Agrobacterium-mediated Transient Expression Research Articles

Plant-based Vaccines

The pandemic of novel coronavirus disease-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has stimulated scientists from different backgrounds to gear up on developing vaccines against the virus. Several antigenic epitopes of the virus have the potential to induce an immunogenic response, among which viral spike protein (“S” protein) is considered to be the most suitable vaccine candidate. In this review, the latest progress in the field of plant molecular pharming (PMF), its application, limitations, and commercial initiatives toward the production of the SARS-CoV-2 vaccine have been discussed. Vaccine production by PMF has gained considerable attention these days and can be used for large-scale commercial production of antigenic proteins, antibodies, and other biopharmaceuticals. New age plant breeding techniques facilitated by CRISPR-Cas-based genome editing technology and next-generation sequencing methods also help to achieve greater precision and rapidity. Several unique advantages are offered by plant-based vaccine production techniques over that of the microbial or mammalian cell culture system. Virus-like particles and Agrobacterium-mediated transient somatic expression systems have a high potential for the large scale, cost-effective, and robust production of plant-derived vaccines against SARS-CoV-2.

Agrobacterium-Mediated Transient Transformation of Marchantia Liverworts.

Agrobacterium-mediated transient gene expression is a rapid and useful approach for characterizing functions of gene products in planta. However, the practicability of the method in the model liverwort Marchantia polymorpha has not yet been thoroughly described. Here we report a simple and robust method for Agrobacterium-mediated transient transformation of Marchantia thalli and its applicability. When thalli of M. polymorpha were co-cultured with Agrobacterium tumefaciens carrying β-glucuronidase (GUS) genes, GUS staining was observed primarily in assimilatory filaments and rhizoids. GUS activity was detected 2 days after infection and saturated 3 days after infection. We were able to transiently co-express fluorescently tagged proteins with proper localizations. Furthermore, we demonstrate that our method can be used as a novel pathosystem to study liverwort-bacteria interactions. We also provide evidence that air chambers support bacterial colonization.

Transient Production of Human β-Glucocerebrosidase With Mannosidic-Type N-Glycan Structure in Glycoengineered Nicotiana benthamiana Plants.

Gaucher disease is an inherited lysosomal storage disorder caused by a deficiency of functional enzyme β-glucocerebrosidase (GCase). Recombinant GCase has been used in enzyme replacement therapy to treat Gaucher disease. Importantly, the terminal mannose N-glycan structure is essential for the uptake of recombinant GCase into macrophages via the mannose receptor. In this research, recombinant GCase was produced using Agrobacterium-mediated transient expression in both wild-type (WT) and N-acetylglucosaminyltransferase I (GnTI) downregulated Nicotiana benthamiana (ΔgntI) plants, the latter of which accumulates mannosidic-type N-glycan structures. The successfully produced functional GCase exhibited GCase enzyme activity. The enzyme activity was the same as that of the conventional mammalian-derived GCase. Notably, N-glycan analysis revealed that a mannosidic-type N-glycan structure lacking plant-specific N-glycans (β1,2-xylose and α1,3-fucose residues) was predominant in all glycosylation sites of purified GCase produced from ΔgntI plants. Our research provides a promising alternative plant line as a host for the production of recombinant GCase with a mannosidic-type N-glycan structure. This glycoengineered plant might be applicable to the production of other pharmaceutical proteins, especially mannose receptor targeted protein, for therapeutic uses.

A Meloidogyne graminicola Pectate Lyase Is Involved in Virulence and Activation of Host Defense Responses.

Plant-parasitic nematodes secrete an array of cell-wall-degrading enzymes to overcome the physical barrier formed by the plant cell wall. Here, we describe a novel pectate lyase gene Mg-PEL1 from M. graminicola. Quantitative real-time PCR assay showed that the highest transcriptional expression level of Mg-PEL1 occurred in pre-parasitic second-stage juveniles, and it was still detected during the early parasitic stage. Using in situ hybridization, we showed that Mg-PEL1 was expressed exclusively within the subventral esophageal gland cells of M. graminicola. The yeast signal sequence trap system revealed that it possessed an N-terminal signal peptide with secretion function. Recombinant Mg-PEL1 exhibited hydrolytic activity toward polygalacturonic acid. Rice plants expressing RNA interference vectors targeting Mg-PEL1 showed an increased resistance to M. graminicola. In addition, using an Agrobacterium-mediated transient expression system and plant immune response assays, we demonstrated that the cell wall localization of Mg-PEL1 was required for the activation of plant defense responses, including programmed plant cell death, reactive oxygen species (ROS) accumulation and expression of defense-related genes. Taken together, our results indicated that Mg-PEL1 could enhance the pathogenicity of M. graminicola and induce plant immune responses during nematode invasion into plants or migration in plants. This provides a new insight into the function of pectate lyases in plants-nematodes interaction.

