An active DNA transposon nDart causing leaf variegation and mutable dwarfism and its related elements in rice

While characterized mutable alleles caused by DNA transposons have been abundant in maize since the discovery of Dissociation conferring variegation by Barbara McClintock, only a few mutable alleles have been described in rice even though the rice genome contains various transposons. Here, we show that a spontaneous mutable virescent allele, pyl-v, is caused by the disruption of the nuclear-coded essential chloroplast protease gene, OsClpP5, due to insertion of a 607-bp non-autonomous DNA transposon, non-autonomous DNA-based active rice transposon one (nDart1), belonging to the hAT superfamily. The transposition of nDart1 can be induced by crossing with a line containing an autonomous element, aDart, and stabilized by segregating out of aDart. We also identified a novel mutable dwarf allele thl-m caused by an insertion of nDart1. The japonica cultivar Nipponbare carries no aDart, although it contains epigenetically silenced Dart element(s), which can be activated by 5-azacytidine. Nipponbare bears four subgroups of about 3.6-kb Dart-like sequences, three of which contain potential transposase genes, and around 3.6-kb elements without an apparent transposase gene, as well as three subgroups of about 0.6-kb nDart1-related elements that are all internal deletions of the Dart-like sequences. Both nDart1 and 3.6-kb Dart-like elements were also present in indica varieties 93-11 and Kasalath. nDart1 appears to be the most active mutagen among nDart1-related elements contributing to generating natural variations. A candidate for an autonomous element, aDart, and a possible application of nDart1 for transposon tagging are discussed.     

Transposon display for active DNA transposons in rice

Transposon display (TD) is a powerful technique to identify the integration site of transposons in gene tagging as a functional genomic tool for elucidating gene function. Although active endogenous DNA transposons have been used extensively for gene tagging in maize, only two active endogenous DNA transposons in rice have been identified, the 0.43-kb element mPing of the MITE family and the 0.6-kb nDart element of the hAT family. The nDart transposition was shown to be induced by crossing with a line containing its autonomous element aDart and stabilized by segregating aDart under natural growth conditions, while mPing-related elements were shown to transpose in cultured cells, plants regenerated from an anther culture, and gamma-ray-irradiated plants. No somaclonal variation should occur in nDart-promoted gene tagging because no tissue culture was involved in nDart activation. As an initial step to develop an effective tagging system using nDart in rice, we tried to visualize GC-rich nDart-related elements comprising 18 nDart-related sequences of 0.6-kb and 63 nDart-related elements longer than 2 kb in Nipponbare by TD. Comparing the observed bands in TD with the anticipated virtual bands of the nDart-related elements based upon the available rice genome sequence, we have improved our TD protocol by optimizing the PCR amplification conditions and are able to visualize approximately 87% of the anticipated bands produced from the nDart-related elements. To compare the visualization efficiency of these nDart-related elements with that of 50 mPing elements and a unique Ping sequence in Nipponbare, we also tried to visualize the mPing-related elements; all mPing-related elements are easily visualized. Based on these results, we discuss the parameters affecting the visualization efficiencies of these rice DNA transposons. We also discuss the utilization of nDart elements in gene tagging for functional genomics in rice.     

Transposition and target preferences of an active nonautonomous DNA transposon nDart1 and its relatives belonging to the hAT superfamily in rice

The nonautonomous nDart1 element in the hAT superfamily is one of a few active DNA transposons in rice. Its transposition can be induced by crossing with a line containing an active autonomous element, aDart1, and stabilized by segregating aDart1. No somaclonal variation should occur in nDart1-promoted gene tagging because no tissue culture is involved in nDart1 activation. By transposon display analysis, we examined the activities of nDart1-related elements in the selfed progeny of a mutable virescent pyl-v plant containing aDart1. Although various nDart1-related elements are present in the rice genome, only nDart1-3 subgroup elements, nDart1-0 and nDart1-3 in particular, were found to be transposed frequently and integrated into various sites almost all over the genome, and a fraction of the transposed elements were found to be transmitted to the next generation. More than half of the newly integrated elements were identified as nDart1-0. Analysis of the newly inserted sites revealed that the nDart1-3 subgroup elements were predominantly integrated into single-copy regions. More than 60% of the transposed elements were inserted into the genic regions that comprise putative coding regions and their 0.5-kb flanking segments, and approximately two-thirds of them were within the 0.5-kb area in front of the putative initiation codons, i.e., promoter-proximal genic regions. These characteristic features of nDart1-3 subgroup elements seem to be suitable for developing an efficient and somaclonal variation-free gene tagging system for rice functional genomics.     

