N. benthamiana and N. tabacum plants were grown in soil in a controlled environment under a 16 h/8 h day/night cycle.

To obtain the plasmid encoding NbAELP fused to the DYKDDDDKDYKDVDDYKDDDDK (3xFLAG) sequence (Ueda et al., 2011), a 3'-fragment of NbAELP containing the 3xFLAG encoding sequence was amplified using NbAELP(BglII+) and FL_SalI_r primers and subsequently digested with BglII and SalI. The 3xFLAG encoding sequence was generated after annealing the primers FL2_SalI_r and FL2_SalI_d, resulting in a fragment with overhangs corresponding to SalI and PstI “sticky” ends. These fragments were inserted into 35S-NbAELP via BglII/PstI sites to generate the 35S-NbAELP:3xFLAG plasmid. To obtain the proNbAELP (1000):GUS plasmid, NheI and SacI sites were introduced at the 5′- and the 3′-ends of proNbAELP, respectively, through PCR with primers “proNbAELP (NheI+)” and “proNbAELP (SacI−).” The proNbAELP (1,000) NheI/SacI fragment and the GUS-35S(term) fragment flanked with SacI/EcoRI sites was cloned into pBIN19, previously digested with XbaI/EcoRI, to generate the proNbAELP(1000):GUS construct. To obtain the proNbAELP(1500):GUS variant, the upstream region of proNbAELP was amplified using the primers “proNbAELP(SbfI+)” and “proNbAELP (HindIII−)” and subsequently inserted with the proNbAELP(1000):GUS fragment, flanked with HindIII/EcoRI sites, into pBIN19, previously digested with SbfI/EcoRI. To obtain proNbAELP(500):GUS, the 3′-proximal ~500 nt fragment of proNbAELP, digested with SalI/SacI, and the GUS-35S(term) fragment, flanked with SacI/EcoRI, were inserted into pBIN19 via SalI/EcoRI sites.

The proNtPME sequence was amplified using the primer pairs “PMEpr (HindIII+)” and “PMEpr (NcoI−)” and subsequently cloned into the pAL-TA vector (Evrogen, Russia). The obtained proNtPME fragment, with flanking 5′-HindIII and 3′-NcoI sites and the fragment containing GUS-35S(term), digested with NcoI/EcoRI, was inserted into pCampia1300 (CAMBIA, Australia) via HindIII/EcoRI sites, resulting in the proNtPME:GUS construct. For proNtPME:GFP construct, the second used fragment was GFP-35S(term) flanked with NcoI/EcoRI sites.

A full list of the oligonucleotides used for cloning is presented in Table S1.

The 5′-RACE of GUS cDNA from proNbAELP:GUS transgenic plants and PME cDNA from wild-type N. tabacum plants was performed using the Mint RACE cDNA amplification set (Evrogen, Russia) according to manufacturer's instructions. The following gene-specific primers were used: “GUS rev1”; “GUS rev2”; “PME_rev1”; “PME_rev2”; and “PME_rev3” (Table S1).

Genomic DNA was isolated from plant tissues using the ZR Plant/Seed DNA MiniPrep™ kit (Zymo Research, USA). A GenomeWalker™ Universal Kit (Clontech, Takara) was used for two rounds of “genome walking” according to the manufacturer's instructions. To identify the NbAELP promoter region, the first round of walking was performed using the following oligonucleotides: G05_Rev6, G05_Rev7, and G05_Rev8; the second round of walking was performed using the oligonucleotides G05_Rev10, G05_Rev11, and G05_Rev12 (Table S1). The promoter region fragment was amplified using primers G05_Rev7 and G05_prom_Dir2 and subsequently cloned into the pAL-TA plasmid (Evrogen, Russia).

To identify the NtPME promoter region, the first round of walking was performed using the following oligonucleotides: PME_rev4, PME_rev5 and PME_rev6; the second round of walking was performed using the oligonucleotides PME_rev7, PME_rev8 and PME_rev9 (Table S1). The promoter region fragment was isolated using primers PME_rev6 and PMEpr_D and subsequently cloned into the pAL-TA plasmid (Evrogen, Russia).

