A Highly Efficient Agrobacterium-Mediated Method for Transient Gene Expression and Functional Studies in Multiple Plant Species

Abstract

Although the use of stable transformation technology has led to great insight into gene function, its application in high-throughput studies remains arduous. Agro-infiltration have been widely used in species such as Nicotiana benthamiana for the rapid detection of gene expression and protein interaction analysis, but this technique does not work efficiently in other plant species, including Arabidopsis thaliana . As an efficient high-throughput transient expression system is currently lacking in the model plant species A. thaliana , we developed a method that is characterized by high efficiency, reproducibility, and suitability for transient expression of a variety of functional proteins in A. thaliana and 7 other plant species, including Brassica oleracea , Capsella rubella , Thellungiella salsuginea , Thellungiella halophila , Solanum tuberosum , Capsicum annuum , and N. benthamiana . Efficiency of this method was independently verified in three independent research facilities, pointing to the robustness of this technique. Furthermore, in addition to demonstrating the utility of this technique in a range of species, we also present a case study employing this method to assess protein–protein interactions in the sucrose biosynthesis pathway in Arabidopsis .

On the Detection and Functional Significance of the Protein–Protein Interactions of Mitochondrial Transport Proteins

“…However, several studies have consistently shown that yeast ADP/ATP carriers are monomeric in structure and function [44]. As the mitochondrial respiratory chain supercomplexes have been investigated by BN-PAGE under different conditions [78], this method could be used to detect the dynamics of the mitochondrial carrier protein complex. In addition, several mitochondrial carriers were also investigated by BN-PAGE, such as the 2-oxoglutarate carrier [52], the dicarboxylate carrier [51], and the tricarboxylate carrier [50].…”

Regulation of Arabidopsis photoreceptor CRY2 by two distinct E3 ubiquitin ligases

“…These results demonstrate that LRBs directly and specifically interact with photoexcited and phosphorylated CRY2. We next examined LRB1-CRY2 and LRB2-CRY2 interactions in vivo, using the BiFC (Bimolecular Fluorescence Complementation) assay in Arabidopsis cells 34 To further test the hypothesis that the Cul3 LRBs and Cul4 COP1-SPAs E3 ubiquitin ligases catalyze blue light-dependent CRY2 ubiquitination, we performed three independent experiments to investigate the activity of LRBs and COP1 in the blue light-induced polyubiquitination of CRY2, using the IP/immunoblot assays. In the first two experiments, we prepared transgenic plants overexpressing Flag-GFP-tagged CRY2 (FGFP-CRY2) in the wild-type (WT), the strong (lrb1lrb2-1lrb3) or weak (lrb1lrb2-2lrb3) alleles of the lrb123 triple mutant ( Fig.…”

“…Western blots were performed as described above. Primary antibodies used here were anti-Flag (1:1500, cat # F1804, Sigma), anti-Myc (1:5000, cat # 05-724, Millipore) and anti-HA (1:5000, cat # 12013819001, Roche), and secondary antibodies used were anti-Mouse-HRP (1:10000, cat # 31430, ThermoFisher) and anti-Rabbit-HRP (1:10000, cat # 31460, ThermoFisher).Bimolecular fluorescence complementation (BiFC) assayBiFC assays in Arabidopsis plants were performed as previously described methods with minor modifications34 . Briefly, Agrobacteria AGL0 transformed with BiFC plasmids were grown in LB medium with 40 μM Acetosyringone and 1% Glucose in 28°C shaker overnight.…”

Molecular Pharming to Support Human Life on the Moon, Mars, and Beyond

“…From the perspective of molecular pharming, lettuce 52,53 serves as a fast-growing crop with a small cultivation footprint in which the edible biomass is also the expressible biomass capable of producing pharmaceuticals. Potato 54,55 represents a slow-growing crop that has the advantage of distinct edible biomass (tubers) and expressible biomass (leaves); molecular pharming would not significantly impact the total available food resource. Leaves detached from the intact plant are capable of providing comparable pharmaceutical yield to those from the intact plant [56][57][58] .…”