Agrobacterium-mediated transient transfection of Nicotiana benthamiana remains the most popular method for rapid synthesis of heterologous proteins in plants; yet relatively little is known about agrobacterial population stability, physiological state or plasmid maintenance following infiltration into N. benthamiana leaves. Developing a better understanding of post-infiltration agrobacterium populations is important for designing new tools and strategies to exploit the agrobacterium–plant interaction using synthetic biology. In this study, we developed molecular tools and methods for monitoring and manipulating agrobacteria within leaf tissue. This capability may support the development of engineered agrobacteria for diverse applications such as reporting on plant physiology or synthesising additional metabolites while residing in the leaf, in parallel with plant metabolism. Nicotiana benthamiana is a preferred host for heterologous protein expression due to innate hyper-susceptibility to transfection and tolerance to infiltration of leaf tissue with agrobacteria (Bally et al., 2015), which transfer linear DNA (T-DNA) to the plant nucleus via a type IV secretion system and a set of chaperone proteins encoded by the vir genes. Transferred DNA is transcriptionally active and can direct high levels of heterologous protein synthesis. Transfection occurs within the first day after infiltrating leaves with a suspension of agrobacteria, and the titre of plant-synthesised heterologous protein typically peaks after 2–10 days before declining due to a combination of plant defence responses including RNA silencing and proteolysis (Grosse-Holz et al., 2018). Though transfection occurs on the same day as infiltration, viable agrobacteria are still present in leaves when plants are harvested for downstream processing (Knödler et al., 2023). To monitor agrobacteria post-infiltration, Agrobacterium fabrum GV3101::pMP90 (previously known as A. tumefaciens GV3101::pMP90 (De Saeger et al., 2021)) was co-transformed with a binary vector for transfecting plant cells with a T-DNA encoding overexpression of a red fluorescent protein (pGGDNR_mCherry, where the pGGDNR vector is a modification of pCAMBIA1301) and a second plasmid encoding constitutive intracellular overexpression of a green fluorescent protein (GFP) within the agrobacterium (pSEVA431_pH-tdGFP, where agrobacterial expression of pH-tdGFP is controlled by a synthetic bacterial promoter, J23100) (Figure 1a). Predicted transcription and translation initiation rates for pH-tdGFP under the control of different promoters used in this study are included in Table S1. Intracellular expression of pH-tdGFP by A. fabrum was sufficient to enable visual monitoring of A. fabrum populations during N. benthamiana infiltration and transfection (Figure 1b, Figure S1). Further details of plasmid design are provided in Appendix S1. Leaf tissue was sampled daily for 8 days post-infiltration. Leaf discs (1 cm diameter) were homogenised in phosphate buffered saline with 0.1% (w/v) Triton X-100 (3 μL extraction buffer per mg fresh sample weight), and green and red fluorescence signals corresponding to pH-tdGFP and mCherry were measured in 10 μL samples of leaf homogenate (complete details of all experimental methods are provided in the Appendix S1). The titre of heterologous mCherry protein synthesised by transfected plant cells peaked on the second day post-infiltration before steadily declining (Figure 1c); yet synthesis of pH-tdGFP by A. fabrum increased linearly for 8 days (Figure 1d). Nucleic acid staining has previously been used to pre-stain agrobacteria prior to infiltration and then directly visualise their attachment to plant cells (Simmons et al., 2012), but long-term monitoring of agrobacteria was not possible with this method due to loss of fluorescence in the first 72 h post-infiltration. The expression data presented in Figure 1d indicate that A. fabrum present in the leaf was not only viable but retained sufficient metabolic activity to support a linear increase in pH-tdGFP expression. Furthermore, the plasmid encoding pH-tdGFP overexpression was maintained by agrobacteria in the leaf for at least 8 days in the absence of antibiotic selection. Retention of the plasmid in the absence of selective pressure may be due to slow agrobacterial growth rates within leaf tissue limiting the opportunity for plasmid loss. The discovery that agrobacteria can overexpress heterologous proteins for more than a week after infiltrating leaf tissue opens the possibility of implementing additional genetic programmes in agrobacteria post-infiltration. Samples of A. fabrum were extracted from leaf tissue at 0, 2, 4, 6 and 8 days post-infiltration and inoculated into a minimal defined culture medium to further evaluate agrobacterial viability and maintenance of the pH-tdGFP expression plasmid. Culture media included rifampicin and gentamycin to minimise growth of non-agrobacterial strains but excluded spectinomycin and kanamycin required for selection of pSEVA431_pH-tdGFP and pGGDNR_mCherry plasmids. The maximum growth rate of A. fabrum after extraction from leaf tissue was 0.095 h−1 ± 0.028 (mean ± standard deviation) and there was no significant difference in maximum growth rate between untransformed A. fabrum or strains bearing pSEVA431_pH-tdGFP, pGGDNR_mCherry or both plasmids (Figure S2). There was also no significant difference in lag phase (the time taken to reach maximum growth rate) between samples taken 2, 4, 6 or 8 days post-infiltration (one-way ANOVA), suggesting that agrobacteria have a comparable physiological state across this sampling period, characterised by very low growth rate but with sufficient metabolic activity to support heterologous protein expression. Only samples extracted and inoculated into liquid culture on day zero immediately post-infiltration took significantly longer (one-way ANOVA P < 0.0001) to adapt to growth in minimal defined liquid medium and reach the maximum growth rate (Figure S3). Expression of pH-tdGFP continued in liquid cultures after extraction from leaves, and after 36 h of cultivation, there was no significant difference in pH-tdGFP titre between samples extracted from leaves 2, 4, 6 or 8 days post-infiltration (Figure S4). The same was observed for mCherry expression with strains transformed with the pGGDNR_mCherry plasmid (where mCherry expression was controlled by the cauliflower mosaic virus 35S promoter, which is known to drive low levels of expression in agrobacteria (Jacob et al., 2002)) (Figure S5). These data demonstrate