Dedifferentiation-mediated changes in transposition behavior make the Activator transposon an ideal tool for functional genomics in rice

Abstract

There is an inverse relationship between the level of cytosine methylation in genomic DNA and the activity of plant transposable elements. Increased transpositional activity is seen during early plant development when genomic methylation patterns are first erased and then reset. Prolonging the period of hypomethylation might therefore result in an increased transposition frequency, which would be useful for rapid genome saturation in transposon-tagged plant lines. We tested this hypothesis using transgenic rice plants containing Activator (Ac) from maize. R1 seeds from an Ac-tagged transgenic rice line were either directly germinated and grown to maturity, or induced to dedifferentiate in vitro, resulting in cell lines that were subsequently regenerated into multiple mature plants. Both populations were then analyzed for the presence, active reinsertion and amplification of Ac. Plants from each population showed excision-reinsertion events to both linked and unlinked sites. However, the frequency of transposition in plants regenerated from cell lines was more than nine-fold greater than that observed in plants germinated directly from seeds. Other aspects of transposon behavior were also markedly affected. For example, we observed a significantly larger proportion of transposition events to unlinked sites in cell line-derived plants. The tendency for Ac to insert into transcribed DNA was not affected by dedifferentiation. The differences in Ac activity coincided with a pronounced reduction in the level of genomic cytosine methylation in dedifferentiated cell cultures. We used the differential transposon behavior induced by dedifferentiation in the cell-line derived population for direct applications in functional genomics and validated the approach by recovering Ac insertions in a number of genes. Our results demonstrate that obtaining multiple Ac insertions is useful for functional annotation of the rice genome

Cost-effective production of a vaginal protein microbicide to prevent HIV transmission

“…We have previously demonstrated that the methylation clock in plants can be reset by dedifferentiation, resulting in significant increases in levels of transgene expression (39). By subjecting immature zygotic embryos from one 2G12 highly expressing line to dedifferentiation and subsequent regeneration, we were able to not only boost accumulation levels by 30-40% compared with plants restricted to the sexual reproduction cycle but also to eliminate most of the seed-to-seed variation.…”

Epigenetics in plant tissue culture

“…The transposable element Activator (Ac) in maize looses DNA methylation and becomes activated upon dedifferentiation (Brettell and Dennis 1991). The same occurs in transgenic rice plants containing Ac from maize (Kohli et al 2004). In maize, the activation is passed on for two sexual generations (Brettell and Dennis 1991).…”

“…In contrast, genetic changes occur at random. It should be noted that in a somaclonal population the same genetic change may be found in many individuals but then the mutational event happened early during the tissue culture period and was clonally multiplied into a number of descendants (Brettell and Dennis 1991;Kohli et al 2004). …”

Particle bombardment and the genetic enhancement of crops: myths and realities

“…For example, Kohli et al (2001Kohli et al ( , 2004 have produced a large population of transgenic rice plants tagged with the maize Ac transposon. They found that this population was suitable for saturation mutagenesis and the rapid PCR-based cloning of interrupted genes using unique barcode elements present in the DNA cassette used for transformation (Kohli et al 2001).…”