Abstract
Cotton leaf curl Burewala virus (CLCuBuV), belonging to the genus Begomovirus, possesses single-stranded monopartite DNA genome. The bidirectional promoters representing Rep and coat protein (CP) genes of CLCuBuV were characterized and their efficacy was assayed. Rep and CP promoters of CLCuBuV and 35S promoter of Cauliflower mosaic virus (CaMV) were fused with β-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes. GUS activity in individual plant cells driven by Rep, CP and 35S promoters was estimated using real-time PCR and fluorometric GUS assay. Histochemical staining of GUS in transformed tobacco (Nicotiana tabacum cv. Xanthi) leaves showed highest expression driven by Rep promoter followed by 35S promoter and CP promoter. The expression level of GUS driven by Rep promoter in transformed tobacco plants was shown to be two to four-fold higher than that of 35S promoter, while the expression by CP promoter was slightly lower. Further, the expression of GFP was monitored in agroinfiltrated leaves of N. benthamiana, N. tabacum and cotton (Gossypium hirsutum) plants using confocal laser scanning microscopy. Rep promoter showed strong consistent transient expression in tobacco and cotton leaves as compared to 35S promoter. The strong constitutive CLCuBuV Rep promoter developed in this study could be very useful for high level expression of transgenes in a wide variety of plant cells.
Highlights
Members of the family Geminiviridae, commonly called as geminiviruses, are characterized by having single-stranded, circular DNA genome of 2.5–3.0 kb, which is either monopartite or bipartite and encapsidated within virions having geminate morphology
We have compared the expression patterns of GUS gene driven by Cotton leaf curl Burewala virus (CLCuBuV) replication such as AC1 (Rep) and CLCuBuV coat protein (CP) promoters with CaMV35S promoter in transformed transgenic tobacco plants
The highest expression of GUS gene was obtained using the CLCuBuV Rep promoter followed by the Cauliflower mosaic virus (CaMV) 35S promoter and the lowest activity was doi:10.1371/journal.pone.0121656.g006
Summary
Members of the family Geminiviridae, commonly called as geminiviruses, are characterized by having single-stranded, circular DNA genome of 2.5–3.0 kb, which is either monopartite or bipartite and encapsidated within virions having geminate morphology. Monopartite begomoviruses have one genomic component (DNA-A) with six open reading frames (ORFs). Most monopartite begomoviruses are associated with satellite molecule called betasatellite which is often required for the development of wild type symptom in naturally-infected host plant. It requires a helper begomovirus for its replication, systemic infection and whitefly vector-mediated transmission [5,6,7,8]. Some begomovirus betasatellite disease complexes are associated with a nanovirus like component named alphasatellite [8,9,10,11]
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