A one-time inducible transposon for creating knockout mutants

The maize transposon Ac can move to a new location within the genome to create knockout mutants in transgenic plants. In rice, Ac transposon is very active but sometimes undergoes further transposition and leaves an empty mutated gene. Therefore, we developed a one-time transposon system by locating one end of the transposon in the intron of the Ac transposase gene, which is under the control of the inducible promoter (PR-1a). Treatment with salicylic acid induced transposition of this transposon, COYA, leading to transposase gene breakage in exons. The progeny plants inheriting the transposition events become stable knockout mutants, because no functional transposase could be yielded. The behavior of COYA was analyzed in single-copy transgenic rice plants. We determined the expression of the modified transposase gene and its ability to trigger transposition events in transgenic rice plants. The COYA element thus exhibits potential for development of an inducible transposon system suitable for gene isolation in heterologous plant species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Ac transposition in transgenic tomato plants

Summary As an initial step towards developing a transposon mutagenesis system in tomato, the maize transposable element Ac was transformed into tomato plants via Agrobacterium tumefaciens. Southern analysis of leaf tissue indicated that in nine out of eleven transgenic plants, Ac excised from the T-DNA and reintegrated into new chromosomal locations. The comparison of Ac banding pattern in different leaves of the same primary transformant provided evidnece for transposition during later stages of transgenic plant development. There was no evidence of Ds mobilization in tomato transformants.     

Developing a transposon tagging system to isolate rust-resistance genes from flax

Summary A line of flax, homozygous for four genes controlling resistance to flax rust, was transformed with T-DNA vectors carrying the maize transposable elements Ac and Ds to assess whether transposition frequency would be high enough to allow transposon tagging of the resistance genes. Transposition was much less frequent in flax than in Solanaceous hosts such as tobacco, tomato and potato. Transposition frequency in callus tissue, but not in plants, was increased by modifications to the transposase gene of Ac. Transactivation of the excision of a Ds element was achieved by expressing a cDNA copy of the Ac transposase gene from the Agrobacterium T-DNA 2 promoter. Progeny of three plants transformed with Ac and 15 plants transformed with Ds and the transposase gene, were examined for transposition occurring in the absence of selection. Transposition was observed in the descendants of only one plant which contained at least nine copies of Ac. Newly transposed Ac elements were observed in 25–30% of the progeny of some members of this family and one active Ac element was located 28.8 (SE=6.3) map units from the L ⁶ rust-resistance gene. This family will be potentially useful in our resistance gene tagging program.     

Frequency and distance of transposition of a modifiedDissociation element in transgenic tobacco

Effective transposon tagging with theAc/Ds system in heterologous plant species relies on the accomplishment of a potentially high transposon-induced mutation frequency. The primary parameters that determine the mutation frequency include the transposition frequency and the transposition distance. In addition, the development of a generally applicable transposon tagging strategy requires predictable transposition behaviour. We systematically analysedDs transposition frequencies andDs transposition distances in tobacco. An artificialDs element was engineered with reporter genes that allowed transposon excision and integration to be monitored visually. To analyse the variability ofDs transposition between different tobacco lines, eight single copy T-DNA transformants were selected. Fortrans-activation of theDs elements, differentAc lines were used carrying an unmodifiedAc ⁺ element, an immobilizedsAc element and a stableAc element under the control of a heterologous chalcone synthas (chsA) promoter. With allAc elements, eachDs line showed characteristic and heritable variegation patterns at the seedling level. SimilarDs line-specificity was observed for the frequency by whichDs transpositions were germinally transmitted, as well as for the distances of theDs transpositions. ThesAc element induced transposition ofDs late in plant development, resulting in low germinal transposition frequencies (0.37%) and high incidences of independent transposition (83%). The majority of theseDs elements (58%) transposed to genetically closed linked sites (10 cM).     

Introduction and transposition of the maize transposable element Ac in rice (Oryza sativa L.).

Summary To develop a transposon tagging system in an important cereal plant, rice (Oryza sativa L.), the maize transposable element Ac (Activator) was introduced into rice protoplasts by electroporation. We employed a phenotypic assay for excision of Ac from the selectable hph gene encoding resistance to hygromycin B. Southern blot analysis of hygromycin B-resistant calli showed that the Ac element can transpose from the introduced hph gene into the rice chromosomes. Sequence analysis of several Ac excision sites in the hph gene revealed sequence alterations characteristic of the excision sites of this plant transposable element. The Ac element appears to be active during development of transgenic rice plants from calli. Moreover, hybridization patterns of different leaves from the same plant indicated that some Ac elements are stable whereas others are able to transpose further during development of leaves. The results indicate that the introduced Ac element can transpose efficiently in transgenic rice plants.     