Harnessing a Transient Gene Expression System in Nicotiana benthamiana to Explore Plant Agrochemical Transporters.

Functional characterization of plant agrichemical transporters provided an opportunity to discover molecules that have a high mobility in plants and have the potential to increase the amount of pesticides reaching damage sites. Agrobacterium-mediated transient expression in tobacco is simple and fast, and its protein expression efficiency is high; this system is generally used to mediate heterologous gene expression. In this article, transient expression of tobacco nicotine uptake permease (NtNUP1) and rice polyamine uptake transporter 1 (OsPUT1) in Nicotiana benthamiana was performed to investigate whether this system is useful as a platform for studying the interactions between plant transporters and pesticides. The results showed that NtNUP1 increases nicotine uptake in N. benthamiana foliar discs and protoplasts, indicating that this transient gene expression system is feasible for studying gene function. Moreover, yeast expression of OsPUT1 apparently increases methomyl uptake. Overall, this method of constructing a transient gene expression system is useful for improving the efficiency of analyzing the functions of plant heterologous transporter-encoding genes and revealed that this system can be further used to study the functions of transporters and pesticides, especially their interactions.

Effects of lighting conditions on Agrobacterium-mediated transient expression of recombinant hemagglutinin in detached Nicotiana benthamiana leaves inoculated with a deconstructed viral vector

Effects of lighting conditions on Agrobacterium-mediated transient expression of recombinant hemagglutinin in detached Nicotiana benthamiana leaves inoculated with a deconstructed viral vector

Comparative potato genome editing: Agrobacterium tumefaciens-mediated transformation and protoplasts transfection delivery of CRISPR/Cas9 components directed to StPPO2 gene

Delivery of the CRISPR/Cas9 components to the plant cells is a key step in its application as a genome editing tool. Here, we compared Agrobacterium-mediated transformation and protoplast transfection with CRISPR/Cas9 components for potato genome editing. Two sgRNAs were designed to simultaneously direct Cas9 to the StPPO2 gene encoding for a tuber polyphenol oxidase (PPO). A binary vector (CR-PPO) was utilized for either Agrobacterium-mediated transformation or for transient expression in protoplasts, while ribonucleoprotein complexes (RNP-PPO) were additionally assayed in protoplasts. Editing efficiency varied, yielding 9.6%, 18.4% and 31.9% of edited lines from Agrobacterium-mediated transformation, RNP-PPO and CR-PPO transient expression in protoplasts, respectively. Furthermore, only the CR-PPO transient expression resulted in lines edited in all four StPPO2 alleles, observed in 46% of the edited lines and confirmed by tuber PPO activity and enzymatic browning analysis. Lines with on-target DNA insertions were found from all three approaches and characterized by sequencing. The dual-sgRNA strategy resulted in a low incidence of the targeted deletion, likely due to contrasting efficiencies between sgRNAs, that was partially evident in the in silico analysis. Our results demonstrate that gene editing efficiency in potato depends on the CRISPR/Cas9 delivery strategy and provide insights to consider when selecting the appropriate methodology. We compared the StPPO2 gene editing outcomes and efficiencies through Agrobacterium-mediated transformation and protoplasts transfection with DNA or RNPs, and demonstrated that genome editing efficiency depends on the CRISPR/Cas9 delivery approach in potato.

The role of chaperone complex HSP90-SGT1-RAR1 as the associated machinery for hybrid inviability between Nicotiana gossei Domin and N. tabacum L.

