The Tobamoviral Movement Protein: A "Conditioner" to Create a Favorable Environment for Intercellular Spread of Infection.

During their evolution, viruses acquired genes encoding movement protein(s) (MPs) that mediate the intracellular transport of viral genetic material to plasmodesmata (Pd) and initiate the mechanisms leading to the increase in plasmodesmal permeability. Although the current view on the role of the viral MPs was primarily formed through studies on tobacco mosaic virus (TMV), the function of its MP has not been fully elucidated. Given the intercellular movement of MPs independent of genomic viral RNA (vRNA), this characteristic may induce favorable conditions ahead of the infection front for the accelerated movement of the vRNA (i.e. the MP plays a role as a "conditioner" of viral intercellular spread). This idea is supported by (a) the synthesis of MP from genomic vRNA early in infection, (b) the Pd opening and the MP transfer to neighboring cells without formation of the viral replication complex (VRC), and (c) the MP-mediated movement of VRCs beyond the primary infected cell. Here, we will consider findings that favor the TMV MP as a "conditioner" of enhanced intercellular virus movement. In addition, we will discuss the mechanism by which TMV MP opens Pd for extraordinary transport of macromolecules. Although there is no evidence showing direct effects of TMV MP on Pd leading to their dilatation, recent findings indicate that MPs exert their influence indirectly by modulating Pd external and structural macromolecules such as callose and Pd-associated proteins. In explaining this phenomenon, we will propose a mechanism for TMV MP functioning as a conditioner for virus movement.

Plasmodesmata Conductivity Regulation: A Mechanistic Model.

Plant cells form a multicellular symplast via cytoplasmic bridges called plasmodesmata (Pd) and the endoplasmic reticulum (ER) that crosses almost all plant tissues. The Pd proteome is mainly represented by secreted Pd-associated proteins (PdAPs), the repertoire of which quickly adapts to environmental conditions and responds to biotic and abiotic stresses. Although the important role of Pd in stress-induced reactions is universally recognized, the mechanisms of Pd control are still not fully understood. The negative role of callose in Pd permeability has been convincingly confirmed experimentally, yet the roles of cytoskeletal elements and many PdAPs remain unclear. Here, we discuss the contribution of each protein component to Pd control. Based on known data, we offer mechanistic models of mature leaf Pd regulation in response to stressful effects.

The biological activity of bispecific trastuzumab/pertuzumab plant biosimilars may be drastically boosted by disulfiram increasing formaldehyde accumulation in cancer cells.

Studies of breast cancer therapy have examined the improvement of bispecific trastuzumab/pertuzumab antibodies interacting simultaneously with two different epitopes of the human epidermal growth factor receptor 2 (HER2). Here, we describe the creation and production of plant-made bispecific antibodies based on trastuzumab and pertuzumab plant biosimilars (bi-TPB-PPB). Using surface plasmon resonance analysis of bi-TPB-PPB antibodies binding with the HER2 extracellular domain, we showed that the obtained Kd values were within the limits accepted for modified trastuzumab and pertuzumab. Despite the ability of bi-TPB-PPB antibodies to bind to Fcγ receptor IIIa and HER2 oncoprotein on the cell surface, a proliferation inhibition assay did not reveal any effect until α1,3-fucose and ß1,2-xylose in the Asn297-linked glycan were removed. Another approach to activating bi-TPB-PPB may be associated with the use of disulfiram (DSF) a known aldehyde dehydrogenase 2 (ALDH2) inhibitor. We found that disulfiram is capable of killing breast cancer cells with simultaneous formaldehyde accumulation. Furthermore, we investigated the capacity of DSF to act as an adjuvant for bi-TPB-PPB antibodies. Although the content of ALDH2 mRNA was decreased after BT-474 cell treatment with antibodies, we only observed cell proliferation inhibiting activity of bi-TPB-PPB in the presence of disulfiram. We concluded that disulfiram can serve as a booster and adjuvant for anticancer immunotherapy.

Plant-Made Antibodies: Properties and Therapeutic Applications.

