Multiplicity of the Agrobacterium Infection of Nicotiana benthamiana for Transient DNA Delivery.

Biological DNA transfer into plant cells mediated by Agrobacterium represents one of the most powerful tools for the engineering and study of plant systems. Transient expression of transfer DNA (T-DNA) in particular enables rapid testing of gene products and has been harnessed for facile combinatorial expression of multiple genes. In analogous mammalian cell-based gene expression systems, a clear sense of the multiplicity of infection (MOI) allows users to predict and control viral transfection frequencies for applications requiring single versus multiple transfection events per cell. Despite the value of Agrobacterium-mediated transient transformation of plants, MOI has not been quantified. Here, we analyze the Poisson probability distribution of the T-DNA transfer in leaf pavement cells to determine the MOI for the widely used model system Agrobacterium GV3101/Nicotiana benthamiana. These data delineate the relationship between an individual Agrobacterium strain infiltration OD600, plant cell perimeter, and leaf age, as well as plant cell coinfection rates. Our analysis establishes experimental regimes where the probability of near-simultaneous delivery of >20 unique T-DNAs to a given plant cell remains high throughout the leaf at infiltration OD600 above ∼0.2 for individual strains. In contrast, single-strain T-DNA delivery can be achieved at low strain infiltration OD600: at OD600 0.02, we observe that ∼40% of plant cells are infected, with 80% of those infected cells containing T-DNA product from just a single strain. We anticipate that these data will enable users to develop new approaches to in-leaf library development using Agrobacterium transient expression and reliable combinatorial assaying of multiple heterologous proteins in a single plant cell.

Psychometric Indicators of the Pro-environmental Attitudes' Questionnaire: Colombian Version.

The detrimental effect of human behavior on the environment is undeniable. Attitudes are recognized as a predictor of the pro-environmental behavior; therefore, having good quality tools in Colombia to measure them is strategic to assess interventions. This study aims to establish psychometric indicators for the pro-environmental attitudes questionnaire (PEAQ) Colombian version to a sample of 415 volunteers (53% women and 47% men) aged 18-70 years (M = 40.28; SD = 14.06). We used the 28-item PEAQ already linguistically adapted for Colombia. We applied the following questionnaires: Environmental awareness (EA) (11 items), environmental values (EV) (4 items), and the pro-environmental at work questionnaire (PEWQ) (31 items). We used a one-parameter Rasch model and Winsteps program to assess the PEAQ's one-dimensionality and item statistics by gender, and estimated Spearman's rho coefficient between the PEAQ scores and the scales for concurrent validity. The PEAQ in this study has 24 items because 4 items did not fit into the Rasch model criteria. Its one-dimensionality was supported by an explained variance (43%) and the first residual variance (12%). The coefficients, α = 0.95 and Ω = 0.95; Rasch for persons = 0.90; and Rasch for items = 0.95. The correlation between the PEAQ and the EC, EV, and PEWQ scales were Spearman's rho coefficient = 0.859 (p ≤ 0.001), 0.795 (p ≤ 0.001), and 0.885 (p ≤ 0.001), respectively. Thus, the PEAQ Colombian version's psychometric indicators support it as a valid and reliable instrument to measure pro-environmental attitudes in this country.

Specific Packaging of Custom RNA Molecules into Cowpea Mosaic Virus-like Particles.

Recent discoveries in the dynamics of genome replication and packaging in the plant virus Cowpea mosaic virus (CPMV) has led to the development of a novel method for specifically packaging an RNA molecule of choice into virus-like particles (VLPs) of CPMV. Thanks to modern gene synthesis and molecular cloning methods, the DNA sequence corresponding to an RNA sequence of interest can be cloned into a suitable expression plasmid for transient expression in plants. We describe here a method for ensuring that this RNA sequence will be packaged within VLPs of CPMV in plant cells by replication-dependent RNA packaging. This requires co-expression of the CPMV replication machinery alongside the CPMV coat protein precursor. These components are co-expressed in the leaves of the Nicotiana benthamiana plant and this co-expression results in the production of large quantities of VLPs that contain the RNA sequence of choice. These VLPs are easy to extract and purify from the plant tissue, and are stable for months in refrigerated conditions. These VLPs can then be used for a variety of different applications, such as RNA delivery or control reagents in RT-qPCR.

A Replicating Viral Vector Greatly Enhances Accumulation of Helical Virus-Like Particles in Plants.