Agrobacterium-mediated transformation of N. benthamiana was performed using the conventional leaf disc method (Horsch and Klee, 1986). Tobacco leaf discs were incubated with A. tumefaciens containing the binary vector for 24 h at 26°C in darkness. The infected discs were transferred to regeneration medium (MS medium, supplemented with 1 mg/l 6-benzyladenine and 0.1 mg/l α-naphthalene acetic acid) containing 700 mg/l of cefotaxime and 100 mg/l of kanamycin for selection. Kanamycin-resistant shoots were rooted on selective MS medium containing 100 mg/l of kanamycin. Transgenic T₀ plants were obtained from N. benthamiana discs and characterized through PCR and GUS activity analyses.

N. benthamiana plants were mechanically inoculated with TMV virions (100 μg/ml) in 50 mM sodium phosphate buffer, pH 7.0, in the presence of celite, as described previously (Dorokhov et al., 1981).

The Agrobacterium tumefaciens strain GV3101 was transformed with individual binary constructs and grown at 28°C in LB medium supplemented with 50 mg/l rifampicin, 25 mg/l gentamycin and 50 mg/l kanamycin. The Agrobacterium from an overnight culture were resuspended in 10 mM MES buffer (pH 5.5) supplemented with 10 mM MgSO₄ and adjusted to a final OD₆₀₀ of 0.1. Agroinjection was performed using nearly fully expanded N. benthamiana leaves attached to the intact plant. A bacterial suspension was infiltrated into the leaf tissue using a 2-ml syringe, after which the plants were grown under greenhouse conditions at 24°C and a 16 h/8 h light/dark photoperiod. The fluorescent cells were counted after 2–3 days of storage in a growth chamber at 24°C under a 16 h/8 h light/dark photoperiod. The bacterial growth was measured by macerating five leaf discs of 1 cm² from the inoculated tissue of each sample in 10 mM MgCl₂, plating the serial dilutions on nutrient agar plates, and counting the colony-forming units (cfu).

The experiments were performed in accordance with the protocol (Ueki and Citovsky, 2011).

The leaf fragments were fixed in 0.1 M Sörensen phosphate buffer (pH 7.4) containing 4 % glutaraldehyde (SPI) for 1.5 h at room temperature, postfixed with 1% OsO₄ (Sigma) for 2 h and embedded in Epon 812 (Fluka). Semi-thin (1–3 μm) transverse sections were cut using an LKB Ultratome III and stained in solution containing 0.5% methylene blue and 0.5% sodium tetraborate at 60°C for 2 min. The preparations were examined using an AxioVert 200M microscope (Carl Zeiss) equipped with an AxioCam MRc digital camera. For brightness and contract correction, and final presentation, all images were transferred into Adobe Photoshop (Adobe Systems).

For Western blot analysis, the proteins from agroinjected leaves were divided into S30, P30, P1, and CW crude fractions according to Deom et al. (1990) with modifications. Briefly, frozen plant material was ground to a powder in liquid nitrogen followed by addition of 3 volumes of ice-cold modified GB buffer (100 mM Tris, pH 8.0, 0.4 M sucrose, 10 mM KCL, 5 mM MgCl₂, 10 mM β-mercaptoethanol). The obtained slurry was filtered through a double-layered Miracloth (Millipore/Merck). The material retained on the filter was collected and washed (30–60 min incubation followed by centrifugation at 1,000 × g) 5–8 times with GB-buffer supplemented with 0.1% Triton X-100 (the final wash was performed without Triton X-100) to obtain the CW-enriched fraction. The filtrate was centrifuged at 1,000 × g for 10 min to obtain the P1 (pellet enriched with nuclei) fraction. Supernatant was further divided into S30 (supernatant enriched with soluble proteins) and P30 (pellet enriched with membrane non-soluble proteins) fractions after centrifugation at 30,000 × g for 30 min. Pellets from CW, P1 and P30 fractions were resuspended in one volume of 1xPBS. To obtain the fraction of apoplast proteins the leaf was infiltrated with buffer containing 100 mM Tris pH 7.0, 150 mM NaCl, 20 mM CaCl₂, 200 mM mannitol, 0.05% Triton X-100. Then the leaf segments were put into the 0.5 ml tube with the perforated bottom which was inserted into 1.5 ml tube. The apoplast proteins fraction was obtained by centrifugation at 5,000 × g for 5 min.

