Marc-Andre D'aoust

Antisense inhibition of tomato fruit sucrose synthase decreases fruit setting and the sucrose unloading capacity of young fruit.

The role of sucrose synthase (SuSy) in tomato fruit was studied in transgenic tomato (Lycopersicon esculentum) plants expressing an antisense fragment of fruit-specific SuSy RNA (TOMSSF) under the control of the cauliflower mosaic virus 35S promoter. Constitutive expression of the antisense RNA markedly inhibited SuSy activity in flowers and fruit pericarp tissues. However, inhibition was only slight in the endosperm and was undetectable in the embryo, shoot, petiole, and leaf tissues. The activity of sucrose phosphate synthase decreased in parallel with that of SuSy, but acid invertase activity did not increase in response to the reduced SuSy activity. The only effect on the carbohydrate content of young fruit was a slight reduction in starch accumulation. The in vitro sucrose import capacity of fruits was not reduced by SuSy inhibition at 23 days after anthesis, and the rate of starch synthesized from the imported sucrose was not lessened even when SuSy activity was decreased by 98%. However, the sucrose unloading capacity of 7-day-old fruit was substantially decreased in lines with low SuSy activity. In addition, the SuSy antisense fruit from the first week of flowering had a slower growth rate. A reduced fruit set, leading to markedly less fruit per plant at maturity, was observed for the plants with the least SuSy activity. These results suggest that SuSy participates in the control of sucrose import capacity of young tomato fruit, which is a determinant for fruit set and development.

Transient Expression of Antibodies in Plants Using Syringe Agroinfiltration

The improvements in agroinfiltration methods for plant-based transient expression now allow the production of significant amounts of recombinant proteins in a matter of days. While vacuum-based agroinfiltration has been brought to large scale to meet the cost, speed and surge capacity requirements for vaccine and therapeutic production, the more accessible and affordable syringe agroinfiltration procedure still represents a fast and high-yielding approach to recombinant protein production at lab scale. The procedure exemplified here has proven its reproducibility and high-yield capacity for the production of proteins with varying levels of complexity, including monoclonal antibodies.

Upstream regulatory regions from the maize Sh1 promoter confer tissue-specific expression of the ,-glucuronidase gene in tomato

Abstract A promoter fusion (Sh35) combining upstream regulatory regions from the maize Sh1 promoter with a truncated 35S promoter, Δ9035 (–90 to +8) has been compared with the original Sh1 promoter for its capacity to promote expression of the β-glucuronidase (GUS) gene in stably transformed tomato plants. For both promoters, very faint GUS expression was detected in the vegetative tissues, and no expression was detected in the fruit pericarp tissues. However, in the seed, Sh1 promoted low GUS expression but Sh35 directed 25-fold higher GUS expression. For both constructs, the profile of GUS expression was similar to that of endogenous sucrose synthase activity, but maximal GUS activity was reached 15 days after the peak of sucrose synthase activity.

PLANT EXPRESSION SYSTEM

A plant expression system and methods for expressing a protein of interest in a plant are provided. The plant expression system comprises a first nucleic acid sequence regulatory region sequence, operatively linked with a one or more than one comovirus enhancer, a nucleotide sequence of interest, one or more than one geminivirus amplification elements, and a second nucleic acid encoding a geminivirus replicase. The method of producing a protein of interest in a plant, involves introducing the plant expression system into a plant, or portion of the plant, and incubating the plant or the portion of the plant under conditions that permit the expression of the nucleotide sequence and producing the protein of interest.

Leaf proteome rebalancing in Nicotiana benthamiana for upstream enrichment of a transiently expressed recombinant protein.

