A decade ago, the value of Nicotiana benthamiana as a tool for plant molecular biologists was beginning to be appreciated. Scientists were using it to study plant-microbe and protein-protein interactions, and it was the species of choice with which to activate plasmid-encoded viruses, screen for gene functions with virus-induced gene silencing (VIGS), and transiently express genes by leaf agroinfiltration. However, little information about the species' origin, diversity, genetics, and genomics was available, and biologists were asking the question of whether N. benthamiana is a second fiddle or virtuoso. In this review, we look at the increased knowledge about the species and its applications over the past decade. Although N. benthamiana may still be the sidekick to Arabidopsis, it shines ever more brightly with realized and yet-to-be-exploited potential.

A single lineage of Nicotiana benthamiana is widely used as a model plant(1) and has been instrumental in making revolutionary discoveries about RNA interference (RNAi), viral defence and vaccine production. It is peerless in its susceptibility to viruses and its amenability in transiently expressing transgenes(2,3). These unparalleled characteristics have been associated both positively and negatively with a disruptive insertion in the RNA-dependent RNA polymerase 1 gene, Rdr1(4-6). For a plant so routinely used in research, the origin, diversity and evolution of the species, and the basis of its unusual abilities, have been relatively unexplored. Here, by comparison with wild accessions from across the spectrum of the species' natural distribution, we show that the laboratory strain of N. benthamiana is an extremophile originating from a population that has retained a mutation in Rdr1 for ∼0.8 Myr and thereby traded its defence capacity for early vigour and survival in the extreme habitat of central Australia. Reconstituting Rdr1 activity in this isolate provided protection. Silencing the functional allele in a wild strain rendered it hypersusceptible and was associated with a doubling of seed size and enhanced early growth rate. These findings open the way to a deeper understanding of the delicate balance between protection and vigour.

The transient leaf assay in Nicotiana benthamiana is widely used in plant sciences, with one application being the rapid assembly of complex multigene pathways that produce new fatty acid profiles. This rapid and facile assay would be further improved if it were possible to simultaneously overexpress transgenes while accurately silencing endogenes. Here, we report a draft genome resource for N. benthamiana spanning over 75% of the 3.1 Gb haploid genome. This resource revealed a two-member NbFAD2 family, NbFAD2.1 and NbFAD2.2, and quantitative RT-PCR (qRT-PCR) confirmed their expression in leaves. FAD2 activities were silenced using hairpin RNAi as monitored by qRT-PCR and biochemical assays. Silencing of endogenous FAD2 activities was combined with overexpression of transgenes via the use of the alternative viral silencing-suppressor protein, V2, from Tomato yellow leaf curl virus. We show that V2 permits maximal overexpression of transgenes but, crucially, also allows hairpin RNAi to operate unimpeded. To illustrate the efficacy of the V2-based leaf assay system, endogenous lipids were shunted from the desaturation of 18∶1 to elongation reactions beginning with 18∶1 as substrate. These V2-based leaf assays produced ∼50% more elongated fatty acid products than p19-based assays. Analyses of small RNA populations generated from hairpin RNAi against NbFAD2 confirm that the siRNA population is dominated by 21 and 22 nt species derived from the hairpin. Collectively, these new tools expand the range of uses and possibilities for metabolic engineering in transient leaf assays.

Bananas are a staple food source and a major export commodity worldwide. The Cavendish dessert banana is a triploid AAA genome type and accounts for around 47% of global production. Being essentially sterile, genetic modification is perhaps the only pathway available to improve this cultivar. In this study, we used the CRISPR/Cas9 gene editing system to deliver a self-cleaving polycistronic guide RNA (gRNA) designed to target exon 1 of the Phytoene desaturase (PDS) gene in the Cavendish cultivar “Williams”. Genotyping of 19 independent events showed a 100% PDS modification rate primarily in the form of insertions (1–105 nt) or deletions (1–55 nt) (indels) at the predicted cleavage site. Tri-allelic disruptive modifications were observed in 63% of plants and resulted in both albinism and dwarfing. Pale green (16%) and wildtype green (21%) phenotypes generally correlated with in-frame indels in at least one of the three PDS alleles. Editing efficiency was dependent on both target site selection and Cas9 abundance. This is the first report of a highly effective CRISPR/Cas9 modification system using a polycistronic gRNA in Cavendish banana. Such an editing platform will be of considerable utility for the development of disease resistance and novel agro-traits in this commercially important cultivar into the future.

Expressing double-stranded RNA (dsRNA) in transgenic plants to silence essential genes within herbivorous pests is referred to as trans-kingdom RNA interference (TK-RNAi) and has emerged as a promising strategy for crop protection. However, the dicing of dsRNA into siRNAs by the plant’s intrinsic RNAi machinery may reduce this pesticidal activity. Therefore, genetic constructs, encoding ∼200 nt duplex-stemmed-hairpin (hp) RNAs, targeting the acetylcholinesterase gene of the cotton bollworm, Helicoverpa armigera, were integrated into either the nuclear or the chloroplast genome of Nicotiana benthamiana. Undiced, full-length hpRNAs accumulated in transplastomic lines of N. benthamiana and conferred strong protection against H. armigera herbivory while the hpRNAs of nuclear-transformed plants were processed into siRNAs and gave more modest anti-feeding activity. This suggests that there is little or no RNAi machinery or activity in the chloroplast, that hpRNAs produced within this organelle do not enter the cytoplasm, and that oral delivery of chloroplast-packaged intact hpRNA is a more effective means of delivering TK-RNAi than using nuclear encoded hpRNAs. This contrasts with a recently reported correlation between siRNA expression and effectiveness of TK-RNAi targeting the chitinase gene of H. armigera, but is consistent with reports of efficient TK-RNAi by dsRNA generated in chloroplasts by converging promoters flanking a pest gene sequence and from very small (21 nt-stem) hpRNAs resembling artificial miRNAs. Here we demonstrate that hpRNAs, constructed along the conventional design principles of plant RNAi constructs but integrated into the chloroplast genome, are stable and effective over multiple generations, and hold the promise of providing durable pest resistance in crops.

Introducing a new trait into a crop through conventional breeding commonly takes decades, but recently developed genome sequence modification technology has the potential to accelerate this process. One of these new breeding technologies relies on an RNA-directed DNA nuclease (CRISPR/Cas9) to cut the genomic DNA, in vivo, to facilitate the deletion or insertion of sequences. This sequence specific targeting is determined by guide RNAs (gRNAs). However, choosing an optimum gRNA sequence has its challenges. Almost all current gRNA design tools for use in plants are based on data from experiments in animals, although many allow the use of plant genomes to identify potential off-target sites. Here, we examine the predictive uniformity and performance of eight different online gRNA-site tools. Unfortunately, there was little consensus among the rankings by the different algorithms, nor a statistically significant correlation between rankings and in vivo effectiveness. This suggests that important factors affecting gRNA performance and/or target site accessibility, in plants, are yet to be elucidated and incorporated into gRNA-site prediction tools.

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.

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