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.     

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.     

Development of an efficient two-element transposon tagging system in Arabidopsis thaliana

Summary Modified Ac and Ds elements, in combination with dominant markers (to facilitate monitoring of excision, reinsertion and segregation of the elements) were introduced into Arabidopsis thaliana ecotype Landsberg erecta. The frequencies of somatic and germinal transactivation of the Ds elements were monitored using a streptomycin resistance assay. Transactivation was significantly higher from a stable Ac (sAc) carrying a 537 by deletion of the CpG-rich 5 untranslated leader of the transposase mRNA than from a wild-type sAc. However, substitution of the central 1.77 kb of the transposase open reading frame (ORF) with a hygromycin resistance marker did not alter the excision frequency of a Ds element. -Glucuronidase (GUS) or iaaH markers were linked to the transposase source to allow the identification of plants in which the transposase source had segregated away from the transposed Ds element, eliminating the possibility of somatic or germinal re-activation. Segregation of the excision marker, Ds and sAc was monitored in the progeny of plants showing germinal excision of Ds. 29% of the plants inheriting the excision marker carried a transposed Ds element.     

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.     

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).     

Use of Ac as an insertional mutagen in Arabidopsis

A pilot-scale transposon mutagenesis experiment using a modified autonomous Activator (Ac) element, AcΔNael, was carried out in Arabidopsis thaliana. Four different transformants carrying Ac elements in different and defined genomic locations were used to generate 1000 plants carrying approximately 500 independent germinal transposition events. These plants were then selfed and the 1000 families screened in tissue culture and soil for phenotypic mutants. Fifty different families segregated mutations in their progeny. Preliminary Southern blot analysis of 29 families which segregated mutant progeny, showed that 28 had a transposed Ac. Six of the families were further tested for linkage between the transposed Ac and the mutant phenotype, and instability of the putatively tagged locus. Two of the mutants were shown to be tagged as they were tightly linked to a transposed Ac, and somatic and germinal reversion was associated with loss of Ac. One other mutant locus was shown to be closely linked to a transposed Ac, and therefore was likely to be tagged. The remaining three mutations were not tagged as they were not linked to a transposed Ac. In two of the tagged mutants Ac had transposed to closely linked sites, while in a third mutant the co-segregating Ac had transposed to a site which was not tightly linked to the donor T-DNA. Multiple insertions into the DIF1 locus were found, due to the preferential transposition of Ac to a linked site.     

The maize transposable element Ac is mobile in the legume Lotus japonicus

To evaluate the prospects for transposon mutagenesis in the autogamous diploid legume Lotus japonicus, the behaviour of the maize transposable element Ac was analysed in the progeny of 38 independent transgenic plants. The conditions for monitoring donor site excision using histochemical localization of -glucuronidase activity or the alternative spectinomycin resistance assay were established, and used to follow Ac mobility through two generations. Somatic excision was monitored as variegated cotyledons in the T₂ generation and germinal excision events were scored in segregating T₃ families as complete -glucuronidase-mediated staining of cotyledons or as a fully green spectinomycin-resistant phenotype. Using these assays an average germinal excision frequency of 12% was estimated in the T₃ offspring from variegated plants. The fidelity of the excision assays was ascertained by comparing the frequency of germinal excision to the frequency of Ac reinsertion at new positions of the genome. Transposition of Ac in 42% of the plants and detection of the characteristic Ac insertion/excision footprints suggests that insertion mutagenesis with the autonomous maize Activator element is feasible in Lotus japonicus. Parameters influencing Ac behaviour, such as dosage, position effects and modification of the element itself, were also investigated comparing homozygous and hemizygous plants from the same family and by analysing different transformants.Abbreviations W white - V variegated - FG fully green - FB fully blue - aadA spectinomycin adenyltransferase     