Site specific cytosine methylation in rice nonautonomous transposable element <Emphasis Type="Italic">nDart</Emphasis>

The mobile nonautonomous element nDart, which is active in intact rice plants, exhibits locus specific transposition. Due to the high homogeneity of nDart elements, the locus specificity of nDart transposition might be controlled by factors other than genetic differences. In this study, we elucidated the regulation of the locus specificity of nDart transposition. The difference of transpositional activities in 10 nDart elements among rice varieties exhibiting nDart transposition was clearly correlated with the methylation state of nDart elements. Both hyper- and hypo-methylated nDart elements were inactive, while site specific methylation in both subterminal regions was identified in active nDart loci. The specific methylation sites contain the pentamer motif GCC/ACG. The repeated motifs in the subterminal region of nDart elements may contribute to the stable secondary structure of nDart elements with low free energy. Our results suggested that site specific cytosine methylation may loosen the stable secondary structure of the nDart element to allow it to bind TPase, which then perform the excision of nDart elements from genomic loci.     

Examination of transpositional activity of nDart1 at different stages of rice development

As a useful tool to elucidate gene functions, a rice transposon tagging line has been developed using an active endogenous DNA transposon, nDart1. It was highly desirable to evaluate the transposition timing and frequency of the nDart1 elements during rice development to facilitate the generation of an efficient mutant isolation system. Comparison of the detected new insertions at different stages of rice development by transposon display analysis demonstrated that the last heading tiller carry a higher number of nDart1 elements than the main culm. Moreover, it was revealed that the last heading tiller could produce progeny that carried more new insertions of nDart1 elements, mainly as a result of the accumulation of somatic insertions in the parental plant. This report demonstrates that late tillers increase the probability of producing independent mutant lines.     

Distribution and mapping of an active autonomous <Emphasis Type="Italic">aDart</Emphasis> element responsible for mobilizing nonautonomous <Emphasis Type="Italic">nDart1</Emphasis> transposons in cultivated rice varieties

An endogenous 0.6-kb rice DNA transposon, nDart1, has been identified as a causative element of a spontaneous mutable virescent allele pyl-v conferring pale-yellow leaves with dark-green sectors in the seedlings, due to somatic excision of nDart1 integrated into the OsClpP5 gene encoding the nuclear-coded chloroplast protease. As the transposition of nDart1 depends on the presence of an active autonomous aDart element in the genome, the plants exhibiting the leaf variegation carry the active aDart element. As several mutable alleles caused by nDart1 insertions have subsequently been identified, nDart1-promoted gene tagging has been proven to be an effective system. At present, the nDart/aDart system appears to be the only endogenous rice DNA transposon system whose transposition activity can be controlled under natural growth conditions without any artificial treatments, including tissue cultures. To apply the nDart/aDart tagging system in various cultivated rice varieties, we explored the presence and distribution of an active autonomous aDart element in 19 temperate japonica, 30 tropical japonica, and 51 indica varieties. Only eight temperate japonica varieties were found to bear a single copy of an active aDart element, and no aDart activity could be detected in the indica varieties examined. Six of seven japonica varieties appear to carry the active aDart element at the identical site on chromosome 6, whereas the remaining one contains aDart on chromosome 5. Leaf variegations in the plants with the mutable pyl-v allele and the excision frequencies of endogenous nDart1 elements indicated that the aDart element on chromosome 6 is more active than that on chromosome 5. The findings described here are an important step in the development of a new and efficient nDart1-promoted gene-tagging system in various rice cultivars. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A rice Tc1/mariner-like element transposes in yeast

