Mobilized retrotransposon Tos17 of rice by alien DNA introgression transposes into genes and causes structural and methylation alterations of a flanking genomic region

摘要

Retrotransposons, also called class I mobile elements, transpose via reverse transcription of RNA intermediates, and are major genomic components in eukaryotes. The most prominent retrotransposons in plants are the LTR (long terminal repeat) retroelements that can be further divided into copia- and gypsy-like classes (reviewed by Kumar and Bennetzen 1999; Bennetzen 2000). Although functionally intact, LTR-retrotransposons usually contain sequences encoding all proteins required for their retrotransposition, but they are largely quiescent during normal growth and development (Wessler 1996; Grandbastien 1998; Kumar and Bennetzen 1999). Nevertheless, accumulating evidence indicates that some of the plant retrotransposons are inducible; that is, they are responsive to, and often transcriptionally and/or transpositionally activated by various biotical and abiotical stresses such as wounding, pathogen attack, cell culture induction, environmental cues (e.g. drought) and interspecies introgression (Hirochika 1997; Hirochika et al. 1996; Grandbastien 1998; Kalendar et al. 2000; Liu and Wendel 2000). Because some of these stresses are prevalent in natural plant populations, and because uncontrolled retrotranspositions could potentially disrupt the host's genome, it is not surprising that plant genomes have evolved genetic and/or epigenetic mechanisms to tightly control their activity. On the other hand, even limited activation and mobilization of retrotransposons may have bearing on the host genome, particularly if the elements transpose into or near functional genes. Apart from gene inactivation, the insertion of LTR elements may also cause changes in structure and/or epigenetic states of the flanking sequences and lead to alterations in expression (Whitelaw and Martin 2001; Kashkush et al. 2003).Tos17 is an endogenous copia-like retrotransposon in rice (Hirochika et al. 1996). The number of copies of Tos17 is very low, ranging from 1 to 4 in various cultivars growing under normal conditions, but can be significantly elevated by tissue culture (Hirochika et al. 1996; Hirochika 1997). It was found that Tos17 is active only during callus culture and immediately becomes inactive upon plant regeneration (Hirochika et al. 1996Hirochika 1997, 2001), thus suggesting developmentally regulated efficient element silencing. Nevertheless, the mechanism for silencing of Tos17 is not clear, neither is its possible influence on flanking sequences after retrotransposition.We have found that Tos17 could also be mobilized by sexual introgression into the rice genome of genomic DNA from wild rice (Zizania latifolia); copy number of the element in some introgression lines was markedly increased though insertion sites have not been isolated (Liu and Wendel 2000). Also, similar to the case in tissue culture (Hirochika et al. 1996), Tos17 was also likely inactivated after retrotransposition in the introgression lines (Liu and Wendel 2000).To confirm mobilization of Tos17 by introgression, and to study the possible role of cytosine methylation on the element's activity, particularly the potential influence of element insertion on structure and epigenetic state of the target sequences, we selected an introgression line (Tong35) wherein Tos17 was activated but its transposition was very limited, hence indicating rapid and efficient repression of its activity. The isolation of all possible genomic regions flanking Tos17 (original and newly transposed) in this line, has enabled the confirmation of element mobilization induced by alien DNA introgression, and the analysis on genomic structure and methylation status of both Tos17 itself and its targeted flanking sequences. We report that sequence structure and cytosine methylation state of all copies of Tos17 itself in the introgression line remain the same hypermethylation as that of the rice parent. In contrast, heritable alterations in both structure and methylation level of one of the three low-copy sequences f