Design of an artificial transcriptional system for production of high levels of recombinant proteins in tobacco (Nicotiana benthamiana).

Plants have recently received much attention as a means of producing recombinant proteins because they are easy to grow at a low cost and at a large scale. Although many plant protein expression systems have been developed, there remains a need for improved systems that deliver high yields of recombinant proteins. Transcription of the recombinant gene is a key step in increasing the yield of recombinant proteins. However, revealed strong promoters, terminators, and transcription factors that have been identified do not necessarily lead to high level production of recombinant proteins. Thus, in this study, a robust expression system was designed to produce high levels of recombinant protein consisting of a novel hybrid promoter, FM'M-UD, coupled with an artificial terminator, 3PRt. FM'M-UD contained fragments from three viral promoters (the promoters of Mirabilis mosaic caulimovirus (MMV) full-length transcript, the MMV subgenomic transcript, and figwort mosaic virus subgenomic transcript) and two types of cis-acting elements (four GAL4 binding sites and two zinc finger binding sites). The artificial terminator, 3PRt, consisted of the PINII and 35S terminators plus RB7, a matrix attachment region. The FM'M-UD promoter increased protein levels of reporters GFP, RBD : SD1 (part of S protein from SARS-CoV-2), and human interleukin-6 (hIL6) by 4-6-fold, 2-fold, and 6-fold, respectively, relative to those of the same reporters driven by the CaMV 35S promoter. Furthermore, when the FM'M-UD/3PRt expression cassette was expressed together with GAL4/TAC3d2, an artificial transcription factor that bound the GAL4 binding sites in FM'M-UD, levels of hIL6 increased by 10.7-fold, relative to those obtained from the CaMV 35S promoter plus the RD29B terminator. Thus, this novel expression system led to the production of a large amount of recombinant protein in plants.

SARS-CoV-2 spike trimer vaccine expressed in Nicotiana benthamiana adjuvanted with Alum elicits protective immune responses in mice.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has spurred rapid development of vaccines as part of the public health response. However, the general strategy used to construct recombinant trimeric severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) proteins in mammalian cells is not completely adaptive to molecular farming. Therefore, we generated several constructs of recombinant S proteins for high expression in Nicotiana benthamiana. Intramuscular injection of N. benthamiana-expressed Sct vaccine (NSct Vac) into Balb/c mice elicited both humoral and cellular immune responses, and booster doses increased neutralizing antibody titres. In human angiotensin-converting enzyme knock-in mice, two doses of NSct Vac induced anti-S and neutralizing antibodies, which cross-neutralized Alpha, Beta, Delta and Omicron variants. Survival rates after lethal challenge with SARS-CoV-2 were up to 80%, without significant body weight loss, and viral titres in lung tissue fell rapidly, with no infectious virus detectable at 7-day post-infection. Thus, plant-derived NSct Vac could be a candidate COVID-19 vaccine.

The B1 Domain of Streptococcal Protein G Serves as a Multi-Functional Tag for Recombinant Protein Production in Plants.

Plants have long been considered a cost-effective platform for recombinant production. A recently recognized additional advantage includes the low risk of contamination of human pathogens, such as viruses and bacterial endotoxins. Indeed, a great advance has been made in developing plants as a "factory" to produce recombinant proteins to use for biopharmaceutical purposes. However, there is still a need to develop new tools for recombinant protein production in plants. In this study, we provide data showing that the B1 domain of Streptococcal protein G (GB1) can be a multi-functional domain of recombinant proteins in plants. N-terminal fusion of the GB1 domain increased the expression level of various target proteins ranging from 1.3- to 3.1-fold at the protein level depending on the target proteins. GB1 fusion led to the stabilization of the fusion proteins. Furthermore, the direct detection of GB1-fusion proteins by the secondary anti-IgG antibody eliminated the use of the primary antibody for western blot analysis. Based on these data, we propose that the small GB1 domain can be used as a versatile tag for recombinant protein production in plants.

Production of a Bacteria-like Particle Vaccine Targeting Rock Bream (Oplegnathus fasciatus) Iridovirus Using Nicotiana benthamiana.