BACKGROUND: A cost-effective plant platform for therapeutic monoclonal antibody production is both flexible and scalable. Plant cells have mechanisms for protein synthesis and posttranslational modification, including glycosylation, similar to those in animal cells. However, plants produce less complex and diverse Asn-attached glycans compared to animal cells and contain plant-specific residues. Nevertheless, plant-made antibodies (PMAbs) could be advantageous compared to those produced in animal cells due to the absence of a risk of contamination from nucleic acids or proteins of animal origin. OBJECTIVE: In this review, the various platforms of PMAbs production are described, and the widely used transient expression system based on Agrobacterium-mediated delivery of genetic material into plant cells is discussed in detail. RESULTS: We examined the features of and approaches to humanizing the Asn-linked glycan of PMAbs. The prospects for PMAbs in the prevention and treatment of human infectious diseases have been illustrated by promising results with PMAbs against human immunodeficiency virus, rotavirus infection, human respiratory syncytial virus, rabies, anthrax and Ebola virus. The pre-clinical and clinical trials of PMAbs against different types of cancer, including lymphoma and breast cancer, are addressed. CONCLUSION: PMAb biosafety assessments in patients suggest that it has no side effects, although this does not completely remove concerns about the potential immunogenicity of some plant glycans in humans. Several PMAbs at various developmental stages have been proposed. Promise for the clinical use of PMAbs is aimed at the treatment of viral and bacterial infections as well as in anti-cancer treatment.

Methanol in Plant Life.

Until recently, plant-emitted methanol was considered a biochemical by-product, but studies in the last decade have revealed its role as a signal molecule in plant-plant and plant-animal communication. Moreover, methanol participates in metabolic biochemical processes during growth and development. The purpose of this review is to determine the impact of methanol on the growth and immunity of plants. Plants generate methanol in the reaction of the demethylation of macromolecules including DNA and proteins, but the main source of plant-derived methanol is cell wall pectins, which are demethylesterified by pectin methylesterases (PMEs). Methanol emissions increase in response to mechanical wounding or other stresses due to damage of the cell wall, which is the main source of methanol production. Gaseous methanol from the wounded plant induces defense reactions in intact leaves of the same and neighboring plants, activating so-called methanol-inducible genes (MIGs) that regulate plant resistance to biotic and abiotic factors. Since PMEs are the key enzymes in methanol production, their expression increases in response to wounding, but after elimination of the stress factor effects, the plant cell should return to the original state. The amount of functional PMEs in the cell is strictly regulated at both the gene and protein levels. There is negative feedback between one of the MIGs, aldose epimerase-like protein, and PME gene transcription; moreover, the enzymatic activity of PMEs is modulated and controlled by PME inhibitors (PMEIs), which are also induced in response to pathogenic attack.

Human Endogenous Formaldehyde as an Anticancer Metabolite: Its Oxidation Downregulation May Be a Means of Improving Therapy.

Malignant cells are characterized by an increased content of endogenous formaldehyde formed as a by-product of biosynthetic processes. Accumulation of formaldehyde in cancer cells is combined with activation of the processes of cellular formaldehyde clearance. These mechanisms include increased ALDH and suppressed ADH5/FDH activity, which oncologists consider poor and favorable prognostic markers, respectively. Here, the sources and regulation of formaldehyde metabolism in cancer cells are reviewed. The authors also analyze the participation of oncoproteins such as fibulins, FGFR1, HER2/neu, FBI-1, and MUC1-C in the control of genes related to formaldehyde metabolism, suggesting the existence of two mutually exclusive processes in cancer cells: 1) production and 2) oxidation and elimination of formaldehyde from the cell. The authors hypothesize that the study of the anticancer properties of disulfiram and alpha lipoic acid - which affect the balance of formaldehyde in the body - may serve as the basis of future anticancer therapy.

The Antioxidant Cofactor Alpha-Lipoic Acid May Control Endogenous Formaldehyde Metabolism in Mammals.