Aliquotes from all fractions were analyzed through SDS-polyacrylamide gel electrophoresis and blotted onto polyvinylidene difluoride membranes (GE Healthcare). For GFP, NbAELP, FLAG or TMV MP detection, the membranes were probed with corresponding antibodies: goat anti-GFP antibodies conjugated with horseradish peroxidase (Rockland Immunochemicals), mouse anti-FLAG monoclonal antibodies (Sigma), mouse polyclonal antibodies against recombinant NbAELP or TMV MP. Anti-mouse antibodies conjugated with horseradish peroxidase (Rockland Immunochemicals) were used as secondary antibodies. The bands were visualized using the chemiluminescence ECL kit (GE Healthcare). The densitometry of the GFP bands on the Western blots was performed using open-access ImageJ software.

The PME activity in plant samples was quantified through a gel diffusion assay according to Dorokhov et al. (2012). Briefly, the tissue samples were flash-frozen in liquid nitrogen and homogenized in 3 volumes of extraction buffer (1 M NaCl, 2.5 mM phenylmethylsulfonyl fluoride, 0.1 M citrate and 0.2 M sodium phosphate, dibasic, pH 7.0). The homogenate was centrifuged at 16,000 g at 4°C, and the protein concentration in the recovered supernatant was determined using the Bio-Rad protein assay kit. Approximately 30 μl of the homogenate was subsequently loaded onto a 2% (w/v) agarose gel containing 0.1% of 90% esterified pectin (Sigma) in a Petri dish. The gels were incubated for 16 h at 28°C, rinsed with water, and stained for 45 min at room temperature with 0.05% (w/v) ruthenium red dye (Sigma), which stains de-esterified pectin. The diameter of each stained zone was measured to the nearest 0.1 mm using calipers. The amount of PME activity (in nkatals) was calculated based on the standard curve of the log-transformed enzyme activity versus the stained zone diameter generated using a commercial-grade orange peel PME (Sigma).

The methanol emitted by proNbAELP:GUS transgenic plants was measured in the headspace of hermetically sealed jars as previously described (Dorokhov et al., 2012). The methanol content was determined through gas chromatography. Methanol emissions was expressed as mg of methanol per 1 g fresh leaf weight.

The experimental procedures included a 6 h-incubation of proNbAELP:GUS transgenic plants with methanol vapors (4 mg of methanol per 20-L sealed dessicator), followed by storage under greenhouse conditions after 18 h and qRT-PCR to examine GUS mRNA accumulation.

GUS activity was determined using a previously described method (Jefferson et al., 1987) and measured in relative light units. GUS activity was normalized to the protein concentration estimated using a Bio-Rad protein assay kit. The mean values (with SE bars) for 3 to 10 independent experiments are shown.

Dried leaf samples (30 mg) were hydrolyzed with 1 ml of 1M hydrochloric acid (100°C for 2.5 h). The resulting solution was centrifuged for 10 min at 14,000 × g. To 0.5 ml of the supernatant was diluted with 1.5 ml of water and loaded to a reversed-phase concentrating cartridge (Diasorb C16), the first 1.8 ml was discarded and the next 0.2 ml was collected. To 20 μl of a mixture of standards at a concentration of 1 g/L of each carbohydrate (external standard) and test solution, 20 μl of an internal standard solution (glucosamine solution with a concentration of 1 g/l) was added and evaporated on a SpeedVac vacuum centrifugal evaporator with heating in a polypropylene tube. To the dried sample, 20 μl of a 0.5 M solution of PMP (1-phenyl-3-methyl-5-pyrazolone) in methanol and 20 μl of 0.3 M KOH was added, shaken thoroughly and incubated at 70°C for 2 h. The sample was neutralized by the addition of 20 μl of 0.3 M hydrochloric acid and the excess of the PMP reagent was extracted twice with 500 μl of benzene. The residue was evaporated on a SpeedVac with heating and dissolved in 500 μl of acetonitrile/water (1:9). The test mixture and analytical samples (injected vol 10 μL) were analyzed by reversed-phase HPLC in a gradient mode on a Luna C18(2) 4.6 × 250 mm (5 μm) column with the mobile phase A—water, B—acetonitrile and D—100 mM potassium hydrogen phosphate in water (pH 9.12) at a flow rate of 1 ml/min, a temperature of 25°C and with UV detection at 260 nm using a gradient chromatograph Agilent 1,100 with photodiode array detector. Gradient program: 50% D (constant), 8–16% B (5 min), 16–16% B (8 min), 16–30% B (4 min). Total analysis time is 20.65 min. Collection and processing of chromatograms was carried out with ChemStation (Agilent) and AutoChrom1200 (ACDlabs) programs.

