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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 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.

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: β-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.

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.

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.

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 (TMV) and resistance to bacteria. In this study, we revealed that mRNA levels of the methanol-inducible gene encoding 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 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 promoter sequence (proNbAELP) into the genome resulted in the co-suppression of the natural gene expression, accompanied by a reduction in the mRNA content and increased PME synthesis. Knockdown of resulted in high activity of PME in the cell wall and a decrease in the leaf glucose level, creating unfavorable conditions for reproduction in injected leaves. Our results showed that NbAELP is capable of binding the TMV movement protein (MP ) and is likely to affect the cellular nucleocytoplasmic transport, which may explain the sensitivity of knockdown plants to TMV. Although NbAELP was primarily detected in the cell wall, the influence of this protein on cellular mRNA levels might be associated with reduced transcriptional activity of the gene in the nucleus. To confirm this hypothesis, we isolated the gene promoter (proNtPME) and showed the inhibition of proNtPME-directed and 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 expression, which results in reversion of mRNA level and methanol emission to levels found in the intact plant.

A coordinated and generalized plant response to adverse environmental factors largely depends on the proper and finely-tuned regulation of intercellular transport via plasmodesmata (PD). However, the knowledge of the whole network of PD-controlling mechanisms is far from complete. Earlier, a cellular factor, Kunitz peptidase inhibitor-like protein (KPILP), that affects PD gating and plays a proviral role, was identified in Nicotiana benthamiana plants. Here we characterized its homolog from N. tabacum, NtKPILP, which is hardly detectable in leaves of intact plants, in contrast to roots, flowers and seeds where NtKPILP is highly expressed. However, its mRNA accumulation in leaves increases in response to various stresses, including viral infection. NtKPILP was demonstrated to affect chloroplast functioning. Using the virus-induced gene silencing approach, we have shown that NtKPILP downregulation negatively affects intercellular transport of macromolecules, inducing callose deposition at PD and suppressing beta-1,3-glucanase mRNA accumulation. Together, the obtained results indicate that NtKPILP is a viral infection-responsive cellular factor that is involved in PD permeability regulation, sharing thus the features of KPILPs from other Nicotiana species.

The mechanical damage of plant tissues leads to the activation of methanol production and its release into the atmosphere. The gaseous methanol or vapors emitted by the damaged plant induce resistance in neighboring intact plants to bacterial pathogens but create favorable conditions for viral infection spread. Among the Nicotiana benthamiana methanol-inducible genes (MIGs), most are associated with plant defense and intercellular transport. Here, we characterize NbMIG21, which encodes a 209 aa protein (NbMIG21p) that does not share any homology with annotated proteins. NbMIG21p was demonstrated to contain a nucleolus localization signal (NoLS). Colocalization studies with fibrillarin and coilin, nucleolus and Cajal body marker proteins, revealed that NbMIG21p is distributed among these subnuclear structures. Our results show that recombinant NbMIG21 possesses DNA-binding properties. Similar to a gaseous methanol effect, an increased NbMIG21 expression leads to downregulation of the nuclear import of proteins with nuclear localization signals (NLSs), as was demonstrated with the GFP-NLS model protein. Moreover, upregulated NbMIG21 expression facilitates tobacco mosaic virus (TMV) intercellular transport and reproduction. We identified an NbMIG21 promoter (PrMIG21) and showed that it is methanol sensitive; thus, the induction of NbMIG21 mRNA accumulation occurs at the level of transcription. Our findings suggest that methanol-activated NbMIG21 might participate in creating favorable conditions for viral reproduction and spread.

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. 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. We conclude that PMT is active in suppression of cell proliferation and tumor growth.

Plants are a promising platform for recombinant protein production. Here we propose a novel approach to increase the level of viral vector-mediated recombinant protein synthesis. This approach is based on the hypothesis that antiviral protection is weakened during the antibacterial cellular response. We suggested that introduced to the cell foreign nuclear localized proteins, including effectors such as bacterial nucleomodulins, can interfere with the import of cellular nuclear proteins and launch antibacterial defense reactions, creating favorable conditions for cytoplasmic virus reproduction. Here, we performed synthesis of an artificial nuclear protein—red fluorescent protein (mRFP) fused with a nuclear localization sequence (NLS)—in plant cells as a mimetic of a bacterial effector. Superproduction of mRFP:NLS induced Nicotiana benthamiana γ-thionin (NbγThio) mRNA accumulation. Both NLS-containing protein synthesis and increased NbγThio expression stimulated reproduction of the viral vector based on the genome of crucifer-infecting tobacco mosaic virus (crTMV) in N. benthamiana leaves. We isolated the NbγThio gene promoter (PrγThio) and showed that PrγThio activity sharply increased in response to massive synthesis of GFP fused with NLS. We conclude that NLS-induced PrγThio activation and increased accumulation of Nbγthio mRNA led to the stimulation of GFP expression from crTMV: GFP vector in the transient expression system.

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.