Department of Microbiology, North Carolina State University, Raleigh, NC 27695-7615, USA1
Author for correspondence: Tim Petty. Fax +1 919 515 7867. e-mail tim_petty{at}ncsu.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() |
---|
![]() |
Main text |
---|
![]() ![]() ![]() ![]() |
---|
|
Interestingly, although a recognizable CLE-like sequence is found upstream from the AR1 and/or BR1 ORFs in many begomoviruses, including TGMV, it is not universally present. One of the viruses which lacks a CLE is BGMV. Whether the late gene promoters of CLE-containing and CLE-lacking viruses are functionally equivalent has not previously been tested. However, al2 mutants of TGMV or BGMV can be complemented to some extent by the heterologous A component during co-infection of their common host Nicotiana benthamiana, and hybrid viruses with the AL2 ORF exchanged between BGMV and TGMV are viable (Gillette et al., 1998 ). These observations suggested that the AL2 proteins of TGMV and BGMV can function equivalently, despite the absence of a CLE in BGMV. Here we have evaluated the relationship between the late gene promoters of BGMV and those of the better characterized virus, TGMV. Promoter activities were measured directly in the presence or absence of each AL2 protein using transient gene expression assays in protoplasts. We also analysed the phenotypes in planta of hybrid viruses in which the ARi or BRi non-coding regions were exchanged between BGMV and TGMV.
To test directly whether the AL2 proteins of BGMV or TGMV could trans-activate the BGMV late gene promoters, which lack the CLE, we used a reporter gene expression assay. Reporter plasmids were constructed in which restriction fragments that encompassed the ARi or BRi non-coding sequences from TGMV or BGMV, together with CR and additional upstream flanking DNA (solid arcs in Fig. 1a), were used to drive expression of a firefly luciferase gene. These DNA sequences are similar in extent to those tested in other, comparable studies (Sunter & Bisaro, 1992
). Protoplasts were prepared from N. benthamiana leaves (Jones et al., 1990
) and aliquots of 2·5x105 cells were transfected with 2·5 µg of a reporter plasmid, either alone or together with 10 µg of a plasmid from which either BGMV AL2 or TGMV AL2 was constitutively expressed (Sunter & Bisaro, 1992
). After incubation for 72 h under continuous light, the protoplasts were lysed, and luciferase activity and total protein concentration were determined. Each promoter was analysed in at least three independent experiments, and representative results are presented (Fig. 1b
). This analysis showed directly that BGMV is similar to TGMV in that its AR1 and BR1 promoters are trans-activated by the AL2 protein. We also confirmed that the AL2 proteins of each virus were able to trans-activate the AR1 and BR1 promoters of the other. These results extend previous demonstrations of heterologous complementation between begomovirus AL2 proteins and promoters (Sunter et al., 1994
) to include those of BGMV, the type member of the genus. Although other studies have implicated the CLE in trans-activation (Ruiz-Medrano et al., 1999
), our results show that this motif per se is not a necessary component of all AL2-responsive begomovirus promoters.
The transient reporter gene expression assays indicated that overall the AR1 and BR1 promoters of TGMV and BGMV functioned similarly in leaf mesophyll protoplasts. To confirm these results and extend them to virus-infected whole plants, we also constructed and analysed hybrid viruses in which the ARi or BRi non-coding DNA sequences were exchanged on SspIXbaI or SspISnaBI restriction fragments, respectively (Fig. 1a). These non-coding sequences contain proximal elements of the AR1 and BR1 promoters (Petty et al., 1988
; Sunter & Bisaro, 1989
, 1992
). Whether the adjacent upstream sequences also contribute to promoter activity is currently unknown, but their exchange was not attempted because CR contains virus-specific elements of the replication origin (Fontes et al., 1994
). Each hybrid DNA component was constructed in a recombinant plasmid as a partial tandem dimer. Hybrids in the genetic background of TGMV were designated TB-ARi (plasmid pTBARSX) and TB-BRi (plasmid pTG1.2B
R), and those in the background of BGMV were designated BT-ARi (plasmid pGTARSX) and BT-BRi (plasmid pGTBRSS). Plasmids containing partial tandem dimers of wild-type TGMV and BGMV DNA components were described previously (Fontes et al., 1994
).
The phenotypes of the resulting hybrid viruses were determined after inoculation of N. benthamiana by microprojectile bombardment with recombinant plasmids, as described previously (Schaffer et al., 1995 ). Each hybrid A component was co-inoculated with wild-type DNA B from the appropriate virus, and each hybrid B component was co-inoculated with wild-type DNA A. Wild-type TGMV elicits strong symptoms of infection in N. benthamiana, and by comparison those induced by the TGMV-based hybrid viruses TB-ARi and TB-BRi were attenuated. On directly inoculated leaves, both hybrids produced smaller chlorotic lesions than wild-type TGMV, and on systemically infected leaves the symptoms consisted of mild rugosity, epinasty and vein yellowing. In contrast to TGMV, wild-type BGMV produces asymptomatic systemic infections of N. benthamiana (Petty et al., 1995
). Similar to wild-type BGMV, the BT-ARi hybrid produced completely asymptomatic systemic infection in this host. In contrast, BT-BRi gave rise to small, pale chlorotic lesions on directly inoculated leaves, and also occasionally to some slight epinasty of the systemically infected leaves.