Total RNA was extracted from plant tissues using TriReagent (MRC) according to the manufacturer's instructions. The RNA concentration was determined using a Nanodrop ND-1000 spectrophotometer (Isogen Life Sciences). All RNA samples had a 260:280 absorbance ratio between 1.9 and 2.1. The synthesis of the first strand, followed by real-time qPCR, was performed as described in Dorokhov et al. (2012). Briefly, 0.1 mg of random hexamers and 0.1 mg of oligo-dT primer were added to 2 mg of total RNA to obtain cDNA through reverse transcription performed using Superscript II reverse-transcriptase (Invitrogen), according to the manufacturer's protocol. Real-time quantitive PCR was carried out using the iCycler iQ real-time PCR detection system (Bio-Rad). Target genes were detected using sequence-specific primers (Table S2) and Eva Green master mix (Syntol) according to the manufacturer's instructions. Each sample was run in triplicate, and a non-template control was added to each run. A minimum of five biological replicates were performed.

Student's t-test was performed using Excel (Microsoft). P-values < 0.05 were considered significant.

Sequence data from this article can be obtained from the EMBL data library under accession numbers HG937605 (N. benthamiana AELP promoter region) and HG937606 (Nicotiana tabacum PME promoter region).

The analysis of the amino acid sequence of NbAELP classified it in the family of mutarotases (aldose 1-epimerases, EC 5.1.3.3) (Figure S1). High-resolution X-ray structure of the galactose mutarotase from Lactococcus lactis indicates the presence of an active site in its structure, which is an open cleft responsible for anchoring the sugar and includes Arg71, His96, His170, Asp243, and Glu304 (Thoden and Holden, 2002; Thoden et al., 2002). All these amino acids are present in the sequence of NbAELP and other AELPs from plants of the Solanaceae family. The similarity between NbAELP and the mammalian mutarotase was also confirmed by the ability of the antibodies obtained for the recombinant 6xHis tag containing NbAELP reacted with the porcine kidney mutarotase (Figure 1A). Unlike the mutarotases of bacteria (L. lactis) and mammals (porcine kidney), NbAELP contains a signal sequence that is also predicted for AELP of other plants of the Solanaceae family (tobacco, potato) (Figure S1). Western blot analysis of the leaf protein reveals NbAELP in membrane-containing P30 and the cell wall (CW) fractions (Figure 1A). However, due to the high homology between different AELPs, antibodies against NbAELP could cross-react with other members of this family, such as the glycoprotein gp40, which is highly abundant in N. benthamiana cells, therefore we examined the subcellular localization of NbAELP with a 3xFLAG tag fused to its C-terminus (Figure S2). The 35S-NbAELP:3xFLAG construct was introduced into the leaves of N. benthamiana plants using agroinjection; after 72 h, the subcellular leaf fractions were analyzed for NbAELP:3xFLAG accumulation. Western blot analysis revealed that the NbAELP:3xFLAG was primarily present in the CW (Figure 1B) and apoplasts (Figure 1C). The NbAELP:3xFLAG was also slightly detected in the P30 membrane and P1 nuclear fractions (Figure 1B). Notably, these results did not preclude the presence of small amounts of NbAELP or its fragments in the nucleus.