The production of plant helical virus-like particles (VLPs) via plant-based expression has been problematic with previous studies suggesting that an RNA scaffold may be necessary for their efficient production. To examine this, we compared the accumulation of VLPs from two potexviruses, papaya mosaic virus and alternanthera mosaic virus (AltMV), when the coat proteins were expressed from a replicating potato virus X- based vector (pEff) and a non-replicating vector (pEAQ-HT). Significantly greater quantities of VLPs could be purified when pEff was used. The pEff system was also very efficient at producing VLPs of helical viruses from different virus families. Examination of the RNA content of AltMV and tobacco mosaic virus VLPs produced from pEff revealed the presence of vector-derived RNA sequences, suggesting that the replicating RNA acts as a scaffold for VLP assembly. Cryo-EM analysis of the AltMV VLPs showed they had a structure very similar to that of authentic potexvirus particles. Thus, we conclude that vectors generating replicating forms of RNA, such as pEff, are very efficient for producing helical VLPs.

Improving plant transient expression through the rational design of synthetic 5' and 3' untranslated regions.

BACKGROUND: The growing field of plant molecular farming relies on expression vectors that allow high yields of recombinant proteins to be produced through transient gene expression. While numerous expression vectors currently exist for this purpose, there are very few examples of systematic efforts to improve upon these. Moreover, the current generation of expression systems makes use of naturally-occurring regulatory elements, typically selected from plant viruses, to maximise yields. This study aims to use rational design to generate synthetic sequences that can rival existing ones. RESULTS: In this work, we present the rational design of novel synthetic 5' and 3' untranslated regions (UTRs) which can be used in various combinations to modulate accumulation levels of transiently-expressed recombinant proteins. Using the pEAQ-HT expression vector as a point of comparison, we show that pre-existing expression systems can be improved by the deployment of rationally designed synthetic UTRs. Notably, we show that a suite of short, synthetic 5'UTRs behave as expression enhancers that outperform the HT 5'UTR present in the CPMV-HT expression system. Furthermore, we confirm the critical role played by the 3'UTR of cowpea mosaic virus RNA-2 in the performance of the CPMV-HT system. Finally, we use the knowledge obtained from these results to develop novel expression vectors (named pHRE and pHREAC) that equal or outperform pEAQ-HT in terms of recombinant protein yield. These new vectors are also domesticated for the use of certain Type IIS restriction enzymes, which allows for quicker cloning and straightforward assessment of different combinations of UTRs. CONCLUSIONS: We have shown that it is possible to rationally design a suite of expression modulators in the form of synthetic UTRs. We have created novel expression vectors that allow very high levels of recombinant protein expression in a transient expression context. This will have important consequences for future efforts to develop ever-better plant transient overexpression vectors for research or industrial applications.

Inside or outside? A new collection of Gateway vectors allowing plant protein subcellular localization or over-expression.

Transient expression of proteins based on agro-infiltration techniques has proven very efficient and straightforward to study the intrinsic properties of proteins. The level of protein expression has been enhanced by the use of vector plasmids containing virus-derived sequences and the cloning step has been facilitated by recombination technologies. The pEAQ-HT-DEST series of vectors fulfilling these improvements are vectors of choice. However, they lack the possibility to directly and easily fuse the protein of interest to a fluorescent tag or to address it to the secretion pathway. In the present work we describe the production of 15 pEAQ-HT-DEST1-based plasmids designed to use the Gateway® cloning technology and to generate high levels of fluorescent fusion protein by agro-infiltration, in planta. This collection of plasmids includes binary vectors allowing N-terminal or C-terminal fusion to the bright tags EGFP or TagRFP for cytoplasmic accumulation or secretion and represents therefore a valuable tool for subcellular localization or biochemical studies. A viral protein, the blue fluorescent protein TagBFP, the green fluorescent protein variant T-Sapphire and an Arabidopsis protein were transiently expressed in N. benthamiana to demonstrate the potential of these vectors.

Improving agroinfiltration-based transient gene expression in Nicotiana benthamiana.

