Date Published: February 23, 2017
Publisher: Public Library of Science
Author(s): Joshua G. Philips, Fatima Naim, Michał T. Lorenc, Kevin J. Dudley, Roger P. Hellens, Peter M. Waterhouse, A.L.N. Rao.
Nicotiana benthamiana is employed around the world for many types of research and one transgenic line has been used more extensively than any other. This line, 16c, expresses the Aequorea victoria green fluorescent protein (GFP), highly and constitutively, and has been a major resource for visualising the mobility and actions of small RNAs. Insights into the mechanisms studied at a molecular level in N. benthamiana 16c are likely to be deeper and more accurate with a greater knowledge of the GFP gene integration site. Therefore, using next generation sequencing, genome mapping and local alignment, we identified the location and characteristics of the integrated T-DNA. As suggested from previous molecular hybridisation and inheritance data, the transgenic line contains a single GFP-expressing locus. However, the GFP coding sequence differs from that originally reported. Furthermore, a 3.2 kb portion of a transposon, appears to have co-integrated with the T-DNA. The location of the integration mapped to a region of the genome represented by Nbv0.5scaffold4905 in the http://www.benthgenome.com assembly, and with less integrity to Niben101Scf03641 in the http://www.solgenomics.net assembly. The transposon is not endogenous to laboratory strains of N. benthamiana or Agrobacterium tumefaciens strain GV3101 (MP90), which was reportedly used in the generation of line 16c. However, it is present in the popular LBA4404 strain. The integrated transposon sequence includes its 5’ terminal repeat and a transposase gene, and is immediately adjacent to the GFP gene. This unexpected genetic arrangement may contribute to the characteristics that have made the 16c line such a popular research tool and alerts researchers, taking transgenic plants to commercial release, to be aware of this genomic hitchhiker.
Reporter genes have been profoundly important in advancing biological research. In plants, one of the first widely adopted reporter systems utilised beta glucuronidase (GUS) , which converts a soluble clear substrate to an insoluble blue precipitate. Unfortunately, this is a destructive histochemical technique. The more recent discovery and application of the green fluorescent protein (GFP) from Aequorea victoria allows reporter assays to be live, continuous and non-destructive, and has revolutionised molecular science. GFP was first used in bacteria and animal cells but was rapidly adopted by plant researchers following addition of plant regulatory signals and codon optimisation. The pioneering plant expression constructs, such as mGFP4, mGFP5, and mGFP5-ER [2, 3], have been used extensively in both monocotyledon and dicotyledon plants. For many of these species, different research groups have produced their own GFP-expressing lines and rarely has a single transgenic GFP-expressing line of a species been embraced by a whole research sector. However, the 16c line of N. benthamiana, from David Baulcombe’s laboratory, is the notable exception. It was generated alongside three other lines (GFP8, GFP17b, and GFPY) , which have not been widely distributed, but 16c has been cited in more than 750 publications. These include: exploring virus-plant interactions, RNAi, mobile signals, florigens, grafting, viroids, protein structure and function, protein-protein interactions, human and avian viruses, and silencing suppressors. We have already published and made available the genome and transcriptome sequences of N. benthamiana [5–7] (www.benthgenome.com), and believe that reporting further details about the location and characteristics of the T-DNA insertion in the 16c line will aid the plant research community.
Over the last decade, Arabidopsis thaliana and Oryza sativa have been the popular model species for studying molecular mechanisms in plant biology. Both are diploid, have fully sequenced small genomes, are easy to transform and have a broad range of available mutants. The T-DNA insertion lines of A. thaliana, in particular, have been very useful. N. benthamiana has been used extensively for transient gene expression in the study of many mechanisms, but much less so using stable transgenesis because of the complexity of its allopolyploid genome, lack of genomic sequence information, and the dearth of well-defined mutants . However, there is now considerable interest in how mechanisms operate in polyploid genomes, as most crop species are polyploids. There are also two independent draft genome assemblies of N. benthamiana (benthgenome.com & solgenomics.net) from an Australian and American research group, respectively, and CRISPR-Cas technology appears capable of efficiently generating targeted N. benthamiana mutants [15, 16]. The GFP gene in 16c has been used in many studies including those on the induction and maintenance of epigenetic changes [17, 18], and as a reporter for post-transcriptional gene silencing . However, many of these assays have relied on incomplete or inaccurate details of the GFP locus. In order to furnish researchers with more accurate details for future studies utilising 16c, we determined the sequence and location of this locus and discovered some unexpected details. We confirmed, at a sequence level, the assertion that the GFP transgene is in a single locus and that it is highly expressed when compared to the NPTII selectable marker gene. We determined that the GFP transgene has the ER targeting and retention signals of mGFP5-ER and not the untargeted GFP design of mGFP5, as reported in the original description . We also identified a sequence variation that alters an amino acid residue within the GFP protein that is not a reported change in mGFP5-ER arising from mGFP4-ER.