Viral diseases are extremely widespread infections that change constantly through mutations. To produce vaccines against viral diseases, transient expression systems are employed, and Nicotiana benthamiana (tobacco) plants are a rapidly expanding platform. In this study, we developed a recombinant protein vaccine targeting the major capsid protein (MCP) of iridovirus fused with the lysine motif (LysM) and coiled-coil domain of coronin 1 (ccCor1) for surface display using Lactococcus lactis. The protein was abundantly produced in N. benthamiana in its N-glycosylated form. Total soluble proteins isolated from infiltrated N. benthamiana leaves were treated sequentially with increasing ammonium sulfate solution, and recombinant MCP mainly precipitated at 40-60%. Additionally, affinity chromatography using Ni-NTA resin was applied for further purification. Native structure analysis using size exclusion chromatography showed that recombinant MCP existed in a large oligomeric form. A minimum OD600 value of 0.4 trichloroacetic acid (TCA)-treated L. lactis was required for efficient recombinant MCP display. Immunogenicity of recombinant MCP was assessed in a mouse model through enzyme-linked immunosorbent assay (ELISA) with serum-injected recombinant MCP-displaying L. lactis. In summary, we developed a plant-based recombinant vaccine production system combined with surface display on L. lactis.

Plant-based, adjuvant-free, potent multivalent vaccines for avian influenza virus via Lactococcus surface display.

Influenza epidemics frequently and unpredictably break out all over the world, and seriously affect the breeding industry and human activity. Inactivated and live attenuated viruses have been used as protective vaccines but exhibit high risks for biosafety. Subunit vaccines enjoy high biosafety and specificity but have a few weak points compared to inactivated virus or live attenuated virus vaccines, especially in low immunogenicity. In this study, we developed a new subunit vaccine platform for a potent, adjuvant-free, and multivalent vaccination. The ectodomains of hemagglutinins (HAs) of influenza viruses were expressed in plants as trimers (tHAs) to mimic their native forms. tHAs in plant extracts were directly used without purification for binding to inactivated Lactococcus (iLact) to produce iLact-tHAs, an antigen-carrying bacteria-like particle (BLP). tHAs BLP showed strong immune responses in mice and chickens without adjuvants. Moreover, simultaneous injection of two different antigens by two different formulas, tHAH5N6 + H9N2 BLP or a combination of tHAH5N6 BLP and tHAH9N2 BLP, led to strong immune responses to both antigens. Based on these results, we propose combinations of plant-based antigen production and BLP-based delivery as a highly potent and cost-effective platform for multivalent vaccination for subunit vaccines.

A Tonoplast-Associated Calcium-Signaling Module Dampens ABA Signaling during Stomatal Movement.

Stomatal movement, critical for photobiosynthesis, respiration, and stress responses, is regulated by many factors, among which abscisic acid (ABA) is critical. Early events of ABA signaling involve Ca2+ influx and an increase of cytoplasmic calcium ([Ca2+]cyt). Positive regulators of this process have been extensively studied, whereas negative regulators are obscure. ABA-induced stomatal closure involves K+ flux and vacuolar convolution. How these processes are connected with Ca2+ is not fully understood. We report that pat10-1, a null mutant of Arabidopsis (Arabidopsis thaliana) PROTEIN S-ACYL TRANSFERASE10 (PAT10), is hypersensitive to ABA-induced stomatal closure and vacuolar convolution. A similar phenotype was observed in cbl2;cbl3, the double mutant of CBL2 and CBL3, whose tonoplast association depends on PAT10. Functional loss of the PAT10-CBL2/CBL3 system resulted in enhanced Ca2+ influx and [Ca2+]cyt elevation. Promoting vacuolar K+ accumulation by overexpressing NHX2 suppressed ABA-hypersensitive stomatal closure and vacuolar convolution of the mutants, suggesting that PAT10-CBL2/CBL3 positively mediates vacuolar K+ accumulation. We have identified CBL-interacting protein kinases (CIPKs) that mediate CBL2/CBL3 signaling during ABA-induced stomatal movement. Functional loss of the PAT10-CBL2/3-CIPK9/17 system in guard cells enhanced drought tolerance. We propose that the tonoplast CBL-CIPK complexes form a signaling module that negatively regulates ABA signaling during stomatal movement.

Overexpression of Nogo receptor 3 (NgR3) correlates with poor prognosis and contributes to the migration of epithelial cells of nasopharyngeal carcinoma patients.