A key factor influencing the yield of biopharmaceuticals in plants is the ratio of recombinant to host proteins in crude extracts. Postextraction procedures have been devised to enrich recombinant proteins before purification. Here, we assessed the potential of methyl jasmonate (MeJA) as a generic trigger of recombinant protein enrichment in Nicotiana benthamiana leaves before harvesting. Previous studies have reported a significant rebalancing of the leaf proteome via the jasmonate signalling pathway, associated with ribulose 1,5-bisphosphate carboxylase oxygenase (RuBisCO) depletion and the up-regulation of stress-related proteins. As expected, leaf proteome alterations were observed 7 days post-MeJA treatment, associated with lowered RuBisCO pools and the induction of stress-inducible proteins such as protease inhibitors, thionins and chitinases. Leaf infiltration with the Agrobacterium tumefaciens bacterial vector 24 h post-MeJA treatment induced a strong accumulation of pathogenesis-related proteins after 6 days, along with a near-complete reversal of MeJA-mediated stress protein up-regulation. RuBisCO pools were partly restored upon infiltration, but most of the depletion effect observed in noninfiltrated plants was maintained over six more days, to give crude protein samples with 50% less RuBisCO than untreated tissue. These changes were associated with net levels reaching 425 μg/g leaf tissue for the blood-typing monoclonal antibody C5-1 expressed in MeJA-treated leaves, compared to less than 200 μg/g in untreated leaves. Our data confirm overall the ability of MeJA to trigger RuBisCO depletion and recombinant protein enrichment in N. benthamiana leaves, estimated here for C5-1 at more than 2-fold relative to host proteins.

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.

Cell wall biochemical alterations during Agrobacterium-mediated expression of hemagglutinin-based influenza virus-like vaccine particles in tobacco

Summary Influenza virus‐like particles (VLPs) have been shown to induce a safe and potent immune response through both humoral and cellular responses. They represent promising novel influenza vaccines. Plant‐based biotechnology allows for the large‐scale production of VLPs of biopharmaceutical interest using different model organisms, including Nicotiana benthamiana plants. Through this platform, influenza VLPs bud from the plasma membrane and accumulate between the membrane and the plant cell wall. To design and optimize efficient production processes, a better understanding of the plant cell wall composition of infiltrated tobacco leaves is a major interest for the plant biotechnology industry. In this study, we have investigated the alteration of the biochemical composition of the cell walls of N. benthamiana leaves subjected to abiotic and biotic stresses induced by the Agrobacterium‐mediated transient transformation and the resulting high expression levels of influenza VLPs. Results show that abiotic stress due to vacuum infiltration without Agrobacterium did not induce any detectable modification of the leaf cell wall when compared to non infiltrated leaves. In contrast, various chemical changes of the leaf cell wall were observed post‐Agrobacterium infiltration. Indeed, Agrobacterium infection induced deposition of callose and lignin, modified the pectin methylesterification and increased both arabinosylation of RG‐I side chains and the expression of arabinogalactan proteins. Moreover, these modifications were slightly greater in plants expressing haemagglutinin‐based VLP than in plants infiltrated with the Agrobacterium strain containing only the p19 suppressor of silencing.

pH gradient mitigation in the leaf cell secretory pathway alters the defense response of Nicotiana benthamiana to agroinfiltration

Partial neutralization of the Golgi lumen pH by ectopic expression of influenza virus M2 proton channel stabilizes acid-labile and protease-susceptible recombinant proteins in the plant cell secretory pathway. Here, we assessed the impact of M2 channel expression on the proteome of Nicotiana benthamiana leaf tissue infiltrated with the bacterial gene vector Agrobacterium tumefaciens, keeping in mind the key role of pH homeostasis on secreted protein processing and the involvement of protein secretion processes in plant cells upon microbial challenge. The proteomes of leaves agroinfiltrated with an empty vector or with an M2 channel-encoding vector were compared with the proteome of non-infiltrated leaves using a iTRAQ quantitative proteomics procedure. Leaves infiltrated with the empty vector had a low soluble protein content compared to non-infiltrated leaves, associated with a strong decrease of photosynthesis-associated proteins (including Rubisco) and a parallel increase of stress-related secreted proteins (including pathogenesis-related proteins, protease inhibitors and molecular chaperones). M2 expression partly compromised these alterations of the proteome to restore original soluble protein and Rubisco contents, associated with higher levels of translation-associated (ribosomal) proteins and reduced levels of stress-related proteins in the apoplast. Proteome changes in M2-expressing leaves were determined both transcriptionally and post-transcriptionally, to alter the steady-state levels of proteins not only along the secretory pathway but also in other cellular compartments including the chloroplast, the cytoplasm, the nucleus and the mitochondrion. These data illustrate the cell-wide influence of Golgi lumen pH homeostasis on the leaf proteome of N. benthamiana plants responding to microbial challenge. They underline in practice the relevance of carefully considering the eventual off-target effects of accessory proteins used to modulate specific cellular or metabolic functions in plant protein biofactories.

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

Summary 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.