It can be assumed that the synthesis of NbAELP, which affects the functioning of the cell, should be strictly regulated in the intact leaf. We performed an analysis of the NbAELP mRNA in the tissues of the intact N. benthamiana plant. Thus, we selected N. benthamiana plants of different ages (Figure S3), including 2 (seedling stage), 6, 8, and 20 (flowering plants) weeks, and analyzed the NbAELP mRNA content of their leaves and roots. Figure 2 shows the increase in the NbAELP mRNA level during the growth of the plant from the seedling to the flowering stage. Although plant growth is accompanied by a modification of the CW, the increase of NbAELP mRNA content in leaves (Figure 2A) and roots (Figure 2B) is markedly higher in comparison to PME mRNA. Albeit the NbAELP mRNA content in the leaves increases with age, its content in the mature plant is much lower than in the roots.

Assuming the involvement of NbAELP in viral pathogenesis, we hypothesized that during TMV infection there is a direct correlation between the synthesis of the viral proteins and the accumulation of NbAELP mRNA. To verify this assumption, we infected N. tabacum plants with TMV and measured the amount of AELP mRNA in different parts of the infected leaves. The typical symptom of systemic TMV infection on tobacco is mosaic (Figure S4): dark green islands on the leaves with negligible TMV particles number and the yellow-green areas that are characterized by massive virus accumulation (Moore et al., 2001). Our analysis showed that AELP mRNA content increased in infected leaves compared with the intact leaf. Conversely, the content of PME mRNA decreased in both the dark green islands and the yellow-green areas of the mosaic leaf. Moreover, we observed the drop in the mRNA levels of the chloroplast genes of the AtpC (ATP synthase gamma chain, chloroplastic) and RCA (Ribulose bisphosphate carboxylase/oxygenase activase) genes in the dark green islands (Figure 3).

We concluded that NbAELP in leaves manifests itself as a pathogenesis-related protein involved in the plant-virus interaction.

Previously, we showed that injury induced PME mRNA synthesis leads to the formation of methanol, which in turn stimulates the accumulation of NbAELP mRNA (Dorokhov et al., 2012). This suggests that the NbAELP gene promoter is probably sensitive to methanol. To check this hypothesis, we isolated the N. benthamiana chromosomal DNA portion with a length of 1,563 base pairs upstream of the NbAELP start codon (proNbAELP) and performed a bioinformatics analysis of the sequence. We predicted potential cis-regulatory elements that control the gene stress response (Figure S5). In the second step, we created a series of proNbAELP-based binary vectors with varying lengths of the proNbAELP region followed by the GUS sequence (Figure 4A, top). The results showed that although proNbAELP directs GUS synthesis, it is inferior to the CaMV 35S promoter, which is considered a strong constitutive promoter for achieving high levels of gene expression in dicot plants. The levels of GUS increased with the length of proNbAELP sequence (Figure 4A, bottom).

To assess whether methanol activates proNbAELP, we created transgenic N. benthamiana plants expressing GUS under the control of proNbAELP. The transgenic plants were incubated in a methanol-rich atmosphere for 6 h and were subsequently maintained under greenhouse conditions (Figure 4B, top). Following this, the accumulation of GUS mRNA was analyzed using qRT-PCR. As shown in Figure 4B (bottom), the treatment with methanol dramatically increased the level of GUS mRNA.

We concluded that proNbAELP is sensitive to gaseous methanol and can mediate the increased synthesis of GUS mRNA in the leaves of transgenic proNbAELP-GUS plants in response to methanol.

The analysis of proNbAELP-GUS transgenic N. benthamiana plants revealed an extremely low content of endogenous NbAELP mRNA (Figure S6). The reduced NbAELP mRNA levels likely reflect NbAELP gene silencing induced by the transgenic proNbAELP DNA sequence and/or the 5′ non-translated NbAELP mRNA sequence, which is located upstream of the GUS open reading frame. The analysis of the transcription start site (TSS) localization revealed a 24-nt region of homology between the 5′ non-translated regions of NbAELP and proNbAELP-GUS mRNAs (Figure S7).

