Phosphinothricin Acetyltransferase Activity Research Articles

Resistance to glufosinate is proportional to phosphinothricin acetyltransferase expression and activity in LibertyLink(®) and WideStrike(®) cotton.

Main conclusionInsertion of the gene encoding phosphinothricin acetyltransferase (PAT) has resulted in cotton plants resistant to the herbicide glufosinate. However, the lower expression and commensurate reduction in PAT activity is a key factor in the low level of injury observed in theWideStrike®cotton and relatively high level of resistance observed in LibertyLink®cotton.LibertyLink® cotton cultivars are engineered for glufosinate resistance by overexpressing the bar gene that encodes phosphinothricin acetyltransferase (PAT), whereas the insect-resistant WideStrike® cultivars were obtained using the similar pat gene as a selectable marker. The latter cultivars carry some level of resistance to glufosinate which enticed certain farmers to select this herbicide for weed control with WideStrike® cotton. The potency of glufosinate on conventional FM 993, insect-resistant FM 975WS, and glufosinate-resistant IMACD 6001LL cotton cultivars was evaluated and contrasted to the relative levels of PAT expression and activity. Conventional cotton was sensitive to glufosinate. The single copy of the pat gene present in the insect-resistant cultivar resulted in very low RNA expression of the gene and undetectable PAT activity in in vitro assays. Nonetheless, the presence of this gene provided a good level of resistance to glufosinate in terms of visual injury and effect on photosynthetic electron transport. The injury is proportional to the amount of ammonia accumulation. The strong promoter associated with bar expression in the glufosinate-resistant cultivar led to high RNA expression levels and PAT activity which protected this cultivar from glufosinate injury. While the insect-resistant cultivar demonstrated a good level of resistance to glufosinate, its safety margin is lower than that of the glufosinate-resistant cultivar. Therefore, farmers should be extremely careful in using glufosinate on cultivars not expressly designed and commercialized as resistant to this herbicide.

Unintended consequences: high phosphinothricin acetyltransferase activity related to reduced fitness in barley

Selectable markers used in plant transformation, such as the bar gene, have been chosen for selection efficacy and for the absence of pleiotropic effects. Recent research has suggested that expression of bar in Arabidopsis affects the transcriptome, and in at least one barley cultivar, high bar expression has been associated with severe fitness reductions. However, the observed sensitivity in barley was thought to be an isolated case associated with a specific genetic stock. In this study, fitness reductions were observed in some near-isogenic lines in the background of the cultivar Garnet when the bar locus was homozygous. The fitness of the near-isogenic lines was correlated with the levels of bar mRNA in the donor parents. Previous studies had shown heritability of bar expression in F1 barley hybrids, so these results suggested that Garnet barley is intolerant of high phosphinothricin acetyltransferase activity. Phosphinothricin acetyltransferase activities were determined for eight near-isogenic lines that showed a range of phenotypes (apparently normal to abnormal and nonviable) when homozygous for bar. Fitness was clearly and negatively correlated with phosphinothricin acetyltransferase activity. These results suggest genetic variability for sensitivity to bar expression in barley, and the possibility of negative but non-obvious effects in apparently tolerant cultivars cannot be ruled out. Similar problems may be associated, but not yet detected, with other selectable markers. The potential for pleiotropic effects of selectable markers can be avoided by transformation strategies that enable the elimination of selectable markers.

L-Methionine Sulfoximine, but Not Phosphinothricin, Is a Substrate for an Acetyltransferase (Gene PA4866) from Pseudomonas aeruginosa: Structural and Functional Studies