Behavior of the maize transposable element Activator in Daucus carota

Summary We previously described the introduction of the maize Ac element in carrot hairy roots where it was shown to transpose. Further studies on this system allowed us to observe complete excision of the element from its original insertion site on the AcT-DNA. Analysis of hairy root subcultures derived from single root tips resolved the chimeric status created by transposition of Ac establishing the unicellular origin of secondary root meristems. Studies with restriction enzymes revealed a specific methylation pattern in the vicinity of the empty donor site left after Ac excision. This process, although not of general occurrence, was probably associated with the excision/transposition process. The activity of Ac in different hairy root transformants as judged by the rate of excision from the original insertion site appeared to be maintained during a period of ca. 2 years, and there was no evidence for bursts of transposition occurring during somatic embryogenesis. The data obtained support the hypothesis that excision is a concerted process involving all the copies of Ac present in a given cell.     

Germinal Excision and Reinsertion Frequencies of the Mobile Element Ds Transposed from Two Unlinked T-DNA Loci in Tomato

Acceptor sites of unlinked transposed Ds element from two T-DNA loci in tomato were mapped. Experimental data obtained from TC₁ progeny testing were employed for estimation of germinal excision frequency (GEF) of Ds element and frequency of its reinsertion (FR). The donor T-DNAs 1481J and 1601D, containing a 35S:NPT transformation marker, a 35S:BAR or nos:BAR excision marker conferring phosphinothricine resistance and a Ds element in the 5 untranslated leader of the nos (or 35S): BAR gene, were located on chromosome 7 and 8, respectively. Ds transposition was induced by 105121 T-DNA carrying stabilized Ac (sAc) which provides a source of transposase and 2:GUS marker conferring -glucuronidase activity. Tomato plants harbouring the Ds in 1481J or 1601D locus and sAc were crossed and F₁D, were crossed individually as seed parents to wild-type plants to generate TC₁ progenies. TC₁ seed was germinated on phosphinothricine (Basta)-containing medium, and individual seedlings carrying a transposed Ds and lacking sAc were identified by PCR (to detect the Ds) on phosphinothricine resistant individuals that lacked -glucuronidase activity. From segregation ratio in TC₁ the germinal excision and reinsertion frequencies of the Ds element were estimated for individual F₁ plants. A total of 14560 TC₁ seedlings of 1481J and 16195 TC₁ seedlings of 1601D was analyzed. We observed high variation between individual plants as regards both GEF and FR despite of donor locus (1481J or 1601D), however, the average germinal excision frequencies as well as average frequencies of reinsertion were very similar for both donor loci: GEF_1481J = 24 %, GEF_1501D = 25 %, FR_1481J = 42 %, FR_1601D = 46 %.     

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.     

Analysis of the frequency of inheritance of transposed Ds elements in Arabidopsis after activation by a CaMV 35S promoter fusion to the Ac transposase gene

The Ac/Ds transposon system of maize shows low activity in Arabidopsis. However, fusion of the CaMV 35S promoter to the transposase gene (35S::TPase) increases the abundance of the single Ac mRNA encoded by Ac and increases the frequency of Ds excision. In the experiments reported here it is examined whether this high excision frequency is associated with efficient re-insertion of the transposon. This was measured by using a Ds that carried a hygromycin resistance gene (HPT) and was inserted within a streptomycin resistance gene (SPT). Excision of Ds therefore gives rise to streptomycin resistance, while hygromycin resistance is associated with the presence of a transposed Ds or with retention of the element at its original location. Self-fertilisation of most individuals heterozygous for Ds and 35S::TPase produced many streptomycin-resistant (strep^r) progeny, but in many of these families a small proportion of strep^r seedlings were also resistant to hygromycin (hyg^r). Nevertheless, 70% of families tested did give rise to at least one strep^r, hyg^r seedling, and over 90% of these individuals carried a transposed Ds. In contrast, the Ac promoter fusion to the transposase gene (Ac::TPase) produced fewer strep^rhyg^r progeny, and only 53% of these carried a transposed Ds. However, a higher proportion of the strep^r seedlings were also hyg^r than after activation by 35S::TPase. We also examined the genotype of strep^r, hyg^r seedlings and demonstrated that after activation by 35S::TPase many of these were homozygous for the transposed Ds, while this did not occur after activation by Ac::TPase. From these and other data we conclude that excisions driven by 35S::TPase usually occur prior to floral development, and that although a low proportion of strep^r progeny plants inherit a transposed Ds, those that do can be efficiently selected with an antibiotic resistance gene contained within the element. Our data have important implications for transposon tagging strategies in transgenic plants and these are discussed.     