retention of both plasmids for at least a week in the absence of selective pressure. Three different inducible promoter systems were tested to evaluate whether transgene expression could be chemically induced in agrobacteria resident in N. benthamiana leaves. Salicylate-inducible (Meyer et al., 2019) (P_nahRAM), anhydrotetracycline-inducible (Meyer et al., 2019) (P_tetR) and 3-hydroxybenzoate-inducible (Martínez-García et al., 2023) (P_xyls/Pm) promoters were coupled to the pH-tdGFP reporter. Promoter performance was initially characterised in A. fabrum in liquid culture. The greatest dynamic range and total pH-tdGFP signal were observed with the salicylate-inducible promoter P_nahRAM (Figure 1e and Figure S6), where the pH-tdGFP signal was induced >80-fold upon application of 100 μM sodium salicylate, and the total signal was more than double that observed with synthetic constitutive promoters P_J23100 and P_J23111. Expression from the P_xylS/Pm promoter (induced with 1 mM 3-hydroxybenzoate) was comparable to the P_J23100 constitutive promoter, though high background expression was observed in the uninduced state (Figure S6). Weak anhydrotetracycline-inducible expression was observed with P_tetR (Figure 1e and Figure S4). Agrobacteria in the A. fabrum C58 lineage frequently develop spontaneous resistance to tetracyclines (Luo and Farrand, 1999), which could be a potential cause of the minimal expression observed in P_tetR strains. The P_nahRAM promoter was selected for testing in plant infiltration experiments due to its high dynamic range. Agrobacterium fabrum transformed with P_nahRAM was infiltrated into N. benthamiana leaves. Three days post-infiltration, a 100 μM solution of sodium salicylate was infiltrated into the same leaves, and pH-tdGFP expression was monitored with daily sampling. Three days after salicylate treatment, expression of pH-tdGFP had increased by more than fivefold (6 days after the initial infiltration of agrobacteria into N. benthamiana leaves) (Figure 1f). Inducible reporter protein expression was not observed in N. benthamiana leaves when using the anhydrotetracycline (P_tetR) or 3-hydroxybenzoate (P_xylS/Pm)-inducible promoters (Figure S7). Poor induction may be due in part to the catabolism of chemical inducers by the plant host and removal from leaves via vascular transport. The low induction responses observed relative to those seen in agrobacterial liquid cultures highlight the need for very strong inducible promoters with a high dynamic range, such as P_nahRAM, or promoters that are inducible via environmental signals rather than small molecules. Salicylic acid is upregulated during a variety of plant stress responses, and exogenous application of salicylic acid is associated with increased pathogen defence in N. benthamiana (Jiang et al., 2021). Despite the possibility of enhanced pathogen defence, salicylate-induced pH-tdGFP synthesis by A. fabrum was comparable to that observed with the strong constitutive promoter P_J23100, and significantly more than the background signal from the pSEVA231 negative control (Figure 1f). Subjecting N. benthamiana to drought stress also induced a >2-fold increase in pH-tdGFP expression by A. fabrum bearing the P_nahRAM plasmid (Figure S8). Agrobacterium fabrum is capable of inducible and stable protein overexpression in leaves for several days post-infiltration, beyond the typical sampling window for plant-based transient protein expression. This opens new opportunities for engineering host–microbe interactions in leaf tissue, such as endowing agrobacteria with new functions as a sensor and reporter of host metabolism and product formation, or as a new compartment contributing metabolites or co-products. WH, ZL and JBB contributed to all experiments, design, analysis and drafting the manuscript. KM and SM contributed to experiments. CT supported confocal microscopy. RES, PW and FS contributed to design and drafting the manuscript. This work was supported by the Australian Research Council Centre of Excellence in Synthetic Biology (project CE200100029). Confocal microscopy analyses were performed at the Central Analytical Research Facility operated by Research Infrastructure (QUT). The pSEVA231 and pSEVA431 plasmids were a generous gift from the Standard European Vector Architecture collection (https://seva-plasmids.com). The data that supports the findings of this study are available in the supplementary material of this article. Appendix S1 Materials and methods. Table S1 Plasmid information. Figure S1 Agrobacterial pH-tdGFP and plant-based mCherry fluorescence. Figure S2 Growth rate of A. fabrum extracted from leaves. Figure S3 Lag phase of A. fabrum extracted from leaves. Figure S4 Expression of pH-tdGFP by A. fabrum in liquid culture after extraction from N. benthamiana leaves. Figure S5 Expression of mCherry by A. fabrum in liquid culture after extraction from N. benthamiana leaves. Figure S6 Inducible expression of pH-tdGFP by A. fabrum in liquid culture. Figure S7 Expression of pH-tdGFP regulated by different promoters in A. fabrum within N. benthamiana leaves, six days post-infiltration. Figure S8 Expression of pH-tdGFP in A. fabrum within leaves of drought stressed N. benthamiana, seven days post-infiltration. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Nicotiana benthamiana is an invaluable model plant and biotechnology platform with a ~3 Gb allotetraploid genome. To further improve its usefulness and versatility, we have produced high-quality chromosome-level genome assemblies, coupled with transcriptome, epigenome, microRNA and transposable element datasets, for the ubiquitously used LAB strain and a related wild accession, QLD. In addition, single nucleotide polymorphism maps have been produced for a further two laboratory strains and four wild accessions. Despite the loss of five chromosomes from the ancestral tetraploid, expansion of intergenic regions, widespread segmental allopolyploidy, advanced diploidization and evidence of recent bursts of Copia pseudovirus (Copia) mobility not seen in other Nicotiana genomes, the two subgenomes of N. benthamiana show large regions of synteny across the Solanaceae. LAB and QLD have many genetic, metabolic and phenotypic differences, including disparate RNA interference responses, but are highly interfertile and amenable to genome editing and both transient and stable transformation. The LAB/QLD combination has the potential to be as useful as the Columbia-0/Landsberg errecta partnership, utilized from the early pioneering days of Arabidopsis genomics to today.

