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Nicotiana benthamiana has long served as a crucial plant material extensively used in plant physiology research, particularly in the field of plant pathology, because of its high susceptibility to plant viruses. Additionally, it serves as a production platform to test vaccines and other valuable substances. Among its approximately 3.1 Gb genome, 57 583 genes have been annotated within a 61 Mb region. We created a comprehensive and easy-to-use platform to use transcriptomes for modern annotation. These tools allow to visualize gene expression profiles, draw molecular evolutionary phylogenetic trees of gene families, perform functional enrichment analyses, and facilitate output downloads. To demonstrate their utility, we analyzed the gene expression profiles of enzymes within the nicotine biosynthesis pathway, a secondary metabolic pathway characteristic of the Nicotiana genus. Using the developed tool, expression profiles of the nicotine biosynthesis pathway genes were generated. The expression patterns of eight gene groups in the pathway were strongly expressed in the roots and weakly expressed in leaves and flowers of N. benthamiana. The results were consistent with the established gene expression profiles in Nicotiana tabacum and provided insights into gene family composition and expression trends. The compilation of this database tool can facilitate genetic analysis of N. benthamiana in the future., (© 2024 The Author(s). The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Nicotiana benthamiana is predominantly distributed in arid habitats across northern Australia. However, none of six geographically isolated accessions shows obvious xerophytic morphological features. To investigate how these tender-looking plants withstand drought, we examined their responses to water deprivation, assessed phenotypic, physiological, and cellular responses, and analysed cuticular wax composition and wax biosynthesis gene expression profiles. Results showed that the Central Australia (CA) accession, globally known as a research tool, has evolved a drought escape strategy with early vigour, short life cycle, and weak, water loss-limiting responses. By contrast, a northern Queensland (NQ) accession responded to drought by slowing growth, inhibiting flowering, increasing leaf cuticle thickness, and altering cuticular wax composition. Under water stress, NQ increased the heat stability and water impermeability of its cuticle by extending the carbon backbone of cuticular long-chain alkanes from c. 25 to 33. This correlated with rapid upregulation of at least five wax biosynthesis genes. In CA, the alkane chain lengths (c. 25) and gene expression profiles remained largely unaltered. This study highlights complex genetic and environmental control over cuticle composition and provides evidence for divergence into at least two fundamentally different drought response strategies within the N. benthamiana species in < 1 million years., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Tomato Yellow Leaf Curl Virus (TYLCV) is one of the most devastating pathogens of tomato, worldwide. It is vectored by the globally prevalent whitefly, Bemisia tabaci, and is asymptomatic in a wide range of plant species that act as a virus reservoir. The most successful crop protection for tomato in the field has been from resistance genes, of which five loci have been introgressed fromwild relatives. Of these, the Ty-1/Ty-3 locus, which encodes an RNA-dependent RNA polymerase 3 (RDR3), has been the most effective. Nevertheless, several TYLCV strains that break this resistance are beginning to emerge, increasing the need for new sources of resistance. Here we use segregation analysis and CRISPR-mediated gene dysfunctionalisation to dissect the differential response of two isolates of Nicotiana benthamiana to TYLCV infection. Our study indicates the presence of a novel non-RDR3, but yet to be identified, TYLCV resistance gene in a wild accession of N. benthamiana . This gene has the potential to be incorporated into tomatoes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Hayashi, Souvan, Bally, de Felippes and Waterhouse.)

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., (© 2023. The Author(s).)

To be properly expressed, genes need to be accompanied by a terminator, a region downstream of the coding sequence that contains the information necessary for the maturation of the mRNA 3' end. The main event in this process is the addition of a poly(A) tail at the 3' end of the new transcript, a critical step in mRNA biology that has important consequences for the expression of genes. Here, we review the mechanism leading to cleavage and polyadenylation of newly transcribed mRNAs and how this process can affect the final levels of gene expression. We give special attention to an aspect often overlooked, the effect that different terminators can have on the expression of genes. We also discuss some exciting findings connecting the choice of terminator to the biogenesis of small RNAs, which are a central part of one of the most important mechanisms of regulation of gene expression in plants., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

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., (© 2022 John Wiley & Sons Ltd.)

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., (© 2022. Crown.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 de Felippes, Shand and Waterhouse.)

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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.

