Replication of tomato golden mosaic virus DNA B in transgenic plants expressing open reading frames (ORFs) of DNA A: requirement of ORF AL2 for production of single-stranded DNA

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Abstract
Tomato golden mosaic geminivirus has a genome of two single-stranded (ss) DNA components, A and B. An almost identical ‘common’ region in DNA A and DNA B is thought to contain sequence elements controlling replication and transcription. Hence investigation of sequences important for DNA replication by in vitro mutagenesis is complicated by possible effects on the transcription of genes for replication proteins. To overcome this problem, transgenic plants expressing open reading frames (ORFs) of DNA A from an enhanced cauliflower mosaic virus 35S RNA promoter were constructed and tested for their ability to support the replication of DNA B and DNA B mutants. The results show that plants transgenic for ORF AL1 are able to support the replication of the double-stranded (ds) forms of DNA B, but that ORF AL2 is required in addition to produce ssDNA B. ORFs AL3, BL1 or BR1 were not required for replication of ds or ssDNA B. To the best of our knowledge this is the first time that essential replication proteins of ds or ssDNA B. To the best of our knowledge this is the first time that essential replication proteins of a geminivirus have been expressed constitutively from a plant genome withoutgiving rise to replication DNA A molecules, thereby allowing DNA B to replicate alone. Such transgenic plants should enable not only the mutational analysis of sequence elements within the replication origin region, but also the construction of a new generation of vectors for gene amplification in plants, based on a minimal virus replicon.