Sequence-specific gene silencing by transitive signals in plants

This work contributes to a research field that aims at unraveling the process of RNA silencing, in particular of transitive silencing. In the first, application-oriented part of the thesis, we examined the potential of transitive silencing as a tool for functional genomics, and in the second, more fundamental part, we focused on factors that influence the efficiency of transitive silencing, such as the processivity of the key enzyme RDR6, the length of homology between primary and secondary target, and the presence of viroid sequences in the primary target. Frequently used methods that exploit RNA silencing for gene discovery in plants are hairpin-induced or virus-induced gene silencing. Transitivity could be an alternative silencing technology, but in plants it has not been shown yet whether amplification products are able to induce silencing of endogenous targets. To address the first goal of this thesis, we developed a transitive XYZ system in Nicotiana tabacum (Chapter 3; Van Houdt et al., 2003) and in Arabidopsis thaliana (Chapter 4). In both plant species we analyzed the ability of a sense transgene IR locus (X) to induce transitivity along a transgenic primary target (Y), resulting in silencing of a transgenic secondary target (Z) without homology to the silencing inducer. In tobacco, we also investigated the methylation status of the silencing inducer X and of the target sequences (Chapter 3). In Arabidopsis, we examined whether the in trans-silenced Y transcripts can produce a transitive silencing signal that is able to induce silencing of an endogenous gene target (Chapter 4). The second goal of this work was to clarify some characteristics of transitive silencing. VIGS has been shown to spread over a distance of at least 1000 nt from the 5’ end to the 3’ end of the target mRNA, while 3’ to 5’ spreading can extend at least through 332 nt, with a possible limit of 600 nt (Petersen and Albrechtsen, 2005; Vaistij et al., 2002). We examined the extent of 3’ to 5’ spreading induced by the IR locus X and looked at the influence of an increasing distance between primary and secondary target sequences in a primary target Y on the onset of transitive silencing of a transgenic target Z (Chapter 5). In the same study, we investigated the influence of the length of sequence homology between primary and secondary target on the frequency and efficiency of transitive silencing of an endogenous target. Finally, we examined whether inserting a viroid sequence into a primary target Y inhibited transitive silencing induced by IR locus X (Chapter 6), because results from the research group of M. Wassenegger recently demonstrated in tobacco that viroid-induced silencing did not induce spreading along a GFP-viroid fusion transgene (Vogt et al., 2004).

Grantor dc:publisher

Ghent University. Faculty of Sciences

Year dc:date

2006

Author dc:creator

• Bleys, Annick

Contributors dc:contributor

• Depicker, Anna

• Biology and Life Sciences

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

eng

Identifier

https://biblio.ugent.be/publication/469332
https://biblio.ugent.be/publication/469332/file/1878340

OAI identifier oai:identifier

oai:archive.ugent.be:469332