Optimization and Utilization of &lt;em&gt;Agrobacterium&lt;/em&gt;-mediated Transient Protein Production in &lt;em&gt;Nicotiana&lt;/em&gt;

Shamloul, Moneim; Trusa, Jason; Mett, Vadim; Yusibov, Vidadi

doi:10.3791/51204-v

Cited by 12 publications

(11 citation statements)

References 26 publications

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“…This method of introducing vector sequences typically utilizes a high density of Agrobacterium cells to increase the coverage of infected host cells. However, higher concentrations of Agrobacteria can be detrimental to leaf tissue, leading to biotic stresses and even necrosis, resulting in undesirably low yields of heterologous proteins (Shamloul et al ., 2014 ). Thus, further improvements to the expression system, allowing the vectors to more rapidly move cell‐to‐cell to cover all available leaf tissue, would potentially allow for reduced concentrations of Agrobacteria to be utilized.…”

Section: Discussionmentioning

confidence: 99%

“…For this purpose, WT and transgenic N. benthamiana seeds were first germinated in the dark for 5 days, and young seedlings were transferred into individual pots and grown in a Conviron walk‐in growth chamber (GR Series, Controlled Environments Inc.) at 24 °C, 70% humidity, and under 16 h light/8 h dark conditions. For vacuum infiltration, plants were grown for 5–6 weeks in a hydroponic system using rockwool as the support matrix (Shamloul et al ., 2014 ).…”

Section: Methodsmentioning

confidence: 99%

“…Infiltrated plants were covered and incubated in a walk‐in growth chamber overnight and uncovered 12–16 h later. Agrobacteria carrying plasmid constructs encoding Yellow fever virus and B. anthracis antigens were introduced into N. benthamiana by vacuum infiltration, as described previously using A. tumefaciens cultures at an OD 600nm = 0.5 for the target and OD 600nm = 0.1 for the P1/HC‐Pro silencing suppressor of Turnip mosaic virus (Shamloul et al ., 2014 ). Transgenic N. benthamiana plants were generated by the transformation of leaf discs (Horsch et al ., 1985 ).…”

Section: Methodsmentioning

confidence: 99%

“…Agrobacteria carrying plasmid constructs encoding Yellow fever virus and B. anthracis antigens were introduced into N. benthamiana by vacuum infiltration, as described previously using A. tumefaciens cultures at an OD 600nm = 0.5 for the target and OD 600nm = 0.1 for the P1/HC-Pro silencing suppressor of Turnip mosaic virus ª 2022 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 21, 635-645 (Shamloul et al, 2014). Transgenic N. benthamiana plants were generated by the transformation of leaf discs (Horsch et al, 1985).…”

Section: Agrobacterium-mediated Infiltration and Plant Transformationmentioning

confidence: 99%

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Facilitating viral vector movement enhances heterologous protein production in an established plant system

Wang

Prokhnevsky

Skarjinskaia

et al. 2023

Plant Biotechnology Journal

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Summary Molecular farming technology using transiently transformed Nicotiana plants offers an economical approach to the pharmaceutical industry to produce an array of protein targets including vaccine antigens and therapeutics. It can serve as a desirable alternative approach for those proteins that are challenging or too costly to produce in large quantities using other heterologous protein expression systems. However, since cost metrics are such a critical factor in selecting a production host, any system‐wide modifications that can increase recombinant protein yields are key to further improving the platform and making it applicable for a wider range of target molecules. Here, we report on the development of a new approach to improve target accumulation in an established plant‐based expression system that utilizes viral‐based vectors to mediate transient expression in Nicotiana benthamiana. We show that by engineering the host plant to support viral vectors to spread more effectively between host cells through plasmodesmata, protein target accumulation can be increased by up to approximately 60%.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Agrobacterium-mediated Infiltration and Plant Transformationmentioning

confidence: 99%

See 2 more Smart Citations

Facilitating viral vector movement enhances heterologous protein production in an established plant system