SQUAMOSA PROMOTER BINDING-LIKE (SPL) proteins constitute a large family of transcription factors known to play key roles in growth and developmental processes, including juvenile-to-adult and vegetative-to-reproductive phase transitions. This makes SPLs interesting targets for precision breeding in plants of the Nicotiana genus used as e.g. recombinant biofactories. We report the identification of 49 SPL genes in Nicotiana tabacum cv. K326 and 43 SPL genes in Nicotiana benthamiana LAB strain, which were classified into eight phylogenetic groups according to the SPL classification in Arabidopsis. Exon-intron gene structure and DNA-binding domains were highly conserved between homeologues and orthologues. Thirty of the NbSPL genes and 33 of the NtSPL genes were found to be possible targets of microRNA 156. The expression of SPL genes in leaves was analysed by RNA-seq at three different stages, revealing that genes not under miR156 control were in general constitutively expressed at high levels, whereas miR156-regulated genes showed lower expression, often developmentally regulated. We selected the N. benthamiana SPL13_1a gene as target for a CRISPR/Cas9 knock-out experiment. We show here that a full knock-out in this single gene leads to a significant delay in flowering time, a trait that could be exploited to increase biomass for recombinant protein production.

Inter-tissue communication is instrumental to coordinating the whole-body level behaviour for complex multicellular organisms. However, little is known about the regulation of inter-tissue information exchange. Here we carried out genetic screens for root-to-shoot mobile silencing in Arabidopsis plants with a compromised small RNA-mediated gene silencing movement rate and identified radical-induced cell death 1 (RCD1) as a critical regulator of root-shoot communication. RCD1 belongs to a family of poly (ADP-ribose) polymerase proteins, which are highly conserved across land plants. We found that RCD1 coordinates symplastic and apoplastic movement by modulating the sterol level of lipid rafts. The higher superoxide production in rcd1-knockout plants resulted in lower plasmodesmata (PD) frequency and altered PD structure in the symplasm of the hypocotyl cortex. Furthermore, the mutants showed increased lateral area of tracheary pits, which reduced axial movement. Our study highlights a novel mechanism through which root-to-shoot long-distance signalling can be modulated both symplastically and apoplastically.