Gene knockdown reveals its function and influence on the biosynthetic processes in the plant. The virus spreading is closely related to the transport of photoassimilates because the PD capacity increase leads to the outflow of sugars from the source leaves to the sink leaves. To assess the potential effect of NbAELP on sugar metabolism, we tested the glucose content in the leaves of the proNbAELP-GUS transgenic N. benthamiana plants compared with the wild-type. The glucose content of the transgenic plants with reduced NbAELP mRNA content appeared to be significantly lower (Figure 5A) than control, indicating the role of NbAELP in regulating carbohydrate metabolism.

Biologically, a decrease in glucose levels creates unfavorable conditions for the propagation of the bacterial pathogen (Yamada et al., 2016). To test this assumption, we introduced a suspension of A. tumefaciens into the leaf and tested the reproduction of these bacteria. Figure 5B shows that the viability of bacteria introduced into the transgenic leaf almost two times lower.

Thus, in proNbAELP-GUS transgenic N. benthamiana plants, low glucose in the leaves is correlated to the resistance to A. tumefaciens bacteria.

Reproduction of the virus depends on the efficacy of viral RNA replication and its intercellular movement. Previously, we showed that methanol enhances the reproduction of TMV, which is associated with the ability of β-1,3-glucanase and NbAELP to stimulate PD gating (Dorokhov et al., 2012). It can be assumed that the direct interaction of TMV MP with β-1,3-glucanase and NbAELP plays an important role in these effects. To verify this assumption, we used the renatured blot overlay binding assay. Figure 6A shows that in vitro TMV MP interacts with NbAELP, but does not interact with β-1,3-glucanase, suggesting that differential mechanisms govern the involvement of NbAELP in TMV pathogenesis.

The properties of NbAELP to bind TMV MP and promote PD gating suggest its involvement in TMV reproduction as an MP carrier (Lee et al., 2003). Previously, we showed that NbAELP might increase TMV-directed GFP accumulation due to viral reproduction (Dorokhov et al., 2012). It could be assumed that plants with NbAELP knockdown would also be resistant to both TMV and bacteria. However, contrary to our expectations, proNbAELP-GUS transgenic N. benthamiana plants were highly sensitive to the virus. Figure 6B shows that inoculation with the TMV suspension of the transgenic leaf resulted in a much greater accumulation of viral RNA compared with the leaves of wild-type plants.

The enhanced ability of plants with NbAELP knockdown to allow TMV reproduction suggests that NbAELP has an additional ability to influence TMV infection.

We suggested that like tobacco gp40, NbAELP participates in nucleocytoplasmic transport (Heese-Peck and Raikhel, 1998). To determine whether NbAELP functions in the import of nuclear proteins, we used an experimental approach based on the import of GFP fused to the NLS from a nuclear animal protein prothymosin alpha (NLS^pTα). Figures 7A,B show that NbAELP agroinjection prevented the nuclear trafficking of GFP:NLS^pTα. The presence of GFP in the cytoplasm was not associated with the degradation of GFP:NLS^pTα or the appearance of NLS-lacking GFP. Western blot analysis showed the high stability of GFP:NLS^pTα fusion proteins (data not shown).

It can be assumed that the NbAELP-induced disruption of nucleocytoplasmic transport could lead to a decrease in plant cell resistance to the foreign nucleic acid and could increase the efficiency of leaf cells transfection. Figure 7C shows that NbAELP increased the number of GFP:NLS^pTα-containing cells as expected. The quantification of the observed cells with GFP fluorescence revealed a two-fold increase in number of GFP–containing cells per cm² compared with the control (Figure 7D). We conclude that the overexpression of NbAELP can disrupt the nucleocytoplasmic traffic and suppress cell resistance against foreign nucleic acids for example silencing.

