Identification of sequences required for AL2-mediated activation of the tomato golden mosaic virus-yellow vein BR1 promoter

A 108 bp sequence has been identified in the tomato golden mosaic virus-yellow vein (yvTGMV) B component that is necessary and sufficient for AL2-mediated activation of the BR1 promoter. The sequence appears to have a bipartite arrangement, with elements located between −144 to −77 and −59 to −36 from the transcription start site, with both being required for activation by AL2. These sequences are located upstream of a TATA box and bind nuclear proteins from spinach, tomato and Arabidopsis. These sequences are also capable of binding Arabidopsis PPD2, which has been shown previously to interact with the yvTGMV coat protein (CP) promoter. We have identified two putative transcription factor-binding sites (CCAAT and GTGANTG10) that are conserved in sequences necessary for activation of the yvTGMV BR1, as well as the yvTGMV and cabbage leaf curl virus (CabLCV) CP promoters, which are all activated by AL2. The yvTGMV BR1 promoter exhibits AL2-independent expression in vascular tissue, similar to the yvTGMV, CabLCV and spinach curly top virus CP promoters. Together, this further confirms a common regulatory mechanism for AL2-mediated activation of bipartite begomovirus promoters.

Distinct Viral Sequence Elements Are Necessary for Expression of Tomato golden mosaic virus Complementary Sense Transcripts That Direct AL2 and AL3 Gene Expression

Transient expression studies using Nicotiana benthamiana protoplasts and plants have identified sequences important for transcription of complementary sense RNAs derived from Tomato golden mosaic virus (TGMV) DNA component A that direct expression of AL2 and AL3. Transcription of two complementary sense RNAs, initiating at nucleotides 1,935 (AL1935) and 1,629 (AL1629), is directed by unique sequences located upstream of each transcription initiation site. One element is located between 28 and 124 nucleotides (nt) upstream of the AL1935 transcription start site, which differs from a second element located 150 nt downstream, between 129 and 184 nt upstream of the AL1629 transcription start site. Transcription initiation at nucleotide 1,935 is lower than that at nucleotide 1,629 as determined by run-on transcription assays, and the resulting transcript is only capable of expressing AL3. The transcript initiating at nucleotide 1,629 is capable of directing expression of both AL2 and AL3, although expression of AL3 is up to fourfold greater than that for AL2. Nuclear factors purified from tobacco suspension cells bind to sequences upstream of both AL1935 and AL1629, correlating with the ability of these sequences to direct gene expression. Thus, in tobacco, regulatory sequences direct transcription of two unique TGMV messenger RNAs that differentially express AL2 and AL3.

Tomato yellow spot virus, a tomato-infecting begomovirus from Brazil with a closer relationship to viruses from Sida sp., forms pseudorecombinants with begomoviruses from tomato but not from Sida

Geminiviruses are characterized by a circular, single-stranded DNA genome and twinned icosahedral particles. Begomoviruses (whitefly-transmitted geminiviruses) are a major constraint to crop production worldwide. In Brazil, tomato-infecting begomoviruses emerged as serious pathogens over the last 10 years, due to the introduction of a new biotype of the insect vector. Tomato yellow spot virus (ToYSV) is a newly described begomovirus originally isolated from tomato, but phylogenetically closer to viruses from Sida sp. A study was performed to determine the viability of pseudorecombinants formed between the DNA components of ToYSV and other weed- and tomato-infecting begomoviruses from Brazil. Despite its closer relationship to weed-infecting viruses, ToYSV was only capable of forming viable pseudorecombinants with tomato viruses. An infectious pseudorecombinant formed between ToYSV DNA-A and tomato crinkle leaf yellows virus (TCrLYV) DNA-B induced severe symptoms in Nicotiana benthamiana. This was attributed, at least in part, to the fact that the origins of replication of both components had identical Rep-binding sequences. However, this was not the case for another infectious pseudorecombinant formed between tomato golden mosaic virus (TGMV) DNA-A and ToYSV DNA-B, which have different Rep-binding sequences. These results reinforce the notion that pseudorecombinant formation cannot be explained solely on the basis of phylogenetic relationships and conserved iteron sequences, and suggest that the TGMV Rep protein may be more versatile in terms of recognizing heterologous DNA components than that of ToYSV.

Functional equivalence of late gene promoters in bean golden mosaic virus with those in tomato golden mosaic virus

In the bipartite geminivirus tomato golden mosaic virus (TGMV), the activity of late gene promoters is up-regulated by the multifunctional viral protein AL2. Cis-acting sequences required for AL2-mediated promoter responses have not been well characterized. However, nucleotide sequence analysis has implicated a motif termed the conserved late element (CLE). The CLE is present in TGMV and many other begomoviruses, although it is not ubiquitous. Here we analysed the regulation of late gene expression in bean golden mosaic virus (BGMV), one of the begomoviruses which lacks the CLE. Transient reporter gene assays showed that BGMV late gene promoters were trans-activated in Nicotiana benthamiana protoplasts, both by the homologous BGMV AL2 protein and by the heterologous TGMV AL2 protein. The BGMV AL2 protein also trans-activated TGMV late gene promoters. Consistent with these results, we found that hybrid viruses with the late gene promoters exchanged between BGMV and TGMV were viable in planta.