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. 2014 Apr;15(3):265-74.
doi: 10.1111/mpp.12090. Epub 2013 Dec 5.

Transgenic expression of pectin methylesterase inhibitors limits tobamovirus spread in tobacco and Arabidopsis

Vincenzo Lionetti  1 , Alessandro Raiola, Felice Cervone, Daniela Bellincampi
Affiliations

Transgenic expression of pectin methylesterase inhibitors limits tobamovirus spread in tobacco and Arabidopsis

Vincenzo Lionetti et al. Mol Plant Pathol. 2014 Apr.

Erratum in

  • Mol Plant Pathol. 2014 Aug;15(6):646

Abstract

Plant infection by a virus is a complex process influenced by virus-encoded factors and host components which support replication and movement. Critical factors for a successful tobamovirus infection are the viral movement protein (MP) and the host pectin methylesterase (PME), an important plant counterpart that cooperates with MP to sustain viral spread. The activity of PME is modulated by endogenous protein inhibitors (pectin methylesterase inhibitors, PMEIs). PMEIs are targeted to the extracellular matrix and typically inhibit plant PMEs by forming a specific and stable stoichiometric 1:1 complex. PMEIs counteract the action of plant PMEs and therefore may affect plant susceptibility to virus. To test this hypothesis, we overexpressed genes encoding two well-characterized PMEIs in tobacco and Arabidopsis plants. Here, we report that, in tobacco plants constitutively expressing a PMEI from Actinidia chinensis (AcPMEI), systemic movement of Tobacco mosaic virus (TMV) is limited and viral symptoms are reduced. A delayed movement of Turnip vein clearing virus (TVCV) and a reduced susceptibility to the virus were also observed in Arabidopsis plants overexpressing AtPMEI-2. Our results provide evidence that PMEIs are able to limit tobamovirus movement and to reduce plant susceptibility to the virus.

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Figures

Figure 1
Figure 1
SPPGIP‐AcPMEI gene construct and expression of Actinidia chinensis pectin methylesterase inhibitor (AcPMEI) in transgenic tobacco lines. (a) Schematic diagram of AcPMEI with the secretory signal peptide of bean polygalacturonase inhibitor protein 1 (SPPGIP) and schematic representation of the expression construct, pBI121Ω‐SPPGIP‐AcPMEI, used for plant transformation. Expression of SPPGIP‐AcPMEI was driven by the Cauliflower mosaic virus (CaMV) 35S promoter (35S), the Ω leader translation enhancer and the nopaline synthase (nos) terminator. (b) Representative Western blot analysis performed using polyclonal antibodies generated against purified AcPMEI; M, molecular weight marker; WT, wild‐type. (c) Level of AcPMEI expression in transgenic lines determined by Western blot analysis using ImageJ software. The fold change was relative to line 3‐2. Bars represent the average ± standard deviation of three independent experiments. Asterisk indicates significant difference according to Student's t‐test (P < 0.05).
Figure 2
Figure 2
Pectin methylesterase (PME) activity and cell wall methylesterification in leaves of wild‐type (WT) and transformed tobacco plants. PME activity in leaves (a) and in leaf blade and midvein (b) of WT and transformed plants. (c) Degree of cell wall methylesterification in leaves of WT and transformed plants. Data represent average ± standard deviation (n = 6). The experiments were repeated three times with similar results. The different letters indicate datasets significantly different according to analysis of variance (ANOVA) followed by Tukey's test (P < 0.05).
Figure 3
Figure 3
Accumulation of Tobacco mosaic virus‐green fluorescent protein (TMV‐GFP) in leaves of wild‐type (WT) and transformed plants. (a) GFP fluorescence of infection foci in inoculated TMV‐GFP tobacco leaves at 9 days post‐inoculation (dpi). Inoculated leaves were photographed under illumination by ultraviolet light. (b) The average area of TMV‐GFP infection foci ± standard error at 9 dpi. At least 100 spots from 10 leaves were measured in each of three independent experiments. The different letters indicate datasets significantly different according to analysis of variance (ANOVA) followed by Tukey's test (P < 0.01).
Figure 4
Figure 4
Delayed systemic movement of Tobacco mosaic virus (TMV) and symptoms in wild‐type (WT) and transgenic tobacco plants. (a) TMV symptoms in systemic leaves of WT and Actinidia chinensis pectin methylesterase inhibitor (AcPMEI)‐transformed plants (line 3‐2) at 8 days post‐inoculation (dpi). (b) Representative Western blot analysis indicating TMV‐CP accumulation in the upper systemic leaves of infected WT and transformed lines at the indicated days post‐inoculation. (c) Coat protein (CP) accumulation expressed as a percentage of the maximal amount of CP measured in WT plants. Data represent average ± standard error (n = 3); the experiment was repeated three times with similar results.
Figure 5
Figure 5
Pectin methylesterase (PME) activity in wild‐type (WT) and transgenic tobacco plants at early stages of Tobacco mosaic virus (TMV) infection. The PME activity was quantified at the indicated hours post‐inoculation (hpi); M, mock‐inoculated plants; TMV, virus‐inoculated plants. Data represent average ± standard deviation (n = 6). The experiment was repeated twice with similar results. The different letters indicate datasets significantly different according to analysis of variance (ANOVA) followed by Tukey's test (P < 0.05).
Figure 6
Figure 6
Detection of recombinant Turnip vein clearing virus (TVCV)‐MPHis after nickel affinity column chromatography by sodium dodecylsulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) (a) and Western blot analysis (b). M, molecular weight marker; lane 1, proteins from Escherichia coli  M15 (negative control); lane 2, proteins from E. coli  M15 transformed with TVCV‐MPHis. Arrows indicate the purified movement protein (MP). Pectin methylesterase (PME) activity in fractions eluted from affinity chromatography on a Sepharose column conjugated with TVCV‐MPHis (c) and from a blank Sepharose column (d): 1, intercellular washing fluids; 2, flow‐through; 3, washing; 4, proteins eluted from the columns.
Figure 7
Figure 7
Systemic accumulation of Turnip vein clearing virus‐coat protein (TVCV‐CP) in rosette leaves of wild‐type (WT) and pectin methylesterase inhibitor‐overexpressing Arabidopsis (AtPMEI‐2) plants. (a) Representative sodium dodecylsulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) indicating TVCV‐CP accumulation (arrow) in systemic leaves of infected WT and AtPMEI‐2 plants at the indicated days post‐inoculation. (b) CP accumulation is expressed as a percentage of the maximal amount of CP measured in WT plants. Data represent average ± standard error (n = 3). The experiment was repeated three times with similar results.
Figure 8
Figure 8
Pectin methylesterase (PME) activity in wild‐type (WT) and transgenic Arabidopsis plants at early stages of Turnip vein clearing virus (TVCV) infection. PME activity was quantified at the indicated hours post‐inoculation (hpi); M, mock‐inoculated plants; TVCV, virus‐inoculated plants. Data represent the average ± standard deviation (n = 6). The experiment was repeated twice with similar results. The different letters indicate datasets significantly different according to analysis of variance (ANOVA) followed by Tukey's test (P < 0.05).
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