The behaviour of the autonomous maize transposable element En/Spm in Arabidopsis thaliana allows efficient mutagenesis

The behavior of the autonomous maize transposable element En/Spm of maize was studied in Arabidopsis. Transgenic Arabidopsis plants carrying En-1 elements were propagated for 12 generations using a single seed descent procedure. The distribution and activity of the En-1 element was monitored using Southern DNA hybridisations in generations 1, 6 and 12. In the first generation the highest number of En-1 insertions per line was 7, which increased to 20 in generation 12. The average number of En-1 insertions increased only slightly in the population, due to a gradual accumulation of segregants that lost the transposable element. During the development of the En-1 mutagenised population the element remained active even in the high-copy lines. In situ hybridisation demonstrated that multiple En-1 insertions were distributed over all Arabidopsis chromosomes. From the initial En-1 mutagenised populations many unstable gene mutations were recovered, indicating that En-1 can be used as a efficient tool for gene tagging in Arabidopsis.     

Spotting factor (Spf) from the Spotted-dilute system in maize is a member of the En/Spm controlling element family

The Spotted-dilute controlling element system in maize involves an autonomous Spotting factor (Spf), and a receptor at the r1 locus haplotype R1-r(spotted dilute2). Its relationship with other maize transposable element systems is poorly characterized. Through development of a genetic tester that carries receptors for both the Spotted-dilute and the En/Spm controlling element systems, we determined that both receptors respond equally to Spf and En/Spm and that Spf is therefore a member of the En/Spm family of controlling elements.     

Efficient insertional mutagenesis in rice using the maize En/Spm elements

We have developed a novel system for insertional mutagenesis in rice (Oryza sativa) based on the maize (Zea mays) enhancer/suppressor mutator (En/Spm) element. In this system, a single T-DNA construct with Spm-transposase and the non-autonomous defective suppressor mutator (dSpm) element is used in conjunction with green fluorescent protein (GFP) and Discosoma sp. Red Fluorescence Protein (DsRed) fluorescent markers to select unlinked stable transpositions of dSpm. Using this system, we could demonstrate high frequencies of unlinked germinal transposition of dSpm in rice. Analysis of dSpm flanking sequences from 353 stable insertion lines revealed that the dSpm insertions appear to be widely distributed on rice chromosomes with a preference for genic regions (70%). The dSpm insertions appear to differ from Activator-Dissociation (Ac-Ds) elements in genomic distribution and exhibit a greater fraction of unlinked transpositions when compared with Ds elements. The results obtained in this study demonstrate that the maize En/Spm element can be used as an effective tool for functional genomics in rice and can complement efforts using other insertional mutagens. Further, the efficacy of the non-invasive fluorescence-based selection system is promising for its application to other crops.     

Definition and characterization of an artificial En/Spm-based transposon tagging system in transgenic tobacco

A transposon tagging system for heterologous hosts, based on the maize En/Spm transposable element, was developed in transgenic tobacco. In this system, the two En-encoded trans-acting factors necessary for excision are expressed by fusing their cDNAs to the CaMV 35S promoter. The dSpm receptor component is inserted in the 5-untranslated leader of the bar gene. Germinal revertants can therefore be selected by seed germination on L-PPT-containing medium or by spraying seedlings with the herbicide Basta. Using this bar-based excision reporter construct, an average frequency of germinal excision of 10.1% was estimated for dSpm-S, an En/Spm native internal deletion derivative. Insertion of En-foreign sequences in a receptor, such as a DHFR selectable marker gene in dSpm-DHFR, does not abolish its capacity to transpose. However, dSpm-DHFR has a lower frequency of somatic and germinal excision than dSpm-S. Revertants carrying a transposed dSpm-DHFR element can be selected with methotrexate. Germinal excision is frequently associated with reinsertion but, as in maize, dSpm has a tendency to integrate at chromosomal locations linked to the donor site. Concerning the timing of excision, independent germinal transpositions are often found within a single seed capsule. All activity parameters analysed suggest that transposon tagging with this system in heterologous hosts should be feasible.     

Gene Marker Loss Induced by the Transposable Element, En, in Maize

The En/Spm transposable element system in maize includes the functional element, En/Spm and the receptor element I/dSpm. An En receptor has been found that shows En-induced breakage. This En-responsive receptor (designated 1836518) is located on the short arm of chromosome 9, proximal to Wx. In the presence of En, markers distal to the receptor show a loss of gene expression. Kernels heterozygous for aleurone and endosperm marker genes have a variegated appearance. The hypothesis is advanced that this variegation represents a physical loss of the chromosome segments carrying the genes distal to the receptor position. It is the first case of an En-controlled breakage event.     

Increased Ac excision (iae): Arabidopsis thaliana mutations affecting Ac transposition

The maize transposable element Ac is highly active in the heterologous hosts tobacco and tomato, but shows very much reduced levels of activity in Arabidopsis . A mutagenesis experiment was undertaken with the aim of identifying Arabidopsis host factors responsible for the observed low levels of Ac activity. Seed from a line carrying a single copy of the Ac element inserted into the streptomycin phosphotransferase (SPT) reporter fusion, and which displayed typically low levels of Ac activity, were mutagenized using gamma rays. Nineteen mutants displaying high levels of somatic Ac activity, as judged by their highly variegated phenotypes, were isolated after screening the M₂ generation on streptomycin-containing medium. The mutations fall into two complementation groups, iae1 and iae2 , are unlinked to the SPT::Ac locus and segregate in a Mendelian fashion. The iae1 mutation is recessive and the iae2 mutation is semi-dominant. The iae1 and iae2 mutants show 550- and 70-fold increases, respectively, in the average number of Ac excision sectors per cotyledon. The IAE1 locus maps to chromosome 2, whereas the SPT:: Ac reporter maps to chromosome 3. A molecular study of Ac activity in the iae1 mutant confirmed the very high levels of Ac excision predicted using the phenotypic assay, but revealed only low levels of Ac re-insertion. Analyses of germinal transposition in the iae1 mutant demonstrated an average germinal excision frequency of 3% and a frequency of independent Ac re-insertions following germinal excision of 22%. The iae mutants represent a possible means of improving the efficiency of Ac/Ds transposon tagging systems in Arabidopsis , and will enable the dissection of host involvement in Ac transposition and the mechanisms employed for controlling transposable element activity.     

