Scale-Up of Agrobacterium-Mediated Transient Protein Expression in Bioreactor-Grown Nicotiana glutinosa Plant Cell Suspension Culture (original) (raw)
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A rapid Agrobacterium -mediated Arabidopsis thaliana transient assay system
Plant Molecular Biology Reporter, 2004
Stable transformation of Arabidopsis thaliana is a lengthy process that involves up to 3 mo of plant growth and seed selection. We have developed a rapid, 3-wk transient assay system to test the functionality of cis-regulatory regions controlling expression of a reporter gene in plants before undertaking stable transformation. Two-week-old Arabidopsis seedlings were vacuum-infiltrated with Agrobacterium tumefaciens cultures carrying various upstream regulatory regions controlling uidA (~-glucuronidase [GUS]) expression. Seedlings were fixed and stained for GUS activity 3-5 d following infiltration. Regulatory regions tested in this system include the cauliflower mosaic virus (CaMV) 35S promoter, the upstream regulatory region of ribosomal protein gene L23A-1, and a temperature-inducible regulatory region (HSPIO1B) also from Arabidopsis. The percentage of seedlings positive for GUS activity varied depending on the construct used, with the CaMV 35S promoter producing the highest number of GUS-positive seedlings. Temperature induction treatments elicited increased GUS expression in seedlings transformed with the HSPIO1B regulatory region. Regardless of construct, GUS expression levels were higher in seedlings collected 5 d following Agrobacterium infiltration than those collected 3-4 d postinfiltration.
Biotechnology Progress, 2008
A viral vector based on the bean yellow dwarf virus was investigated for its potential to increase transient gene expression. An intron-containing GUS reporter gene and the cis-acting viral regulatory elements were incorporated in the viral vector and could be complemented by the viral replication-associated proteins provided on a secondary vector. All vectors were delivered to Nicotiana glutinosa plant cell suspension or hairy root cultures via auxotrophic Agrobacterium tumefaciens. Cell culture generated greater yield of reporter gene expression than did root culture, as a result of the limitation imposed on roots to express the protein only in surface tissue containing actively dividing cells. Reporter gene expression increased for cell culture when the reporter gene construct was co-delivered with the construct supplying both viral replication associated proteins (REP and REPA); gene expression decreased when the construct supplying only the viral REP protein was co-delivered. Reporter protein expression increased from 0.091% for the reporter construct alone to 0.22% total soluble protein (% TSP) when the viral Repsupplying vector was co-delivered with the reporter gene construct. Reporter protein was generated 3 days after the initiation of bacterial co-culture, providing for rapid generation of heterologous protein in cell culture.
Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana
Journal of Visualized Experiments, 2014
Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana × N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about twofold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).
Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana
Journal of Visualized Experiments, 2014
Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana × N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about twofold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).
An Agrobacterium-mediated transient gene expression system for intact leaves
Plant Science, 1997
,411 efficient and reproducible AgrohLlc~teriut,l-mediated transient gene expression system for intact leaf tissue was developed. A high level of transient expression was observed when bacteria. which were pretreated in C?Y gene-inducing conditions. were infiltrated into complete leaf tissue. Histochemical /I-glucuronidase assays showed large transgene-expressing sectors comprising of up to 90% of the leaf area. As a consequence of infiltration, the induced bacteria entered into the intercellular spaces, thus enabling T-DNA transfer in all cell layers of the leaf. The protocol was optimized for Phasrolus wlgaris leaves. but similar results were obtained with other plant species. such as P/mroh~.s uc,utifdim. poplar, and tobacco. A /I-glucuronidase chimeric gene interrupted by an intron was used as a marker for histological detection of the sectors. Copyright :CJ 1997 Elsevier Science Ireland Ltd.