The healthy human body contains small amounts of metabolic formaldehyde (FA) that mainly results from methanol oxidation by pectin methylesterase, which is active in a vegetable diet and in the gastrointestinal microbiome. With age, the ability to maintain a low level of FA decreases, which increases the risk of Alzheimer's disease and dementia. It has been shown that 1,2-dithiolane-3-pentanoic acid or alpha lipoic acid (ALA), a naturally occurring dithiol and antioxidant cofactor of mitochondrial α-ketoacid dehydrogenases, increases glutathione (GSH) content and FA metabolism by mitochondrial aldehyde dehydrogenase 2 (ALDH2) thus manifests a therapeutic potential beyond its antioxidant property. We suggested that ALA can contribute to a decrease in the FA content of mammals by acting on ALDH2 expression. To test this assumption, we administered ALA in mice in order to examine the effect on FA metabolism and collected blood samples for the measurement of FA. Our data revealed that ALA efficiently eliminated FA in mice. Without affecting the specific activity of FA-metabolizing enzymes (ADH1, ALDH2, and ADH5), ALA increased the GSH content in the brain and up-regulated the expression of the FA-metabolizing ALDH2 gene in the brain, particularly in the hippocampus, but did not impact its expression in the liver in vivo or in rat liver isolated from the rest of the body. After ALA administration in mice and in accordance with the increased content of brain ALDH2 mRNA, we detected increased ALDH2 activity in brain homogenates. We hypothesized that the beneficial effects of ALA on patients with Alzheimer's disease may be associated with accelerated ALDH2-mediated FA detoxification and clearance.

The Intergenic Interplay between Aldose 1-Epimerase-Like Protein and Pectin Methylesterase in Abiotic and Biotic Stress Control.

The mechanical damage that often precedes the penetration of a leaf by a pathogen promotes the activation of pectin methylesterase (PME); the activation of PME leads to the emission of methanol, resulting in a "priming" effect on intact leaves, which is accompanied by an increased sensitivity to Tobacco mosaic virus (TMV) and resistance to bacteria. In this study, we revealed that mRNA levels of the methanol-inducible gene encoding Nicotiana benthamiana aldose 1-epimerase-like protein (NbAELP) in the leaves of intact plants are very low compared with roots. However, stress and pathogen attack increased the accumulation of the NbAELP mRNA in the leaves. Using transiently transformed plants, we obtained data to support the mechanism underlying AELP/PME-related negative feedback The insertion of the NbAELP promoter sequence (proNbAELP) into the N. benthamiana genome resulted in the co-suppression of the natural NbAELP gene expression, accompanied by a reduction in the NbAELP mRNA content and increased PME synthesis. Knockdown of NbAELP resulted in high activity of PME in the cell wall and a decrease in the leaf glucose level, creating unfavorable conditions for Agrobacterium tumefaciens reproduction in injected leaves. Our results showed that NbAELP is capable of binding the TMV movement protein (MPTMV) in vitro and is likely to affect the cellular nucleocytoplasmic transport, which may explain the sensitivity of NbAELP knockdown plants to TMV. Although NbAELP was primarily detected in the cell wall, the influence of this protein on cellular PME mRNA levels might be associated with reduced transcriptional activity of the PME gene in the nucleus. To confirm this hypothesis, we isolated the N. tabacum PME gene promoter (proNtPME) and showed the inhibition of proNtPME-directed GFP and GUS expression in leaves when co-agroinjected with the NbAELP-encoding plasmid. We hypothesized that plant wounding and/or pathogen attack lead to PME activation and increased methanol emission, followed by increased NbAELP expression, which results in reversion of PME mRNA level and methanol emission to levels found in the intact plant.

Tobamovirus 3'-Terminal Gene Overlap May be a Mechanism for within-Host Fitness Improvement.

Overlapping genes (OGs) are a universal phenomenon in all kingdoms, and viruses display a high content of OGs combined with a high rate of evolution. It is believed that the mechanism of gene overlap is based on overprinting of an existing gene. OGs help virus genes compress a maximum amount of information into short sequences, conferring viral proteins with novel features and thereby increasing their within-host fitness. Analysis of tobamovirus 3'-terminal genes reveals at least two modes of OG organization and mechanisms of interaction with the host. Originally isolated from Solanaceae species, viruses (referred to as Solanaceae-infecting) such as tobacco mosaic virus do not show 3'-terminal overlap between movement protein (MP) and coat protein (CP) genes but do contain open reading frame 6 (ORF6), which overlaps with both genes. Conversely, tobamoviruses, originally isolated from Brassicaceae species (referred to as Brassicaceae-infecting) and also able to infect Solanaceae plants, have no ORF6 but are characterized by overlapping MP and CP genes. Our analysis showed that the MP/CP overlap of Brassicaceae-infecting tobamoviruses results in the following: (i) genome compression and strengthening of subgenomic promoters; (ii) CP gene early expression directly from genomic and dicistronic MP subgenomic mRNA using an internal ribosome entry site (IRES) and a stable hairpin structure in the overlapping region; (iii) loss of ORF6, which influences the symptomatology of Solanaceae-infecting tobamoviruses; and (iv) acquisition of an IRES polypurine-rich region encoding an MP nuclear localization signal. We believe that MP/CP gene overlap may constitute a mechanism for host range expansion and virus adjustment to Brassicaceae plants.

