RNA-mediated transposition of the tobacco retrotransposon Tnt1 in Arabidopsis thaliana.

The tobacco (Nicotiana tabacum) retrotransposon Tnt1 was introduced into Arabidopsis thaliana. In this heterologous host plant species, Tnt1 undergoes an RNA-mediated transposition and creates a 5 bp duplication at the insertion sites. This is the first report of transposition of a retrotransposon after introduction into a heterologous host species. Tnt1 transposed during in vitro regeneration of transformed A.thaliana, but no transposition event was detected as happening in T2 and T3 generation plants. Newly synthesized copies of Tnt1 can integrate into coding regions of the host DNA. Our results open up the possibility of using Tnt1 as a new tool for insertional mutagenesis and functional analysis of plant genomes, in addition to the strategies of T-DNA and transposon tagging.     

Efficient transposition of the Tnt1 tobacco retrotransposon in the model legume Medicago truncatula

The tobacco element, Tnt1, is one of the few active retrotransposons in plants. Its transposition is activated during protoplast culture in tobacco and tissue culture in the heterologous host Arabidopsis thaliana. Here, we report its transposition in the R108 line of Medicago truncatula during the early steps of the in vitro transformation-regeneration process. Two hundred and twenty-five primary transformants containing Tnt1 were obtained. Among them, 11.2% contained only transposed copies of the element, indicating that Tnt1 transposed very early and efficiently during the in vitro transformation process, possibly even before the T-DNA integration. The average number of insertions per transgenic line was estimated to be about 15. These insertions were stable in the progeny and could be separated by segregation. Inspection of the sequences flanking the insertion sites revealed that Tnt1 had no insertion site specificity and often inserted in genes (one out of three insertions). Thus, our work demonstrates the functioning of an efficient transposable element in leguminous plants. These results indicate that Tnt1 can be used as a powerful tool for insertion mutagenesis in M. truncatula.     

Successful gene tagging in lettuce using the Tnt1 retrotransposon from tobacco

The tobacco (Nicotiana tabacum) element Tnt1 is one of the few identified active retrotransposons in plants. These elements possess unique properties that make them ideal genetic tools for gene tagging. Here, we demonstrate the feasibility of gene tagging using the retrotransposon Tnt1 in lettuce (Lactuca sativa), which is the largest genome tested for retrotransposon mutagenesis so far. Of 10 different transgenic bushes carrying a complete Tnt1 containing T-DNA, eight contained multiple transposed copies of Tnt1. The number of transposed copies of the element per plant was particularly high, the smallest number being 28. Tnt1 transposition in lettuce can be induced by a very simple in vitro culture protocol. Tnt1 insertions were stable in the progeny of the primary transformants and could be segregated genetically. Characterization of the sequences flanking some insertion sites revealed that Tnt1 often inserted into genes. The progeny of some primary transformants showed phenotypic alterations due to recessive mutations. One of these mutations was due to Tnt1 insertion in the gibberellin 3beta-hydroxylase gene. Taken together, these results indicate that Tnt1 is a powerful tool for insertion mutagenesis especially in plants with a large genome.     

Distribution dynamics of the Tnt1 retrotransposon in tobacco

Retrotransposons contribute significantly to the size, organization and genetic diversity of plant genomes. Although many retrotransposon families have been reported in plants, to this day, the tobacco Tnt1 retrotransposon remains one of the few elements for which active transposition has been shown. Demonstration that Tnt1 activation can be induced by stress has lent support to the hypothesis that, under adverse conditions, transposition can be an important source of genetic variability. Here, we compared the insertion site preference of a collection of newly transposed and pre-existing Tnt1 copies identified in plants regenerated from protoplasts or tissue culture. We find that newly transposed Tnt1 copies are targeted within or close to host gene coding sequences and that the distribution of pre-existing insertions does not vary significantly from this trend. Therefore, in spite of their potential to disrupt neighboring genes, insertions within or near CDS are not preferentially removed with age. Elimination of Tnt1 insertions within or near coding sequences may be relaxed due to the polyploid nature of the tobacco genome. Tnt1 insertions within or near CDS are thus better tolerated and can putatively contribute to the diversification of tobacco gene function. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Rapid proliferation of the maize transposable element Activator in transgenic tomato.