Immunity cell-surface receptors Ve1 and Ve2 protect against fungi of the genus Verticillium causing early dying, a worldwide disease in many crops. Characterization of microbe-associated molecular pattern immunity receptors has advanced our understanding of disease resistance but signal amplification remains elusive. Here, we report that transgenic plants expressing Ve1 and Ve2 together, reduced pathogen titres by a further 90% compared to plants expressing only Ve1 or Ve2. Confocal and immunoprecipitation confirm that the two receptors associate to form heteromeric complexes in the absence of the ligand and positively regulate signaling. Bioassays show that the Ve1Ve2 complex activates race-specific amplified immunity to the pathogen through a rapid burst of reactive oxygen species (ROS). These results indicate a mechanism by which the composition of a cell-surface receptor heterocomplex may be optimized to increase immunity against devastating plant diseases.

The role of terminators is more commonly associated with the polyadenylation and 3′ end formation of new transcripts. Recent evidence, however, suggests that this regulatory region can have a dramatic impact on gene expression. Nonetheless, little is known about the molecular mechanisms leading to the improvements associated with terminator usage in plants and the different elements in a plant terminator. Here, we identified an element in the Arabidopsis HSP18.2 terminator (tHSP) to be essential for the high level of expression seen for transgenes under the regulation of this terminator. Our molecular analyses suggest that this newly identified sequence acts to improve transcription termination, leading to fewer read-through events and decreased amounts of small RNAs originating from the transgene. Besides protecting against silencing, the tHSP-derived sequence positively impacts splicing efficiency, helping to promote gene expression. Moreover, we show that this sequence can be used to generate chimeric terminators with enhanced efficiency, resulting in stronger transgene expression and significantly expanding the availability of efficient terminators that can be part of good expression systems. Thus, our data make an important contribution toward a better understanding of plant terminators, with the identification of a new element that has a direct impact on gene expression, and at the same time, creates new possibilities to modulate gene expression via the manipulation of 3′ regulatory regions.

Topical application of double-stranded RNA (dsRNA) as RNA interference(RNAi) based biopesticides represents a sustainable alternative to traditional transgenic, breeding-based or chemical crop protection strategies. A key feature of RNAi is its ability to act non-cell autonomously, a process that plays a critical role in plant protection. However, the uptake of dsRNA upon topical application, and its ability to move and act non-cell autonomously remains debated and largely unexplored. Here we show that when applied to a leaf, unprocessed full-length dsRNA enters the vasculature and rapidly moves to multiple distal below ground, vegetative and reproductive tissue types in several model plant and crop hosts. Intact unprocessed dsRNA was detected in the apoplast of leaves, roots and flowers after leaf application and maintained in subsequent new growth. Furthermore, we show mobile dsRNA is functional against root infecting fungal and foliar viral pathogens. Our demonstration of the uptake and maintained movement of intact and functional dsRNA stands to add significant benefit to the emerging field of RNAi-based plant protection.