Nucleic acids were extracted from systemically infected leaves and analysed as described previously (Jeffrey et al., 1996 ). Briefly, DNA concentrations were determined by fluorimetry in the presence of Hoechst 33258 dye, and 2·5 µg aliquots were resolved by electrophoresis and Southern blotting. The blots were hybridized with 32P-labelled viral component-specific probes (Fig. 2a
), and the accumulation of viral DNA (expressed as ng viral DNA/µg total leaf DNA) was determined by PhosphorImager comparison with cloned TGMV double-stranded (ds) DNA standards (Fig. 2b
). This analysis confirmed that each of the hybrid viruses was capable of infecting N. benthamiana systemically. Infectivity of both TGMV and BGMV in N. benthamiana requires BR1 expression (Brough et al., 1988
; Schaffer et al., 1995
), and systemic movement of BGMV in this host additionally requires expression of AR1 (Pooma et al., 1996
), so the heterologous non-coding sequences in TB-BRi, BT-ARi and BT-BRi were clearly functional. Although TGMV ar1 mutants can spread systemically in N. benthamiana (Brough et al., 1988
; Gardiner et al., 1988
), their ability to accumulate single-stranded (ss) DNA is severely compromised (Jeffrey et al., 1996
). In contrast, TB-ARi was efficiently able to accumulate ssDNA (Fig. 2a
, b
), which is consistent with AR1 gene expression being driven by the heterologous BGMV non-coding sequences in this hybrid virus as well.
|
In comparison with wild-type TGMV, changes in the relative molar accumulation of DNAs A and B showed that the TGMV-based hybrid DNA components exhibited significant cis-acting defects in DNA accumulation (Fig. 2b). In contrast to the wild-type TGMV A:B ratio of
1:3, the molar A:B ratio for TB-ARi was
1:10, and for TB-BRi it was
1:1. These altered ratios reflect a decrease of
3-fold in the accumulation of the hybrid A component TB-ARi, and a similar
3-fold decrease in the accumulation of the hybrid B component TB-BRi, relative to wild-type DNAs A and B. Previous studies have identified roles in TGMV DNA replication for CR sequences upstream from and including the stemloop structure (Orozco et al., 1998
). Our results suggest that the efficiency of TGMV DNA replication may also depend on sequences downstream from the stemloop structure, which were replaced in the TB-ARi and TB-BRi hybrid DNA components. Because the relative molar accumulation of the A and B components of the BGMV-based hybrids BT-ARi and BT-BRi did not differ significantly from wild-type BGMV (Fig. 2b
), it appears that determinants of BGMV replication efficiency are not similarly located in this region. It may be significant that in TGMV, but not in BGMV, the sequences which are conserved between DNAs A and B (i.e. CR) extend downstream from the stemloop structure, although the sequence conservation in this region is imperfect (Hamilton et al., 1984
). Further experiments designed specifically to address DNA replication efficiency will be required to test this possibility.
In conclusion, we have shown here that non-coding sequences adjacent to the AR1 and BR1 ORFs of TGMV and BGMV contain functionally equivalent promoter sequences which respond to the AL2 proteins of either virus in transient gene expression assays in N. benthamiana protoplasts. Analysis of hybrid viruses in which the ARi or BRi non-coding regions were exchanged between BGMV and TGMV confirmed that the promoters are also functionally equivalent, at a gross level at least, in virus-infected plants. Taken together, these results suggest that comparison of the BGMV and TGMV promoters may provide a useful experimental system with which to define cis-acting elements required for their common response to AL2.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() |
---|
Brough, C. L., Hayes, R. J., Morgan, A. J., Coutts, R. H. A. & Buck, K. W.(1988). Effects of mutagenesis in vitro on the ability of cloned tomato golden mosaic virus DNA to infect Nicotiana benthamiana plants. Journal of General Virology 69, 503-514.
Fontes, E. P. B., Gladfelter, H. J., Schaffer, R. L., Petty, I. T. D. & Hanley-Bowdoin, L.(1994). Geminivirus replication origins have a modular organization. Plant Cell 6, 405-416.
Frischmuth, T. & Stanley, J.(1991). African cassava mosaic virus DI DNA interferes with the replication of both genomic components. Virology 183, 539-544.[Medline]
Gardiner, W. E., Sunter, G., Brand, L., Elmer, J. S., Rogers, S. G. & Bisaro, D. M.(1988). Genetic analysis of tomato golden mosaic virus: the coat protein is not required for systemic spread or symptom development. EMBO Journal 7, 899-904.