Agrobacterium-mediated transformation of germinating seeds of Arabidopsis thaliana: A non-tissue culture approach

Summary Germinating seeds of Arabidopsis thaliana were cocultivated with an Agrobacterium tumefaciens strain (C58Clrif) carrying the pGV3850:pAK1003 Ti plasmid. This Ti plasmid contains the neomycin phosphotransferase II gene (NPT II) which confers resistance to kanamycin and G418. Seeds (T1 generation) imbibed for 12 h before a 24 h exposure to Agrobacterium gave rise to the highest number of transformed progeny (T2 generation). Over 200 kanamycin-resistant T2 seedlings were isolated. Some of the T2 seedlings and T3 families were characterized for genetic segregation of functional NPT II gene(s), NPT II activity, and the presence of T-DNA inserts (Southern analysis). Ninety percent of the T2 individuals transmitted the resistance factor to the T3 families in a Mendelian fashion. Of the T3 families segregating in a Mendelian fashion (n=111), 62% segregated for one functional insert, 29% for two unlinked or linked functional inserts, 5% for three unlinked inserts, 1% for four unlinked inserts, whereas 3% appeared to be homozygous for the insert(s). The 13 families that did not exhibit Mendelian segregation ratios fell into 2 classes, both of which had a deficiency of kanamycin-resistant seedlings. In the Group I T3 families (n=6) only 0%–2% of the seedlings were resistant to kanamycin (100 mg/l), whereas in the Group II families (n=7) 8%–63% of the seedlings were resistant. All of the kanamycin-resistant plants that were tested were found to possess NPT II activity. Southern analysis revealed that all of the resistant plants contained at least one copy of the T-DNA and that the majority of the plants had multiple inserts. Explants from kanamycin-resistant plants survived and formed callus when cultured on callus-inducing medium containg G418.     

The maize autonomous element Activator (Ac) shows a minimal germinal excision frequency of 0.2%–0.5% in transgenic Arabidopsis thaliana plants

Summary The autonomous mobile element Activator from Zea mays was introduced into Arabidopsis thaliana via Agrobacterium-mediated gene transfer. The use of a chimaeric construct, where the Ac element is located in the leader of the neomycin phosphotransferase (NPT II) gene, enabled the excision of Ac to be monitored by assaying for the reconstitution of NPT II gene activity. Using this approach, the transpositional activity of AC was initially studied in primary transformants. About 50% of the regenerating Ac transformants showed evidence for excision of the element. Reintegration of Ac was confirmed by Southern blot analysis. Transposition events are transmitted to the F1 generation with a minimal frequency of 0.3%. In a few exceptional cases they are detected in a high proportion of the F1 generation. Seedlings from the F2 and F3 generations were assayed for the rate of germinal excisions by scoring for kanamycin resistance. The minimal frequency of germinal excision events amounts to 0.2%–0.5% and hence allows the use of the Ac element for gene tagging purposes in A. thaliana.     

Transposition of the maize transposable element Ac in Solanum tuberosum

Summary The maize transposable element Ac has been introduced into potato via the T-DNA (transferred DNA) of Agrobacterium tumefaciens. Ac was inserted within the untranslated leader region of a neomycin phosphotransferase II (NPT-II) gene such that excision restored NPT-II activity. Two approaches to monitor Ac excision were used. (i) Using an Agrobacterium strain harbouring plasmid pGV3850::pKU3, leaf discs were selected on kanamycin (Km) after exposure to Agrobacterium. (ii) Using a strain containing plasmid pGV3850HPT::pKU3, the leaf discs were selected on hygromycin (Hm) and the resulting shoots were checked for NPT-II expression. Thirteen kanamycin resistant shoots transformed with pGV3850::pKU3 were isolated, suggesting that Ac had excised from the NPT-II gene. Out of 43 hygromycin resistant shoots transformed with pGV3850HPT::pKU3, 22 expressed the NPT-II gene, indicating that Ac had undergone excision in approximately 50% of the hygromycin resistant shoots. Southern analysis revealed that all kanamycin resistant plants contained the DNA restriction fragments expected when Ac excises from the NPT-II gene. The presence of Ac at new locations within the genomic DNA of several transformants was also detected.     

