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: 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

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

<i>cis</i>-3-hexen-1-ol vapors induce methanol emission from leaves.

<p>The upper panel depicts how <i>N. benthamiana</i> leaves were treated with a 3-h exposure to continuous airflow from an evaporator containing <i>cis</i>-3-hexen-1-ol. The bottom panel shows the methanol content in the water trap after the leaf underwent treatment with different concentrations of <i>cis</i>-3-hexen-1-ol. The data shown represent five independent experiments. Standard error bars are indicated. ***, <i>P</i><0.001.</p

Measurement of VOCs in the headspace of <i>PME</i>-transgenic and wounded leaves.

<p>(A–C) Methanol (A), ethylene (B) and <i>cis</i>-3-hexen-1-ol (C) content detected in the headspace of a single wounded <i>N. tabacum</i> leaf or a single <i>PME</i>-transgenic tobacco leaf using the water-drop method. After a 3 h incubation, the wounded <i>N. tabacum</i> and <i>PME</i> transgenic tobacco leaves were removed, and the methanol/<i>cis</i>-3-hexen-1-ol content in the water/decane drop was measured. Ethylene was measured in the headspace air samples. Intact <i>N. tabacum</i> leaves were used as controls in this experiment. The data shown represent five independent experiments. Standard error bars are indicated. ***, <i>P</i><0.001; **, <i>P</i><0.01; n.s., not significantly different (Student's <i>t</i>-test); n.d., not detected.</p

Bacterial growth is suppressed in plants maintained in a flow-through system.

<p>The measurement of <i>R. solanacearum</i> growth in “receiver” (R) plants after 3-h exposure to continuous airflow from various emitters (E): <i>PME</i> transgenic, wounded tobacco, wounded <i>PME-</i>transgenic plants or methanol evaporation in the flow-through system (A). The <i>N. benthamiana</i> “receiver” plants were inoculated by syringe injection using <i>R. solanacearum</i> (10⁶ cfu/ml) inoculum. The diagram (B) shows bacterial growth at inoculated sites on the leaves 4 days after injection with <i>R. solanacearum</i>. Log-transformed data were obtained from six independent samples. ***, <i>P</i><0.001 (Student's <i>t</i>-test).</p

MIG expression levels were measured in the <i>PME</i>-transgenic tobacco line <i>pro1</i>.

<p>The MIG expression levels in the control plants (wild-type tobacco plants) were set to 1. The data shown represent five independent experiments. Standard error bars are indicated. ***, <i>P</i><0.001 (Student's <i>t</i>-test).</p

MIG transcripts accumulation is increased in the leaves of “receiver” plants after 18 h of exposure to methanol emitters.

<p>Wounded <i>N. tabacum</i> (A) or <i>PME</i>-transgenic tobacco plants (B) were used as “emitter” plants, whereas <i>N. benthamiana</i> plants were used as “receivers”, as shown in the schematic diagram (left). The semi-log plots on the right show the measurements of relative mRNA quantities for selected MIGs examined by qPCR. “Receiver” plants maintained with intact tobacco plants were used as the controls for this experiment, and the control expression level was set to 1. The data shown represent five independent experiments. Standard error bars are indicated. ***, <i>P</i><0.001 (Student's <i>t</i>-test).</p

The local movement of crTMV:GFP is facilitated in methanol-treated leaves.

<p>(A) Schematic drawing of the experimental procedures used to measure the local movement of crTMV:GFP in methanol-treated leaves. (B) Quantification of crTMV:GFP foci in the leaves of control and methanol-treated plants at 3 dpi. No fewer than 1000 foci were counted. The data shown represent five independent experiments. Standard error bars are indicated. The unpaired two-tailed Student's <i>t</i>-test <i>P</i>-values for the statistical significance of the difference between the control and methanol-treated plants are indicated. ***, <i>P</i><0.001. (C) Epifluorescent micrographs of crTMV:GFP foci in control (left panel) and methanol-treated (right panel) plants. (D) Box-and-whisker plots show the statistical distribution of the data, and horizontal red lines across the boxes represent the median size of the crTMV:GFP expression foci (µm²×10⁴). The ends of the boxes indicate the 1^st and 3^rd quartiles, respectively. No fewer than 1000 foci were counted. The data shown represent five independent experiments. The statistical significance of the differences between each control and methanol-treated plant was analyzed by ANOVA and calculated to be <i>P</i> = 0.005. (E) MIG co-injection enhances crTMV:GFP vector reproduction. A fluorimetric analysis of GFP accumulation was conducted using leaves grown for 5 days after co-agroinjection with crTMV:GFP and vectors encoding BG, NCAPP and MIG-21. Fluorescence measurements are presented in relative light units. The GFP fluorescence, which was observed in co-agroinjection with empty pBin19, was assigned a value of 1. Empty pBin19 served as a control. The data shown represent 5 independent experiments. Standard error bars are indicated.</p

Methanol and MIGs encoding BG, NCAPP and MIG-21 facilitate cell-to-cell communication.

<p>(A) Single (upper) and multiple (bottom) epidermal cells containing 2×GFP were observed under epifluorescence microscopy after methanol treatment. (B, C) Quantification of 2×GFP movement after methanol treatment (B) and MIG agroinjection (C). No fewer than 1000 cell clusters were counted for each experiment. The data shown represent five independent experiments. Standard error bars are indicated. The unpaired two-tailed Student's <i>t</i>-test <i>P</i>-values for the statistical significance of the difference between the control and methanol-treated plants are indicated.</p