To understand the role of NbAELP in a viral infection, we explored the close, competitive relationship between NbAELP and NbPME. Our results showed that NbAELP mRNA decrease in the proNbAELP-GUS transgenic N. benthamiana plants was related to the increase in NbPME mRNA (Figure 8A), the high level of PME enzymatic activity in the CW (Figure 8B) and the increase in methanol emission (Figure 8C). The long-term presence of high PME levels in CWs can result in dwarfism in plants (Hasunuma et al., 2004). In the present study, the proNbAELP-GUS transgenic N. benthamiana plants showed signs of dwarfism (Figure 9A, Table 1) like those observed with 35S-PME transgenic N. tabacum plants (Figure S8A). Moreover, the high CW PME content leads to an increase in the size of cells in 35S-NtPME transgenic tobacco leaves (Figure S8B) and proNbAELP-GUS transgenic N. benthamiana leaves (Figure 9B, Table 1). In both types of transgenic plants, the leaves and the cuticle were thicker and showed significant starch deposition compared with the wild-type.

Biologically, if there is a relationship between NbAELP and NbPME at the mRNA level, a methanol-induced increase in NbAELP mRNA synthesis should also cause a change in the accumulation of NbPME mRNA within the cells. To directly verify this assumption, N. benthamiana plants were agroinjected with an NbAELP-encoding binary vector (Figure 10A), and the endogenous level of NbPME mRNA was measured using qRT-PCR (Figure 10B). Figure 10C shows the accumulation of NbAELP mRNA and the consequential reduction of endogenous NbPME mRNA in the leaves of 35S-NbAELP agroinjected plants. An unpaired two-tailed Student t-test was used to confirm that there was a statistically significant difference in the NbPME mRNA levels in leaves taken from control plants versus 35S-NbAELP-agroinjected plants. This effect cannot be explained by a non-specific degradation of mRNA because the endogenous mRNA levels of the β-1,3-glucanase gene are sensitive to cellular stress (Dorokhov et al., 2012) and do not increase following agroinjection (Figure 10C).

Thus, the observed relationship between NbAELP and NbPME mRNA levels in proNbAELP-GUS transgenic plants was confirmed by experiments when NbAELP gene overexpression suppressed the accumulation of NbPME mRNA in the cytoplasm.

To unravel the relationship between NbAELP and PME, we hypothesized a mechanism in which NbAELP influences the NbPME mRNA synthesis. To determine whether AELP influences PME gene transcription, we isolated N. tabacum chromosomal DNA nucleotide sequence upstream of the NtPME gene (proNtPME) and located the TSS (Figure S9A). To confirm that the 1.75-kb long DNA region that is upstream of the NtPME gene promotes mRNA synthesis, we constructed a binary vector encoding GUS (Figure S9B) and then measured GUS activity in the leaves of N. benthamiana plants agroinjected with proNtPME-GUS. Figure S9C shows that proNtPME can mediate GUS synthesis, although the activity of this promoter was 5 times lower than that of the strong constitutive CaMV 35S promoter. Thus, we concluded that proNtPME possesses promoter transcriptional activity.

To support the hypothesis that NbAELP may modify the transcriptional activity of proNtPME, we co-injected plants with a binary vector encoding NbAELP and a proNtPME-GFP vector, which had GFP under the control of proNtPME (Figure 11A). Agroinjecting plants with proNtPME-GFP alone did not result in visible leaf fluorescence, but GFP expression could be detected through Western blot analysis. The GFP bands were analyzed using densitometry to estimate the amount of the synthesized GFP. Figure 11B shows that NbAELP inhibited the accumulation of both GFP mRNA and the corresponding protein. Notably, NbAELP did not affect the accumulation of 35S-directed GFP mRNA (data not shown).

In the next series of experiments, we used a construct that had GUS in place of the GFP gene (Figure 11C). N. benthamiana leaves that were simultaneously agroinjected with proNtPME-GUS, and 35S-NbAELP showed decreased proNtPME-driven GUS expression at the mRNA (Figure 11D) and protein (Figure 11E) levels. The control experiments demonstrated no influence of NbAELP on GUS synthesis driven by the 35S promoter (Figure S10).

Thus, we concluded that NbAELP could suppress proNtPME-driven mRNA accumulation, and therefore, downregulates the synthesis of PME mRNA by the feedback principle as shown in Figure 11F.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.