The gene PA4866 from Pseudomonas aeruginosa is documented in the Pseudomonas genome database as encoding a 172 amino acid hypothetical acetyltransferase. We and others have described the 3D structure of this protein (termed pita) [Davies et al. (2005) Proteins: Struct., Funct., Bioinf. 61, 677-679; Nocek et al., unpublished results], and structures have also been reported for homologues from Agrobacterium tumefaciens (Rajashankar et al., unpublished results) and Bacillus subtilis [Badger et al. (2005) Proteins: Struct., Funct., Bioinf. 60, 787-796]. Pita homologues are found in a large number of bacterial genomes, and while the majority of these have been assigned putative phosphinothricin acetyltransferase activity, their true function is unknown. In this paper we report that pita has no activity toward phosphinothricin. Instead, we demonstrate that pita acts as an acetyltransferase using the glutamate analogues l-methionine sulfoximine and l-methionine sulfone as substrates, with Km(app) values of 1.3 +/- 0.21 and 1.3 +/- 0.13 mM and kcat(app) values of 505 +/- 43 and 610 +/- 23 s-1 for l-methionine sulfoximine and l-methionine sulfone, respectively. A high-resolution (1.55 A) crystal structure of pita in complex with one of these substrates (l-methionine sulfoximine) has been solved, revealing the mode of its interaction with the enzyme. Comparison with the apoenzyme structure has also revealed how certain active site residues undergo a conformational change upon substrate binding. To investigate the role of pita in P. aeruginosa, a mutant strain, Depp4, in which pita was inactivated through an in-frame deletion, was constructed by allelic exchange. Growth of strain Depp4 in the absence of glutamine was inhibited by l-methionine sulfoximine, suggesting a role for pita in protecting glutamine synthetase from inhibition.

Production of Herbicide Resistant Transgenic Lily Plants by Particle Bombardment

Calli induced from bulb scales of Lilium X formolongi were bombarded with the plasmid pAct1-F, which harbors the uidA gene encoding β-glucuronidase (GUS) that is driven by the rice actin-1 promoter. Based on the frequency of GUS-expressing cells in the calli, bombardment conditions were optimized. Calli were bombarded with pDM302 harboring the bar gene, encoding phosphinothricin acetyltransferase (PAT) that is driven by the actin-1 promoter, and selected for bialaphos resistance. Regenerated bialaphos-resistant lily plants were obtained six months after bombardment. PCR analysis suggested that some of the bialaphos-resistant plants were transgenic. Transgenic bialaphos-resistant plants displayed PAT activity and resistance to bialaphos-based herbicide.

Stable transformation and tissue culture response in current European winter wheats ( Triticum aestivum L.)

In order to efficiently complement traditional wheat breeding with genetic transformation technology it will be desirable to introduce transgenes into the ideal genetic background. Poor tissue culture performance is limiting the number of wheat genotypes that can be stably transformed. We statistically analysed the tissue culture response of 38 current European winter wheats and discuss genetic factors influencing this trait. Although regenerable callus cultures could be initiated from immature embryos of all 38 winter wheats analysed, the number of regenerated plants per cultured explant differed highly significantly (p<0.01) among genotypes. Ten cultivars with excellent ranking in this parameter were selected for transformation experiments. Independent transgenic plants were recovered from nine winter wheat genotypes with a frequency ranging between 0.2% and 2.0% of the cultured immature embryos after biolistic transfer of the bar gene and bialaphos selection. The nine transformable winter wheat genotypes included a recently released high-yielding, disease-resistant cultivar (cv. Certo), well established cultivars with elite bread-making quality (cv. Tarso, Alidos) and current breeding lines differing in yield, disease resistance and grain quality. Transgene integration and expression were confirmed by Southern blot analysis, polymerase chain reaction, phosphinothricin acetyltransferase activity assay and herbicide application. Transgene expression was stably transmitted to the sexual progeny of all transgenic lines analysed and segregated in a Mendelian fashion in the majority of lines. The introduction of transgenes into the ideal genetic background will allow a thorough evaluation of their crop improvement potential.

Transgene silencing and reactivation in sorghum

Amongst the important cereals, sorghum has been the most recalcitrant to genetic transformation. There are a few reports on sorghum transformation and the majority of these have reported silencing of one of the transgenes. We regenerated plants from two independent transgenic sorghum callus lines that were cotransformed with Ubi promoter :bar and Act1-D promoter :gusA gene constructs using the particle bombardment method. Southern analyses indicated integration of multiple copies of both the transgenes. T0 plants were found to express the bar gene. The gusA gene, however, was silenced. It was possible to activate gusA gene expression in T1 seedlings and in calli derived from immature T1 and T2 embryos by 5-azacytidine (azaC) treatment. In certain cases, spontaneous expression of the gusA gene was observed in T1 and T2 immature embryo-derived calli. Expression of the bar gene, as analyzed by Basta™ tolerance and Phosphinothricin acetyltransferase (PAT) assays, was detected in T0, T1 and T2 plantlets; however, the expression was reduced in the T2 progeny obtained from a homozygous T1 parent. PAT activity was also lower in the immature embryo-derived T2 calli from the same homozygous T1 parent. Again, culture on azaC increased the level of PAT activity in these calli. Moreover, in a separate set of stable transformation experiments, it was possible to recover a much higher than usual number of gusA gene expressing transgenic calli by growing the bombarded tissues in the presence of azaC. Taken together, these results suggest that methylation-based silencing is frequent in sorghum and probably responsible for several cases of transgene inactivation reported earlier for this crop.

