Abstract
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 www.benthgenome.com assembly, and with less integrity to Niben101Scf03641 in the 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.
Highlights
Reporter genes have been profoundly important in advancing biological research
Many of the reads aligned perfectly with the transfer region of the Ti plasmid and formed one contig but none aligned with left border (LB) or right border (RB) sequences (Fig 1)
There are two independent draft genome assemblies of N. benthamiana from an Australian and American research group, respectively, and CRISPR-Cas technology appears capable of efficiently generating targeted N. benthamiana mutants [15, 16]
Summary
One of the first widely adopted reporter systems utilised beta glucuronidase (GUS) [1], which converts a soluble clear substrate to an insoluble blue precipitate. 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) [4], which have not been widely distributed, but 16c has been cited in more than 750 publications. We have already published and made available the genome and transcriptome sequences of N. benthamiana [5,6,7] (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
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