Nicotiana is found predominantly in the Americas and Australia, but also has representatives in Africa and the Pacific Islands. All native Australian Nicotiana species belong to section Suaveolentes. The number of species in this section is uncertain and subject to revision. An example of this uncertainty is the taxonomic status of a South Australian Nicotiana accession colloquially termed 'Corunna'. Here, we report sequences for nuclear and plastid markers for N. sp. Corunna (D.E.Symon 17088) and accessions of two other Australian species, N. burbidgeae and N. benthamiana. Phylogenetic comparison of these sequences with those of other members of Nicotiana places all three taxa in N. section Suaveolentes and shows that 'Corunna' represents a distinct phylogenetic lineage in a well supported clade along with N. goodspeedii, N. maritima, N. amplexicaulis and N. suaveolentes. Phenetic analysis of floral characters also supports recognition of N. sp. Corunna (D.E.Symon 17088) as a distinct species, which we describe here as Nicotiana paulineana Newbigin & P.M.Waterh., sp. nov. The enlarged molecular dataset described here contributes to a better understanding of taxonomic relationships within the section.

For millennia, natural and artificial selection has combined favourable alleles for desirable traits in crop species. While modern plant breeding has achieved steady increases in crop yields over the last century, on the current trajectory we will simply not meet demand by 2045. Novel breeding strategies and sources of genetic variation will be required to sustainably fill predicted yield gaps and meet new consumer preferences. Here, we highlight that stepping up to meet this grand challenge will increasingly require thinking 'beyond the gene'. Significant progress has been made in understanding the contributions of both epigenetic variation and cis-regulatory variation to plant traits. This non-genic variation has great potential in future breeding, synthetic biology and biotechnology applications.

Helicoverpa armigera, the cotton bollworm, is a major insect pest for a wide range of agricultural crops. It causes huge yield losses through feeding damage and increasing the crop’s vulnerability to bacterial and fungal infection. H. armigera has evolved substantial resistance to many different chemical insecticides, prompting the development of transgenic crop plants with alternative insect-resistance-conferring mechanisms. For example, transgenic crops producing Bacillus thuringiensis (Bt) toxins have been very successful. However, there is still a concern about insect populations emerging with resistance to these biopesticides. Novel strategies that give effective protection, without affecting the environment, need to be continuously developed and implemented. Such a strategy is Trans-kingdom RNAi, which is based on making plants express double-stranded (ds) or hairpin (hp) RNA for ingestion by herbivorous pests. The RNA triggers silencing of specific genes within the pest leading to its death or impaired growth. However, the efficacy of the approach appears to depend on the means of delivering the RNA. We will describe new approaches and delivery strategies, including chloroplast-based expression, which greatly enhance the potency of insect protection.

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.

Plant viruses are commonly vectored by flying or crawling animals, such as aphids and beetles, and cause serious losses in major agricultural and horticultural crops. Controlling virus spread is often achieved by minimizing a crop's exposure to the vector, or by reducing vector numbers with compounds such as insecticides. A major, but less obvious, factor not controlled by these measures is Homo sapiens. Here, we discuss the inconvenient truth of how humans have become superspreaders of plant viruses on both a local and a global scale.

All flowering plants have evolved through multiple rounds of polyploidy throughout the evolutionary process. Intergenomic interactions between subgenomes in polyploid plants are predicted to induce chromatin modifications such as histone modifications to regulate expression of gene homoeologs. Nicotiana benthamiana is an ancient allotetraploid plant with ecotypes collected from climatically diverse regions of Australia. Studying the chromatin landscape of this unique collection will likely shed light on the importance of chromatin modifications in gene regulation in polyploids as well its implications in adaptation of plants in environmentally diverse conditions. Generally, chromatin immunoprecipitation and high throughput DNA sequencing (ChIP-seq) is used to study chromatin modifications. However, due to the starchy nature of mature N. benthamiana leaves, previously published protocols were unsuitable. The higher amounts of starch in leaves that co-precipitated with nuclei hindered downstream processing of DNA. Here we present an optimised ChIP protocol for N. benthamiana leaves to facilitate comparison of chromatin modifications in two closely related ecotypes. Several steps of ChIP were optimised including tissue harvesting, nuclei isolation, nuclei storage, DNA shearing and DNA recovery. Commonly available antibodies targeting histone 3 lysine 4 trimethylation (H3K4me3) and histone 3 lysine 9 dimethylation (H3K9me2) histone modifications were used and success of ChIP was confirmed by PCR and next generation sequencing. Collectively, our optimised method is the first comprehensive ChIP method for mature starchy leaves of N. benthamiana to enable studies of chromatin landscape at the genome-wide scale.

