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.

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.

Identification and validation of suitable reference genes that exhibit robust transcriptional stability across many sample types is an absolute requirement of all qRT-PCR experiments. Often, however, only small numbers of reference genes, validated across limited sample types, are available for non-model species. This points to a clear need to assess and validate a wider range of potential reference genes than is currently available. We therefore looked to test and validate a large number of potential reference genes across a wide range of tissue types and treatments to determine the applicability of these reference genes for use in grapevine and other non-model plant species. Potential reference genes were selected based on stability of gene transcription in the model plant species Arabidopsis or due to their common use in the grapevine community. The selected reference genes were analyzed across two datasets consisting of a range of either 'Sauvignon blanc' or 'Pinot noir' tissues. A total of 11 potential reference genes were screened across the two datasets. Gene stability was analyzed by GeNorm, a widely used Excel application, or an ANOVA-based method developed in red clover. Both analysis methods showed that all 11 potential reference genes are stably expressed in the datasets tested, but the rankings of gene stability differed based on the datasets and analysis method used. Furthermore, the transcript stability of these genes, initially identified in Arabidopsis and now validated in grapevine, suggests applicability across a wide range of non-model plant species in addition to their utility in grapevine.

BackgroundAscorbate is a powerful antioxidant in plants and an essential micronutrient for humans. The GDP-L-galactose phosphorylase (GGP) gene encodes the rate-limiting enzyme of the L-galactose pathway—the dominant ascorbate biosynthetic pathway in plants—and is a promising gene candidate for increasing ascorbate in crops. In addition to transcriptional regulation, GGP production is regulated at the translational level through an upstream open reading frame (uORF) in the long 5′-untranslated region (5’UTR). The GGP genes have yet to be identified in bread wheat (Triticum aestivum L.), one of the most important food grain sources for humans.ResultsBread wheat chromosomal groups 4 and 5 were found to each contain three homoeologous TaGGP genes on the A, B, and D subgenomes (TaGGP2-A/B/D and TaGGP1-A/B/D, respectively) and a highly conserved uORF was present in the long 5’UTR of all six genes. Phylogenetic analyses demonstrated that the TaGGP genes separate into two distinct groups and identified a duplication event of the GGP gene in the ancestor of the Brachypodium/Triticeae lineage. A microsynteny analysis revealed that the TaGGP1 and TaGGP2 subchromosomal regions have no shared synteny suggesting that TaGGP2 may have been duplicated via a transposable element. The two groups of TaGGP genes have distinct expression patterns with the TaGGP1 homoeologs broadly expressed across different tissues and developmental stages and the TaGGP2 homoeologs highly expressed in anthers. Transient transformation of the TaGGP coding sequences in Nicotiana benthamiana leaf tissue increased ascorbate concentrations more than five-fold, confirming their functional role in ascorbate biosynthesis in planta.ConclusionsWe have identified six TaGGP genes in the bread wheat genome, each with a highly conserved uORF. Phylogenetic and microsynteny analyses highlight that a transposable element may have been responsible for the duplication and specialized expression of GGP2 in anthers in the Brachypodium/Triticeae lineage. Transient transformation of the TaGGP coding sequences in N. benthamiana demonstrated their activity in planta. The six TaGGP genes and uORFs identified in this study provide a valuable genetic resource for increasing ascorbate concentrations in bread wheat.

Agrobacterium tumefaciens has been foundational in the development of transgenic plants for both agricultural biotechnology and plant molecular research. However, the transformation efficiency and level of transgene expression obtained for any given construct can be highly variable. These inefficiencies often require screening of many lines to find one with consistent and heritable transgene expression. Transcriptional gene silencing is known to affect transgene expression, and is associated with DNA methylation, especially of cytosines in symmetric CG and CHG contexts. While the specificity, heritability and silencing-associated effects of DNA methylation of transgene sequences have been analyzed in many stably transformed plants, the methylation status of transgene sequences in the T-DNA during the transformation process has not been well-studied. Here we used agro-infiltration of the eGFP reporter gene in Nicotiana benthamiana leaves driven by either an AtEF1α-A4 or a CaMV-35S promoter to study early T-DNA methylation patterns of these promoter sequences. The T-DNA was examined by amplicon sequencing following sodium bisulfite treatment using three different sequencing platforms: Sanger sequencing, Ion Torrent PGM, and the Illumina MiSeq. Rapid DNA methylation was detectable in each promoter region just 2–3 days post-infiltration and the levels continued to rapidly accumulate over the first week, then steadily up to 21 days later. Cytosines in an asymmetric context (CHH) were the most heavily and rapidly methylated. This suggests that early T-DNA methylation may be important in determining the epigenetic and transcriptional fate of integrated transgenes. The Illumina MiSeq platform was the most sensitive and robust way of detecting and following the methylation profiles of the T-DNA promoters. The utility of the methods was then used to show a subtle but significant difference in promoter methylation during intron-mediated enhancement. In addition, the method was able to detect an increase in promoter methylation when the eGFP reporter gene was targeted by siRNAs generated by co-infiltration of a hairpin RNAi construct.