Biolistic transformation of haploid isolated microspores of barley (Hordeum vulgare L.)

Transgenic barley plants were produced by the direct delivery of plasmid DNA into isolated microspores of barley cv. Igri using high velocity microprojectiles. The plasmid pAHC25 contained the uidA and bar genes, each under the control of a maize Ubi1 promoter. Bombarded microspores were cultured and selected on solid medium containing varying concentrations (2-5 mg/L) of the Basta herbicide active agent bialaphos. The effectiveness of selection with bialaphos depended on its interaction with the medium component glutamine. Six transgenic plants (R0) were obtained, and the presence of the uidA and bar genes and their integration into nuclear DNA in transformed R0 plants were confirmed by PCR and Southern blot analysis. Phosphinothricin acetyltransferase activity was observed in all six R0 transgenic plants, whereas none showed &beta;-glucuronidase (GUS) activity in histochemical GUS assays. Two of the six R0 plants were haploid and sterile; one of them was trisomic and partially sterile; the remainder were diploid, but one of them was also sterile. Inheritance of the transgenes in progeny of three seed-producing transgenic plants was investigated. Southern blot analysis of genomic DNA from R1 plants showed that the introduced bar and uidA genes were hemizygous and stably cotransmitted to the R1 progeny derived from self-pollination. Analysis of Basta resistance and the integration of the bar gene by PCR analysis in R1 plants indicated that the bar gene was being inherited and expressed as a single dominant trait. Fluorescent in situ hybridization was performed on chromosomes of the trisomic plant to confirm the presence of transgenes in the genome.

Rapid transformation ofMedicago truncatula: regeneration via shoot organogenesis

A rapid transformation and regeneration system has been developed forM. truncatula cv Jemalong (barrel medic) by which it is possible to obtain transgenic plants within 2.5 months. The procedure involvesAgrobacterium-mediated transformation of cotyledon explants coupled with the regeneration of transformed plants via direct organogenesis. To develop the procedure,M. truncatula explants were transformed with the binary plasmid pSLJ525 which carries thebar gene. Thebar gene encodes phosphinothricin acetyl transferase, and transformed plants were selected on media containing phosphinothricin (Ignite, AgrEvo). Transformed plants show phosphinothricin acetyl transferase activity and Southern blot analysis indicates that they carry thebar gene integrated into their genomes. The resistance to phosphinothricin is stable and is inherited by the R1 progeny as a single dominant Mendelian trait. The transgenic plants are highly resistant to the broad spectrum herbicide, Ignite and therefore may also have commercial applications.

Stable transformation of barley callus using biolistic� particle bombardment and the phosphinothricin acetyltransferase (bar) gene

Suspension culture cells of barley (Hordeum vulgare L. cv. Klages) were collectec on filters and bombarded with gold particles carrying the Basta® resistance (bar) gene, or both bar and the β-glucuronidase (uidA) genes in a Biolistic® Particle Accelerator. Filters carrying the bombarded tissues were selected on basal medium with increasing concentrations (maximum of 200 mg I-1) of phosphinothricin. Surviving calli were assayed for the presence of phosphinothricin acetyltransferase activity. All positive calli were shown to have the bar gene integrated into their genome. Nine independent stable transformation events have been analyzed with substantial variation in the integration pattern and transgene copy number. Some albino plantlets were produced, but no mature plants were obtained.

Rapid Production of Fertile Transgenic Plants of Rye (Secale cereale L.)

We have produced transgenic rye plants by the direct delivery of plasmid DNA, containing the uidA and bar genes, into young embryogenic calli using high velocity microprojectiles. Of the fourteen independent callus lines selected on varying concentrations of phosphinothricin (PPT), the active ingredient of the herbicide Basta, six showed phosphinothricin acetyltransferase (PAT) activity. Plants (R0) were regenerated from two putative transformed lines on medium containing Basta. All of the R0 plants recovered from the two lines showed GUS expression (in leaves, roots, pistils and pollen), PAT activity and resistance to topical application of Basta. The presence of the uidA and bar genes and their integration into nuclear DNA in transformed R0 plants and their R1 progeny was confirmed by Southern analysis. Flowering fertile R0 plants were obtained 8–9 months after the initial culture and bombardment. Male and female transmission of the two genes, which segregated as dominant Mendelian traits in R1 plants, was demonstrated.