Construction of an inducible transposon,INAc, to develop a gene tagging system in higher plants

An inducible transposable element, termed INAc (inducible Activator), was constructed for development of a gene tagging system in higher plants. The advantage of such an inducible element is that, unlike the native transposon, its excision can be induced at any time during plant development and the resulting mutants are stable after removal of the inducer. A fusion of the SA inducible promoter (PR-1a) with the Ac transposase gene was inserted together with a hygromycin resistance gene between ca. 400 bp sequences from each end of the maize Ac element, yielding INAc. The INAc element was introduced into tobacco and tomato plants. A high frequency of spontaneous transposition was apparent in primary transformed tomato calli but not in tobacco calli. Treatment of tobacco plants with salicylic acid induced transposition of INAc in both somatic and germinal tissue, with germinal transposition events being revealed by characterization of the progeny of transformed plants whose flowers were exposed to SA. The INAc element thus exhibits potential for development of an inducible transposon system suitable for gene isolation in heterologous plant species.     

In planta transformation of Arabidopsis thaliana

Transformants of Arabidopsis thaliana can be generated without using tissue culture techniques by cutting primary and secondary inflorescence shoots at their bases and inoculating the wound sites with Agrobacterium tumefaciens suspensions. After three successive inoculations, treated plants are grown to maturity, harvested and the progeny screened for transformants on a selective medium. We have investigated the reproducibility and the overall efficiency of this simple in planta transformation procedure. In addition, we determined the T-DNA copy number and inheritance in the transformants and examined whether transformed progeny recovered from the same Agrobacterium-treated plant represent one or several independent transformation events. Our results indicate that in planta transformation is very reproducible and yields stably transformed seeds in 7–8 weeks. Since it does not employ tissue culture, the in planta procedure may be particularly valuable for transformation of A. thaliana ecotypes and mutants recalcitrant to in vitro regeneration. The transformation frequency was variable and was not affected by lower growth temperature, shorter photoperiod or transformation vector. The majority of treated plants gave rise to only one transformant, but up to nine siblings were obtained from a single parental plant. Molecular analysis suggested that some of the siblings originated from a single transformed cell, while others were descended from multiple, independently transformed germ-line cells. More than 90% of the transformed progeny exhibited Mendelian segregation patterns of NPTII and GUS reporter genes. Of those, 60% contained one functional insert, 16% had two T-DNA inserts and 15% segregated for T-DNA inserts at more than two unlinked loci. The remaining transformants displayed non-Mendelian segregation ratios with a very high proportion of sensitive plants among the progeny. The small numbers of transformants recovered from individual T₁ plants and the fact that none of the T₂ progeny were homozygous for a specific T-DNA insert suggest that transformation occurs late in floral development.National Research Council of Canada Publication No. 38003     

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.     

Behaviour of the maize transposable element Ac in Arabidopsis thaliana

The somatic and germinal activity of the maize transposable element, Ac, has been analysed in progeny of 43 transformants of A. thaliana using a streptomycin resistance assay to monitor Ac excision. The ability to assay somatic activity enabled, for the first time, a detailed analysis of Ac activity in individual A. thaliana seedlings to be made. The effects of T-DNA copy number, generation, dosage at each locus, flanking sequences and orientation of the element were compared. The most striking observation was the variability in Ac activity in genotypically identical individuals and the poor penetrance of the variegated phenotype. In general, increasing Ac dosage increased both somatic and germinal excision frequencies. The majority of families from individuals selected as inheriting an excision event carried transposed Ac elements re-integrated in different positions in the genome.