Doctor of Philosophy
School of Biological Sciences
Zahid, Kiran, Understanding the role of RNA-silencing mediated processes in inter-specific hybridisation and origin of Cucumber mosaic virus satellite RNAs, Doctor of Philosophy thesis, School of Biological Sciences, University of Wollongong, 2013. http://ro.uow.edu.au/theses/3948
RNA-silencing is essential to the regulation of many developmental processes in eukaryotes. It functions as a sequence specific defence mechanism against foreign nucleic acid molecules, such as viruses, transposons and transgenes; and regulates gene expression at both transcriptional and post-transcription levels.
Recently, a number of studies in plants have indicated possible roles of RNAsilencing pathways in hybridisation and heterosis. In the first part of the current study, the role of two RNA-silencing mediated processes is explored: regulation of transgene expression and satellite RNA (sat RNA) induced RNA-directed DNA methylation (RdDM), in Nicotiana inter-specific hybrids in comparison to the nonhybrid controls.
The first strategy consisted of determining the difference in transgene expression between Nicotiana hybrids and non-hybrids. The transgenes were found more repressed in hybrids than non-hybrids suggesting that RNA-silencing pathways play role in inter-specific hybridisation-associated gene expression changes. Moreover, my results suggest that transgenes may act like transposons, which are in general more repressed in hybrids than non-hybrids or parental lines; this repression of transposons may free transcriptional machinery for transcription of other genes that allow heterosis.
Sat RNAs are small RNA pathogens in plants that do not encode functional proteins and depend on associated helper viruses for replication, encapsidation, systemic movement and transmission. Despite the growing interest in sat RNAs, the origin of these agents remains unknown.
The second part of the current study explored the abnormal behaviour of a transgene containing viral sat RNA sequence (Sat) in Nicotiana species.While examining the difference in CMV sat RNA induced RdDM between Nicotiana hybrid and non-hybrid, the Sat sequence of the 35S-GUS-Sat transgene was observed to be highly methylated without sat RNA infection. This finding suggested that the tobacco genome encodes siRNAs that are homologous to and direct DNA methylation against the Sat sequence. Northern blot analysis detected 24-nt siRNAs in N. tabacum, N. benthamiana and N. clevelandii plants using Sat probe. Expression of the 35S-GUSSat fusion transgene was found to be repressed in the N. tabacum plants in comparison to 35S-GUS transgene, suggesting that the high level cytosine methylation at the Sat sequence inhibits the expression of the transgene.
The detection of 24-nt CMV sat RNA-like siRNAs led to the final part of this study: searching for CMV sat RNA-like sequences in the Nicotiana genome. Southern blot hybridisation using the CMV sat RNA probe showed the presence of multiple hybridising bands, and PCR amplification using conserved CMV sat RNA primers detected a number of different variants of the CMV sat RNA-like sequences in the Nicotiana species. Taken together, the results suggest that the CMV sat RNA is derived from RNA molecules transcribed from a repetitive sequence in the Nicotiana genome and provides a plausible model for the origin of viral sat RNAs in general.