Two cultured cell lines (GTH4 and GTH4S) of a Nicotiana interspecific F1 hybrid (N. gossei×N. tabacum) were comparatively analyzed to find genetic factors related to hybrid inviability. Both cell lines proliferated at 37°C, but after shifting to 26°C, GTH4 started to die similar to the F1 hybrid seedlings, whereas GTH4S survived. As cell death requires de novo expression of genes and proteins, we compared expressed protein profiles between the two cell lines, and found that NgSGT1, a cochaperone of the chaperone complex (HSP90-SGT1-RAR1), was expressed in GTH4 but not in GTH4S. Agrobacterium-mediated transient expression of NgSGT1, but not NtSGT1, induced cell death in leaves of N. tabacum, suggesting its possible role in hybrid inviability. Cell death in N. tabacum was also induced by transient expression of NgRAR1, but not NtRAR1. In contrast, transient expression of any parental combinations of three components revealed that NgRAR1 promoted cell death, whereas NtRAR1 suppressed it in N. tabacum. A specific inhibitor of HSP90, geldanamycin, inhibited the progression of hypersensitive response-like cell death in GTH4 and leaf tissue after agroinfiltration. The present study suggested that components of the chaperone complex are involved in the inviability of Nicotiana interspecific hybrid.

Split-luciferase Complementation Imaging Assay to Study Protein-protein Interactions in Nicotiana benthamiana.

The experimental identification of protein-protein interactions (PPIs) is critical to understand protein function. Thus, a plethora of sensitive and versatile approaches have been developed to detect PPIs in vitro or in vivo, such as protein pull-down, yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP), and bimolecular fluorescence complementation (BiFC) assays. The recently established split-luciferase complementation (Split-LUC) imaging assay has several advantages compared to other approaches to detect PPIs in planta: it is a relatively simple and fast method to detect PPIs in vivo; the results are quantitative, with high sensitivity and low background; it measures dynamic PPIs in real-time; and it requires limited experimental materials and instrumentation. In this assay, the amino-terminal and carboxyl-terminal halves of the luciferase enzyme are fused to two proteins of interest (POIs), respectively; the luciferase protein is reconstituted when two POIs interact with each other, giving rise to a measurable activity. Here, we describe a protocol for the Split-LUC imaging assay using a pair of modified gateway-compatible vectors upon Agrobacterium-mediated transient expression in Nicotiana benthamiana. With this setup, we have successfully confirmed a series of interactions among virus-plant proteins, virus-virus proteins, plant-plant proteins, or bacteria-plant proteins in N. benthamiana.

Identification and Characterization of a Novel Protein Disulfide Isomerase Gene (MgPDI2) from Meloidogyne graminicola.

Protein disulfide isomerase (PDI) is a multifunctional enzyme that catalyzes rate-limiting reactions such as disulfide bond formation, isomerization, and reduction. There is some evidence that indicates that PDI is also involved in host-pathogen interactions in plants. In this study, we show that the rice root-knot nematode, Meloidogyne graminicola, has evolved a secreted effector, MgPDI2, which is expressed in the subventral esophageal glands and up-regulated during the early parasitic stage of M. graminicola. Purified recombinant MgPDI2 functions as an insulin disulfide reductase and protects plasmid DNA from nicking. As an effector, MgPDI2 contributes to nematode parasitism. Silencing of MgPDI2 by RNA interference in the pre-parasitic second-stage juveniles (J2s) reduced M. graminicola multiplication and also increased M. graminicola mortality under H2O2 stress. In addition, an Agrobacterium-mediated transient expression assay found that MgPDI2 caused noticeable cell death in Nicotiana benthamiana. An intact C-terminal region containing the first catalytic domain (a) with an active motif (Cys-Gly-His-Cys, CGHC) and the two non-active domains (b and b′) is required for cell death induction in N. benthamiana. This research may provide a promising target for the development of new strategies to combat M. graminicola infections.

Optimization of Agrobacterium-Mediated Transient Expression of Immunocytokine Based on Human Interferon-α-2b and Anti-HER2 Antibody in Nicotiana Benthamiana

Optimizing the composition of an agroinfiltration solution is one way to boost the efficiency of the transient expression of the target protein in plants by using Agrobacterium tumefaciens. We use the design of the experiment to investigate the effects that some chemical additives introduced into the agroinfiltration medium have on the yield of the target immunocytokine. It is shown that acetosyringone (at 600 μM) and the surfactant Pluronic-68 (at 0.2%) markedly increase the yield of the target protein; lipoic acid (0–5 μM) and povidone (0–0.5 g/L) do not have a significant effect on this parameter; and ascorbic acid at concentrations of 25–50 mM significantly lowers the yield. Using an agroinfiltration solution with the specific composition developed in this study, the yield of the target protein is found to be more than 500 μg/g, which is sufficiently high for the practical production of recombinant proteins in plants.