An Alternative Nested Reading Frame May Participate in the Stress-Dependent Expression of a Plant Gene.

Although plants as sessile organisms are affected by a variety of stressors in the field, the stress factors for the above-ground and underground parts of the plant and their gene expression profiles are not the same. Here, we investigated NbKPILP, a gene encoding a new member of the ubiquitous, pathogenesis-related Kunitz peptidase inhibitor (KPI)-like protein family, that we discovered in the genome of Nicotiana benthamiana and other representatives of the Solanaceae family. The NbKPILP gene encodes a protein that has all the structural elements characteristic of KPI but in contrast to the proven A. thaliana KPI (AtKPI), it does not inhibit serine peptidases. Unlike roots, NbKPILP mRNA and its corresponding protein were not detected in intact leaves, but abiotic and biotic stressors drastically affected NbKPILP mRNA accumulation. In search of the causes of suppressed NbKPILP mRNA accumulation in leaves, we found that the NbKPILP gene is "matryoshka," containing an alternative nested reading frame (ANRF) encoding a 53-amino acid (aa) polypeptide (53aa-ANRF) which has an amphipathic helix (AH). We confirmed ANRF expression experimentally. A vector containing a GFP-encoding sequence was inserted into the NbKPILP gene in frame with 53aa-ANRF, resulting in a 53aa-GFP fused protein that localized in the membrane fraction of cells. Using the 5'-RACE approach, we have shown that the expression of ANRF was not explained by the existence of a cryptic promoter within the NbKPILP gene but was controlled by the maternal NbKPILP mRNA. We found that insertion of mutations destroying the 53aa-ANRF AH resulted in more than a two-fold increase of the NbKPILP mRNA level. The NbKPILP gene represents the first example of ANRF functioning as a repressor of a maternal gene in an intact plant. We proposed a model where the stress influencing the translation initiation promotes the accumulation of NbKPILP and its mRNA in leaves.

Metabolic methanol: molecular pathways and physiological roles.

Methanol has been historically considered an exogenous product that leads only to pathological changes in the human body when consumed. However, in normal, healthy individuals, methanol and its short-lived oxidized product, formaldehyde, are naturally occurring compounds whose functions and origins have received limited attention. There are several sources of human physiological methanol. Fruits, vegetables, and alcoholic beverages are likely the main sources of exogenous methanol in the healthy human body. Metabolic methanol may occur as a result of fermentation by gut bacteria and metabolic processes involving S-adenosyl methionine. Regardless of its source, low levels of methanol in the body are maintained by physiological and metabolic clearance mechanisms. Although human blood contains small amounts of methanol and formaldehyde, the content of these molecules increases sharply after receiving even methanol-free ethanol, indicating an endogenous source of the metabolic methanol present at low levels in the blood regulated by a cluster of genes. Recent studies of the pathogenesis of neurological disorders indicate metabolic formaldehyde as a putative causative agent. The detection of increased formaldehyde content in the blood of both neurological patients and the elderly indicates the important role of genetic and biochemical mechanisms of maintaining low levels of methanol and formaldehyde.

Dietary methanol regulates human gene activity.

