The widely used Nicotiana benthamiana 16c line has an unusual T-DNA integration pattern including a transposon sequence

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.

An Insight into T-DNA Integration Events in Medicago sativa

“…Possible ways to improve the quality of insertions in the alfalfa genome can be envisaged: Launching the T-DNA from the Agrobacterium chromosome may reduce the risk of transferring sequences belonging to the binary vector (including antibiotic resistance genes for bacterial selection); however, engineering the bacterial chromosome adds complication to the procedures, and a decrease in the transformation efficiency may result [ 27 ]; it should also be considered that rare cases of transfer of sequences belonging to Agrobacterium chromosome are documented [ 10 , 11 , 12 ]; Increasing the number of LBs and VirG gene or including negative selectable marker genes in the VB would improve the correct processing of the borders and allow counter selection of events that include VB sequences [ 3 , 30 , 36 , 50 , 67 ]; Using plant-derived sequences for vector construction (e.g., plant-derived SMGs, cisgenesis) [ 68 , 69 , 70 ] can relieve the perceived risk of genetically modified plants. The application of new breeding techniques such as genome editing is also offering new tools for precise modification of the alfafa genome.…”

“…Launching the T-DNA from the Agrobacterium chromosome may reduce the risk of transferring sequences belonging to the binary vector (including antibiotic resistance genes for bacterial selection); however, engineering the bacterial chromosome adds complication to the procedures, and a decrease in the transformation efficiency may result [ 27 ]; it should also be considered that rare cases of transfer of sequences belonging to Agrobacterium chromosome are documented [ 10 , 11 , 12 ];…”

“…The main findings can be summarized as follows: Upon activation of the Vir gene cascade the ssT-DNA is released by the action of VirD1/VirD2 endonuclease complex; such mechanism involves the transfer of the sequence between the RB (5′-end) and LB (3′-end) to the host cells through a type IV secretion system [ 6 , 7 ]. Sequences exceeding the borders and belonging to the so-called vector backbone (VB) are frequently transferred along with the ssT-DNA [ 3 ]; other bacterial DNA may also enter the plant cell, including plasmid DNA [ 8 , 9 ] and chromosomal DNA [ 10 , 11 , 12 ]. Coordinated activity of bacterial and host proteins are necessary for infection and T-DNA integration, the latter largely relying on host factors [ 13 , 14 ].…”

“…Sequences exceeding the borders and belonging to the so-called vector backbone (VB) are frequently transferred along with the ssT-DNA [ 3 ]; other bacterial DNA may also enter the plant cell, including plasmid DNA [ 8 , 9 ] and chromosomal DNA [ 10 , 11 , 12 ].…”

Are the current gRNA ranking prediction algorithms useful for genome editing in plants?

“…A standard construct, pCas9-GFP ( Fig 1A), encoding a SpCas9 protein under the control of a double 35S promoter and two gRNAs from the tRNA delivery system [6], was tested for its ability to edit the integrated mGFP5-ER reporter gene in transgenic line 16c of N.benthamiana [22,23]. The tRNA system was used to express more than one gRNA from a single Pol III promoter.…”