Wang

Prokhnevsky

Skarjinskaia

et al. 2023

Plant Biotechnology Journal

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“…Agrobacterium strains containing a binary construct were cultured overnight in LB liquid medium. The bacterial cells were collected by centrifugation and resuspended in induction medium (1× MS salt, 10 mM MES, 200 μM acetosyringone, and 2% sucrose) (Shamloul et al ., 2014) for 6 h incubation with shaking at 30°C. Agrobacterium cells were then resuspended in infiltration buffer (1× MS, 10 mM MES, and 200 μM acetosyringone) and adjusted to an optical density at an OD 600nm of 0.8.…”

Section: Methodsmentioning

confidence: 99%

Engineered Agrobacterium improves transformation by mitigating plant immunity detection

Yang

Felix

et al. 2023

New Phytologist

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Summary Agrobacterium tumefaciens microbe‐associated molecular pattern elongation factor Tu (EF‐Tu) is perceived by orthologs of the Arabidopsis immune receptor EFR activating pattern‐triggered immunity (PTI) that causes reduced T‐DNA‐mediated transient expression. We altered EF‐Tu in A. tumefaciens to reduce PTI and improved transformation efficiency. A robust computational pipeline was established to detect EF‐Tu protein variation in a large set of plant bacterial species and identified EF‐Tu variants from bacterial pathogen Pseudomonas syringae pv. tomato DC3000 that allow the pathogen to escape EFR perception. Agrobacterium tumefaciens strains were engineered to substitute EF‐Tu with DC3000 variants and examined their transformation efficiency in plants. Elongation factor Tu variants with rarely occurred amino acid residues were identified within DC3000 EF‐Tu that mitigates recognition by EFR. Agrobacterium tumefaciens strains were engineered by expressing DC3000 EF‐Tu instead of native agrobacterial EF‐Tu and resulted in decreased plant immunity detection. These engineered A. tumefaciens strains displayed an increased efficiency in transient expression in both Arabidopsis thaliana and Camelina sativa. The results support the potential application of these strains as improved vehicles to introduce transgenic alleles into members of the Brassicaceae family.

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Optimization of the human colorectal carcinoma antigen GA733-2 production in tobacco plants

Park

Kim

et al. 2021

Plant Biotechnol Rep

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The colorectal carcinoma-associated protein GA733-2 is one of the representative candidate protein for the development of plant-derived colorectal cancer vaccine. Despite of its significant importance for colorectal vaccine development, low efficiency of GA733-2 production limits its wide applications. To improve productivity of GA733-2 in plants, we here tested multiple factors that affect expression of recombinant GA733-2 (rGA733-2) and rGA733 fused to fragment crystallizable (Fc) domain (rGA733-Fc) protein. The rGA733-2 and rGA733-Fc proteins were highly expressed when the pBINPLUS vector system was used for transient expression in tobacco plants. In addition, the length of interval between rGA733-2 and left border of T-DNA affected the expression of rGA733 protein. Transient expression analysis using various combinations of Agrobacterium tumefaciens strains (C58C1, LBA4404, and GV3101) and tobacco species (Nicotiana tabacum cv. Xanthi nc and Nicotiana benthamiana) revealed that higher accumulation of rGA733-2 and rGA733-Fc proteins were obtained by combination of A. tumefaciens LBA4404 and Nicotiana benthamiana. Transgenic plants generated by introduction of the rGA733-2 and rGA733-Fc expression cassettes also significantly accumulated corresponding recombinant proteins. Bioactivity and stability of the plant-derived rGA733 and rGA733-Fc were evaluated by further in vitro assay, western blot and N-glycosylation analysis. Collectively, we here suggest the optimal condition for efficient production of functional rGA733-2 protein in tobacco system.

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Optimization and Utilization of <em>Agrobacterium</em>-mediated Transient Protein Production in <em>Nicotiana</em>

Cited by 12 publications

References 26 publications

Facilitating viral vector movement enhances heterologous protein production in an established plant system

Facilitating viral vector movement enhances heterologous protein production in an established plant system

Engineered Agrobacterium improves transformation by mitigating plant immunity detection

Optimization of the human colorectal carcinoma antigen GA733-2 production in tobacco plants

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