AgroLux: bioluminescent Agrobacterium to improve molecular pharming and study plant immunity.

Agroinfiltration in Nicotiana benthamiana is widely used to transiently express heterologous proteins in plants. However, the state of Agrobacterium itself is not well studied in agroinfiltrated tissues, despite frequent studies of immunity genes conducted through agroinfiltration. Here, we generated a bioluminescent strain of Agrobacterium tumefaciens GV3101 to monitor the luminescence of Agrobacterium during agroinfiltration. By integrating a single copy of the lux operon into the genome, we generated a stable 'AgroLux' strain, which is bioluminescent without affecting Agrobacterium growth in vitro and in planta. To illustrate its versatility, we used AgroLux to demonstrate that high light intensity post infiltration suppresses both Agrobacterium luminescence and protein expression. We also discovered that AgroLux can detect Avr/Cf-induced immune responses before tissue collapse, establishing a robust and rapid quantitative assay for the hypersensitive response (HR). Thus, AgroLux provides a non-destructive, versatile and easy-to-use imaging tool to monitor both Agrobacterium and plant responses.

pH Gradient Mitigation in the Leaf Cell Secretory Pathway Attenuates the Defense Response of Nicotiana benthamiana to Agroinfiltration.

Partial neutralization of the Golgi lumen pH by the ectopic expression of influenza virus M2 proton channel is useful to stabilize acid-labile recombinant proteins in plant cells, but the impact of pH gradient mitigation on host cellular functions has not been investigated. Here, we assessed the unintended effects of M2 expression on the leaf proteome of Nicotiana benthamiana infiltrated with the bacterial gene vector Agrobacterium tumefaciens. An isobaric tags for relative and absolute quantification quantitative proteomics procedure was followed to compare the leaf proteomes of plants agroinfiltrated with either an "empty" vector or an M2-encoding vector. Leaves infiltrated with the empty vector had a low soluble protein content compared to noninfiltrated control leaves, associated with increased levels of stress-related proteins but decreased levels of photosynthesis-associated proteins. M2 expression partly compromised these effects of agroinfiltration to restore soluble protein content in the leaf tissue, associated with restored levels of photosynthesis-associated proteins and reduced levels of stress-related proteins in the apoplast. These data illustrate the cell-wide influence of the Golgi lumen pH homeostasis on the leaf proteome of N. benthamiana responding to microbial challenge. They also underline the relevance of assessing the eventual unintended effects of accessory proteins used to modulate specific cellular or metabolic functions in plant protein biofactories.

Activity-based proteomics reveals nine target proteases for the recombinant protein-stabilizing inhibitor SlCYS8 in Nicotiana benthamiana.

Co-expression of protease inhibitors like the tomato cystatin SlCYS8 is useful to increase recombinant protein production in plants, but key proteases involved in protein proteolysis are still unknown. Here, we performed activity-based protein profiling to identify proteases that are inhibited by SlCYS8 in agroinfiltrated Nicotiana benthamiana. We discovered that SlCYS8 selectively suppresses papain-like cysteine protease (PLCP) activity in both apoplastic fluids and total leaf extracts, while not affecting vacuolar-processing enzyme and serine hydrolase activity. A robust concentration-dependent inhibition of PLCPs occurred in vitro when purified SlCYS8 was added to leaf extracts, indicating direct cystatin-PLCP interactions. Activity-based proteomics revealed that nine different Cathepsin-L/-F-like PLCPs are strongly inhibited by SlCYS8 in leaves. By contrast, the activity of five other Cathepsin-B/-H-like PLCPs, as well as 87 Ser hydrolases, was unaffected by SlCYS8. SlCYS8 expression prevented protein degradation by inhibiting intermediate and mature isoforms of granulin-containing proteases from the Resistant-to-Desiccation-21 (RD21) PLCP subfamily. Our data underline the key role of endogenous PLCPs on recombinant protein degradation and reveal candidate proteases for depletion strategies.

Recombinant protein susceptibility to proteolysis in the plant cell secretory pathway is pH-dependent.

Cellular engineering approaches have been proposed to mitigate unintended proteolysis in plant protein biofactories, involving the design of protease activity-depleted environments by gene silencing or in situ inactivation with accessory protease inhibitors. Here, we assessed the impact of influenza virus M2 proton channel on host protease activities and recombinant protein processing in the cell secretory pathway of Nicotiana benthamiana leaves. Transient co-expression assays with M2 and GFP variant pHluorin were first conducted to illustrate the potential of proton export from the Golgi lumen to promote recombinant protein yield. A fusion protein-based system involving protease-sensitive peptide linkers to attach inactive variants of tomato cystatin SlCYS8 was then designed to relate the effects of M2 on protein levels with altered protease activities in situ. Secreted versions of the cystatin fusions transiently expressed in leaf tissue showed variable 'fusion to free cystatin' cleavage ratios, in line with the occurrence of protease forms differentially active against the peptide linkers in the secretory pathway. Variable ratios were also observed for the fusions co-expressed with M2, but the extent of fusion cleavage was changed for several fusions, positively or negatively, as a result of pH increase in the Golgi. These data indicating a remodelling of endogenous protease activities upon M2 expression confirm that the stability of recombinant proteins in the plant cell secretory pathway is pH-dependent. They suggest, in practice, the potential of M2 proton channel to modulate the stability of protease-susceptible secreted proteins in planta via a pH-related, indirect effect on host resident proteases.