The availability of recent state-of-the-art long-read sequencing technologies has significantly increased the ease and speed of producing high-quality plant genome assemblies. A wide variety of genome-related software tools are now available and they are typically benchmarked using microbial or model eukaryotic genomes such as Arabidopsis and rice. However, many plant species have much larger and more complex genomes than these, and the choice of tools, parameters, and/or strategies that can be used is not always obvious. Thus, we have compared the metrics of assemblies generated by various pipelines to discuss how assembly quality can be affected by two different assembly strategies. First, we focused on optimizing read preprocessing and assembler variables using eight different de novo assemblers on five different Pacific Biosciences long-read datasets of diploid and tetraploid species. Then, we examined a single scaffolding tool (quickmerge) that has been employed for the postprocessing step. We then merged the outputs from multiple assemblies to produce a higher quality consensus assembly. Then, we benchmarked the assemblies for completeness and accuracy (assembly metrics and BUSCO), computer memory, and CPU times. Two lightweight assemblers, Miniasm/Minimap/Racon and WTDBG, were deemed good for novice users because they involved smaller required learning curves and light computational resources. However, two heavyweight tools, CANU and Flye, should be the first choice when the goal is to achieve accurate and complete assemblies. Our results will provide valuable guidance in future plant genome projects and beyond.

Summary The cotton bollworm, Helicoverpa armigera , is a major insect pest for a wide range of agricultural crops. It causes significant yield loss through feeding damage and by increasing the crop’s vulnerability to bacterial and fungal infections. Although expression of Bacillus thuringiensis (Bt) toxins in transgenic crops has been very successful in protecting against insect pests, including H. armigera , field‐evolved resistance has occurred in multiple species. To manage resistant populations, new protection strategies must be continuously developed. Trans‐kingdom RNA interference (TK‐RNAi) is a promising method for controlling herbivorous pests. TK‐RNAi is based on delivering dsRNA or hairpin RNA containing essential insect gene sequences to the feeding insect. The ingested molecules are processed by the insect’s RNAi machinery and guide it to silence the target genes. Recently, TK‐RNAi delivery has been enhanced by expressing the ds‐ or hpRNAs in the chloroplast. This compartmentalizes the duplexed RNA away from the plant’s RNAi machinery, ensuring that it is delivered in an unprocessed form to the insect. Here, we report another alternative approach for delivering precursor anti‐insect RNA in plants. Insect pre‐microRNA (pre‐miR) transcripts were modified to contain artificial microRNAs (amiRs), targeting insect genes, and expressed in transgenic Nicotiana benthamiana plants. These modified pre‐miRs remained largely unprocessed in the plants, and H. armigera feeding on leaves from these plants had increased mortality, developmental abnormalities and delayed growth rates. This shows that plant‐expressed insect pre‐amiRs (plin‐amiRs) are a new strategy of protecting plants against herbivorous insects.

Transgenes have become essential to modern biology, being an important tool in functional genomic studies and also in the development of biotechnological products. One of the major challenges in the generation of transgenic lines concerns the expression of transgenes, which, compared to endogenes, are particularly susceptible to silencing mediated by small RNAs (sRNAs). Several reasons have been put forward to explain why transgenes often trigger the production of sRNAs, such as the high level of expression induced by commonly used strong constitutive promoters, the lack of introns, and features resembling viral and other exogenous sequences. However, the relative contributions of the different genomic elements with respect to protecting genes from the silencing machinery and their molecular mechanisms remain unclear. Here, we present the results of a mutagenesis screen conceived to identify features involved in the protection of endogenes against becoming a template for the production of sRNAs. Interestingly, all of the recovered mutants had alterations in genes with proposed function in transcription termination, suggesting a central role of terminators in this process. Indeed, using a GFP reporter system, we show that, among different genetic elements tested, the terminator sequence had the greatest effect on transgene-derived sRNA accumulation and that a well-defined poly(A) site might be especially important. Finally, we describe an unexpected mechanism, where transgenes containing certain intron/terminator combinations lead to an increase in the production of sRNAs, which appears to interfere with splicing.

