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. 2012 Sep;13(7):775-84.
doi: 10.1111/j.1364-3703.2012.00786.x. Epub 2012 Feb 17.

Abiotic stress responses promote Potato virus A infection in Nicotiana benthamiana

Taina Suntio  1 , Kristiina Mäkinen
Affiliations

Abiotic stress responses promote Potato virus A infection in Nicotiana benthamiana

Taina Suntio et al. Mol Plant Pathol. 2012 Sep.

Abstract

The effect of abiotic stress responses on Potato virus A (PVA; genus Potyvirus) infection was studied. Salt, osmotic and wounding stress all increased PVA gene expression in infected Nicotiana benthamiana leaves. According to the literature, an early response to these stresses is an elevation in cytosolic Ca(2+) concentration. The infiltration of 0.1 m CaCl(2) into the infected leaf area enhanced the translation of PVA RNA, and this Ca(2+) -induced effect was more profound than that induced solely by osmotic stress. The inhibition of voltage-gated Ca(2+) channels within the plasma membrane abolished the Ca(2+) effect, suggesting that Ca(2+) had to be transported into the cytosol to affect viral gene expression. This was also supported by a reduced wounding effect in the presence of the Ca(2+) -chelating agent ethylene glycol tetraacetic acid (EGTA). In the absence of viral replication, the intense synthesis of viral proteins in response to Ca(2+) was transient. However, a Ca(2+) pulse administered at the onset of wild-type PVA infection enhanced the progress of infection within the locally infected leaf, and the virus appeared earlier in the systemic leaves than in the control plants. This suggests that the cellular environment was thoroughly modified by the Ca(2+) pulse to support viral infection. One message of this study is that the sensing of abiotic stress, which leads to cellular responses, probably via Ca(2+) signalling, associated with enhanced virus infection, may lead to higher field crop losses. Therefore, the effect of abiotic stress on plant viral infection warrants further analysis.

