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RNA viral vectors for improved Agrobacterium-mediated transient expression of heterologous proteins in Nicotiana benthamiana cell suspensions and hairy roots.

ABSTRACT: BACKGROUND: Plant cell suspensions and hairy root cultures represent scalable protein expression platforms. Low protein product titers have thus far limited the application of transient protein expression in these hosts. The objective of this work was to overcome this limitation by harnessing A. tumefaciens to deliver replicating and non-replicating RNA viral vectors in plant tissue co-cultures. RESULTS: Replicating vectors derived from Potato virus X (PVX) and Tobacco rattle virus (TRV) were modified to contain the reporter gene ?-glucuronidase (GUS) with a plant intron to prevent bacterial expression. In cell suspensions, a minimal PVX vector retaining only the viral RNA polymerase gene yielded 6.6-fold more GUS than an analogous full-length PVX vector. Transient co-expression of the minimal PVX vector with P19 of Tomato bushy stunt virus or HC-Pro of Tobacco etch virus to suppress post-transcriptional gene silencing increased GUS expression by 44 and 83%, respectively. A non-replicating vector containing a leader sequence from Cowpea mosaic virus (CPMV-HT) modified for enhanced translation led to 70% higher transient GUS expression than a control treatment. In hairy roots, a TRV vector capable of systemic movement increased GUS accumulation by 150-fold relative to the analogous PVX vector. Histochemical staining for GUS in TRV-infected hairy roots revealed the capacity for achieving even higher productivity per unit biomass. CONCLUSIONS: For the first time, replicating PVX vectors and a non-replicating CPMV-HT vector were successfully applied toward transient heterologous protein expression in cell suspensions. A replicating TRV vector achieved transient GUS expression levels in hairy roots more than an order of magnitude higher than the highest level previously reported with a viral vector delivered by A. tumefaciens.

SUBMITTER: Larsen JS

PROVIDER: S-EPMC3403893 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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RNA viral vectors for improved Agrobacterium-mediated transient expression of heterologous proteins in Nicotiana benthamiana cell suspensions and hairy roots.

Larsen Jeffrey S JS Curtis Wayne R WR

BMC biotechnology 20120506

<h4>Background</h4>Plant cell suspensions and hairy root cultures represent scalable protein expression platforms. Low protein product titers have thus far limited the application of transient protein expression in these hosts. The objective of this work was to overcome this limitation by harnessing A. tumefaciens to deliver replicating and non-replicating RNA viral vectors in plant tissue co-cultures.<h4>Results</h4>Replicating vectors derived from Potato virus X (PVX) and Tobacco rattle virus ...[more]

PMID: 22559055

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Project description:BackgroundAgrobacterium tumefaciens-based transient assays have become a common tool for answering questions related to protein localization and gene expression in a cellular context. The use of these assays assumes that the transiently transformed cells are observed under relatively authentic physiological conditions and maintain 'normal' sub-cellular behaviour. Although this premise is widely accepted, the question of whether cellular organization and organelle morphology is altered in Agrobacterium-infiltrated cells has not been examined in detail. The first indications of an altered sub-cellular environment came from our observation that a common laboratory strain, GV3101(pMP90), caused a drastic increase in stromule frequency. Stromules, or 'stroma-filled-tubules' emanate from the surface of plastids and are sensitive to a variety of biotic and abiotic stresses. Starting from this observation, the goal of our experiments was to further characterize the changes to the cell resulting from short-term bacterial infestation, and to identify the factor responsible for eliciting these changes.ResultsUsing a protocol typical of transient assays we evaluated the impact of GV3101(pMP90) infiltration on chloroplast behaviour and morphology in Nicotiana benthamiana. Our experiments confirmed that GV3101(pMP90) consistently induces stromules and alters plastid position relative to the nucleus. These effects were found to be the result of strain-dependant secretion of cytokinin and its accumulation in the plant tissue. Bacterial production of the hormone was found to be dependant on the presence of a trans-zeatin synthase gene (tzs) located on the Ti plasmid of GV3101(pMP90). Bacteria-derived cytokinins were also correlated with changes to both soluble sugar level and starch accumulation.ConclusionAlthough we have chosen to focus on how transient Agrobacterium infestation alters plastid based parameters, these changes to the morphology and position of a single organelle, combined with the measured increases in sugar and starch content, suggest global changes to cell physiology. This indicates that cells visualized during transient assays may not be as 'normal' as was previously assumed. Our results suggest that the impact of the bacteria can be minimized by choosing Agrobacterium strains devoid of the tzs gene, but that the alterations to sub-cellular organization and cell carbohydrate status cannot be completely avoided using this strategy.

Project description:BackgroundAgroinfiltration 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.ResultsUsing 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 (?-glucuronidase) expression were established. The effects of chemical additives in the liquid infiltration media were investigated and acetosyringone (500 ?M), the antioxidant lipoic acid (5 ?M), 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.ConclusionsIn 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.

Dataset Information

RNA viral vectors for improved Agrobacterium-mediated transient expression of heterologous proteins in Nicotiana benthamiana cell suspensions and hairy roots.

Publications

RNA viral vectors for improved Agrobacterium-mediated transient expression of heterologous proteins in Nicotiana benthamiana cell suspensions and hairy roots.

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