Catharanthus roseus is an important medicinal plant with a capacity to synthesize >130 monoterpinoid indole alkaloids (MIA). Many of these compounds, including the chemotherapeutics vinblastine, vincristine and their chemical derivatives have high economic value with diverse applications. Vincristine is the only effective antileukaemic drug that can drastically reduce white blood cell counts. Since the 1950s, it has increased the survival rate of children with leukaemia from 20% to 80% (Nejat et al., 2015). However, due to its scarcity, vincristine is one of the most expensive plant-derived compounds on the market and supply cannot meet demand (Kumar et al., 2013). Total chemical synthesis of vinblastine and vincristine is inefficient due to their structural complexity and stereochemistry and industrial production relies on the extraction of precursors from C. roseus in which the MIAs accumulate in trace amounts; for example 5.0 and 0.5 ppm for vinblastine and vincristine, respectively. Accordingly, recent attention has shifted to the development of new approaches to elevate MIA levels in C. roseus. The MIA biosynthetic pathway, however, is intricate and composed of around 30 to 50 enzymatic steps with multifaceted metabolic regulation involving intracellular compartmentalization, different cell types and tissue differentiation, making it a complex target for metabolic engineering (Dugé de Bernonville et al., 2015, Nejat et al., 2015). Furthermore, unravelling the MIA biosynthetic pathway and its regulation in C. roseus has been hindered by a paucity of technologies to express candidate genes in planta. With the exception of a virus-induced gene silencing (VIGS) system (Liscombe and O'Connor, 2011), genomic approaches that enable gene functions to be explored in vivo have been most effective in (and often limited to) hairy root and cell suspension cultures. These systems lack the required level of cyto- and tissue-differentiation essential for the expression of MIA pathway genes and, therefore, do not truly reflect MIA biosynthesis and its regulation in planta (Liscombe and O'Connor, 2011). To overcome this, we have utilized the replication machinery of a C. roseus-infecting geminivirus to develop an autonomously replicating viral expression system that provides consistent and high gene expression in C. roseus leaves. Geminiviruses are small, circular, single-stranded DNA viruses that infect monocotyledonous and dicotyledonous plants in tropical and subtropical regions. The family Geminiviridae is divided into nine genera, of which the Begomovirus genus is the largest consisting of both monopartite and bipartite genome types (Hanley-Bowdoin et al., 2013). Geminiviruses replicate their genomes exclusively in the nucleus of the infected host cell via a rolling circle replication process (Figure 1b) initiated at a consensus stem loop structure located in the intergenic region (IR). The IR also serves to direct divergent transcription of both virion and complementary sense genes. A coat protein (CP) forms the viral capsid and mediates vector transmission, V2 and AV2 function as anti-defence proteins to inhibit post-transcriptional gene silencing (PTGS) and the Replication initiator protein (Rep) initiates viral replication. Begomoviruses encode three additional gene products, the transcriptional activator protein (TrAP; and the related C2 protein) interfere with transcriptional gene silencing (TGS) and PTGS, the replication enhancer protein (REn) is involved in viral replication and the C4 protein counteracts PTGS (Hanley-Bowdoin et al., 2013). In 2012, the complete nucleotide sequence of a bipartite begomovirus was isolated from C. roseus, the virus was named Catharanthus yellow mosaic virus (CaYMV) (Ilyas et al., 2013). We designed a CaYMV-based replicating vector system for transient gene expression in C. roseus; illustrated in Figure 1a. The system contains the CaYMV Rep, Ren and TrAP genes under the transcriptional control of their native promoter sequences and located between repeats of the CaYMV IR. A cauliflower mosaic virus (CaMV) 35S 3′ UTR was inserted downstream of REn to effectively terminate transcription. The CP gene was not included in order to render the system non-infectious. Outside the replicative unit, but within the T-DNA, was located an expression cassette containing the CaYMV AV2 gene under the transcriptional control of an enhanced CaMV 35S promoter (2X35S), potato gbbs gene intron IV and alfalfa mosaic virus translational enhancer. The AV2 gene was included in the expression system based on its anti-defence role during virus infection. AV2 was placed outside of the replication unit to moderate its expression as AV2 proteins can trigger a hypersensitive response (Mubin et al., 2010). A multiple cloning site (MCS) was included in the replication unit in order