Excision of the En/Spm transposable element of Zea mays requires two element-encoded proteins.

An excision assay system for En/Spm was developed in transgenic tobacco. The characteristics of excision and integration are similar to the natural system of Zea mays. In this transgenic model system two En/Spm encoded trans-acting functions, TNPA and TNPD, are required for excision. A biochemical model for transposition is proposed that might also be applicable to other transposable elements.     

Diversity of LTR-retrotransposons and <Emphasis Type="Italic">Enhancer/Suppressor</Emphasis><Emphasis Type="Italic">Mutator</Emphasis>-like transposons in cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz)

Cassava (Manihot esculenta Crantz), though a major world crop with enormous potential, is very under studied. Little is known about its genome structure and organisation. Transposable elements have a key role in the evolution of genome structure, and can be used as important tools in applied genetics. This paper sets out to survey the diversity of members of three major classes of transposable element within the cassava genome and in relation to similar elements in other plants. Members of two classes of LTR-retrotransposons, Ty1/copia-like and Ty3/gypsy-like, and of Enhancer/Suppressor Mutator (En/Spm)-like transposons were isolated and characterised. Analyses revealed 59 families of Ty1/copia, 26 families of Ty3/gypsy retrotransposons, and 40 families of En/Spm in the cassava genome. In the comparative analyses, the predicted amino acid sequences for these transposon classes were compared with those of related elements from other plant species. These revealed that there were multiple lineages of Ty1/copia-like retrotransposons in the genome of cassava and suggested that vertical and horizontal transmission as the source of cassava Mecops may not be mutually exclusive. For the Ty3/gypsy elements network, two groups of cassava Megyps were evident including the Arabidopsis Athila lineage. However, cassava En/Spm-like elements (Meens) constituted a single group within a network of plant En/Spm-like elements. Hybridisation analysis supported the presence of transposons in the genome of cassava in medium (Ty3/gypsy and En/Spm) to high (Ty1/copia) copy numbers. Thus the cassava genome was shown to contain diverse members of three major classes of transposable element; however, the different classes exhibited contrasting evolutionary histories.     

Genetic and molecular analysis of the Enhancer (En) transposable element system of Zea mays

A newly isolated, unstable mutation wx-844::En-1 of Zea mays was proven to be caused by the insertion of the autonomous transposable element En into the Waxy (Wx) gene. Molecular analysis revealed that En-1 is 8.4 kb long, has a 13-bp long perfect inverted repeat at its termini and generates a 3-bp target site duplication. En-1 is integrated into an intron located approximately in the middle of the transcribed region of the Wx gene. Structural evidence is presented indicating that a receptor component (Inhibitor) can arise by internal deletion of an autonomous En element.     

Successful PCR-based reverse genetic screens using an En-1-mutagenised Arabidopsis thaliana population generated via single-seed descent

 The development of an Arabidopsis population via single-seed descent is described which includes 3,000 lines that carry approximately 15,000 independent insertions of the autonomous maize element En-1. A PCR strategy is outlined which allows the recovery of En-1-insertion mutants among this population in any random gene sequence of Arabidopsis thaliana. The method employs PCR reactions on pooled DNA. Positive amplification using a target-specific primer and an En-1-specific primer on row, column and single-tray pools identifies the putative insertion mutant. In a control experiment two insertion mutants of the PIN gene were successfully identified. In addition, a new independent insertion in the PIN gene was detected which was transmitted to the next generation and showed co-segregation with the pin phenotype. These examples demonstrate that the inheritance of inserts of the autonomous element En-1 is stable enough to make a proper genetic analysis feasible in a genomic background with multiple En-1 inserts. Received: 20 March 1998 / Accepted: 31 March 1998     

Multiple independent defective suppressor-mutator transposon insertions in Arabidopsis: a tool for functional genomics.

A new system for insertional mutagenesis based on the maize Enhancer/Suppressor-mutator (En/Spm) element was introduced into Arabidopsis. A single T-DNA construct carried a nonautonomous defective Spm (dSpm) element with a phosphinothricin herbicide resistance (BAR) gene, a transposase expression cassette, and a counterselectable gene. This construct was used to select for stable dSpm transpositions. Treatments for both positive (BAR) and negative selection markers were applicable to soil-grown plants, allowing the recovery of new transpositions on a large scale. To date, a total of 48,000 lines in pools of 50 have been recovered, of which approximately 80% result from independent insertion events. DNA extracted from these pools was used in reverse genetic screens, either by polymerase chain reaction (PCR) using primers from the transposon and the targeted gene or by the display of insertions whereby inverse PCR products of insertions from the DNA pools are spotted on a membrane that is then hybridized with the probe of interest. By sequencing PCR-amplified fragments adjacent to insertion sites, we established a sequenced insertion-site database of 1200 sequences. This database permitted a comparison of the chromosomal distribution of transpositions from various T-DNA locations.