The average mass of CMG2-Fc per kg leaf fresh weight was 717 and 874 mg/kg leaf FW for the Kifunensine and Kifunensine samples, respectively . The control group expression level was consistent with previous results. These data suggest that the addition of kifunensine in the agro-infiltration process was not detrimental to transient protein production, and in this case, it resulted in a 22% increase in CMG2-Fc yield. This allows the use of kifunensine for modification of glycosylation profiles without compromising protein expression. Total soluble protein content of whole leaf extract of Kifunensine and Kifunensine groups were similar as shown in Figure 1, which indicates that kifunensine does not have significant impact on plant protein synthesis in general.In this study, the influences of one-time kifunensine vacuum infiltration on the expression level, N-glycan profile of a recombinant protein, mobile vertical grow tables namely CMG2-Fc, produced transiently in N. benthamiana plants were evaluated in both whole-leaf extract and AWF. We found that kifunensine had a positive impact on protein production when supplied in the agroinfiltration solution; specifically, we observed a 22% increase of protein expression with kifunensine treatment condition, presumably owing to its suppressing effects on ER-associated degradation pathway.
This finding is consistent with previous observations in multiple mammalian cell culture systems, and there is no reason to suspect that this will not be the case for other eukaryotic systems, including plant systems. Plants were monitored visually throughout the incubation period, and there were no significant phenotypical differences between kifunensine-treated and control groups.In the case of a whole-plant study, Roychowdhury et al. have shown that the yield of recombinant cholera toxin B sub-unit dropped by 30% and 75% when kifunensine was supplied at 5 µM hydroponically for 3 days and 5 days post agroinfiltration, respectively. In contrast, we observed slight increase in protein yield when kifunensine was infiltrated in leaf tissue instead of being supplied hydroponically. Thus, the lower protein yield they observed may have resulted from continued application in the hydroponic solution, and it is eliminated when kifunensine was supplied directly to leaf tissue through vacuum infiltration. In addition, in the hydroponic study, the target protein was retained in ER, while the model protein in our study and other cell culture studies were targeted for secretion. This difference in protein targeting may also play a role in protein yield changes upon kifunensine treatment. Kifunensine is known to inhibit enzymatic activity of class I α-mannosidases, and thus should stop mannose trimming in the first place to yield single Man9 N-glycan structures. However, we observed multiple oligomannose-type N-glycans with mannose residues ranging from 3 to 9, although the most abundant structure was Man9.
This observation is consistent with cell culture kifunensine studies, where multiple oligomannose-type N-glycans were detected under kifunensine treatment. This could potentially due to the difference in inhibition efficacy of kifunensine towards class I α-mannosidases isoforms, which results in an incomplete inhibition of mannose trimming from Man9 structure. Also taking enzyme kinetics into consideration, depending on the ER concentrations of Man9 glycoprotein substrate, class I α-mannosidases, and kifunensine, enzymatic mannose trimming from Man9 could take place even if the amount of active ER α-mannosidase I is low. Although mannose trimming was not completely inhibited at Man9 structure, this method still showed the ability to significantly modify glycosylation using a simple bio-processing approach. It is likely that Man9 abundance can be further increased if treated with higher concentration of kifunensine, but it is not necessary if the goal is to eliminate the production of plant-specific complex N-glycans. Although in this case, CMG2-Fc can be purified easily from whole-leaf extracts through a one-step purification with Protein A chromatography, in many cases, multiple steps of chromatography are required to purify a target protein from a large pool of host native proteins when a highly selective affinity tag is not present. This could result in low protein yield and difficulties to achieve high purity, which is typically required for therapeutic recombinant protein products. Targeting proteins to the apoplast allows the collection of target protein in AWF, which contains much lower levels of plant native proteins than whole-leaf extract since only secreted proteins are collected, thus lowers the downstream process complexity. In this case, CMG2-Fc purity and concentration increased by 3.9-folds and 4.4-folds, respectively, when collected in AWF versus in whole-leaf extract.
A similar trend was observed in kifunensine-treated samples, which confirms that kifunensine does not affect protein secretion, allowing secretion of CMG2-Fc with oligomannose-type glycoforms. The increase in purity and concentration was consistent with a previous study on harvesting a target protein from plant AWF. Hence, AWF collection is a feasible method for recombinant protein harvesting, which avoids contamination with intracellular host cell proteins, and is particularly valuable when target protein is hard to purify. Together with kifunensine treatment, apoplast-targeted recombinant protein without any plant-specific glycoforms can be transiently produced in N. benthamiana, and likely in other plants as well. Products can be collected at high concentration and purity from AWF, containing predominantly oligomannose-type N-glycans. Further studies should focus on determining how long the inhibition effect of kifunensine lasts after the one-time vacuum infiltration by monitoring the protein glycoform profile at multiple time points after vacuum infiltration, and the threshold concentration of kifunensine that results in a complete N-glycan shift from plant complex-type to oligomannose-type for other glycoproteins, particularly those with more N-linked glycosylation sites. In addition, the protein expression kinetics should be compared between kifunensine-treated and untreated groups to maximize target protein yield. Depending on the desired glycoform, this method can also be applied to other N-glycan processing inhibitors such as castanospermine, deoxynojirimycin, and swainsonine.The transient expression of CMG2-Fc is achieved by whole-plant vacuum infiltration of N. benthamiana with Agrobacterium tumefaciens EHA 105 containing binary vector for CMG2-Fc expression and Agrobacterium tumefaciens containing the pBIN binary vector for expression of the RNA silencing suppressor P19 from Tomato bushy stunt virus. Each A. tumefaciens strain was cultured separately in 20 mL of Luria-Bertani media with selection antibiotics for 18 h in the dark at 28 ◦C, on an orbital shaker at 250 rpm. Then, 8 mL of each culture is transferred to 250 mL LB media and incubated for 18 h in the dark at 28 ◦C at 250 rpm shaking to further amplify the cell population. The A. tumefaciens cells were collected by centrifugation at 1800× g for 30 min at 15 ◦C, and the cell density was quantified through absorbance measurement at 600 nm. Both A. tumefaciens strains were resuspended into infiltration buffer with a final cell density of 0.4 for each strain . Six-week old N. benthamiana plants were turned upside down and submerged into the agrobacteria suspension, followed with vacuum infiltration for 1 min after vacuum pressure reached 20 inches Hg. Infiltrated plants were then incubated in a growth chamber at 20 ◦C and 90% humidity for 6 days. All leaves were cut from the petioles were used for AWF collection or stored at −80 ◦C for whole leaf protein extraction.Microplate wells were coated with unlabeled Protein A at a concentration of 0.05 mg/mL in phosphate-buffered saline at 37 ◦C for 1 h, mobile vertical farm and then blocked with 5% nonfat dry milk in PBS buffer. Crude plant extract and purified CMG2-Fc standards were added to wells and incubated at 37 ◦C for 1 h. CMG2-Fc bound to Protein A on the plate was detected by adding 50 µL of horseradish peroxidase -labeled goat anti-human IgG at concentration of 0.4 µg/mL and incubated for 1 h at 37 ◦C.