The Tc1/mariner transposable element superfamily is widely distributed in animal and plant genomes. However, no active plant element has been previously identified. Nearly identical copies of a rice (Oryza sativa) Tc1/mariner element called Osmar5 in the genome suggested potential activity. Previous studies revealed that Osmar5 encoded a protein that bound specifically to its own ends. In this report, we show that Osmar5 is an active transposable element by demonstrating that expression of its coding sequence in yeast promotes the excision of a nonautonomous Osmar5 element located in a reporter construct. Element excision produces transposon footprints, whereas element reinsertion occurs at TA dinucleotides that were either tightly linked or unlinked to the excision site. Several site-directed mutations in the transposase abolished activity, whereas mutations in the transposase binding site prevented transposition of the nonautonomous element from the reporter construct. This report of an active plant Tc1/mariner in yeast will provide a foundation for future comparative analyses of animal and plant elements in addition to making a new wide host range transposable element available for plant gene tagging.     

Position effect of the excision frequency of the Antirrhinum transposon Tam3: implications for the degree of position-dependent methylation in the ends of the element

We identified eight independent Tam3 copies residing in the same Antirrhinum majus genome. All the copies showed excision at 15 °C, but not at 25 °C. Under conditions promoting excision, each copy appeared to transpose in the leaves and flower lobes with a nearly constant frequency, whereas individual transposition abilities varied widely: the most active copy had an excision frequency more than 100-fold greater than that of the least active one. Despite the different transposition abilities, the structures of the eight Tam3 copies were almost identical. These results made it clear that the transpositional ability of Tam3 is regulated by chromosomal position, but they do not imply position-dependent transposase activity. The position effect of the Tam3 transposition was found to be correlated to the methylation state of the copy's end regions: DNA methylation in the Tam3 end regions tended to suppress the excision activity, and the degree of methylation was dependent on the chromosomal position. Our results also provide evidence of de novo methylation provoked by transposition of the endogenous element. We propose a mechanism of transpositional regulation of plant transposons that responds to the degree of methylation as determined by chromosomal position.     

Germinal Transpositions of the Maize Element Dissociation from T-DNA Loci in Tomato

We have analyzed the pattern of germinal transpositions of artificial Dissociation (Ds) transposons in tomato. T-DNA constructs carrying Ds were transformed into tomato, and the elements were trans-activated by crossing to lines transformed with a stabilized Activator (sAc) that expressed the transposase gene. The sAc T-DNA carried a GUS gene to monitor its segregation. The Ds elements were inserted in a marker gene so that excision from the T-DNA could be monitored. The Ds elements also carried a genetic marker that was intended to be used for reinsertion selection of the elements after excision. Unfortunately, this gene was irreversibly inactivated on crossing to sAc. Germinal excision frequencies of Ds averaged 15-40%, but there was large variation between and within plants. Southern hybridization analysis of stable transposed Ds elements indicated that although unique transpositions predominate, early transposition events can lead to large clonal sectors in the germline of developing plants and to sibling offspring carrying the same transposition event. Multiple germinal transpositions from three different loci were examined for uniqueness, and 15 different transpositions were identified from each of three T-DNA loci that carried a single independent Ds. These were mapped relative to the donor T-DNA loci, and for each locus 70-80% of the transposed elements were closely linked to the donor site.     

A transgenic system for generation of transposon Ac/Ds-induced chromosome rearrangements in rice

The maize Activator (Ac)/Dissociation (Ds) transposable element system has been used in a variety of plants for insertional mutagenesis. Ac/Ds elements can also generate genome rearrangements via alternative transposition reactions which involve the termini of closely linked transposons. Here, we introduced a transgene containing reverse-oriented Ac/Ds termini together with an Ac transposase gene into rice (Oryza sativa ssp. japonica cv. Nipponbare). Among the transgenic progeny, we identified and characterized 25 independent genome rearrangements at three different chromosomal loci. The rearrangements include chromosomal deletions and inversions and one translocation. Most of the deletions occurred within the T-DNA region, but two cases showed the loss of 72 kilobase pairs (kb) and 79 kb of rice genomic DNA flanking the transgene. In addition to deletions, we obtained chromosomal inversions ranging in size from less than 10 kb (within the transgene DNA) to over 1 million base pairs (Mb). For 11 inversions, we cloned and sequenced both inversion breakpoints; in all 11 cases, the inversion junctions contained the typical 8 base pairs (bp) Ac/Ds target site duplications, confirming their origin as transposition products. Together, our results indicate that alternative Ac/Ds transposition can be an efficient tool for functional genomics and chromosomal manipulation in rice.     