The sugarcane ShMYB78 transcription factor activates suberin biosynthesis in Nicotiana benthamiana.

A sugarcane MYB present in the culm induces suberin biosynthesis and is involved both with fatty acid and phenolics metabolism. Few transcription factors have been described as regulators of cell wall polymers deposition in C4 grasses. Particularly, regulation of suberin biosynthesis in this group of plants remains poorly understood. Here, we showed that the sugarcane MYB transcription factor ShMYB78 is an activator of suberin biosynthesis and deposition. ShMYB78 was identified upon screening genes whose expression was upregulated in sugarcane internodes undergoing suberization during culm development or triggered by wounding. Agrobacterium-mediated transient expression of ShMYB78 in Nicotiana benthamiana leaves induced the ectopic deposition of suberin and its aliphatic and aromatic monomers. Further, the expression of suberin-related genes was induced by ShMYB78 heterologous expression in Nicotiana benthamiana leaves. ShMYB78 was shown to be a nuclear protein based on its presence in sugarcane internode nuclear protein extracts, and protoplast transactivation assays demonstrated that ShMYB78 activates the promoters of the sugarcane suberin biosynthetic genes β-ketoacyl-CoA synthase (ShKCS20) and caffeic acid-O-methyltransferase (ShCOMT). Our results suggest that ShMYB78 may be involved in the transcriptional regulation of suberin deposition, from fatty acid metabolism to phenylpropanoid biosynthesis, in sugarcane internodes.

Genome-Wide Identification of Apple Ubiquitin SINA E3 Ligase and Functional Characterization of MdSINA2.

SINA (Seven in absentia) proteins are a small family of ubiquitin ligases that play important roles in regulating plant growth and developmental processes as well as in responses to diverse types of biotic and abiotic stress. However, the characteristics of the apple SINA family have not been previously studied. Here, we identified 11 MdSINAs members in the apple genome based on their conserved, N‐terminal RING and C-terminal SINA domains. We also reconstructed a phylogeny of these genes; characterized their chromosomal location, structure, and motifs; and identified two major groups of MdSINA genes. Subsequent qRT-PCR analyses were used to characterize the expression of MdSINA genes in various tissues and organs, and levels of expression were highest in leaves. MdSINAs were significantly induced under ABA and carbon- and nitrate-starvation treatment. Except for MdSINA1 and MdSINA7, the other MdSINA proteins could interact with each other. Moreover, MdSINA2 was found to be localized in the nucleus using Agrobacterium-mediated transient expression. Western-blot analysis showed that MdSINA2 accumulated extensively under light, decreased under darkness, and became insensitive to light when the RING domain was disrupted. Finally, ABA-hypersensitive phenotypes were confirmed by transgenic calli and the ectopic expression of MdSINA2 in Arabidopsis. In conclusion, our results suggest that MdSINA genes participate in the responses to different types of stress, and that MdSINA2 might act as a negative regulator in the ABA stress response.

A simple and efficient Agrobacterium-mediated transient expression system to dissect molecular processes in Brassica rapa and Brassica napus.

The family Brassicaceae is a source of important crop species, including Brassica napus (oilseed rape), Brassica oleracea, and B. rapa, that is used globally for oil production or as a food source (e.g., pak choi or turnip). However, despite advances in recent years, including genome sequencing, a lack of established tools tailored to the study of Brassica crop species has impeded efforts to understand their molecular processes in greater detail. Here, we describe the use of a simple Agrobacterium‐mediated transient expression system adapted to B. rapa and B. napus that could facilitate study of molecular and biochemical events in these species. We also demonstrate the use of this method to characterize the N‐degron pathway of protein degradation in B. rapa. The N‐degron pathway is a subset of the ubiquitin‐proteasome system and represents a mechanism through which proteins may be targeted for degradation based on the identity of their N‐terminal amino acid residue. Interestingly, N‐degron‐mediated processes in plants have been implicated in the regulation of traits with potential agronomic importance, including the responses to pathogens and to abiotic stresses such as flooding tolerance. The stability of transiently expressed N‐degron reporter proteins in B. rapa indicates that its N‐degron pathway is highly conserved with that of Arabidopsis thaliana. These findings highlight the utility of Agrobacterium‐mediated transient expression in B. rapa and B. napus and establish a framework to investigate the N‐degron pathway and its roles in regulating agronomical traits in these species.Significance statementWe describe an Agrobacterium‐mediated transient expression system applicable to Brassica crops and demonstrate its utility by identifying the destabilizing residues of the N‐degron pathway in B. rapa. As the N‐degron pathway functions as an integrator of environmental signals, this study could facilitate efforts to improve the robustness of Brassica crops.