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., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

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., (© 2020 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Transitivity in plants is a mechanism that produces secondary small interfering RNAs (siRNAs) from a transcript targeted by primary small RNAs (sRNAs). It expands the silencing signal to additional sequences of the transcript. The process requires RNA-dependent RNA polymerases (RDRs), which convert single-stranded RNA targets into a double-stranded (ds) RNA, the precursor of siRNAs and is critical for effective and amplified responses to virus infection. It is also important for the production of endogenous secondary siRNAs, such as phased siRNAs (phasiRNAs), which regulate several genes involved in development and adaptation. Transitivity on endogenous transcripts is very specific, utilizing special primary sRNAs, such as miRNAs with unique features, and particular ARGONAUTEs. In contrast, transitivity on transgene and virus (exogenous) transcripts is more generic. This dichotomy of responses implies the existence of a mechanism that differentiates self from non-self targets. In this work, we examine the possible mechanistic process behind the dichotomy and the intriguing counter-intuitive directionality of transitive sequence-spread in plants., (Copyright © 2020 de Felippes and Waterhouse.)

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., Competing Interests: The authors have declared that no competing interests exist.

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.

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.

RNA interference (RNAi) was discovered almost 20 years ago and has been exploited worldwide to silence genes in plants and animals. A decade later, it was found that transforming plants with an RNAi construct targeting an insect gene could protect the plant against feeding by that insect. Production of double-stranded RNA (dsRNA) in a plant to affect the viability of a herbivorous animal is termed trans-kingdom RNAi (TK-RNAi). Since this pioneering work, there have been many further examples of successful TK-RNAi, but also reports of failed attempts and unrepeatable experiments. Recently, three laboratories have shown that producing dsRNA in a plant's chloroplast, rather than in its cellular cytoplasm, is a very effective way of delivering TK-RNAi. Our review examines this potentially game-changing approach and compares it with other transgenic insect-proofing schemes. © 2018 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2018 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

RNA silencing is a powerful tool deployed by plants against viral infection and abnormal gene expression. Plant viruses have evolved a suite of silencing suppressors for counter-defense, which are also widely used to boost transcript and protein accumulation in transient assays. However, only wild type silencing suppressor proteins have been reported to date. Here we demonstrate that P0 of Potato leafroll virus (PLRV), PLP0, can be split into two proteins that only show silencing suppression activity upon co-expression. We cloned each of these proteins in two different constructs and transiently co-infiltrated them in N. benthamiana leaves. We expressed a fluorescent protein from one of the vectors and observed that cells expressing both halves of PLP0 suppressed gene silencing. Further, we showed that Q system of Neurospora crassa, based on co-expression of a transcription activator and inhibitor, is functional in agroinfiltrated leaves of N. benthamiana. Q system combined with the split PLP0 system showed very tight co-expression of Q system's transcriptional activator and inhibitor. Altogether, our experiments demonstrate a functioning conditional silencing suppressor system and its potential as a powerful tool for transient expression in N. benthamiana leaves, as well as the application of the Q system in plants.

In plants, microRNAs (miRNAs) have evolved in parallel to the protein-coding genes that they target for expression regulation, and miRNA-directed gene expression regulation is central to almost every cellular process. MicroRNA, miR163, is unique to the Arabidopsis genus and is processed into a 24-nucleotide (nt) mature small regulatory RNA (sRNA) from a single precursor transcript transcribed from a single locus, the MIR163 gene. The MIR163 locus is a result of a recent inverted duplication event of one of the five closely related S-ADENOSYL-METHYLTRANSFERASE genes that the mature miR163 sRNA targets for expression regulation. Currently, however, little is known about the role of the miR163/S-ADENOSYL-METHYLTRANSFERASE regulatory module in response to biotic stress. Here, we document the expression domains of MIR163 and the S-ADENOSYL-METHYLTRANSFERASE target genes following fusion of their putative promoter sequences to the β-glucuronidase (GUS) reporter gene and subsequent in planta expression. Further, we report on our phenotypic and molecular assessment of Arabidopsis thaliana plants with altered miR163 accumulation, namely the mir163-1 and mir163-2 insertion knockout mutants and the miR163 overexpression line, the MIR163-OE plant. Finally, we reveal miR163 accumulation and S-ADENOSYL-METHYLTRANSFERASE target gene expression post treatment with the defence elicitors, salicylic acid and jasmonic acid, and following Fusarium oxysporum infection, wounding, and herbivory attack. Together, the work presented here provides a comprehensive new biological insight into the role played by the Arabidopsis genus-specific miR163/S-ADENOSYL-METHYLTRANSFERASE regulatory module in normal A. thaliana development and during the exposure of A. thaliana plants to biotic stress.