Methanol (MeOH) is considered to be a poison in humans because of the alcohol dehydrogenase (ADH)-mediated conversion of MeOH to formaldehyde (FA), which is toxic. Our recent genome-wide analysis of the mouse brain demonstrated that an increase in endogenous MeOH after ADH inhibition led to a significant increase in the plasma MeOH concentration and a modification of mRNA synthesis. These findings suggest endogenous MeOH involvement in homeostasis regulation by controlling mRNA levels. Here, we demonstrate directly that study volunteers displayed increasing concentrations of MeOH and FA in their blood plasma when consuming citrus pectin, ethanol and red wine. A microarray analysis of white blood cells (WBC) from volunteers after pectin intake showed various responses for 30 significantly differentially regulated mRNAs, most of which were somehow involved in the pathogenesis of Alzheimer's disease (AD). There was also a decreased synthesis of hemoglobin mRNA, HBA and HBB, the presence of which in WBC RNA was not a result of red blood cells contamination because erythrocyte-specific marker genes were not significantly expressed. A qRT-PCR analysis of volunteer WBCs after pectin and red wine intake confirmed the complicated relationship between the plasma MeOH content and the mRNA accumulation of both genes that were previously identified, namely, GAPDH and SNX27, and genes revealed in this study, including MME, SORL1, DDIT4, HBA and HBB. We hypothesized that human plasma MeOH has an impact on the WBC mRNA levels of genes involved in cell signaling.

Endogenous methanol regulates mammalian gene activity.

We recently showed that methanol emitted by wounded plants might function as a signaling molecule for plant-to-plant and plant-to-animal communications. In mammals, methanol is considered a poison because the enzyme alcohol dehydrogenase (ADH) converts methanol into toxic formaldehyde. However, the detection of methanol in the blood and exhaled air of healthy volunteers suggests that methanol may be a chemical with specific functions rather than a metabolic waste product. Using a genome-wide analysis of the mouse brain, we demonstrated that an increase in blood methanol concentration led to a change in the accumulation of mRNAs from genes primarily involved in detoxification processes and regulation of the alcohol/aldehyde dehydrogenases gene cluster. To test the role of ADH in the maintenance of low methanol concentration in the plasma, we used the specific ADH inhibitor 4-methylpyrazole (4-MP) and showed that intraperitoneal administration of 4-MP resulted in a significant increase in the plasma methanol, ethanol and formaldehyde concentrations. Removal of the intestine significantly decreased the rate of methanol addition to the plasma and suggested that the gut flora may be involved in the endogenous production of methanol. ADH in the liver was identified as the main enzyme for metabolizing methanol because an increase in the methanol and ethanol contents in the liver homogenate was observed after 4-MP administration into the portal vein. Liver mRNA quantification showed changes in the accumulation of mRNAs from genes involved in cell signalling and detoxification processes. We hypothesized that endogenous methanol acts as a regulator of homeostasis by controlling the mRNA synthesis.

Cell wall methanol as a signal in plant immunity.

Cell wall pectin forms a matrix around the cellulose-xyloglucan network that is composed of rhamnogalacturonan I, rhamnogalacturonan II, and homogalacturonan (HG), a major pectic polymer consisting of α-1,4-linked galacturonic acids. HG is secreted in a highly methyl-esterified form and selectively de-methyl-esterified by pectin methylesterases (PMEs) during cell growth and pathogen attack. The mechanical damage that often precedes the penetration of the leaf by a pathogen promotes the activation of PME, which in turn leads to the emission of methanol (MeOH), an abundant volatile organic compound, which is quickly perceived by the intact leaves of the damaged plant, and the neighboring plants. The exposure to MeOH may result in a "priming" effect on intact leaves, setting the stage for the within-plant, and neighboring plant immunity. The emission of MeOH by a wounded plant enhances the resistance of the non-wounded, neighboring "receiver" plants to bacterial pathogens and promotes cell-to-cell communication that facilitates the spread of viruses in neighboring plants.

Methanol may function as a cross-kingdom signal.