Lymph node metastasis (N classification) is one of the most important prognostic factors of nasopharyngeal carcinoma (NPC), and nerve involvement is associated with the transition of the N category in NPC patients. Although the nervous system has been reported to participate in many types of cancer progression, its functions in NPC progression remains unknown. Through analysis of gene profiling data, we demonstrate an enrichment of genes associated with neuronal development and differentiation in NPC tissues and cell lines. Among these genes, Nogo receptor 3 (NgR3), which was originally identified in the nervous system and plays a role in nerve development and regeneration, was inappropriately overexpressed in NPC cells and tissues. Immunohistochemical analysis demonstrated that the overexpression of NgR3 was correlated with poor prognosis in NPC patients. Overexpression of NgR3 promoted, and knocking down NgR3 inhibited, NPC cell migration and invasion in vitro and metastasis in vivo. The ability of NgR3 to promote cell migration was triggered by the downregulation of E-cadherin and enhanced cytoskeletal rearrangement and cell polarity, which were correlated with the activation of focal adhesion kinase (FAK). Collectively, NgR3 is a novel indicator of poor outcomes in NPC patients and plays an important role in driving the progression of NPC. These results suggest a potential link between the nervous system and NPC progression. KEY MESSAGES: Genes involved in the neuronal biological process are enriched in nasopharyngeal carcinoma. Overexpression of NgR3 correlates with poor prognosis of nasopharyngeal carcinoma. NgR3 promotes NPC cell migration by downregulating E-cadherin. NgR3 promotes NPC cell polarity and enhances the formation of NPC cell pseudopodia by activating FAK/Src pathway.

Development and Validation of a Serum Biomarker Panel for the Detection of Esophageal Squamous Cell Carcinoma through RNA Transcriptome Sequencing.

Serum tumor markers for the diagnosis of esophageal squamous cell carcinoma (ESCC) have low sensitivity. This study aims to identify new serum markers for ESCC diagnosis from RNA sequencing (RNA-seq) data. RNA-seq was performed using six pairs of ESCC and matched normal tissues. The candidates for ESCC were screened from the differentially expressed genes. The candidates were analyzed by ELISA from the serum of a test group and a validation group. Real-time PCR, Western blotting and immunohistochemistry were used to detect the expression of the candidates in tumor cell lines and tumor tissues. Ten genes were selected from the RNA-seq data. Serum levels of ADAM12, CHI3L1, MMP13 and SPP1 were significantly higher in the ESCC patients than in the healthy controls. A diagnostic model combining CHI3L1, MMP13, and SPP1 was established. The area under the curve (AUC) values for serum CHI3L1, MMP13, and SPP1 and the diagnostic model for discriminating ESCC patients from controls were 0.732, 0.881, 0.661 and 0.928, respectively. In the validation cohort, the AUC values were 0.753, 0.789, 0.696 and 0.843, respectively. Moreover, the AUC of the model for classifying patients with early ESCC was 0.918 in the test group and 0.857 in the validation group. Overexpression of CHI3L1, MMP13 and SPP1 was observed in the tumor cell lines and tissues. The diagnostic model composed of CHI3L1, MMP13 and SPP1 discriminates ESCC patients with high sensitivity. Our data highlight the potential of this diagnostic model for the noninvasive diagnosis of ESCC.

Adaptor Protein-3-Dependent Vacuolar Trafficking Involves a Subpopulation of COPII and HOPS Tethering Proteins.

Plant vacuoles are versatile organelles critical for plant growth and responses to environment. Vacuolar proteins are transported from the endoplasmic reticulum via multiple routes in plants. Two classic routes bear great similarity to other phyla with major regulators known, such as COPII and Rab5 GTPases. By contrast, vacuolar trafficking mediated by adaptor protein-3 (AP-3) or that independent of the Golgi has few recognized cargos and none of the regulators. In search of novel regulators for vacuolar trafficking routes and by using a fluorescence-based forward genetic screen, we demonstrated that the multispan transmembrane protein, Arabidopsis (Arabidopsis thaliana) PROTEIN S-ACYL TRANSFERASE10 (PAT10), is an AP-3-mediated vacuolar cargo. We show that the tonoplast targeting of PAT10 is mediated by the AP-3 complex but independent of the Rab5-mediated post-Golgi trafficking route. We also report that AP-3-mediated vacuolar trafficking involves a subpopulation of COPII and requires the vacuolar tethering complex HOPS. In addition, we have identified two novel mutant alleles of AP-3δ, whose point mutations interfered with the formation of the AP-3 complex as well as its membrane targeting. The results presented here shed new light on the vacuolar trafficking route mediated by AP-3 in plant cells.

Epstein-Barr Virus-Induced VEGF and GM-CSF Drive Nasopharyngeal Carcinoma Metastasis via Recruitment and Activation of Macrophages.