The plant viral family Luteoviridae is divided into three genera: Luteovirus , Polerovirus and Enamovirus . Without assistance from another virus, members of the family are confined to the cells of the host plant's vascular system. The first open reading frame (ORF) of poleroviruses and enamoviruses encodes P0 proteins which act as silencing suppressor proteins (VSRs) against the plant's viral defense-mediating RNA silencing machinery. Luteoviruses, such as barley yellow dwarf virus-PAV (BYDV-PAV), however, have no P0 to carry out the VSR role, so we investigated whether other proteins or RNAs encoded by BYDV-PAV confer protection against the plant's silencing machinery. Deep-sequencing of small RNAs from plants infected with BYDV-PAV revealed that the virus is subjected to RNA silencing in the phloem tissues and there was no evidence of protection afforded by a possible decoy effect of the highly abundant subgenomic RNA3. However, analysis of VSR activity among the BYDV-PAV ORFs revealed systemic silencing suppression by the P4 movement protein, and a similar, but weaker, activity by P6. The closely related BYDV-PAS P4, but not the polerovirus potato leafroll virus P4, also displayed systemic VSR activity. Both luteovirus and the polerovirus P4 proteins also showed transient, weak local silencing suppression. This suggests that systemic silencing suppression is the principal mechanism by which the luteoviruses BYDV-PAV and BYDV-PAS minimize the effects of the plant's anti-viral defense.

Quantitative proteomics strategies - which are playing important roles in the expanding field of plant molecular systems biology - are traditionally designated as either hypothesis driven or non-hypothesis driven. Many of these strategies aim to select individual peptide ions for tandem mass spectrometry (MS/MS), and to do this mixed hypothesis driven and non-hypothesis driven approaches are theoretically simple to implement. In-depth investigations into the efficacies of such approaches have, however, yet to be described. In this study, using combined samples of unlabeled and metabolically 15 N-labeled Arabidopsis thaliana proteins, we investigate the mixed use of targeted data acquisition (TDA) and data dependent acquisition (DDA) - referred to as TDA/DDA - to facilitate both hypothesis driven and non-hypothesis driven quantitative data collection in individual LC-MS/MS experiments. To investigate TDA/DDA for hypothesis driven data collection, 7 miRNA target proteins of differing size and abundance were targeted using inclusion lists comprised of 1558 m/z values, using 3 different TDA/DDA experimental designs. In samples in which targeted peptide ions were of particularly low abundance (i.e., predominantly only marginally above mass analyser detection limits), TDA/DDA produced statistically significant increases in the number of targeted peptides identified (230 ± 8 versus 80 ± 3 for DDA; p = 1.1 × 10 -3 ) and quantified (35 ± 3 versus 21 ± 2 for DDA; p = 0.038) per experiment relative to the use of DDA only. These expected improvements in hypothesis driven data collection were observed alongside unexpected improvements in non-hypothesis driven data collection. Untargeted peptide ions with m/z values matching those in inclusion lists were repeatedly identified and quantified across technical replicate TDA/DDA experiments, resulting in significant increases in the percentages of proteins repeatedly quantified in TDA/DDA experiments only relative to DDA experiments only (33.0 ± 2.6% versus 8.0 ± 2.7%, respectively; p = 0.011). These results were observed together with uncompromised broad-scale MS/MS data collection in TDA/DDA experiments relative to DDA experiments. Using our observations we provide guidelines for TDA/DDA method design for quantitative plant proteomics studies, and suggest that TDA/DDA is a broadly underutilized proteomics data acquisition strategy.

Viral infection triggers a range of plant responses such as the activation of the RNA interference (RNAi) pathway. The double-stranded RNA binding (DRB) proteins DRB3 and DRB4 are part of this pathway and aid in defending against DNA and RNA viruses, respectively. Using live cell imaging, we show that DRB2, DRB3, and DRB5 relocate from their uniform cytoplasmic distribution to concentrated accumulation in nascent viral replication complexes (VRC) that develop following cell invasion by viral RNA. Inactivation of the DRB3 gene in Arabidopsis by T-DNA insertion rendered these plants less able to repress RNA viral replication. We propose a model for the early stages of virus defense in which DRB2, DRB3, and DRB5 are invasion sensors that relocate to nascent VRC, where they bind to viral RNA and inhibit virus replication.

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.

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.