Recently, we demonstrated that leaf wounding results in the synthesis of pectin methylesterase (PME), which causes the plant to release methanol into the air. Methanol emitted by a wounded plant increases the accumulation of methanol-inducible gene mRNA and enhances antibacterial resistance as well as cell-to-cell communication, which facilitates virus spreading in neighboring plants. We concluded that methanol is a signaling molecule involved in within-plant and plant-to-plant communication. Methanol is considered to be a poison in humans because of the alcohol dehydrogenase (ADH)-mediated conversion of methanol into toxic formaldehyde. However, recent data showed that methanol is a natural compound in normal, healthy humans. These data call into question whether human methanol is a metabolic waste product or whether methanol has specific function in humans. Here, to reveal human methanol-responsive genes (MRGs), we used suppression subtractive hybridization cDNA libraries of HeLa cells lacking ADH and exposed to methanol. This design allowed us to exclude genes involved in formaldehyde and formic acid detoxification from our analysis. We identified MRGs and revealed a correlation between increases in methanol content in the plasma and changes in human leukocyte MRG mRNA levels after fresh salad consumption by volunteers. Subsequently, we showed that the methanol generated by the pectin/PME complex in the gastrointestinal tract of mice induces the up- and downregulation of brain MRG mRNA. We used an adapted Y-maze to measure the locomotor behavior of the mice while breathing wounded plant vapors in two-choice assays. We showed that mice prefer the odor of methanol to other plant volatiles and that methanol changed MRG mRNA accumulation in the mouse brain.We hypothesize that the methanol emitted by wounded plants may have a role in plant-animal signaling. The known positive effect of plant food intake on human health suggests a role for physiological methanol in human gene regulation.

Airborne signals from a wounded leaf facilitate viral spreading and induce antibacterial resistance in neighboring plants.

Many plants release airborne volatile compounds in response to wounding due to pathogenic assault. These compounds serve as plant defenses and are involved in plant signaling. Here, we study the effects of pectin methylesterase (PME)-generated methanol release from wounded plants ("emitters") on the defensive reactions of neighboring "receiver" plants. Plant leaf wounding resulted in the synthesis of PME and a spike in methanol released into the air. Gaseous methanol or vapors from wounded PME-transgenic plants induced resistance to the bacterial pathogen Ralstonia solanacearum in the leaves of non-wounded neighboring "receiver" plants. In experiments with different volatile organic compounds, gaseous methanol was the only airborne factor that could induce antibacterial resistance in neighboring plants. In an effort to understand the mechanisms by which methanol stimulates the antibacterial resistance of "receiver" plants, we constructed forward and reverse suppression subtractive hybridization cDNA libraries from Nicotiana benthamiana plants exposed to methanol. We identified multiple methanol-inducible genes (MIGs), most of which are involved in defense or cell-to-cell trafficking. We then isolated the most affected genes for further analysis: ß-1,3-glucanase (BG), a previously unidentified gene (MIG-21), and non-cell-autonomous pathway protein (NCAPP). Experiments with Tobacco mosaic virus (TMV) and a vector encoding two tandem copies of green fluorescent protein as a tracer of cell-to-cell movement showed the increased gating capacity of plasmodesmata in the presence of BG, MIG-21, and NCAPP. The increased gating capacity is accompanied by enhanced TMV reproduction in the "receivers". Overall, our data indicate that methanol emitted by a wounded plant acts as a signal that enhances antibacterial resistance and facilitates viral spread in neighboring plants.

Plant-made trastuzumab (herceptin) inhibits HER2/Neu+ cell proliferation and retards tumor growth.

BACKGROUND: Plant biotechnology provides a valuable contribution to global health, in part because it can decrease the cost of pharmaceutical products. Breast cancer can now be successfully treated by a humanized monoclonal antibody (mAb), trastuzumab (Herceptin). A course of treatment, however, is expensive and requires repeated administrations of the mAb. Here we used an Agrobacterium-mediated transient expression system to produce trastuzumab in plant cells. METHODOLOGY/PRINCIPAL FINDINGS: We describe the cloning and expression of gene constructs in Nicotiana benthamiana plants using intron-optimized Tobacco mosaic virus- and Potato virus X-based vectors encoding, respectively, the heavy and light chains of trastuzumab. Full-size antibodies extracted and purified from plant tissues were tested for functionality and specificity by (i) binding to HER2/neu on the surface of a human mammary gland adenocarcinoma cell line, SK-BR-3, in fluorescence-activated cell sorting assay and (ii) testing the in vitro and in vivo inhibition of HER-2-expressing cancer cell proliferation. We show that plant-made trastuzumab (PMT) bound to the Her2/neu oncoprotein of SK-BR-3 cells and efficiently inhibited SK-BR-3 cell proliferation. Furthermore, mouse intraperitoneal PMT administration retarded the growth of xenografted tumors derived from human ovarian cancer SKOV3 Her2+ cells. CONCLUSIONS/SIGNIFICANCE: We conclude that PMT is active in suppression of cell proliferation and tumor growth.

