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Genomic regions of tomato leaf curl virus DNA satellite required for replication and for satellite-mediated delivery of heterologous DNAs

Dongmei Li^1,2,

¹ CSIRO Plant Industry, PO Box 350, Glen Osmond, SA 5064, Australia
² School of Agriculture, Food and Wine, The University of Adelaide, Waite Campus, SA 5064, Australia
³ Plant Protection Department, College of Agriculture, Shiraz University, Shiraz, Iran
⁴ Department of Plant Protection, School of Agriculture, Zanjan University, Zanjan, Iran

Correspondence
M. Ali Rezaian
ali.rezaian{at}adelaide.edu.au

Tomato leaf curl virus (TLCV) satellite DNA (sat-DNA) is a 682 nt, circular, single-stranded molecule that lacks an open reading frame (ORF) or an apparent promoter. It contains binding motifs for the TLCV replication-associated protein, but these are dispensable for replication. To identify the regions of the sat-DNA critical for replication, the entire sequence was scanned by deletion/replacement mutagenesis. Transient assays using Nicotiana benthamiana revealed that sequences within nt 296–35 (through nt 682) are essential for replication. Sequence deletions and replacements between nt 35 and 296 were tolerated but with a significant loss of infectivity, indicating that genome size strongly influences replication efficiency. Within the permissible region, inserts of 100–700 nt were retained in transient assays although with a slight reduction in replication. In addition, sat-DNA constructs containing short non-viral DNAs replicated and spread in tobacco plants, indicating their potential as gene-delivery vectors.

Present address: CSIRO Molecular & Health Technologies, 11 Julius Avenue, Riverside Corporate Park, Delhi Rd, NSW 2113, Australia.

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