Characterization and mapping of Ds—GUS-T-DNA lines for targeted insertional mutagenesis

The transposition patterns of the Ds —GUS transposon T-DNA in 23 independent single-copy lines have been characterized and the map positions of 10 of them on three of the five Arabidopsis chromosomes are reported. Using overexpressed Activator ( Ac ) elements as a transposase source, it was found that the primary determinant of transposition frequency is the insertion site of the Ac -T-DNA. Neither the structure of the transposon T-DNA nor, in most cases, its insertion site have a significant effect on transposition frequency. Both the frequency and timing of transposition are influenced by the parent through which the transposon and transposase T-DNAs are transmitted. Overall, nearly 75% of plants in which excision has occurred bear a reinserted element and very short-range transpositions predominate, underlining the advantage of using mapped transposons for insertional mutagenesis.     

The ITR binding domain of the Mariner Mos-1 transposase

Mariner-like elements are widespread eukaryotic transposons, but Mos-1 is the only natural element that is known to be active. Little is known about the biochemistry of mariner transposition. The first step in the process is the binding of the transposase to the 5' and 3' inverted terminal repeats (ITRs) of the element. Using the 3' ITR of the element, we have determined the binding properties of a recombinant Mos-1 transposase produced in bacteria, and we have used deletion derivatives to localize the minimal ITR binding domain between amino acids 1 and 141. Its features and structure indicate that it differs from the ITR binding domain of the transposase encoded by Tc1-related elements.     

Transposition behavior of nonautonomous a <Emphasis Type="Italic">hAT</Emphasis> superfamily transposon <Emphasis Type="Italic">nDart</Emphasis> in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Transposable elements (TEs) have a significant impact on the evolution of gene function and genome structures. An endogenous nonautonomous transposable element nDart was discovered in an albino mutant that had an insertion in the Mg-protoporphyrin IX methyltransferase gene in rice. In this study, we elucidated the transposition behavior of nDart, the frequency of nDart transposition and characterized the footprint of nDart. Novel independent nDart insertions in backcrossed progenies were detected by DNA blotting analysis. In addition, germinal excision of nDart occurred at very low frequency compared with that of somatic excision, 0–13.3%, in the nDart1-4(3-2) and nDart1-A loci by a locus-specific PCR strategy. A total of 253 clones from somatic excision at five nDart loci in 10 varieties were determined. nDart rarely caused deletions beyond target site duplication (TSD). The footprint of nDart contained few transversions of nucleotides flanking to both sides of the TSD. The predominant footprint of nDart was an 8-bp addition. Precise excision of nDart was detected at a rate of only 2.2%, which occurred at two loci among the five loci examined. Furthermore, the results in this study revealed that a highly conserved mechanism of transposition is involved between maize Ac/Ds and rice Dart/nDart, which are two-component transposon systems of the hAT superfamily transposons in plant species.     

Identification of an active transposon in intact rice plants

A transposable element that is active in intact plants has been identified in rice (Oryza sativa L.). The 607-bp element itself, termed nonautonomous DNA-based active rice transposon (nDart), has no coding capacity. It was found inserted in the gene encoding Mg-protoporphyrin IX methyltransferase in a chlorophyll-deficient albino mutant isolated from backcross progeny derived from a cross between wild-type japonica varieties. The nDart has 19-bp terminal inverted repeats (TIRs) and, when mobilized, generates an 8-bp target-site duplication (TSD). At least 13 nDart elements were identified in the genome sequence of the japonica cultivar Nipponbare. Database searches identified larger elements, termed DNA-based active rice transposon (Dart) that contained one ORF for a protein that contains a region with high similarity to the hAT dimerization motif. Dart shares several features with nDart, including identical TIRs, similar subterminal sequences and the generation of an 8-bp TSD. These shared features indicate that the nonautonomous element nDart is an internal deletion derivative of the autonomous element Dart. We conclude that these active transposon systems belong to the hAT superfamily of class II transposons. Because the transposons are active in intact rice plants, they should be useful tools for tagging genes in studies of functional genomics.Electronic Supplementary Material Supplementary material is available for this article at