Comparison of Transient Protein Expression in Tobacco Leaves and Plant Suspension Culture
Biotechnology Progress, 2008
Transient gene expression is being developed to provide a more rapid means of assessing plant tissues as a protein production platform without the labor-intensive and time-consuming process of generating stably transformed transgenic plants. Transient expression of the gus-intron reporter gene was facilitated in three different tobacco species. Two different approaches to T-DNA delivery were compared: (1) infiltration of a prototrophic strain of Agrobacterium into leaves and (2) coculture of plant cell suspension cultures with an Agrobacterium auxotroph. Wounding of plant tissues with a wire brush prior to infiltration had a large positive impact on Nicotiana benthamiana leaves but not for Nicotiana tabacum or Nicotiana glutinosa. The best expression level achieved by leaf infiltration was in N. benthamiana (0.025% total soluble protein). A cell suspension culture line of N. glutinosa achieved an expression level greater than 0.04% TSP. The tissue culture-based technique therefore provides improved levels of transient expression under aseptic conditions to facilitate improvements in expression by control of the plant cell culture and Agrobacterium coculture environments.
Biotechnology and applied biochemistry, 2004
A transient gene-expression system was developed and used to characterize promoter strength, to verify suitability of bacterial gene modifications for expression in plant cells, and to express active antibody molecules. The system is based on suspension tobacco cells transformed by Agrobacterium in a transient way. Conditions such as pre-culture of tobacco cells and the co-cultivation period were identified as determinants to achieve high expression levels. Under established conditions the activity strength of CaMV (cauliflower mosaic virus) 35 S and ToMoTV (tomato mottle taino virus) AL1 promoters were compared. A modified cry gene sequence from Bacillus thuringiensis was expressed and detected by Western-blot analysis. A monoclonal antibody against anti-(hepatitis B virus surface antigen) was produced in such quantities as to allow testing of biological activity and preliminary characterization.
Optimization of Agrobacterium-mediated transformation in Lycopersicon esculentum. L. Mill
Plant Cell Biotechnology and Molecular Biology, 2009
The Agrobacterium-mediated transient assay is a relatively rapid technique and a promising approach for assessing the expression of a gene of interest. Despite the successful application of this transient expression system in several plant species, it is not well understood in spinach. In this study, we analyzed various factors, including infiltration method, Agrobacterium strain and density, and coinfiltration of an RNA silencing suppressor (p19), that affect transient expression following agroinfiltration in spinach. To evaluate the effects of these factors on the transient expression system, we used the b-glucuronidase (GUS) reporter gene construct pB7WG2D as a positive control. The vacuum-based infiltration method was much more effective at GUS gene expression than was the syringe-based infiltration method. Among the three Agrobacterium strains examined (EHA105, LBA4404, and GV2260), infiltration with the GV2260 strain suspension at a final optical cell density (OD 600) of 1.0 resulted in the highest gene expression. Furthermore, co-expression of suppressor p19 also increased the efficiency and duration of gene expression and protein accumulation. The results indicate that the use of optimized conditions for transient gene expression could be a simple, rapid, and effective tool for functional genomics in spinach.
Objective. Assess transient gene expression of GUS in cassava (Manihot esculenta Crantz) leaves using Agrobacterium tumefaciens infiltration. Materials and methods. A. tumefaciens strains GV3101 and AGL1 containing pCAMBIA1305.2 were used to evaluate transient gene expression of β-glucuronidase (GUS). A. tumefaciens infiltration (agroinfiltration) was made using both leaves from in vitro and 1 month old greenhouse plants. Leaves were incubated in X-GLUC buffer, stained and photographed to detect GUS activity. Results. Agroinfiltration assays showed GUS transient expression in leaves of cassava varieties widely cultivated in the north coast and eastern savannah, MCOL2215 (Venezuelan) and CM6438-14 (Vergara), respectively. A. tumefaciens agressive strain AGL1 showed high efficiency inducing GUS expression in cassava leaves. Conclusions. We recommend using A. tumefaciens agressive strain AGL1 for agroinfiltration to assess transient expression in cassava leaves.
Agrobacterium -mediated transient expression of vacuolar GFPs in Petunia leaves and petals
Plant Biosystems - An International Journal Dealing with all Aspects of Plant Biology, 2008
Transient expression assays are useful for saving time in studies of gene function, particularly ones expressed in fruit. Transient expression system of a reporter gene in immature fruit of banana was developed. Agrobacterium tumefaciens, whose plasmid contains an intron-containing β-glucuronidase (gusA) gene under regulatory control of the CaMV 35S promoter, was vacuum-infiltrated into sliced fruits and co-cultured. GUS histochemical assay was performed three days after co-cultivation, and a high level of GUS expression was observed. This transient expression system is useful for routine transient assays to validate genes expressed in banana fruit.