Transgenic engineering of plants is important in both basic and applied research. However, the expression of a transgene can dwindle over time as the plant's small (s)RNA-guided silencing pathways shut it down. The silencing pathways have evolved as antiviral defence mechanisms, and viruses have co-evolved viral silencing-suppressor proteins (VSPs) to block them. Therefore, VSPs have been routinely used alongside desired transgene constructs to enhance their expression in transient assays. However, constitutive, stable expression of a VSP in a plant usually causes pronounced developmental abnormalities, as their actions interfere with endogenous microRNA-regulated processes, and has largely precluded the use of VSPs as an aid to stable transgene expression. In an attempt to avoid the deleterious effects but obtain the enhancing effect, a number of different VSPs were expressed exclusively in the seeds of Arabidopsis thaliana alongside a three-step transgenic pathway for the synthesis of arachidonic acid (AA), an ω-6 long chain polyunsaturated fatty acid. Results from independent transgenic events, maintained for four generations, showed that the VSP-AA-transformed plants were developmentally normal, apart from minor phenotypes at the cotyledon stage, and could produce 40% more AA than plants transformed with the AA transgene cassette alone. Intriguingly, a geminivirus VSP, V2, was constitutively expressed without causing developmental defects, as it acts on the siRNA amplification step that is not part of the miRNA pathway, and gave strong transgene enhancement. These results demonstrate that VSP expression can be used to protect and enhance stable transgene performance and has significant biotechnological application., (© 2015 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Expression of the F-Box protein Leaf Curling Responsiveness (LCR) is regulated by microRNA, miR394, and alterations to this interplay in Arabidopsis thaliana produce defects in leaf polarity and shoot apical meristem organization. Although the miR394-LCR node has been documented in Arabidopsis, the identification of proteins targeted by LCR F-box itself has proven problematic. Here, a proteomic analysis of shoot apices from plants with altered LCR levels identified a member of the Latex Protein (MLP) family gene as a potential LCR F-box target. Bioinformatic and molecular analyses also suggested that other MLP family members are likely to be targets for this post-translational regulation. Direct interaction between LCR F-Box and MLP423 was validated. Additional MLP members had reduction in protein accumulation, in varying degrees, mediated by LCR F-Box. Transgenic Arabidopsis lines, in which MLP28 expression was reduced through an artificial miRNA technology, displayed severe developmental defects, including changes in leaf patterning and morphology, shoot apex defects, and eventual premature death. These phenotypic characteristics resemble those of Arabidopsis plants modified to over-express LCR Taken together, the results demonstrate that MLPs are driven to degradation by LCR, and indicate that MLP gene family is target of miR394-LCR regulatory node, representing potential targets for directly post-translational regulation mediated by LCR F-Box. In addition, MLP28 family member is associated with the LCR regulation that is critical for normal Arabidopsis development., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Horizontal gene transfer (HGT) is known to be a major force in genome evolution. The acquisition of genes from viruses by eukaryotic genomes is a well-studied example of HGT, including rare cases of non-retroviral RNA virus integration. The present study describes the integration of cucumber mosaic virus RNA-1 into soybean genome. After an initial metatranscriptomic analysis of small RNAs derived from soybean, the de novo assembly resulted a 3029-nt contig homologous to RNA-1. The integration of this sequence in the soybean genome was confirmed by DNA deep sequencing. The locus where the integration occurred harbors the full RNA-1 sequence followed by the partial sequence of an endogenous mRNA and another sequence of RNA-1 as an inverted repeat and allowing the formation of a hairpin structure. This region recombined into a retrotransposon located inside an exon of a soybean gene. The nucleotide similarity of the integrated sequence compared to other Cucumber mosaic virus sequences indicates that the integration event occurred recently. We described a rare event of non-retroviral RNA virus integration in soybean that leads to the production of a double-stranded RNA in a similar fashion to virus resistance RNAi plants., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Small open reading frames (sORFs) are an often overlooked feature of plant genomes. Initially found in plant viral RNAs and considered an interesting curiosity, an increasing number of these sORFs have been shown to encode functional peptides or play a regulatory role. The recent discovery that many of these sORFs initiate with start codons other than AUG, together with the identification of functional small peptides encoded in supposedly noncoding primary miRNA transcripts (pri-miRs), has drastically increased the number of potentially functional sORFs within the genome. Here we review how advances in technology, notably ribosome profiling (RP) assays, are complementing bioinformatics and proteogenomic methods to provide powerful ways to identify these elusive features of plant genomes, and highlight the regulatory roles sORFs can play., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In Arabidopsis thaliana, efficient microRNA (miRNA) production requires DICER-LIKE1 (DCL1) with the assistance of a partnering protein, DOUBLE-STRANDED RNA BINDING1 (DRB1) or DRB2. The presence of either of these DRB proteins is crucial to determine the mode of action of a miRNA; i.e., cleavage or translation inhibition. Here we studied the structural determinants for the role of DRB1 and DRB2 in the miRNA pathway. We developed a series of chimeric vectors encoding different functional domains of DRB1 and DRB2, and expressed these in the drb1 mutant background in Arabidopsis under the control of the native DRB1 promoter. Complementation of the drb1 developmental phenotype was used to assess the biological role that each functional domain of DRB1 and DRB2 mediates in the miRNA-guided transcript cleavage pathway. The DRB1 amino acid sequence differs considerably to that of DRB2, and analysis of drb1 transgenic lines revealed that the first dsRNA-binding domains of DRB1 and DRB2 are functionally similar; in contrast, the dsRBD2 of DRB1 and DRB2 appear functionally distinct. Our bioinformatic analysis further suggests that the C-terminal domain of DRB2 mediates a functional role in the miRNA pathway, whereas its counterpart in DRB1 is known to be dispensable. Our results provide evidence for the differences between DRB1 and DRB2 proteins in vivo, which may be essential for the selection of the miRNA regulatory mechanisms, and suggest that these features are conserved among land plants.