Fusion of the inducible promoter of the PR-1a gene to the Activator transposase gene can transactive excision of a non-autonomous transposable element by external and by internal stimuli

The open reading frame coding for the transposase gene of the maize transposon Activator (Ac) was expressed in transgenic tobacco plants under the control of the promoter of the inducible gene for pathogenesis-related protein 1a (PR-1a). Excision of a non-autonomous transposable element (Ds) from chimeric β-glucuronidase (GUS) and luciferase reporter gene constructs was employed to analyze the induction of the Ac transposase by external and by internal stimuli. Applying the GUS histochemical assay, Ds excision events were detected in leaves, stems, and roots after treatment of regenerating shoots with salicylic acid (SA). Varying the SA induction procedure led to different Ds excision patterns in leaves and in roots. Furthermore, Ds excision events were also observed in non-treated, older transgenic plants in the green leaves, but not in germinal cells. Thus, the PR-1a promoter/Ac transposase gene fusion, together with the improved methods for induction of this chimeric gene, may provide a valuable tool for studying basic mechanisms of Ac transposition and for developing modified transposable element systems suitable for gene tagging in higher plants.     

Enhanced frequency of transposition of the maize transposable elementActivator following excision from T-DNA inPetunia hybrida

Many of the systems currently employed for heterologous transposon tagging in plants rely on an excision assay to monitor transposon activity. We have used the streptomycin phosphotransferase (SPT) reporter system to assayAc activity inPetunia hybrida. In other species, such as tobacco orArabidopsis, excision ofAc from the SPT gene in sporogenous tissue gives rise to streptomycin-resistant seedlings in the following generation. The frequency of fully streptomycin-resistant seedlings in petunia was low (0.4%) but molecular analysis of these indicated that the actual excision frequency may be as low as 0.05%. This indicates that the SPT assay is not a reliable selection criterion for germinal excision in petunia. Extensive molecular screening for reinsertion ofAc was consistent with a low primary transposition frequency (0%–0.6%). In contrast to these findings, the progeny of confirmed germinal transpositions for three independent transformants showed frequent transposition to new sites (9.5%–17.0%). This suggests a high frequency of secondary transposition compared with primary transposition from the T-DNA. Segregation analysis indicates that the high transposition activity is closely associated with transposed copies ofAc. No evidence was found for an altered methylation state forAc following transposition. The implications of these results for heterologous transposon tagging in petunia are discussed in the context of the reliability of excision reporter systems in general.     

Effects of gene dosage and sequence modification on the frequency and timing of transposition of the maize element Activator (Ac) in tobacco

The effect of Ac copy number on the frequency and timing of germinal transposition in tobacco was investigated using the streptomycin phosphotransferase gene (SPT) as an excision marker. The activity of one and two copies of the element was compared by selecting heterozygous and homozygous progeny of transformants carrying single SPT::Ac inserts. It was observed that increasing gene copy not only increases the transposition frequency, but also occasionally alters the timing of transposition such that earlier events are obtained. The result is that some homozygous plants generate multiple streptomycin resistant progeny carrying the same transposed Ac (trAc) element. We have also investigated the effect of modification of the sequence in the region around 82 bp downstream of the polyadenylation site and 177 bp from the 3 end of the element on germinal excision frequencies. Alteration of three bases to create a BglII site at this location caused a minor decrease in germinal excision events, but insertion of four bases to create a Cla I site caused a 10-fold decrease in the transposition activity of the Ac element.     

Transposition of the maize autonomous element Activator in transgenic Nicotiana plumbaginifolia plants