Between each of these steps, microplates were washed with PBS with 0.05% v/v of Tween-20. Finally, the protein concentration was quantified by adding 100 µL of TMB substrate . Plates were incubated at room temperature for 10 min, followed by addition of 100 µL 1N HCl to stop the reaction. The TMB substrate reacts with HRP, allowing colorimetric detection of CMG2-Fc levels.Site-specific N-linked glycosylation analysis including measurement of the glycopeptide relative abundance of the protein was analyzed with mass spectrometry. Samples were first reduced with 2 µL of 550 mM dithiothreitol at 65 ◦C for 50 min to break the disulfide bond between cysteine amino acids. Then 4 µL of 450 mM iodoacetamide was added as an alkylation reagent for 25 min. Samples were placed in the dark environment to prevent the loss of IAA efficacy. After denaturing, proteins were digested with 1 µg of trypsin for 18 h in a 37 ◦C water bath. The digestion process was stopped by placing samples at −20 ◦C for around one hour. An Agilent 1290 infinity ultra-high-pressure liquid chromatography system coupled to an Agilent 6495 triple quadrupole mass spectrometer was used for N-glycosylation analysis. For sample separation, the analysis column used on the UHPLC system was an Agilent Eclipse plus C18 column . To protect the analysis column, an Agilent Eclipse plus C18 trap column was used to trap samples first. The mass spectrometer was operated using the dynamic multiple reaction monitoring mode which is a targeted tandem MS mode and Agilent MassHunter Quantitative Analysis B.05.02 software was used for data analysis. In the MRM method, glycopeptides were quantified with the glycopeptide mass used as the precursor ion and the common oxonium fragments with m/z 204.08 and 366.14 used as product ion. The concentration of glycopeptide in ion counts was normalized to the total ion counts of glycopeptides in the sample for the relative abundance calculation. The rhizosphere is the area around the plant root in soil where microorganisms are densely populated and dynamic interactions between the plants and microorganisms occur. These complex interactions and the assemblage of microorganisms are established, shaped, and maintained by the plant root. Plants exude photosynthetically fixed carbons as nutrient sources, secondary metabolites, and signaling molecules to populate root ecological niches with beneficial microorganisms. This “rhizosphere effect” has important implications for plant growth and protections against biotic and abiotic stressors, and for geochemical carbon cycling in soil environments. With the expected rise of severe, climate-change-related weather conditions such as drought and the growing need to increase plant productivity with sustainable agricultural practices, it is vital to gain a mechanistic understanding of the rhizosphere effect and engineer the rhizosphere to improve plant growth and resilience. To understand the mechanisms of the highly dynamic processes occurring in the rhizosphere, in situ interrogation of the system with high spatiotemporal resolution is necessary. However, due to the underground nature of the root system and the sheer complexity of the microbiome in the highly heterogeneous soil environment, studying root and microbial interactions has been challenging.
For example, in a typical rhizosphere analysis experiment, the plant is uprooted from soil in the field or a pot at a defined time point, and the microbiome and other relevant chemicals are sampled in a destructive manner. This practice of uprooting the plant is limiting because the spatial information on microbial community members is lost and the same plant generally cannot be sampled over time. Further, there are many confounding variables, such as chemical reactions with minerals in soil that complicate the analysis and make the studies less reproducible and relevant across different locations and environments. To overcome these experimental hurdles, researchers have designed various types of specialized devices such as rhizotrons and microfluidics devices to improve specific aspects of sampling, analytics, and manipulation of the rhizosphere system, albeit often deviating substantially from the natural system. Fluorescent microscopy is a promising tool for noninvasive in situ imaging of the microbial and root interactions with high spatiotemporal resolution. Developing in situ rhizosphere imaging methods is made especially more relevant as the microbial community colonization of the root and the persistence and succession patterns are likely dynamic and dependent on the developmental stage of the plant. Better knowledge in this can help guide synthetic microbial community inoculation protocols to optimize plant productivity. A notable development of in situ imaging devices is a microfluidic root chip by Massalha et al., in which real-time imaging of the microbial colonization of Arabidopsis thaliana seedlings was captured by high-resolution fluorescent confocal microscopy.