Chronic inflammation induced by persistent microbial infection plays an essential role in tumor progression. Although it is well documented that Epstein-Barr virus (EBV) infection is closely associated with nasopharyngeal carcinoma (NPC), how EBV-induced inflammation promotes NPC progression remains largely unknown. Here, we report that tumor infiltration of tumor-associated macrophages (TAM) and expression of CCL18, the cytokine preferentially secreted by TAM, closely correlate with serum EBV infection titers and tumor progression in two cohorts of NPC patients. In vitro, compared with EBV- NPC cell lines, EBV+ NPC cell lines exhibited superior capacity to attract monocytes and skew them to differentiate to a TAM-like phenotype. Cytokine profiling analysis revealed that NPC cells with active EBV replications recruited monocytes by VEGF and induced TAM by GM-CSF in an NF-κB-dependent manner. Reciprocally, TAM induced epithelial-mesenchymal transition and furthered NF-κB activation of tumor cells by CCL18. In humanized mice, NPC cells with active EBV replications exhibited increased metastasis, and neutralization of CCL18, GM-CSF, and VEGF significantly reduced metastasis. Collectively, our work defines a feed-forward loop between tumor cells and macrophages in NPC, which shows how metastatic potential can evolve concurrently with virus-induced chronic inflammation. Cancer Res; 77(13); 3591-604. ©2017 AACR.

Precocious leaf senescence by functional loss of PROTEIN S-ACYL TRANSFERASE14 involves the NPR1-dependent salicylic acid signaling.

We report here that Arabidopsis PROTEIN S-ACYL TRANSFERASE14 (PAT14), through its palmitate transferase activity, acts at the vacuolar trafficking route to repress salicylic acid (SA) signaling, thus mediating age-dependent but not carbon starvation-induced leaf senescence. Functional loss of PAT14 resulted in precocious leaf senescence and its transcriptomic analysis revealed that senescence was dependent on salicylic acid. Overexpressing PAT14 suppressed the expression of SA responsive genes. Introducing the SA deficient mutants, npr1-5 and NahG, but not other hormonal mutants, completely suppressed the precocious leaf senescence of PAT14 loss-of-function, further supporting the epistatic relation between PAT14 and the SA pathway. By confocal fluorescence microscopy, we showed that PAT14 is localized at the Golgi, the trans-Golg network/early endosome, and prevacuolar compartments, indicating its roles through vacuolar trafficking. By reporter analysis and real time PCRs, we showed that the expression PAT14, unlike most of the senescence associated genes, is not developmentally regulated, suggesting post-transcriptional regulatory mechanisms on its functionality. We further showed that the maize and wheat homologs of PAT14 fully rescued the precocious leaf senescence of pat14-2, demonstrating that the role of PAT14 in suppressing SA signaling during age-dependent leaf senescence is evolutionarily conserved between dicots and monocots.

Arabidopsis RhoGDIs Are Critical for Cellular Homeostasis of Pollen Tubes.

Rhos of plants (ROPs) play a key role in plant cell morphogenesis, especially in tip-growing pollen tubes and root hairs, by regulating an array of intracellular activities such as dynamic polymerization of actin microfilaments. ROPs are regulated by guanine nucleotide exchange factors (RopGEFs), GTPase activating proteins (RopGAPs), and guanine nucleotide dissociation inhibitors (RhoGDIs). RopGEFs and RopGAPs play evolutionarily conserved function in ROP signaling. By contrast, although plant RhoGDIs regulate the membrane extraction and cytoplasmic sequestration of ROPs, less clear are their positive roles in ROP signaling as do their yeast and metazoan counterparts. We report here that functional loss of all three Arabidopsis (Arabidopsis thaliana) GDIs (tri-gdi) significantly reduced male transmission due to impaired pollen tube growth in vitro and in vivo. We demonstrate that ROPs were ectopically activated at the lateral plasma membrane of the tri-gdi pollen tubes. However, total ROPs were reduced posttranslationally in the tri-gdi mutant, resulting in overall dampened ROP signaling. Indeed, a ROP5 mutant that was unable to interact with GDIs failed to induce growth, indicating the importance of the ROP-GDI interaction for ROP signaling. Functional loss of GDIs impaired cellular homeostasis, resulting in excess apical accumulation of wall components in pollen tubes, similar to that resulting from ectopic phosphatidylinositol 4,5-bisphosphate signaling. GDIs and phosphatidylinositol 4,5-bisphosphate may antagonistically coordinate to maintain cellular homeostasis during pollen tube growth. Our results thus demonstrate a more complex role of GDIs in ROP-mediated pollen tube growth.