Protocol for Agrobacterium-mediated transformation of tall fescue and future perspective on the application of genome editing.

Tall fescue (Festuca arundinacea Schreb.) is a major cool-season perennial grass grown for forage and turf. We have obtained transgenic tall fescue by Agrobacterium-mediated transformation to improve agronomically important traits. In our protocol, we use embryogenic calli derived from not only mature seeds but also shoot tips. Although tall fescue cultivars consist of various genotypes with different genetic variation, we can produce transgenic plants at any time with calli induced from shoot tips of in vitro-maintained responsive genotypes. When the hygromycin phosphotransferase gene is used as a selectable marker, transformants are selected by incubation with 100 mg l-1 hygromycin in both selection and regeneration media. Since tall fescue is an anemophilous species, the cultivation of transgenic plants poses the risk of transgenic pollen flow. Recently, it has been reported that genome-edited plants without the integration of foreign DNA fragments can be produced by an Agrobacterium-mediated transient gene expression system. We hope that our protocol will contribute to production of transgene-free genome-edited tall fescue.

An attempt to establish an Agrobacterium-mediated transient expression system in medicinal plants.

Genetic transformation has always been an important method for studying medical plant secondary metabolic regulation, among which stable transformation has a good reproducibility. However, it was time-consuming to obtain a stable transformed hairy root or transgenic plants, which was difficult to satisfy the great demand of researches on medical plant secondary metabolism-related genes. Moreover, Agrobacterium tumefaciens-mediated transient transformation has been extensively applied in studies of functional genes because of its simpleness, low cost, and short period. However, presently, researches on medical plant functional genes commonly used stable genetic transformation and some high-cost and high-difficulty transient transformation methods, such as gene gun and protoplast transformation. Thus, in this study, we selected the seedlings of Nicotiana benthamiana, Salvia miltiorrhiza, and Prunella vulgaris as the experimental material, with the methods of Agrobacterium tumefaciens injection, fast Agrobacterium-mediated seedling transformation (FAST), and FAST and mechanical damage. The results demonstrated that the injection transient transformation system of pCAMBIA1301 vector mediated by A. tumefaciens and the transient transformation of seedling system were not established in S. miltiorrhiza. In addition, the instantaneous transformation system of N. benthamiana and P. vulgaris seedlings was basically set up by FAST method. Besides, using the method of FAST and mechanical damage, the transient genetic transformation system of P. vulgaris seedlings was established for the first time. A. tumefaciens-mediated transient transformation of seedlings with pEAQ vectors provided an effective way and reference for the further study of functional genes of the medicinal plants N. benthamiana and P. vulgaris.

GFP transient expression and silencing in Fragaria x ananassa

BACKGROUND: Stable transformation, transient expression, and post-transcriptional gene silencing (PTGS) are powerful methodologies that allow exploration of gene function. OBJECTIVE: We aimed to apply these methodologies to strawberry leaves. Methods: the binary vectors pBIN19-sgfp, pBICdsGFP and pBIN61-P19 were transferred into A. tumefaciens EHA105 supervirulent strain by electroporation. The sgfp gene silencing was carried out in stably transformed GFP (green fluorescent protein) F. x ananassa Duch. cultivar ‘Pájaro’ strawberry plants by agroinfiltration. GFP-fluorescence was observed using a stereomicroscope (507 nm). RESULTS: We attained a GFP transgenic F. x ananassa plant that expresses the functional protein in all the tissues during a complete and normal life cycle. In planta sgfp transient expression and silencing have also been achieved in F. x ananassa cv. ‘Pájaro’ leaves of wild type and GFP transgenic plants, respectively. Agrobacterium-mediated transient expression was visualized as high intensity green fluorescent spots as early as 7 days post-agroinfiltration (dpa), peaking between 10 and 14 dpa and persisting as long as 24 dpa. A knockdown GFP phenotype was achieved by silencing using a dsGFP hairpin. CONCLUSION: This work contributes significantly to the reverse genetics field in strawberry, might help to gain knowledge in the analysis of functional promoters and thereby allow protein expression and silencing of genes. This will help to develop resistant plants expressing plant defense elicitors or silencing pathogen receptors and/or negative regulators of plant defense.

