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A DNA target of 30 bp is sufficient for RNA-directed DNA methylation

Published online by Cambridge University Press:  01 January 2000

THIERRY PÉLISSIER
Affiliation:
Fraunhofer IUCT, Abteilung für Molekulare Biotechnologie, Grafschaft, Auf dem Aberg 1, D-57392 Schmallenberg, Germany Present address: Biomove, UMR 6547 CNRS, Université Blaise Pascal Clermont-Ferrand II, F-63177 Aubiêre Cedex, France.
MICHAEL WASSENEGGER
Affiliation:
Fraunhofer IUCT, Abteilung für Molekulare Biotechnologie, Grafschaft, Auf dem Aberg 1, D-57392 Schmallenberg, Germany
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Abstract

In higher plants, RNA–DNA interactions can trigger de novo methylation of genomic sequences via a process that is termed RNA-directed DNA methylation (RdDM). In potato spindle tuber viroid (PSTVd)-infected tobacco plants, this process can potentially lead to methylation of all C residues at symmetrical and nonsymmetrical sites within chromosomal inserts that consist of multimers of the 359-bp-long PSTVd cDNA. Using PSTVd cDNA subfragments, we found that genomic targets with as few as 30 nt of sequence complementarity to the viroid RNA are detected and methylated. Genomic sequencing analyses of genome-integrated 30- and 60-bp-long PSTVd subfragments demonstrated that de novo cytosine methylation is not limited to the canonical CpG, CpNpG sites. Sixty-base-pair-long PSTVd cDNA constructs appeared to be densely methylated in nearly all tobacco leaf cells. With the 30-bp-long PSTVd-specific construct, the proportion of cells displaying dense transgene methylation was significantly reduced, suggesting that a minimal target size of about 30 bp is necessary for RdDM. The methylation patterns observed for two different 60-bp constructs further suggested that the sequence identity of the target may influence the methylation mechanism. Finally, a link between viroid pathogenicity and PSTVd RNA-directed methylation of host sequences is proposed.

Type
Research Article
Copyright
© 2000 RNA Society

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