A Bioluminescent Agrobacterium tumefaciens for Imaging Bacterial Metabolic Activity in Planta.

Bioluminescence enables the monitoring of spatiotemporal dynamics and activity of bacterial populations in planta. We here describe a procedure to use AgroLux, a bioluminescent Agrobacterium tumefaciens, as a tool to study bacterial responses upon agroinfiltration. The first method details how to transform bioluminescent AgroLux to carry binary plasmids of interests. Then, a simple agroinfiltration assay for in planta imaging of bioluminescence signals is presented. AgroLux assays will increase our understanding of plant-Agrobacterium interactions and plant immunity and improve molecular farming.

Proteases of Nicotiana benthamiana: an emerging battle for molecular farming.

Molecular farming increasingly uses the tobacco relative Nicotiana benthamiana for production of recombinant proteins through transient expression. Several proteins are produced efficiently with this expression platform, but yields for other proteins are often very low. These low yields are frequently due to endogenous proteases. The latest genome annotations indicate that N. benthamiana encodes for at least 1243 putative proteases that probably act redundantly and consecutively on substrates in different subcellular compartments. Here, we discuss the N. benthamiana protease repertoire that may affect recombinant protein production and recent advances in protease depletion strategies to increase recombinant protein production in N. benthamiana.

Engineering Recombinant Virus-like Nanoparticles from Plants for Cellular Delivery.

Understanding capsid assembly following recombinant expression of viral structural proteins is critical to the design and modification of virus-like nanoparticles for biomedical and nanotechnology applications. Here, we use plant-based transient expression of the Bluetongue virus (BTV) structural proteins, VP3 and VP7, to obtain high yields of empty and green fluorescent protein (GFP)-encapsidating core-like particles (CLPs) from leaves. Single-particle cryo-electron microscopy of both types of particles revealed considerable differences in CLP structure compared to the crystal structure of infection-derived CLPs; in contrast, the two recombinant CLPs have an identical external structure. Using this insight, we exploited the unencumbered pore at the 5-fold axis of symmetry and the absence of encapsidated RNA to label the interior of empty CLPs with a fluorescent bioconjugate. CLPs containing 120 GFP molecules and those containing approximately 150 dye molecules were both shown to bind human integrin via a naturally occurring Arg-Gly-Asp motif found on an exposed loop of the VP7 trimeric spike. Furthermore, fluorescently labeled CLPs were shown to interact with a cell line overexpressing the surface receptor. Thus, BTV CLPs present themselves as a useful tool in targeted cargo delivery. These results highlight the importance of detailed structural analysis of VNPs in validating their molecular organization and the value of such analyses in aiding their design and further modification.

A Chimeric Affinity Tag for Efficient Expression and Chromatographic Purification of Heterologous Proteins from Plants.

The use of plants as expression hosts for recombinant proteins is an increasingly attractive option for the production of complex and challenging biopharmaceuticals. Tools are needed at present to marry recent developments in high-yielding gene vectors for heterologous expression with routine protein purification techniques. In this study, we designed the Cysta-tag, a new purification tag for immobilized metal affinity chromatography (IMAC) of plant-made proteins based on the protein-stabilizing fusion partner SlCYS8. We show that the Cysta-tag may be used to readily purify proteins under native conditions, and then be removed enzymatically to isolate the protein of interest. We also show that commonly used protease recognition sites for linking purification tags are differentially stable in leaves of the commonly used expression host Nicotiana benthamiana, with those linkers susceptible to cysteine proteases being less stable then serine protease-cleavable linkers. As an example, we describe a Cysta-tag experimental scheme for the one-step purification of a clinically useful protein, human α1-antitrypsin, transiently expressed in N. benthamiana. With potential applicability to the variety of chromatography formats commercially available for IMAC-based protein purification, the Cysta-tag provides a convenient means for the efficient and cost-effective purification of recombinant proteins from plant tissues.

Production of a tumour-targeting antibody with a human-compatible glycosylation profile in N. benthamiana hairy root cultures.