Self‐fertile transgenic wheat plants regenerated from isolated scutellar tissues following microprojectile bombardment with two distinct gene constructs†

A system for enhanced induction of somatic embryo‐genesis and regeneration of plants from isolated scutellar tissue of wheat has been developed. This system has been successfully used in the development of a simple and reproducible protocol for the production of self‐fertile transgenic wheat plants. The procedure is rapid resulting in the production of transgenic plantlets within 12 weeks from initiation of cultures and it avoids the need for establishing long‐term callus, cell suspension or protoplast cultures. Somatic embryos regenerated from scutella bombarded with plasmid pBARGUS were selected on L‐phosphinothricin (L‐PPT) to obtain herbicide‐resistant self‐fertile transgenic plants. Phosphinothricin acetyltransferase (PAT) activity was observed at varying levels in 50% of the plants selected on L‐PPT whereas none of the plants showed β‐glucuronidase (GUS) activity. Molecular analysis of PAT‐positive plants confirmed stable integration of both bar and gus genes in R0 and R1 progeny plants. Segregation of the PAT activity and herbicide resistance in R1 progeny plants confirmed the Mendelian inheritance of the bar gene. Additionally, isolated scutella bombarded with plasmid DNA containing a gus::nptII fusion gene driven by a rice actin promoter and its first intron were selected in the presence of geneticin to obtain fully fertile transgenic plants. Functional expression of the fusion gene was demonstrated in transgenic plants by GUS and neomycin phospho‐transferase (NPTII) enzyme assays. Southern blot analysis confirmed the integration of transgenes into the wheat genome. Histochemical GUS staining showed transmission of the fusion gene to floral organs of primary transformants and confirmed Mendelian segregation of the transgene in R1 progeny.

Rapid Production of Transgenic Wheat Plants by Direct Bombardment of Cultured Immature Embryos

We have developed an improved protocol for the rapid and efficient production of transgenic wheat. Three plasmids, each containing the selectable bar gene for resistance to the herbicide Basta and the β-glucuronidase (GUS) reporter gene, were delivered via particle bombardment, directly into immature embryos of two spring and one winter cultivar of wheat four days to two months after culture. Resistant calli were selected on phosphinothricin (PPT) media and screened for histochemical GUS activity. Twelve independent callus lines showing phosphinothricin acetyltransferase (PAT) activity were recovered from the bombardment of 544 explants (374 immature embryos and 170 one or two month old calli). R0 plants were regenerated from seven of these lines, of which so far five have produced R1 progeny, and two of the latter have produced R2 progeny. PAT activity was detected in each of the plants tested from the seven R0 lines, as well as in a 1:1 or 3:1 ratio in R1 plants following cross or self pollination, respectively. Resistance to topical application of Basta was seen in PAT positive plants and transgenic progeny. Molecular analysis by Southern hybridization showed the presence of the bar gene in all PAT positive R0 and R1 plants analyzed. Hybridization of the bar gene probe with high molecular weight DNA further confirmed integration into nuclear DNA. Both male and female transmission of the bar gene, and its segregation as a dominant Mendelian trait in R1 and R2 plants, were demonstrated. Flowering transgenic R0 plants could be obtained in 7–9 months following excision and culture of immature embryos.

Transgenic sorghum plants via microprojectile bombardment.

Transgenic sorghum plants have been obtained after microprojectile bombardment of immature zygotic embryos of a drought-resistant sorghum cultivar, P898012. DNA delivery parameters were optimized based on transient expression of R and C1 maize anthocyanin regulatory elements in scutellar cells. The protocol for obtaining transgenic plants consists of the delivery of the bar gene to immature zygotic embryos and the imposition of bialaphos selection pressure at various stages during culture, from induction of somatic embryogenesis to rooting of regenerated plantlets. One in about every 350 embryos produced embryogenic tissues that survived bialaphos treatment; six transformed callus lines were obtained from three of the eight sorghum cultivars used in this research. Transgenic (T0) plants were obtained from cultivar P898012 (two independent transformation events). The presence of the bar and uidA genes in the T0 plants was confirmed by Southern blot analysis of genomic DNA. Phosphinothricin acetyltransferase activity was detected in extracts of the T0 plants. These plants were resistant to local application of the herbicide Ignite/Basta, and the resistance was inherited in T1 plants as a single dominant locus.