Genome organization and interaction with capsid protein in a multipartite RNA virus

We report the asymmetric reconstruction of the single-stranded RNA (ssRNA) content in one of the three otherwise identical virions of a multipartite RNA virus, brome mosaic virus (BMV). We exploit a sample consisting exclusively of particles with the same RNA content-specifically, RNAs 3 and 4-assembled in planta by agrobacterium-mediated transient expression. We find that the interior of the particle is nearly empty, with most of the RNA genome situated at the capsid shell. However, this density is disordered in the sense that the RNA is not associated with any particular structure but rather, with an ensemble of secondary/tertiary structures that interact with the capsid protein. Our results illustrate a fundamental difference between the ssRNA organization in the multipartite BMV viral capsid and the monopartite bacteriophages MS2 and Qβ for which a dominant RNA conformation is found inside the assembled viral capsids, with RNA density conserved even at the center of the particle. This can be understood in the context of the differing demands on their respective lifecycles: BMV must package separately each of several different RNA molecules and has been shown to replicate and package them in isolated, membrane-bound, cytoplasmic complexes, whereas the bacteriophages exploit sequence-specific "packaging signals" throughout the viral RNA to package their monopartite genomes.

Identification and functional analysis of soybean stearoyl-ACP Δ⁹ desaturase (GmSAD) gene family

Stearoyl-ACP Δ⁹ desaturase (SAD) catalyzes the synthesis of monounsaturated oleic acid or palmitoleic acid in plastids. SAD is the key enzyme to control the ratio of saturated fatty acids to unsaturated fatty acids in plant cells. In order to analyze the regulation mechanism of soybean oleic acid synthesis, soybean (Glycine max) GmSAD family members were genome-wide identified, and their conserved functional domains and physicochemical properties were also analyzed by bioinformatics tools. The spatiotemporal expression profile of each member of GmSADs was detected by qRT-PCR. The expression vectors of GmSAD5 were constructed. The enzyme activity and biological function of GmSAD5 were examined by Agrobacterium-mediated transient expression in Nicotiana tabacum leaves and genetic transformation of oleic acid-deficient yeast (Saccharomyces cerevisiae) mutant BY4389. Results show that the soybean genome contains five GmSAD family members, all encoding an enzyme protein with diiron center and two conservative histidine enrichment motifs (EENRHG and DEKRHE) specific to SAD enzymes. The active enzyme protein was predicted as a homodimer. Phylogenetic analysis indicated that five GmSADs were divided into two subgroups, which were closely related to AtSSI2 and AtSAD6, respectively. The expression profiles of GmSAD members were significantly different in soybean roots, stems, leaves, flowers, and seeds at different developmental stages. Among them, GmSAD5 expressed highly in the middle and late stages of developmental seeds, which coincided with the oil accumulation period. Transient expression of GmSAD5 in tobacco leaves increased the oleic acid and total oil content in leaf tissue by 5.56% and 2.73%, respectively, while stearic acid content was reduced by 2.46%. Functional complementation assay in defective yeast strain BY4389 demonstrated that overexpression of GmSAD5 was able to restore the synthesis of monounsaturated oleic acid, resulting in high oil accumulation. Taken together, soybean GmSAD5 has strong selectivity to stearic acid substrates and can efficiently catalyze the biosynthesis of monounsaturated oleic acid. It lays the foundation for the study of soybean seed oleic acid and total oil accumulation mechanism, providing an excellent target for genetic improvement of oil quality in soybean.

Transient protein expression in tobacco BY-2 plant cell packs using single and multi-cassette replicating vectors

Key messageThis is the first evidence that replicating vectors can be successfully used for transient protein expression in BY-2 plant cell packs.Transient recombinant protein expression in plants and recently also plant cell cultures are of increasing interest due to the speed, safety and scalability of the process. Currently, studies are focussing on the design of plant virus-derived vectors to achieve higher amounts of transiently expressed proteins in these systems. Here we designed and tested replicating single and multi-cassette vectors that combine elements for enhanced replication and hypertranslation, and assessed their ability to express and particularly co-express proteins by Agrobacterium-mediated transient expression in tobacco BY-2 plant cell packs. Substantial yields of green and red fluorescent proteins of up to ~ 700 ng/g fresh mass were detected in the plant cells along with position-dependent expression. This is the first evidence of the ability of replicating vectors to transiently express proteins in BY-2 plant cell packs.