The maize autonomous transposable element Ac was introduced into haploid Nicotiana plumbaginifolia via Agrobacterium tumefaciens transformation of leaf disks. All the regenerated transformants (R0) were diploid and either homozygous or heterozygous for the hygromycin resistance gene used to select primary transformants. The Ac excision frequency was determined using the phenotypic assay of restoration of neomycin phosphotransferase activity and expression of kanamycin resistance among progeny seedlings. Some of the R0 plants segregated kanamycin-resistant seedlings in selfed progeny at a high frequency (34 to 100%) and contained one or more transposed Ac elements. In the primary transformants Ac transposition probably occurred during plant regeneration or early development. Other R0 transformants segregated kanamycin-resistant plants at a low frequency ( 4%). Two transformants of this latter class, containing a unique unexcised Ac element, were chosen for further study in the expectation that their kanamycin resistant progeny would result from independent germinal transposition events. Southern blot analysis of 32 kanamycin-resistant plants (R1 or R2), selected after respectively one or two selfings of these primary transformants, showed that 27 had a transposed Ac at a new location and 5 did not have any Ac element. Transposed Ac copy number varied from one to six and almost all transposition events were independent. Southern analysis of the R2 and R3 progeny of these kanamycin-resistant plants showed that Ac continued to transpose during four generations, and its activity increased with its copy number. The frequency of Ac transposition, from different loci, remained low ( 7%) from R0 to R3 generations when only one Ac copy was present. The strategy of choosing R0 plants that undergo a low frequency of germinal excision will provide a means to avoid screening non-independent transpositions and increase the efficiency of transposon tagging.     

Infrequent transposition of Ac in lettuce,Lactuca sativa

The maize transposable element Activator (Ac) is being used to develop a transposon mutagenesis system in lettuce, Lactuca sativa. Two constructs containing the complete Ac from the waxy-m7 locus of maize were introduced into lettuce and monitored for activity using Southern analysis and PCR amplification of the excision site. No transposition of Ac was detected in over 32 transgenic R₁ plants, although these constructs were known to provide frequent transposition in other species. Also, no transposition was observed in later generations. In subsequent experiments, transposition was detected in lettuce calli using constructs that allowed selection for excision events. In these constructs, the neomycin phosphotransferase II gene was interrupted by either Ac or Ds. Excision was detected as the ability of callus to grow on kanamycin. Synthesis of the transposase from the cDNA of Ac expressed from the T-DNA 2 promoter resulted in more frequent excision of Ds than was observed with the wild-type Ac. No excision was observed with Ds in the absence of the transposase. The excision events were confirmed by amplification of the excision site by PCR followed by DNA sequencing. Excision and reintegration were also confirmed by Southern analysis. Ac/Ds is therefore capable of transposition in at least calli of lettuce.     

Phenotypic assay for excision of the maize controlling element Ac in tobacco

We describe a phenotypic assay designed to detect excision of the maize controlling element Ac from a selectable marker gene, neomycin phosphotransferase II (NPT II). An NPT II gene which expresses kanamycin resistance in tobacco cells, and contains a unique restriction enzyme site in the untranslated leader region, was constructed. Ac, or a defective Ac element (Ac), was inserted into the leader region of this gene. The transposon insertions inactivated the NPT II gene as determined by transient NPT II expression assays. The three plasmids were inserted into the T DNA of Agrobacterium tumefaciens Ti plasmid vectors, and transferred to tobacco protoplasts. The transformed protoplasts were selected with 100 or 200 µg/ml kanamycin. Protoplasts transformed by the NPT II gene interrupted by Ac formed ˜25% as many calli resistant to 100 or 200 µg/ml kanamycin as protoplasts transformed by the uninterrupted NPT II gene. Protoplasts transformed by the NPT II gene interrupted by Ac did not form any calli resistant to 200 µg/ml of kanamycin when transformed under similar conditions. Southern blot hybridization analyses of seven kanamycin-resistant calli or plants obtained after transformation by the NPT II gene interrupted by Ac revealed that in all cases Ac had excised, restoring the structure of the NPT II gene. This assay is therefore useful to monitor the activity of a transposable element such as Ac and to define the regions of this element involved in transposition activity.     

A defined amino acid exchange close to he putative nucleotide binding site is responsible for an oxygen-tolerant variant of the Rhizobium meliloti NifA protein

Summary To study regulation of the (Ds) transposition process in heterologous plant species, the transposase gene of Ac was fused to several promoters that are active late during plant development. These promoters are the flower-specific chalcone synthase A promoter (CHS A), the anther-specific chalcone isomerase B promoter CHI B and the pollen-specific chalcone isomerase A₂ promoter CHI A₂. The modified transposase genes were introduced into a tobacco tester plant. This plant contains Ds stably inserted within the leader sequence of the hygromycin resistance (HPT II) gene. As confirmed with positive control elements, excision of Ds leads to the restoration of a functional HPT II gene and to a hygromycin resistant phenotype. No hygromycin resistance was observed in negative control experiments with Ac derivatives lacking 5 regulatory sequences. Although transactivation of Ds was observed after the introduction of transposase gene fusions in calli, excision in regenerated plants was observed only for the CHS A- or CHI B-transposase gene fusions. With these modified transposase genes, somatic excision frequencies were increased (68%) and decreased (22%), respectively, compared to the situation with the Ac element itself (38%). The shifts in transactivation frequencies were not associated with significant differences in the frequencies of germinally transmitted excision events (approximately 5%). The relative somatic stability of Ds insertions bearing the CHI B-transposase gene fusion suggests the usefulness of this activator element for transposon tagging experiments.     