Gillette, W. K., Meade, T. J., Jeffrey, J. L. & Petty, I. T. D.(1998). Genetic determinants of host-specificity in bipartite geminivirus DNA A components. Virology 251, 361-369.[Medline]
Hamilton, W. D. O., Stein, V. E., Coutts, R. H. A. & Buck, K. W.(1984). Complete nucleotide sequence of the infectious cloned DNA components of tomato golden mosaic virus: potential coding regions and regulatory sequences. EMBO Journal 3, 2197-2205.
Hartitz, M. D., Sunter, G. & Bisaro, D. M.(1999). The tomato golden mosaic virus transactivator (TrAP) is a single-stranded DNA and zinc-binding phosphoprotein with an acidic activation domain. Virology 263, 1-14.[Medline]
Jeffrey, J. L., Pooma, W. & Petty, I. T. D.(1996). Genetic requirements for local and systemic movement of tomato golden mosaic virus in infected plants. Virology 223, 208-218.[Medline]
Jones, R. W., Jackson, A. O. & Morris, T. J.(1990). Defective-interfering RNAs and elevated temperatures inhibit replication of tomato bushy stunt virus in inoculated protoplasts. Virology 176, 539-545.[Medline]
MacDowell, S. W., Coutts, R. H. A. & Buck, K. W.(1986). Molecular characterisation of subgenomic single-stranded and double-stranded DNA forms isolated from plants infected with tomato golden mosaic virus. Nucleic Acids Research 14, 7967-7984.[Abstract]
Noris, E., Jupin, I., Accotto, G. P. & Gronenborn, B.(1996). DNA-binding activity of the C2 protein of tomato yellow leaf curl geminivirus. Virology 217, 607-612.[Medline]
Orozco, B. M., Gladfelter, H. J., Settlage, S. B., Eagle, P. A., Gentry, R. N. & Hanley-Bowdoin, L.(1998). Multiple cis elements contribute to geminivirus origin function. Virology 242, 346-356.[Medline]
Petty, I. T. D., Coutts, R. H. A. & Buck, K. W.(1988). Transcriptional mapping of the coat protein gene of tomato golden mosaic virus. Journal of General Virology 69, 1359-1365.
Petty, I. T. D., Miller, C. G., Meade-Hash, T. J. & Schaffer, R. L.(1995). Complementable and noncomplementable host adaptation defects in bipartite geminiviruses. Virology 212, 263-267.[Medline]
Pooma, W., Gillette, W. K., Jeffrey, J. L. & Petty, I. T. D.(1996). Host and viral factors determine the dispensability of coat protein for bipartite geminivirus systemic movement. Virology 218, 264-268.[Medline]
Rogers, S. G., Bisaro, D. M., Horsch, R. B., Fraley, R. T., Hoffman, N. L., Brand, L., Elmer, J. S. & Lloyd, A. M.(1986). Tomato golden mosaic virus A component DNA replicates autonomously in transgenic plants. Cell 45, 593-600.[Medline]
Ruiz-Medrano, R., Guevara-González, R. G., Argüello-Astorga, G. R., Monsalve-Fonnegra, Z., Herrera-Estrella, L. R. & Rivera-Bustamante, R. F.(1999). Identification of a sequence element involved in AC2-mediated transactivation of the pepper huasteco virus coat protein gene. Virology 253, 162-169.[Medline]
Saunders, K. & Stanley, J.(1995). Complementation of African cassava mosaic virus AC2 gene function in a mixed bipartite geminivirus infection. Journal of General Virology 76, 2287-2292.[Abstract]
Schaffer, R. L., Miller, C. G. & Petty, I. T. D.(1995). Virus and host-specific adaptations in the BL1 and BR1 genes of bipartite geminiviruses. Virology 214, 330-338.[Medline]
Sung, Y. K. & Coutts, R. H. A.(1995). Pseudorecombination and complementation between potato yellow mosaic geminivirus and tomato golden mosaic geminivirus. Journal of General Virology 76, 2809-2815.[Abstract]
Sung, Y. K. & Coutts, R. H. A.(1996). Potato yellow mosaic geminivirus AC2 protein is a sequence non-specific DNA binding protein. FEBS Letters 383, 51-54.[Medline]
Sunter, G. & Bisaro, D. M.(1989). Transcription map of the B genome component of tomato golden mosaic virus and comparison with A component transcripts. Virology 173, 647-655.[Medline]
Sunter, G. & Bisaro, D. M.(1992). Transactivation of geminivirus AR1 and BR1 gene expression by the viral AL2 gene product occurs at the level of transcription. Plant Cell 4, 1321-1331.
Sunter, G. & Bisaro, D. M.(1997). Regulation of a geminivirus coat protein promoter by AL2 protein (TrAP): evidence for activation and derepression mechanisms. Virology 232, 269-280.[Medline]
Sunter, G., Stenger, D. C. & Bisaro, D. M.(1994). Heterologous complementation by geminivirus AL2 and AL3 genes. Virology 203, 203-210.[Medline]
Received 10 July 2000;
accepted 10 November 2000.