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Figures

Figure 1
Figure 1
Schematic diagrams of the different Potato virus A (PVA) constructs used in this work. All constructs are under the control of the Cauliflower mosaic virus 35S promoter. (A) PVAWT is an infectious cDNA copy of wild‐type PVA RNA in which the Renilla luciferase (RLUC) gene is inserted between the NIb and coat protein (CP) cistrons. (B) PVAΔGDD is a cDNA copy of replication‐deficient PVA RNA. In the replication‐deficient mutant, the catalytically active GDD motif of the RNA polymerase NIb is substituted with GAA. (C) The expression construct for monocistronic firefly luciferase (FLUC).
Figure 2
Figure 2
The effect of salt/osmotic stress on viral gene expression. Nicotiana benthamiana plants were co‐infiltrated with Agrobacterium carrying PVAWT and firefly luciferase (FLUC). Salt and osmotic stress was induced by a second infiltration of 0.7, 0.1 or 0.005 m NaCl at 1 day post‐infiltration (DPI). The control (C) did not receive a second infiltration. Samples were collected at 2 and 3 DPI. FLUC (A, B) and normalized PVAWT‐derived Renilla luciferase (RLUC) (C, D) activities. Normalization of the RLUC values is described in the Experimental procedures section. Each column represents the average of 12 parallel plant samples. The error bars report the standard deviation of the mean. (E, F) Fold change of the RLUC and FLUC levels above the nontreated control, which was assigned a relative value of unity.
Figure 3
Figure 3
The effect of mechanical wounding on viral gene expression. Nicotiana benthamiana plants were co‐infiltrated with Agrobacterium carrying PVAWT and firefly luciferase (FLUC). Agrobacterium‐infiltrated leaves were either dusted with carborundum beneath the leaves and wounded by rubbing at 1 day post‐infiltration (DPI) (15 plants) (A) or left without this treatment (15 plants) (B). Samples were collected at 2 and 3 DPI, and Renilla luciferase (RLUC) and FLUC activities were measured. The columns represent the average FLUC activity (A, B) and the normalized PVAWT‐derived RLUC activity (C, D). The error bars represent the standard deviation of the mean. The relative level of wounding‐induced enhancement in FLUC (E) and RLUC (F) gene expression is also shown. The untreated control was assigned the relative value of unity.
Figure 4
Figure 4
The effect of mechanical wounding on viral gene expression in the presence of the Ca2+ chelator ethylene glycol tetraacetic acid (EGTA). Nicotiana benthamiana plants were co‐infiltrated with Agrobacterium carrying PVAWT and firefly luciferase (FLUC). The Agrobacterium‐infiltrated leaves were left without treatment (C; four plants), dusted with carborundum beneath the leaves and wounded by rubbing at 1 day post‐infiltration (DPI) (W; 4 plants) or pre‐infiltrated with 10 mm EGTA 1 h prior to wounding (W + E; four plants). Samples were collected and Renilla luciferase (RLUC) and FLUC activities were determined at 3 DPI. The average FLUC (A) and average normalized PVAWT‐derived RLUC (B) activities are given. The error bar represents the standard deviation of the mean. (C) The relative levels of wounding‐induced enhancement in RLUC gene expression in the presence (W + E) and absence (W) of EGTA. The average value from the nontreated control plants was assigned the relative value of unity.
Figure 5
Figure 5
The effect of CaCl2 infiltration on viral gene expression. Nicotiana benthamiana plants were co‐infiltrated with Agrobacterium carrying PVAWT and firefly luciferase (FLUC). A second infiltration of H2O, 0.1 m CaCl2, 0.005 m CaCl2, 0.15 m NaCl or 0.3 m mannitol (Man) was performed at 1 day post‐infiltration (DPI). No second infiltration was performed for the control (C) plants. Samples were collected at 3 DPI, and FLUC and Renilla luciferase (RLUC) activities were determined. Average FLUC activities (A, C) and normalized average PVAWT‐derived RLUC activities (B, D). Each column represents an average measured from four plants. The error bars represent the standard deviation of the mean. (E) Total protein samples prepared from PVAWT‐infected and 0.005 or 0.1 m CaCl2‐treated leaves were separated on a 12% sodium dodecylsulphate‐polyacrylamide gel electrophoresis (SDS PAGE) gel and blotted onto a poly(vinylidene difluoride) (PVDF) membrane. Immunoblots were probed with anti‐Potato virus A coat protein (PVA CP), anti‐FLUC and anti‐RLUC immunoglobulin Gs (IgGs). The molecular weight standard is on the right and the proteins detected are indicated on the left. The Ponceau‐S‐stained immunoblot membrane serves as a loading control. (F) The third leaf up from the point of infiltration was sampled at 4 DPI and analysed for RLUC activity. Each column represents the average from five sample pools. The error bars represent the standard deviation of the mean.
Figure 6
Figure 6
The effect of time of CaCl2 application on viral gene expression. (A) PVAWT infection and firefly luciferase (FLUC) expression were initiated with Agrobacterium co‐infiltration. In the control (C), there was no subsequent infiltration. In the other samples, CaCl2 (0.1 m) was applied by infiltration either simultaneously with Agrobacterium[0 days post‐infiltration (DPI)] or at 1, 1 + 2, 1.5 and 2 DPI. (A) Each column represents the normalized average PVAWT Renilla luciferase (RLUC) activity at 3 DPI and the error bars represent the standard deviation of the mean. (B) Statistical analysis of the size of the PVAWT infection foci. The diameters of the infection foci were measured with the aid of virus‐derived green fluorescent protein (GFP) fluorescence from samples treated as in (A). The diameter of GFP expression was measured from 259 (C), 116 (0 DPI), 188 (1 DPI), 174 (1 + 2 DPI) and 209 (2 DPI) infection foci. The error bar represents the standard deviation of the mean. (C) Representative infection foci from each sample type visualized by fluorescence microscopy.
Figure 7
Figure 7
Inhibition of Ca2+ pumps located at the plasma membrane by LaCl3 prevents the Ca2+‐induced effect on viral gene expression. Nicotiana benthamiana plants were co‐infiltrated with Agrobacterium carrying PVAWT and firefly luciferase (FLUC). Eighteen hours post‐Agrobacterium infiltration, the plants were infiltrated with either distilled H2O (dH2O) or 1 mm LaCl3, followed by a third infiltration with either dH2O or 0.1 m CaCl2 within 1 h. Four types of PVAWT‐infected samples were collected: dH2O + dH2O, dH2O + CaCl2, LaCl3+ dH2O and LaCl3+ CaCl2 at 3 days post‐infiltration (DPI). The FLUC and Renilla luciferase (RLUC) activities were determined. Each column represents the average FLUC (A) or the normalized average PVAWT‐derived RLUC (B) from four parallel samples, and the error bars represent the standard deviation of the mean.
Figure 8
Figure 8
The effect of CaCl2 on the expression of PVAWT and replication‐deficient PVAΔGDD. Nicotiana benthamiana plants were co‐infiltrated with Agrobacterium carrying either PVAWT or PVAΔGDD and firefly luciferase (FLUC). A second infiltration of either 0.1 or 0.005 m CaCl2 was performed at 1 day post‐infiltration (DPI). The control (C) was not infiltrated for the second time. Samples were collected at 2, 3 and 4 DPI, and the Renilla luciferase (RLUC) and FLUC activities were determined. FLUC activity when co‐infiltrated with PVAWT (A–C) and with PVAΔGDD (G,H). Normalized average PVAWT‐derived (12 plants) (D–F) and PVAΔGDD‐derived (12 plants) (I,J) RLUC activities. The error bars represent the standard deviation of the mean. (K,L) Relative level of enhancement of PVA WT‐ and PVAΔGDD‐derived RLUC activities, respectively, induced by 0.1 or 0.005 m CaCl2. The values from the nontreated control plants were assigned the relative value of unity.
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