Development of Ac and Ds transposon tagging lines for gene isolation in diploid potato

For the development of an efficient transposon tagging strategy it is important to generate populations of plants containing unique independent transposon insertions that will mutate genes of interest. To develop such a transposon system in diploid potato the behavior of the autonomous maize transposable element Ac and the mobile Ds element was studied. A GBSS (Waxy) excision assay developed for Ac was used to monitor excision in somatic starch-forming tissue like tubers and pollen. Excision of Ac results in production of amylose starch that stains blue with iodine. The frequency and patterns of blue staining starch granules on tuber slices enabled the identification of transformants with different Ac activity. After excision the GBSS complementation was usually not complete, probably due to the segment of DNA flanking Ac that is left behind in the GBSS gene. Molecular and phenotypic analyses of 40 primary transformants classified into 4 phenotypic classes revealed reproducible patterns. A very high percentage (32.5%) of the primary transformants clearly showed early excision in the first transformed cell as displayed both by the analysis of the GBSS excision marker gene as well as DNA blot analyses. Genotypes useful for tagging strategies were used for crosses and the frequency of independent germinal transpositions was assessed. In crosses to Ds genotypes, excision of Ds was revealed that correlated to the activity of the Ac genotype. A line displaying Ac amplification to multiple copies conferred a high frequency of independent Ds transpositions. The genotypes described here are useful in somatic insertion mutagenesis aimed at the isolation of tagged mutations in diploid potato.     

Dedifferentiation-mediated changes in transposition behavior make the Activator transposon an ideal tool for functional genomics in rice

There is an inverse relationship between the level of cytosine methylation in genomic DNA and the activity of plant transposable elements. Increased transpositional activity is seen during early plant development when genomic methylation patterns are first erased and then reset. Prolonging the period of hypomethylation might therefore result in an increased transposition frequency, which would be useful for rapid genome saturation in transposon-tagged plant lines. We tested this hypothesis using transgenic rice plants containing Activator (Ac) from maize. R₁ seeds from an Ac-tagged transgenic rice line were either directly germinated and grown to maturity, or induced to dedifferentiate in vitro, resulting in cell lines that were subsequently regenerated into multiple mature plants. Both populations were then analyzed for the presence, active reinsertion and amplification of Ac. Plants from each population showed excision-reinsertion events to both linked and unlinked sites. However, the frequency of transposition in plants regenerated from cell lines was more than nine-fold greater than that observed in plants germinated directly from seeds. Other aspects of transposon behavior were also markedly affected. For example, we observed a significantly larger proportion of transposition events to unlinked sites in cell line-derived plants. The tendency for Ac to insert into transcribed DNA was not affected by dedifferentiation. The differences in Ac activity coincided with a pronounced reduction in the level of genomic cytosine methylation in dedifferentiated cell cultures. We used the differential transposon behavior induced by dedifferentiation in the cell-line derived population for direct applications in functional genomics and validated the approach by recovering Ac insertions in a number of genes. Our results demonstrate that obtaining multiple Ac insertions is useful for functional annotation of the rice genome.These authors contributed equally to the work     

Transposition of the maize autonomous element Activator in transgenic Nicotiana plumbaginifolia plants