Hairy root (HR) cultures derived from Agrobacterium rhizogenes transformation of plant tissues are an advantageous biotechnological manufacturing platform due to the accumulation of recombinant proteins in an otherwise largely protein free culture medium. In this context, HRs descending from transgenic Nicotiana tabacum plants were successfully used for the production of several functional mAbs with plant-type glycans. Here, we expressed the tumor-targeting monoclonal antibody mAb H10 in HRs obtained either by infecting a transgenic N. tabacum line expressing H10 with A. rhizogenes or a glyco-engineered N. benthamiana line (ΔXTFT) with recombinant A. rhizogenes carrying mAb H10 heavy and light chain cDNAs. Selected HR clones derived from both plants accumulated mAb H10 in the culture medium with similar yields (2-3 mg/L). N-glycosylation profiles of antibodies purified from HR supernatant revealed the presence of plant-typical complex structures for N. tabacum-derived mAb H10 and of GnGn structures lacking xylose and fucose for the ΔXTFT-derived counterpart. Both antibody glyco-formats exhibited comparable antigen binding activities. Collectively, these data demonstrate that the co-infection of ΔXTFT Nicotiana benthamiana with recombinant A. rhizogenes is an efficient procedure for the generation of stable HR cultures expressing the tumor-targeting mAb H10 with a human-compatible glycosylation profile, thus representing an important step towards the exploitation of root cultures for the production of 'next generation' human therapeutic antibodies.

Companion Protease Inhibitors for the In Situ Protection of Recombinant Proteins in Plants.

We previously described a procedure for the use of plant protease inhibitors as "companion" accessory proteins to prevent unwanted proteolysis of clinically useful recombinant proteins in leaf crude protein extracts (Benchabane et al. Methods Mol Biol 483:265-273, 2009). Here we describe the use of these inhibitors for the protection of recombinant proteins in planta, before their extraction from leaf tissues. A procedure is first described involving inhibitors co-expressed along-and co-migrating-with the protein of interest in host plant cells. An alternative, single transgene scheme is then described involving translational fusions of the recombinant protein and companion inhibitor. These approaches may allow for a significant improvement of protein steady-state levels in leaves, comparable to yield improvements observed with protease-deficient strains of less complex protein expression hosts such as E. coli or yeasts.

An Accessory Protease Inhibitor to Increase the Yield and Quality of a Tumour-Targeting mAb in Nicotiana benthamiana Leaves.

The overall quality of recombinant IgG antibodies in plants is dramatically compromised by host endogenous proteases. Different approaches have been developed to reduce the impact of endogenous proteolysis on IgGs, notably involving site-directed mutagenesis to eliminate protease-susceptible sites or the in situ mitigation of host protease activities to minimize antibody processing in the cell secretory pathway. We here characterized the degradation profile of H10, a human tumour-targeting monoclonal IgG, in leaves of Nicotiana benthamiana also expressing the human serine protease inhibitor α1-antichymotrypsin or the cysteine protease inhibitor tomato cystatin SlCYS8. Leaf extracts revealed consistent fragmentation patterns for the recombinant antibody regardless of leaf age and a strong protective effect of SlCYS8 in specific regions of the heavy chain domains. As shown using an antigen-binding ELISA and LC-MS/MS analysis of antibody fragments, SlCYS8 had positive effects on both the amount of fully-assembled antibody purified from leaf tissue and the stability of biologically active antibody fragments containing the heavy chain Fc domain. Our data confirm the potential of Cys protease inhibitors as convenient antibody-stabilizing expression partners to increase the quality of therapeutic antibodies in plant protein biofactories.

Modulating secretory pathway pH by proton channel co-expression can increase recombinant protein stability in plants.

Eukaryotic expression systems are used for the production of complex secreted proteins. However, recombinant proteins face considerable biochemical challenges along the secretory pathway, including proteolysis and pH variation between organelles. As the use of synthetic biology matures into solutions for protein production, various host-cell engineering approaches are being developed to ameliorate host-cell factors that can limit recombinant protein quality and yield. We report the potential of the influenza M2 ion channel as a novel tool to neutralize the pH in acidic subcellular compartments. Using transient expression in the plant host, Nicotiana benthamiana, we show that ion channel expression can significantly raise pH in the Golgi apparatus and that this can have a strong stabilizing effect on a fusion protein separated by an acid-susceptible linker peptide. We exemplify the utility of this effect in recombinant protein production using influenza hemagglutinin subtypes differentially stable at low pH; the expression of hemagglutinins prone to conformational change in mildly acidic conditions is considerably enhanced by M2 co-expression. The co-expression of a heterologous ion channel to stabilize acid-labile proteins and peptides represents a novel approach to increasing the yield and quality of secreted recombinant proteins in plants and, possibly, in other eukaryotic expression hosts.