Tackling Heterogeneity: A Leaf Disc-Based Assay for the High-Throughput Screening of Transient Gene Expression in Tobacco

Abstract

Transient Agrobacterium-mediated gene expression assays for Nicotiana tabacum (N. tabacum) are frequently used because they facilitate the comparison of multiple expression constructs regarding their capacity for maximum recombinant protein production. However, for three model proteins, we found that recombinant protein accumulation (rpa) was significantly influenced by leaf age and leaf position effects. The ratio between the highest and lowest amount of protein accumulation (max/min ratio) was found to be as high as 11. Therefore, construct-based impacts on the rpa level that are less than 11-fold will be masked by background noise. To address this problem, we developed a leaf disc-based screening assay and infiltration device that allows the rpa level in a whole tobacco plant to be reliably and reproducibly determined. The prototype of the leaf disc infiltration device allows 14 Agrobacterium-mediated infiltration events to be conducted in parallel. As shown for three model proteins, the average max/min rpa ratio was reduced to 1.4 using this method, which allows for a sensitive comparison of different genetic elements affecting recombinant protein expression.

Animal component‐free Agrobacterium tumefaciens cultivation media for better GMP‐compliance increases biomass yield and pharmaceutical protein expression in Nicotiana benthamiana

“…Following the addition of 200 µM acetosyringone, the infiltration suspension was incubated at ambient temperature (≈22°C) for 2 h. Afterwards, the suspension was either used for a tobacco leaf disc-based assay as previously described [18] or injected into the leaves of intact N. benthamiana plants using a 1-mL needleless syringe. Alternatively intact plants were infiltrated in a desiccator connected to a vacuum membrane pump (Vacuubrand, Wertheim, Germany) at 60 mbar absolute pressure for 1 min.…”

“…Small-scale tests using shake-flask cultures and tobacco leaf discs facilitate high-throughput screening [18]. The benefit of this approach is the reduction of min/max ratios for transient expression in plants from >10 to ≈1 [18].…”

“…The benefit of this approach is the reduction of min/max ratios for transient expression in plants from >10 to ≈1 [18]. The screening method not only facilitated the rapid testing of different peptone preparations but also the implementation of a DoE strategy to identify compositions that significantly increased the OD 600 and thus the biomass yield during the cultivation of A. tumefaciens.…”

“…We used a design-of-experiments (DoE) approach, which has proven useful in similar circumstances [17,18]. Our experiments included the selection of plant-derived peptones to replace animalderived components and the use of fructose instead of the more common sucrose.…”

“…The effect of the new media on the expression levels of these proteins was tested in a leaf disc-based screening format, allowing the rapid comparison of a large number of different plant peptones and media compositions [18]. We confirmed the performance of the final plant peptone Agrobacterium medium (PAM) in terms of bacterial biomass yield and transient protein expression levels by cultivating A. tumefaciens in a 4-L batch-fermentation process followed by the vacuum infiltration of intact N. benthamiana plants.…”