Transient gene expression using syringe agroinfiltration offers a facile and efficient method for various transgenic applications. Nicotiana benthamiana leaves show reliable and high transformation efficiency, but also show some variability in quantitative analysis. We used a nested design in our agroinfiltration experiments to analyze the sources of this variation.
An intron containing firefly luciferase gene was used as an agroinfiltration reporter. Several 6-week-old tobacco plants were infiltrated by upper leaves, after 2 days of transient expression, several samples were punctured from the leaves, and protein extracts from the samples were repeatedly measured for luciferase activity. Interestingly, most of the variation was between sampling points in leaves, with the next important source being different leaves of each plant. Variation between similar experiments, between plants and between remeasurements of extracts can be easily reduced.
Efforts and expenditures on agronomic testing can be optimized when the sources of variation are known. In summary, sample more plants but fewer leaves and more positions on leaves, but run only a few technical replicates.
A wide range of methods and techniques have been used to study promoter activity, gene-protein activity, or protein-protein interactions in vivo to induce transient gene expression in plant cells [1-4]. Protoplast transformation and particle bombardment have the oldest history [ 5 , 6 ] and are still used because of their benefits, despite the disadvantages of being time-consuming and sometimes inefficient [ 7 ]. For example, particle bombardment targets intact tissue to differentiate into different cell and tissue types. Agrobacterium-based transient assays have been increasingly used in recent years [8-10]. Agrobacterium is the oldest [ 11 , 12 ] and still the preferred gene transfer tool for generating stable transformed plants. Agrobacterium interacts with a wide variety of plant cells and injects single-stranded DNA molecules into plant cells via the type IV secretion system, which then translocates to the nucleus, becomes double-stranded, and finally integrates at the chromosome position [ 13 ].
Interestingly, genes located on translocated DNA (T-DNA) are expressed early in the process and, according to the current view, are independent of the integration event . This initial expression is transient and strongly decreases after reaching a peak of approx. 2 days . Transient loss of expression is not due to rapid degradation of nonintegrated T-DNA, but to active silencing. Double infiltration of T-DNA, in which viral suppressor proteins are secreted, prolongs the transient expression for several days, with maximal accumulation occurring 6–7 days after infiltration [ 16 , 17 ].
Agrobacterium-based transient gene expression occurs in a variety of tissues [ 9 ], but the most commonly used target is the mesophyll of expanded leaves. The agrobacterial suspension can be infiltrated into the parenchymal air space by vacuum or syringe, so this method is called “agroinfiltration”. Nicotiana benthamiana leaves in particular have proven to be useful targets for agroinfiltration. A large proportion of N. benthamiana mesophyll cells are transformed by Agrobacterium, and in extreme cases, up to 50% of total soluble leaf proteins [ 18 ] can be encoded by translocated genes. This has led to the use of medicinally active proteins produced by foliar infiltration [19–21]. For research purposes, proteins that are difficult to access in microbial systems have been produced in N. benthamiana, allowing their characterization [22-24] or their activity in plant cells, leading to elucidation of their role or formation in metabolic changes (enzymes). of small molecules of pharmaceutical and commercial interest .
In addition to large-scale protein production, syringe or vacuum agroinfiltration has been used to study protein-protein interactions and plant promoter functions in vivo [ 1 , 26 ]. For quantitative analysis, variations from biological or technical sources limit the precision and statistical power of the analysis. Compared to the use of stably transformed plant lines, transient expression analysis already eliminates variation due to different chromosomal positions and the epigenetic state of the transfected gene. However, many changes remain. In this work, we address the sources of this variation using a hierarchical (nested) experimental design in which the components of experimental variance can be isolated. Our goal was to understand the sources of variation in order to design experiments that are optimal for the effort and cost involved.
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