Transformation and Regeneration of Two Cultivars of Pea (Pisum sativum L.).

A reproducible transformation system was developed for pea (Pisum sativum L.) using as explants sections from the embryonic axis of immature seeds. A construct containing two chimeric genes, nopaline synthase-phosphinothricin acetyl transferase (bar) and cauliflower mosaic virus 35S-neomycin phosphotransferase (nptII), was introduced into two pea cultivars using Agrobacterium tumefaciens-mediated transformation procedures. Regeneration was via organogenesis, and transformed plants were selected on medium containing 15 mg/L of phosphinothricin. Transgenic peas were raised in the glasshouse to produce flowers and viable seeds. The bar and nptII genes were expressed in both the primary transgenic pea plants and in the next generation progeny, in which they showed a typical 3:1 Mendelian inheritance pattern. Transformation of regenerated plants was confirmed by assays for neomycin phosphotransferase and phosphinothricin acetyl transferase activity and by northern blot analyses. Transformed plants were resistant to the herbicide Basta when sprayed at rates used in field practice.

Regeneration of herbicide resistant transgenic rice plants following microprojectile-mediated transformation of suspension culture cells

Suspension cells of Oryza sativa L. (rice) were transformed, by microprojectile bombardment, with plasmids carrying the coding region of the Streptomyces hygroscopicus phosphinothricin acetyl transferase (PAT) gene (bar) under the control of either the 5' region of the rice actin 1 gene (Act1) or the cauliflower mosaic virus (CaMV) 35S promoter. Subsequently regenerated plants display detectable PAT activity and are resistant to BASTA(TM), a phosphinothricin (PPT)-based herbicide. DNA gel blot analyses showed that PPT resistant rice plants contain a bar-hybridizing restriction fragment of the expected size. This report shows that expression of the bar gene in transgenic rice plants confers resistance to PPT-based herbicide by suppressing an increase of ammonia in plants after spraying with the herbicide.

Segregation of transgenes in maize

Progeny recovered from backcrossed transgenic maize tissue culture regenerants (R0) were analyzed to determine the segregation, expression, and stability of the introduced genes. Transgenic A188 x B73 R0 plants (regenerated from embryogenic suspension culture cells transformed by microprojectile bombardment; see [9]) were pollinated with nontransformed B73 pollen. Inheritance of a selectable marker gene, bar, and a nonselectable marker gene, uidA, was analyzed in progeny (R1) representing four independent transformation events. Activity of the bar gene product, phosphinothricin acetyltransferase (PAT), was assessed in plants comprising the four R1 populations. The number of R1 plants containing PAT activity per total number of R1 plants recovered for each population was 2/7, 19/34, 3/14 and 73/73. Molecular analysis confirmed the segregation of bar in three R1 populations and the lack of segregation in one R1 population. Cosegregation analysis indicated genetic linkage of bar and uidA in all four R1 populations. Analysis of numerous R2 plants derived from crossing transformed R1 plants with nontransformed inbreds revealed 1:1 segregation of PAT activity in three of four lines, including the line that failed to segregate in the R1 generation. Integrated copies of bar in one line appeared to be unstable or poorly transmitted.

Transformation of Maize Cells and Regeneration of Fertile Transgenic Plants.

A reproducible system for the generation of fertile, transgenic maize plants has been developed. Cells from embryogenic maize suspension cultures were transformed with the bacterial gene bar using microprojectile bombardment. Transformed calli were selected from the suspension cultures using the herbicide bialaphos. Integration of bar and activity of the enzyme phosphinothricin acetyltransferase (PAT) encoded by bar were confirmed in all bialaphos-resistant callus lines. Fertile transformed maize plants (R0) were regenerated, and of 53 progeny (R1) tested, 29 had PAT activity. All PAT-positive progeny analyzed contained bar. Localized application of herbicide to leaves of bar-transformed R0 and R1 plants resulted in no necrosis, confirming functional activity of PAT in the transgenic plants. Cotransformation experiments were performed using a mixture of two plasmids, one encoding PAT and one containing the nonselected gene encoding [beta]-glucuronidase. R0 plants regenerated from co-transformed callus expressed both genes. These results describe and confirm the development of a system for introduction of DNA into maize.