We have found that the maize transposable element Activator (Ac) can rapidly proliferate when transformed into tomato plants. The fate of transposed Ac elements in self-pollinated progeny of independent transgenic tomato plants was examined by DNA gel blot hybridizations. When a single copy of Ac was introduced into a transformant, the number of copies usually remained low in subsequent generations. In one lineage, however, the number of Ac elements increased from one to more than 15 copies in only two generations. DNA gel blot analyses indicated that the amplified elements were not grossly rearranged. Amplified copies of Ac resided at unique sites in the genome, and segregation analysis indicated that these sites were not tightly linked at one genetic locus. Taken together, these observations indicate that the mechanism of Ac amplification is associated with transposition.     

Transposition Pattern of the Maize Element Ds in Arabidopsis Thaliana

As part of establishing an efficient transposon tagging system in Arabidopsis using the maize elements Ac and Ds, we have analyzed the inheritance and pattern of Ds transposition in four independent Arabidopsis transformants. A low proportion (33%) of plants inheriting the marker used to monitor excision contained a transposed Ds. Selection for the transposed Ds increased this to at least 49%. Overall, 68% of Ds transpositions inherited with the excision marker were to genetically linked sites; however, the distribution of transposed elements varied around the different donor sites. Mapping of transposed Ds elements that were genetically unlinked to the donor site showed that a proportion (3 of 11 tested) integrated into sites which were still physically linked.     

Sexual transmission of transposed activator elements in transgenic tomatoes

The transmission of transposed Ac elements in progeny derived by self-pollination of ten transformed tomato plants has been examined by Southern hybridization analysis. We show that six of these primary transformants have transmitted a transposed Ac to at least one progeny. One of the families was segregating for at least two different insertion events. In five of ten families, progeny were detected that contained a transposed Ac but no donor T-DNA sequences, indicating that a recombination event occurred between the original and new Ac insertion site. Somatic transposition of Ac as late as the R2 generation is evidenced. One family contained an empty donor site fragment but Ac was not detected in either the parent or progeny, indicating Ac was lost in this population early in regeneration. While four of ten families were segregating for aberrant phenotypes, there was no evidence that the mutated gene was linked to a transposed Ac.     

The rice miniature inverted repeat transposable element mPing is an effective insertional mutagen in soybean

Insertional mutagenesis of legume genomes such as soybean (Glycine max) should aid in identifying genes responsible for key traits such as nitrogen fixation and seed quality. The relatively low throughput of soybean transformation necessitates the use of a transposon-tagging strategy where a single transformation event will produce many mutations over a number of generations. However, existing transposon-tagging tools being used in legumes are of limited utility because of restricted transposition (Ac/Ds: soybean) or the requirement for tissue culture activation (Tnt1: Medicago truncatula). A recently discovered transposable element from rice (Oryza sativa), mPing, and the genes required for its mobilization, were transferred to soybean to determine if it will be an improvement over the other available transposon-tagging tools. Stable transformation events in soybean were tested for mPing transposition. Analysis of mPing excision at early and late embryo developmental stages revealed increased excision during late development in most transgenic lines, suggesting that transposition is developmentally regulated. Transgenic lines that produced heritable mPing insertions were identified, with the plants from the highest activity line producing at least one new insertion per generation. Analysis of the mPing insertion sites in the soybean genome revealed that features displayed in rice were retained including transposition to unlinked sites and a preference for insertion within 2.5 kb of a gene. Taken together these findings indicate that mPing has the characteristics necessary for an effective transposon-tagging resource.     

A cloned I-factor is fully functional in Drosophila melanogaster

Summary I-R hybrid dysgenesis in Drosophila melanogaster occurs in female progeny of crosses between reactive strain females and inducer strain males, and is controlled by transposable elements called I-factors. These are 5.3 kb elements that are structurally similar to mammalian LINE elements and other retroposons. We have tested the activity of an I-factor directly, by introducing it into the genome of a reactive strain, using P-element mediated transformation. It confers the complete inducer phenotype on the reactive strain, and can stimulate dysgenesis when transformed males are mated with reactive females. It has transposed in the transformed lines, and we have cloned one of the transposed copies. This is the first time that it has been possible to demonstrate that a particular retroposon is transposition proficient, and to compare donor and transposed elements. We propose a mechanism for I-factor transposition based on these results, and the coding capacity of these elements. We have been unable to detect either autonomous transposition of a complete I-factor from a plasmid injected into reactive strain embryos, or transposition of a marked I-factor when co-injected with a complete element.     

