOX genes into tissue culture cells by particle bombardment and selection on glyphosate-containing medium. Plants have been regenerated both on and off glyphosate selection medium, and are tolerant to normally lethal levels of Roundup^®. Excellent seed set has been obtained from both self and outcross pollinations from both sprayed and unsprayed regenerated plants. Progeny tests have demonstrated normal Mendelian transmission and tolerance to the herbicide for some of the transgenic events.