to insert the transgene of interest. The CaYMV-based cassette was chemically synthesized and mobilized into the binary plasmid pBIN+ to generate vector pBIN+CaYMV (Figure 1a). An expression cassette containing an Orange florescent protein reporter gene sequence and synthetic intron (syntron) under the transcriptional control of a 2X35S promoter was inserted into pBIN+CaYMV as an AsiSI-AvrII restriction fragment to create vector pBIN+CaYMV-Orange (Figure 1b). The syntron was included to prevent Orange expression in Agrobacteria. Following Rep-initiated replicative release, the single-stranded, circular episomal molecule is converted to a double-stranded DNA form via the origin of second strand synthesis and by host polymerases (Figure 1c). This form is both transcriptionally active and serves as a template for further rolling circle amplification resulting in high episomal copy number and high transgene expression levels. The vector pBIN+CaYMV-Orange was mobilized into Agrobacterium tumefaciens (strain AGL1) by electroporation and agroinfiltrated into C. roseus leaves via the lower epidermis with a needle-less syringe, at an OD600 of 1.5. Transient expression of Orange in C. roseus leaves agroinfiltrated with pBIN+CaYMV-Orange was assessed by visualizing florescence with a Dual Florescent Protein (DFP) flashlight and a Royal Blue LED and Yellow filter. Six days after agroinfiltration, fluorescence was compared to wild-type (WT) tissue and leaves infiltrated with a non-replicating vector (pBIN+2X35S-ORANGE). Expression of Orange was significantly higher and more uniform in C. roseus leaves agroinfiltrated with pBIN+CaYMV-Orange (Figure 1d). To prove that high level Orange expression was the result of Rep-mediated replication of the expression unit, the Rep gene sequence in pBIN+CaYMV-Orange was disrupted and the REn coding sequence removed, creating vector pBIN+CaYMV-Orange-NOREP. Minimal Orange florescence was observed in leaf tissue agroinfiltrated with this vector (Figure 1d). To demonstrate replicative release from the vector and the production of CaYMV-Orange episomal DNA forms, outwardly extending primers were designed to amplify a 1100 bp sequence spanning the CaYMV IR. These primers were used in a PCR with DNA extracted from leaves agroinfiltrated with pBIN+CaYMV-Orange and pBIN+CaYMV-Orange-NOREP, 6 days after agroinfiltration (Figure 1e). No PCR product was obtained from WT leaves (Figure 1e, Lane 1). A strong PCR product of ~1100 bp was amplified from leaves agroinfiltrated with pBIN+CaYMV-Orange suggesting extra-chromosomal episome formation and Rep-mediated amplification, whereas a very weak ~1100 bp product was amplified from leaves agroinfiltrated with pBIN+CaYMV-Orange-NOREP, indicating host-mediated recombination and low-level episome formation, independent of Rep (Figure 1e, Lanes 2 and 3, respectively). We have designed an autonomously replicating viral expression platform based on the genome of CaYMV and tailored for high and consistent gene expression in the leaves of C. roseus. Up to this point, the lack of a reliable transgene expression system in this host has been a limitation to better understanding and manipulating MIA biosynthesis and regulation. The CaYMV system could, for example, be used to express candidate genes involved in the transport of MIA intermediates between plant cells and organs, such as a recently proposed but as yet uncharacterized secologanin transporter (Kidd et al., 2019). The platform described here opens up new pathways in MIA research and product development. Our approach also highlights how viruses can be exploited to develop tailored gene expression systems for specific species that are recalcitrant to established gene expression technologies. This work was supported by a discovery early career research award granted to C.M. by the Australian Research Council. The authors have no competing interests. C.M., B.D. and P.W. designed the experiments, C.M. performed the experiments and analysed the results. CM and BD wrote the manuscript. All authors read and approved the final manuscript.

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.

The commercial release of third-generation sequencing technologies (TGSTs), giving long and ultra-long sequencing reads, has stimulated the development of new tools for assembling highly contiguous genome sequences with unprecedented accuracy across complex repeat regions. We survey here a wide range of emerging sequencing platforms and analytical tools for de novo assembly, provide background information for each of their steps, and discuss the spectrum of available options. Our decision tree recommends workflows for the generation of a high-quality genome assembly when used in combination with the specific needs and resources of a project.

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.