The maize autonomous transposable element Ac was introduced into haploid Nicotiana plumbaginifolia via Agrobacterium tumefaciens transformation of leaf disks. All the regenerated transformants (R0) were diploid and either homozygous or heterozygous for the hygromycin resistance gene used to select primary transformants. The Ac excision frequency was determined using the phenotypic assay of restoration of neomycin phosphotransferase activity and expression of kanamycin resistance among progeny seedlings. Some of the R0 plants segregated kanamycin-resistant seedlings in selfed progeny at a high frequency (34 to 100%) and contained one or more transposed Ac elements. In the primary transformants Ac transposition probably occurred during plant regeneration or early development. Other R0 transformants segregated kanamycin-resistant plants at a low frequency (≤ 4%). Two transformants of this latter class, containing a unique unexcised Ac element, were chosen for further study in the expectation that their kanamycin resistant progeny would result from independent germinal transposition events. Southern blot analysis of 32 kanamycin-resistant plants (R1 or R2), selected after respectively one or two selfings of these primary transformants, showed that 27 had a transposed Ac at a new location and 5 did not have any Ac element. Transposed Ac copy number varied from one to six and almost all transposition events were independent. Southern analysis of the R2 and R3 progeny of these kanamycin-resistant plants showed that Ac continued to transpose during four generations, and its activity increased with its copy number. The frequency of Ac transposition, from different loci, remained low (≤ 7%) from R0 to R3 generations when only one Ac copy was present. The strategy of choosing R0 plants that undergo a low frequency of germinal excision will provide a means to avoid screening non-independent transpositions and increase the efficiency of transposon tagging.     

Preferential transposition ofAc to linked sites in Arabidopsis

We have investigated the pattern of transposition of an intact, 4.6-kbAc element inArabidopsis thaliana. Because the trans-acting transposition function (transposase) ofAc is not fully penetrant in Arabidopsis, it is not possible to use it as a diagnostic feature to scoreAc genetically, as has been done in maize and tobacco. Instead, the presence or absence of a transposedAc (trAc) was monitored by Southern blots. Germinal transpositions from the marker SPT::Ac were selected using a streptomycin germination assay and scored for the presence of atrAc. Segregation of thetrAc element and the SPT donor locus was scored in the F₂ progeny of the germinal revertants, and the recombination fraction between thetrAc element and SPT was estimated by the method of maximum likelihood. We have found that, as in maize and tobacco, receptor sites fortrAcs in Arabidopsis tend to be linked to theAc donor locus.     

Enhanced frequency of transposition of the maize transposable elementActivator following excision from T-DNA inPetunia hybrida

Many of the systems currently employed for heterologous transposon tagging in plants rely on an excision assay to monitor transposon activity. We have used the streptomycin phosphotransferase (SPT) reporter system to assayAc activity inPetunia hybrida. In other species, such as tobacco orArabidopsis, excision ofAc from the SPT gene in sporogenous tissue gives rise to streptomycin-resistant seedlings in the following generation. The frequency of fully streptomycin-resistant seedlings in petunia was low (0.4%) but molecular analysis of these indicated that the actual excision frequency may be as low as 0.05%. This indicates that the SPT assay is not a reliable selection criterion for germinal excision in petunia. Extensive molecular screening for reinsertion ofAc was consistent with a low primary transposition frequency (0%–0.6%). In contrast to these findings, the progeny of confirmed germinal transpositions for three independent transformants showed frequent transposition to new sites (9.5%–17.0%). This suggests a high frequency of secondary transposition compared with primary transposition from the T-DNA. Segregation analysis indicates that the high transposition activity is closely associated with transposed copies ofAc. No evidence was found for an altered methylation state forAc following transposition. The implications of these results for heterologous transposon tagging in petunia are discussed in the context of the reliability of excision reporter systems in general.     

Use of the maize transposonsActivator andDissociation to show that phosphinothricin and spectinomycin resistance genes act non-cell-autonomously in tobacco and tomato seedlings

Cell-autonomous genes have been used to monitor the excision of both endogenous transposons in maize andAntirrhinum, and transposons introduced into transgenic plants. In tobacco andArabidopsis, the streptomycin phosphotransferase (SPT) gene reveals somatic excision of the maize transposonActivator (Ac) as green sectors on a white background in cotyledons of seedlings germinated in the presence of streptomycin. Cotyledons of tomato seedlings germinated on streptomycin-containing medium do not bleach, suggesting that a different assay for transposon excision in tomato is desirable. We have tested the use of the spectinomycin resistance (SPEC) gene (aadA) and a Basta resistance (BAR) gene (phosphinothricin acetyltransferase, or PAT) for monitoring somatic excision ofAc in tobacco and tomato. Both genetic and molecular studies demonstrate that genotypically variegated individuals that carry clones of cells from whichAc orDs have excised from either SPEC or BAR genes, can be phenotypically completely resistant to the corresponding antibiotic. This demonstrates that these genes act non-cell-autonomously, in contrast to the SPT gene in tobacco. Possible reasons for this difference are discussed.