BACKGROUND: Agroinfiltration is a simple and effective method of delivering transgenes into plant cells for the rapid production of recombinant proteins and has become the preferred transient expression platform to manufacture biologics in plants. Despite its popularity, few studies have sought to improve the efficiency of agroinfiltration to further increase protein yields. This study aimed to increase agroinfiltration-based transient gene expression in Nicotiana benthamiana by improving all levels of transgenesis. RESULTS: Using the benchmark pEAQ-HT deconstructed virus vector system and the GUS reporter enzyme, physical, chemical, and molecular features were independently assessed for their ability to enhance Agrobacterium-mediated transformation and improve protein production capacities. Optimal Agrobacterium strain, cell culture density and co-cultivation time for maximal transient GUS (ß-glucuronidase) expression were established. The effects of chemical additives in the liquid infiltration media were investigated and acetosyringone (500 µM), the antioxidant lipoic acid (5 µM), and a surfactant Pluronic F-68 (0.002%) were all shown to significantly increase transgene expression. Gene products known to suppress post-transcriptional gene silencing, activate cell cycle progression and confer stress tolerance were also assessed by co-expression. A simple 37 °C heat shock to plants, 1-2 days post infiltration, was shown to dramatically increase GUS reporter levels. By combining the most effective features, a dual vector delivery system was developed that provided approximately 3.5-fold higher levels of absolute GUS protein compared to the pEAQ-HT platform. CONCLUSIONS: In this paper, different strategies were assessed and optimised with the aim of increasing plant-made protein capacities in Nicotiana benthamiana using agroinfiltration. Chemical additives, heat shock and the co-expression of genes known to suppress stress and gene silencing or stimulate cell cycle progression were all proven to increase agroinfiltration-based transient gene expression. By combining the most effective of these elements a novel expression platform was developed capable of producing plant-made protein at a significantly higher level than a benchmark hyper-expression system.

Delivering Cargo: Plant-Based Production of Bluetongue Virus Core-Like and Virus-Like Particles Containing Fluorescent Proteins.

This chapter provides a practical guide to the in planta transient production of bluetongue virus-like particles containing a fluorescent cargo protein. Bluetongue virus (BTV) particles are icosahedral, multishelled entities of a relatively large size. Heterologous expression of the four main structural proteins of BTV results in the assembly of empty virus-like particles which resemble the native virus externally, but are devoid of nucleic acid. The space within the particles is sufficient to allow incorporation of relatively large cargo proteins, such as green fluorescent protein (GFP), by genetic fusion to the structural protein VP3. The method described utilizes the pEAQ vectors for high-level transient expression of such particles in Nicotiana benthamiana.

Virus-Derived Vectors for the Expression of Multiple Proteins in Plants.

This chapter constitutes a practical guide to using the "pEAQ" vector series for transient or stable expression of one or more protein(s) in Nicotiana benthamiana plants. The pEAQ vectors are a series of small binary vectors designed for controlled expression of multiple proteins in plants. To achieve high levels of expression, an expression system based on translational enhancement by the untranslated regions of RNA-2 from cowpea mosaic virus (CPMV), named CPMV-HT, is used. The expression vector pEAQ-HT combines the user-friendly pEAQ plasmid with CPMV-HT to provide a system for high-level expression of proteins in plants.

Fine-tuning levels of heterologous gene expression in plants by orthogonal variation of the untranslated regions of a nonreplicating transient expression system.

A transient expression system based on a deleted version of Cowpea mosaic virus (CPMV) RNA-2, termed CPMV-HT, in which the sequence to be expressed is positioned between a modified 5' UTR and the 3' UTR has been successfully used for the plant-based expression of a wide range of proteins, including heteromultimeric complexes. While previous work has demonstrated that alterations to the sequence of the 5' UTR can dramatically influence expression levels, the role of the 3' UTR in enhancing expression has not been determined. In this work, we have examined the effect of different mutations in the 3'UTR of CPMV RNA-2 on expression levels using the reporter protein GFP encoded by the expression vector, pEAQexpress-HT-GFP. The results showed that the presence of a 3' UTR in the CPMV-HT system is important for achieving maximal expression levels. Removal of the entire 3' UTR reduced expression to approximately 30% of that obtained in its presence. It was found that the Y-shaped secondary structure formed by nucleotides 125-165 of the 3' UTR plays a key role in its function; mutations that disrupt this Y-shaped structure have an effect equivalent to the deletion of the entire 3' UTR. Our results suggest that the Y-shaped secondary structure acts by enhancing mRNA accumulation rather than by having a direct effect on RNA translation. The work described in this paper shows that the 5' and 3' UTRs in CPMV-HT act orthogonally and that mutations introduced into them allow fine modulation of protein expression levels.