Blocking single-stranded transferred DNA conversion to double-stranded intermediates by overexpression of yeast DNA REPLICATION FACTOR A.

Abstract

Agrobacterium tumefaciens delivers its single-stranded transferred DNA (T-strand) into the host cell nucleus, where it can be converted into double-stranded molecules. Various studies have revealed that double-stranded transfer DNA (T-DNA) intermediates can serve as substrates by as yet uncharacterized integration machinery. Nevertheless, the possibility that T-strands are themselves substrates for integration cannot be ruled out. We attempted to block the conversion of T-strands into double-stranded intermediates prior to integration in order to further investigate the route taken by T-DNA molecules on their way to integration. Transgenic tobacco (Nicotiana benthamiana) plants that overexpress three yeast (Saccharomyces cerevisiae) protein subunits of DNA REPLICATION FACTOR A (RFA) were produced. In yeast, these subunits (RFA1-RFA3) function as a complex that can bind single-stranded DNA molecules, promoting the repair of genomic double strand breaks. Overexpression of the RFA complex in tobacco resulted in decreased T-DNA expression, as determined by infection with A. tumefaciens cells carrying the β-glucuronidase intron reporter gene. Gene expression was not blocked when the reporter gene was delivered by microbombardment. Enhanced green fluorescent protein-assisted localization studies indicated that the three-protein complex was predominantly nuclear, thus indicating its function within the plant cell nucleus, possibly by binding naked T-strands and blocking their conversion into double-stranded intermediates. This notion was further supported by the inhibitory effect of RFA expression on the cell-to-cell movement of Bean dwarf mosaic virus, a single-stranded DNA virus. The observation that RFA complex plants dramatically inhibited the transient expression level of T-DNA and only reduced T-DNA integration by 50% suggests that double-stranded T-DNA intermediates, as well as single-stranded T-DNA, play significant roles in the integration process.

• Authors: Mery Dafny-Yelin, Avner Levy, Raz Dafny, Tzvi Tzfira
• Journal: Plant physiology (Plant Physiol) Vol. 167 Issue 1 Pg. 153-63 (Jan 2015) ISSN: 1532-2548 [Electronic] United States
• PMID: 25424309 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.)
• Copyright: © 2015 American Society of Plant Biologists. All Rights Reserved.

Chemical References

• DNA, Bacterial
• DNA, Single-Stranded
• RFA1 protein, S cerevisiae
• RFA2 protein, S cerevisiae
• RPA14 protein, S cerevisiae
• Replication Protein A
• Saccharomyces cerevisiae Proteins
• T-DNA
• RNA Polymerase I

Topics

• Agrobacterium tumefaciens (genetics, physiology)
• DNA, Bacterial (metabolism)
• DNA, Single-Stranded (metabolism)
• Gene Expression
• Plants, Genetically Modified (metabolism)
• RNA Polymerase I (metabolism, physiology)
• Recombination, Genetic
• Replication Protein A (genetics, metabolism, physiology)
• Saccharomyces cerevisiae Proteins (genetics, metabolism, physiology)
• Tobacco (genetics, metabolism, microbiology)
• Two-Hybrid System Techniques