Transposon-mediated single-copy gene delivery leads to increased transgene expression stability in barley

Instability of transgene expression in plants is often associated with complex multicopy patterns of transgene integration at the same locus, as well as position effects due to random integration. Based on maize transposable elements Activator (Ac) and Dissociation (Ds), we developed a method to generate large numbers of transgenic barley (Hordeum vulgare var Golden Promise) plants, each carrying a single transgene copy at different locations. Plants expressing Ac transposase (AcTPase) were crossed with plants containing one or more copies of bar, a selectable herbicide (Basta) resistance gene, located between inverted-repeat Ds ends (Ds-bar). F(1) plants were self-pollinated and the F(2) generation was analyzed to identify plants segregating for transposed Ds-bar elements. Of Ds-bar transpositions, 25% were in unlinked sites that segregated from vector sequences, other Ds-bar copies, and the AcTPase gene, resulting in numerous single-copy Ds-bar plants carrying the transgene at different locations. Transgene expression in F(2) plants with transposed Ds-bar was 100% stable, whereas only 23% of F(2) plants carrying Ds-bar at the original site expressed the transgene product stably. In F(3) and F(4) populations, transgene expression in 81.5% of plants from progeny of F(2) plants with single-copy, transposed Ds-bar remained completely stable. Analysis of the integration site in single-copy plants showed that transposed Ds-bar inserted into single- or low-copy regions of the genome, whereas silenced Ds-bar elements at their original location were inserted into redundant or highly repetitive genomic regions. Methylation of the non-transposed transgene and its promoter, as well as a higher condensation of the chromatin around the original integration site, was associated with plants exhibiting transgene silencing.     

Functional dissection of the cis-acting sequences of the Arabidopsis transposable element Tag1 reveals dissimilar subterminal sequence and minimal spacing requirements for transposition

The Arabidopsis transposon Tag1 has an unusual subterminal structure containing four sets of dissimilar repeats: one set near the 5' end and three near the 3' end. To determine sequence requirements for efficient and regulated transposition, deletion derivatives of Tag1 were tested in Arabidopsis plants. These tests showed that a 98-bp 5' fragment containing the 22-bp inverted repeat and four copies of the AAACCX (X = C, A, G) 5' subterminal repeat is sufficient for transposition while a 52-bp 5' fragment containing only one copy of the subterminal repeat is not. At the 3' end, a 109-bp fragment containing four copies of the most 3' repeat TGACCC, but not a 55-bp fragment, which has no copies of the subterminal repeats, is sufficient for transposition. The 5' and 3' end fragments are not functionally interchangeable and require an internal spacer DNA of minimal length between 238 and 325 bp to be active. Elements with these minimal requirements show transposition rates and developmental control of excision that are comparable to the autonomous Tag1 element. Last, a DNA-binding activity that interacts with the 3' 109-bp fragment but not the 5' 98-bp fragment of Tag1 was found in nuclear extracts of Arabidopsis plants devoid of Tag1.     

A new resource of locally transposed Dissociation elements for screening gene-knockout lines in silico on the Arabidopsis genome

We transposed Dissociation (Ds) elements from three start loci on chromosome 5 in Arabidopsis (Nossen ecotype) by using a local transposition system. We determined partial genomic sequences flanking the Ds elements and mapped the elements' insertion sites in 1,173 transposed lines by comparison with the published genomic sequence. Most of the lines contained a single copy of the Ds element. One-half of the lines contained Ds on chromosome 5; in particular, insertion "hot spots" near the three start loci were clearly observed. In the other lines, the Ds elements were transposed across chromosomes. We found other insertion hot spots at the tops of chromosomes 2 and 4, near nucleolus organizer regions 2 and 4, respectively. Another characteristic feature was that the Ds elements tended to transpose near the chromosome ends and rarely transposed near centromeres. The distribution patterns differed among the three start loci, even though they possessed the same Ds construct. More than one-half of the Ds elements were inserted irregularly into the genome; that is, they did not retain the perfect inverted repeat sequence of Ds nor leave perfect target site duplications. This precise analysis of distribution patterns will contribute to a comprehensive understanding of the transposing mechanism. From these Ds insertion sites, we have constructed a database for screening gene-knockout mutants in silico. In 583 of the 1,173 lines, the Ds elements were inserted into protein-coding genes, which suggests that these lines are gene-knockout mutants. The database and individual lines will be available freely for academic use from the RIKEN Bio-Resource Center (http://www.brc.riken.go.jp/Eng/index.html).