Pol II-directed short RNAs suppress the nuclear export of mRNA.

The synthesis and subsequent nuclear export of non-coding RNA (ncRNA) directed by RNA polymerase (Pol) II is very sensitive to abiotic and biotic external stimuli including pathogen challenges. To assess whether stress-induced ncRNAs may suppress the nuclear export of mRNA, we exploited the ability of Agrobacterium tumefaciens to co-deliver Pol I, II and III promoter-based vectors for the transcription of short (s) ncRNAs, GFP mRNA or genomic RNA of plant viruses (Tobacco mosaic virus, TMV; or Potato virus X, PVX) into the nucleus of Nicotiana benthamiana cells. We showed that, in contrast to Pol I- and Pol III-derived sncRNAs, all tested Pol II-derived sncRNAs (U6 RNA, tRNA or artificial RNAs) resulted in decreased expression of GFP and host mRNA. The level of this inhibitory effect depended on the non-coding transcript length and promoter strength. Short coding RNA (scRNA) can also compete with mRNA for nuclear export. We showed that scRNA, an artificial 117-nt short sequence encoding Elastin-Like peptide element tandems with FLAG sequence (ELF) and the 318-nt N. benthamiana antimicrobial peptide thionin (defensin) gene efficiently decreased GFP expression. The stress-induced export of Pol II-derived sncRNA and scRNA into the cytoplasm via the mRNA export pathway may block nucleocytoplasmic traffic including the export of mRNA responsible for antivirus protection. Consistent with this model, we observed that Pol II-derived sncRNAs as well as scRNA, thionin and ELF strongly enhanced the cytoplasmic reproduction of TMV and PVX RNA.

Trastuzumab-binding peptide display by Tobacco mosaic virus.

Human epidermal growth factor receptor-2 (HER2/neu) is a target for the humanized monoclonal antibody trastuzumab. Recently, trastuzumab-binding peptides (TBP) of HER2/neu that inhibit proliferation of breast cancer cells were identified. We have now studied conditions of efficient assembly in vivo of Tobacco mosaic virus (TMV)-based particles displaying TBP on its surface. The system is based on an Agrobacterium-mediated co-delivery of binary vectors encoding TMV RNA and coat protein (CP) with TBP in its C-terminal extension into plant leaves. We show how the fusion of amino acid substituted TBP (sTBP) to CP via a flexible peptide linker can improve the manufacturability of recombinant TMV (rTMV). We also reveal that rTMV particles with exposed sTBP retained trastuzumab-binding capacity but lost an anti-HER2/neu immunogenic scaffold function. Mouse antibodies against rTMV did not recognize HER2/neu on surface of human SK-BR-3 cells.

Transient expression systems for plant-derived biopharmaceuticals.

In the molecular farming area, transient expression approaches for pharmaceutical proteins production, mainly recombinant monoclonal antibodies and vaccines, were developed almost two decades ago and, to date, these systems basically depend on Agrobacterium-mediated delivery and virus expression machinery. We survey here the current state-of-the-art of this research field. Several vectors have been designed on the basis of DNA- and RNA-based plant virus genomes and viral vectors are used both as single- and multicomponent expression systems in different combinations depending on the protein of interest. The obvious advantages of these systems are ease of manipulation, speed, low cost and high yield of proteins. In addition, Agrobacterium-mediated expression also allows the production in plants of complex proteins assembled from subunits. Currently, the transient expression methods are preferential over any other transgenic system for the exploitation of large and unrestricted numbers of plants in a contained environment. By designing optimal constructs and related means of delivery into plant cells, the overall technology plan considers scenarios that envisage high yield of bioproducts and ease in monitoring the whole spectrum of upstream production, before entering good manufacturing practice facilities. In this way, plant-derived bioproducts show promise of high competitiveness towards classical eukaryotic cell factory systems.