Gene technology regulators receive applications seeking permission for the environmental release of genetically modified (GM) plants, many of which possess beneficial traits such as improved production, enhanced nutrition and resistance to drought, pests and diseases. The regulators must assess the risks to human and animal health and to the environment from releasing these GM plants. One such consideration, of many, is the likelihood and potential consequence of the introduced or modified DNA being transferred to other organisms, including people. While such gene transfer is most likely to occur to sexually compatible relatives (vertical gene transfer), horizontal gene transfer (HGT), which is the acquisition of genetic material that has not been inherited from a parent, is also a possibility considered during these assessments. Advances in HGT detection, aided by next generation sequencing, have demonstrated that HGT occurrence may have been previously underestimated. In this review, we provide updated evidence on the likelihood, factors and the barriers for the introduced or modified DNA in GM plants to be horizontally transferred into a variety of recipients. We present the legislation and frameworks the Australian Gene Technology Regulator adheres to with respect to the consideration of risks posed by HGT. Such a perspective may generally be applicable to regulators in other jurisdictions as well as to commercial and research organisations who develop GM plants.

•In contrast to the prior voluntary system, since 2001, gene technology in Australia has been regulated under a legislated national Gene Technology Regulatory Scheme which is administered by the Gene Technology Regulator.•The Scheme provides science-based assessment of the potential risks of gene technology to the health and safety of people and the environment.•It complements the role of the Australian Therapeutic Goods Administration which regulates all therapeutic products in Australia to ensure they are safe and effective.•Recent reforms to the Scheme contribute to, and anticipate, the continued safe development and delivery of gene-based human therapeutics in Australia as a successful model for other jurisdictions.

γ-glutamyl transferases/transpeptidases (E.C. 2.3.2.2, GGTs) are involved in the catabolism of many compounds that are conjugated to glutathione (GSH), which have a variety of roles. GSH can act as storage and transport vehicle for reduced sulfur; it is involved in the detoxification of xenobiotics and also acts as a redox buffer by utilizing its thiol residue to protect against reactive oxygen species, which accumulate in response to biotic and abiotic stress. Furthermore, many distinctive flavor and aroma compounds in Sauvignon blanc wines originate from odorless C5- and C6-GSH conjugates or their GGT catabolized derivatives. These precursors are then processed into their volatile forms by yeast during fermentation. In many plant species, two or more isoforms of GGTs exist that target GSH-conjugates to either the apoplast or the vacuole. A bioinformatics approach identified multiple GGT candidates in grapevine (Vitis vinifera). However, only a single candidate, VvGGT3, has all the conserved residues needed for GGT activity. This is intriguing given the variety of roles of GSH and GGTs in plant cells. Characterization of VvGGT3 from cv. Sauvignon blanc was then undertaken. The VvGGT3 transcript is present in roots, leaves, inflorescences, and tendril and at equal abundance in the skin, pulp, and seed of mature berries and shows steady accumulation over the course of whole berry development. In addition, the VvGGT3 transcript in whole berries is upregulated upon Botrytis cinerea infection as well as mechanical damage to leaf tissue. VvGGT3-GFP fusion proteins transiently over-expressed in onion cells were used to study subcellular localization. To confirm VvGGT3 activity and localization in vivo, the fluorescent γ-glutamyl-7-amido-4-methylcoumarin substrate was added to Nicotiana benthamiana leaves transiently over-expressing VvGGT3. In combination, these results suggest that the functional VvGGT3 is associated with membrane-like structures. This is not consistent with its closely related functionally characterized GGTs from Arabidopsis, radish and garlic.