Characterization of autonomous Dart1 transposons belonging to the hAT superfamily in rice

Abstract

An endogenous 0.6-kb rice DNA transposon, nDart1-0, was found as an active nonautonomous element in a mutable virescent line, pyl-v, displaying leaf variegations. Here, we demonstrated that the active autonomous element aDart in pyl-v corresponds to Dart1-27 on chromosome 6 in Nipponbare, which carries no active aDart elements, and that aDart and Dart1-27 are identical in their sequences and chromosomal locations, indicating that Dart1-27 is epigenetically silenced in Nipponbare. The identiWcation of aDart in pyl-v was Wrst performed by map-based cloning and by detection of the accumulated transposase transcripts. Subsequently, various transposition activities of the cloned Dart1-27 element from Nipponbare were demonstrated in Arabidopsis. Dart1-27 in Arabidopsis was able to excise nDart1-0 and Dart1-27 from cloned sites, generating footprints, and to integrate into new sites, generating 8-bp target site duplications. In addition to Dart1-27, Nipponbare contains 37 putative autonomous Dart1 elements because their putative transposase genes carry no apparent nonsense or frameshift mutations. Of these, at least four elements were shown to become active aDart elements in transgenic Arabidopsis plants, even though considerable sequence divergence arose among their transposases. Thus, these four Dart1 elements and Dart1-27 in Nipponbare must be potential autonomous elements silenced epigenetically. The regulatory and evolutionary implications of the autonomous Dart1 elements and the development of an eYcient transposon-tagging system in rice are discussed.

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

“…Because Nipponbare does not show the transposition activities of nDart1 and does not contain the active autonomous aDart1 element, we can classify 63 iDart/dDart elements longer than 2 kb in the genome into two categories on the basis of their sequence characteristics, i.e., epigenetically silenced inactive iDart carrying a putative transposase without containing nonsense or frameshift mutations and genetically defective dDart elements bearing apparently defective transposase genes. The presence of iDart1 in Nipponbare was confirmed by demonstrating that several of these iDart1 elements arbitrarily chosen were able to promote the transposition of nDart1-0 in Arabidopsis plants when they were co-transformed with nDart1-0 (Shimatani et al 2009). We could assign the coding capacity of an individual element on the basis of the splicing patterns of mRNAs transcribed from the aDart1 transposase gene ( Fig.…”

“…The results indicate that transposition tagging of Tos17 activated by tissue culture occupies only a small fraction of various causes of somaclonal variation. While endogenous active DNA transposons, which are free from somaclonal variation because no tissue culture is involved in generating insertion mutants, have been extensively used for gene tagging in maize, snapdragon, petunia, and morning glories (May and Martienssen 2003;Chopra et al 2006), only a few active endogenous DNA transposons, mPing, nDart1, dTok, and nDaiZ, have been identified in rice (Nakazaki et al 2003;Fujino et al 2005;Moon et al 2006;Naito et al 2006;Tsugane et al 2006;Nishimura et al 2008;Shimatani et al 2009;Huang et al 2009). Of these active nonautonomous DNA elements, nDart1 appears to be more suitable than the others for transposon tagging in rice because their transposition can be controlled under natural growth conditions, i.e., the transposition of nDart1 can be induced by crossing with a line containing an active autonomous aDart1 element and stabilized by segregating aDart1 (Tsugane et al 2006;Nishimura et al 2008;Shimatani et al 2009).…”

“…We could assign the coding capacity of an individual element on the basis of the splicing patterns of mRNAs transcribed from the aDart1 transposase gene ( Fig. 1; Nishimura et al 2008;Shimatani et al 2009) and basing on the structure of its presumed exon. Nipponbare is likely to carry 35 iDart1, 15 dDart1 including three internal deletion elements, one iDart1-51, two iDart1-52/ 53, one iDart2, six dDart2, one dDart3, and two iDart4 (Takagi et al 2007;Johzuka-Hisatomi et al 2008).…”

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

“…japonica cv. Nipponbare (http://rapdb.dna.affrc.go.jp/), their transpositions were not detected under natural growth conditions without the presence of an active autonomous element, aDart1, containing a transposase gene (Tsugane et al, 2006;Nishimura et al, 2008;Shimatani et al, 2009). The aDart1 was found in specific rice cultivars and the pyl line (Nishimura et al, 2008).…”

“…The transposon-induced variegation patterns in maize kernels (Levy and Walbot, 1990) and Antirrhinum petals (Lister et al, 1993) have been analyzed to investigate the timing and frequency of the endogenous transposons in specific genomic regions. Southern hybridization and a histochemical β-glucuronidase (GUS) assay in transgenic plants have also been utilized to visualize the excision events (Pavingerová et al, 2001;Szeverenyi et al, 2006;Shimatani et al, 2009). …”