Plant microRNAs (miRNAs) operate by guiding the cleavage or translational inhibition of mRNA targets. They act as key gene regulators for development and environmental adaptation, and Dicer-partnering proteins DRB1 and DRB2 govern which form of regulation plays the dominant role. Mutation of Drb1 impairs transcript cleavage, whereas mutation of Drb2 ablates translational inhibition. Regulation of gene expression by miRNA-guided cleavage has been extensively studied, but there is much less information about genes regulated through miRNA-mediated translation inhibition. Here, we compared the proteomes of drb1 and drb2 mutants to gain insight into the indirect effect of the different miRNA regulatory mechanisms in Arabidopsis thaliana. Our results show that miRNAs operating through transcript cleavage regulate a broad spectrum of processes, including catabolism and anabolism, and this was particularly obvious in the fatty acid degradation pathway. Enzymes catalyzing each step of this pathway were upregulated in drb1. In contrast, DRB2-associated translational inhibition appears to be less ubiquitous and specifically aimed toward responses against abiotic or biotic stimuli.

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.

Plant microRNAs (miRNAs) are important regulatory switches. Recent advances have revealed many regulatory layers between the two essential processes, miRNA biogenesis and function. However, how these multilayered regulatory processes ultimately control miRNA gene regulation and connects miRNAs and plant responses with the surrounding environment is still largely unknown. In this opinion article, we propose that the miRNA pathway is highly dynamic and plastic. The apparent flexibility of the miRNA pathway in plants appears to be controlled by a number recently identified proteins and poorly characterized signaling cascades. We further propose that altered miRNA accumulation can be a direct consequence of the rewiring of interactions between proteins that function in the miRNA pathway, an avenue that remains largely unexplored., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

MicroRNAs (miRNAs) are small regulatory RNAs produced by Dicer proteins that regulate gene expression in development and adaptive responses to the environment(1-4). In animals, the degree of base pairing between a miRNA and its target messenger RNA seems to determine whether the regulation occurs through cleavage or translation inhibition(1). In contrast, the selection of regulatory mechanisms is independent of the degree of mismatch between a plant miRNA and its target transcript(5). However, the components and mechanism(s) that determine whether a plant miRNA ultimately regulates its targets by guiding cleavage or translational inhibition are unknown(6). Here we show that the form of regulatory action directed by a plant miRNA is determined by DRB2, a DICER-LIKE1 (DCL1) partnering protein. The dependence of DCL1 on DRB1 for miRNA biogenesis is well characterized(7-9), but we show that it is only required for miRNA-guided transcript cleavage. We found that DRB2 determines miRNA-guided translational inhibition and represses DRB1 expression, thereby allowing the active selection of miRNA regulatory action. Furthermore, our results reveal that the core silencing proteins ARGONAUTE1 (AGO1) and SERRATE (SE) are highly regulated by miRNA-guided translational inhibition. DRB2 has been remarkably conserved throughout plant evolution, raising the possibility that translational repression is the ancient form of miRNA-directed gene regulation in plants, and that Dicer partnering proteins, such as human TRBP, might play a similar role in other eukaryotic systems.