Drought stress acclimation imparts tolerance to Sclerotinia sclerotiorum and Pseudomonas syringae in Nicotiana benthamiana

Abstract

Acclimation of plants with an abiotic stress can impart tolerance to some biotic stresses. Such a priming response has not been widely studied. In particular, little is known about enhanced defense capacity of drought stress acclimated plants to fungal and bacterial pathogens. Here we show that prior drought acclimation in Nicotiana benthamiana plants imparts tolerance to necrotrophic fungus, Sclerotinia sclerotiorum, and also to hemi-biotrophic bacterial pathogen, Pseudomonas syringae pv. tabaci. S. sclerotiorum inoculation on N. benthamiana plants acclimated with drought stress lead to less disease-induced cell death compared to non-acclimated plants. Furthermore, inoculation of P. syringae pv. tabaci on N. benthamiana plants acclimated to moderate drought stress showed reduced disease symptoms. The levels of reactive oxygen species (ROS) in drought acclimated plants were highly correlated with disease resistance. Further, in planta growth of GFPuv expressing P. syringae pv. tabaci on plants pre-treated with methyl viologen showed complete inhibition of bacterial growth. Taken together, these experimental results suggested a role for ROS generated during drought acclimation in imparting tolerance against S. sclerotiorum and P. syringae pv. tabaci. We speculate that the generation of ROS during drought acclimation primed a defense response in plants that subsequently caused the tolerance against the pathogens tested.

Figure 1

Drought acclimation of N. benthamiana plants. Drought stress was imposed on five-week-old plants following gravimetric approach. After all sets of plants reached specific FC’s, they were continued to be maintained at that level for five more days for acclimation (a). Stress levels were confirmed by stress-induced changes in relative water content (RWC) (b); ABA (c) and root-shoot ratio (d) at the end of acclimation period. Each bars represents standard error (n = 6). The one-way analysis of variance (ANOVA) was performed (p = 0.05), and letters above the data points indicate the significance and data points with the same letters are not significantly different.

Figure 2

Sclerotinia sclerotiorum-induced cell death on N. benthamiana plants. Leaves of control and drought acclimated plants were inoculated with potato dextrose agar (PDA) plugs with actively growing S. sclerotiorum cultures. Photographs were taken at 4 dpi (a) and necrotic area was visually scored 0 (no cell death) to 4 (severe cell death) and expressed as percent of each score (b). Five independent plants were analyzed for each treatment (each with 30 inoculation spots). Experiments were repeated twice with reproducible results.

Figure 3

Disease-induced cell death in N. benthamina plants inoculated with Pseudomonas syringae pv. tabaci. Leaves of control and drought acclimated plants were inoculated with P. syringae pv. tabaci and pathogen-induced cell death was visually scored 0 (no cell death) to 5 (100% cell death at the inoculated spot) at 5 dpi. Three independent plants were analyzed for each treatment (each plant with 5 inoculation spots). Experiments were repeated twice with reproducible results.

Figure 4

ROS levels in drought acclimated N. benthamiana plants. The ROS contents were measured in plants maintained at different FC’s at the end of stress period. The O₂⁻ (a) and H₂O₂ (b) were quantified by XTT and scopoletin assay, respectively. Each bars represent the standard error (n = 6). The one-way analysis of variance (ANOVA) was performed (p = 0.05), and letters above the data points indicate the significance and data points with the same letters are not significantly different. (c) Visualization of bacterial growth in methyl viologen (MV) treated N. benthamiana leaves infected with Pseudomonas syringae pv. tabaci. Plants were sprayed with ROS inducer, MV, followed by infection with GFPuv expressing P. syringae pv. tabaci. Photographs were taken under UV light at 4 dpi.

Figure 4

ROS levels in drought acclimated N. benthamiana plants. The ROS contents were measured in plants maintained at different FC’s at the end of stress period. The O₂⁻ (a) and H₂O₂ (b) were quantified by XTT and scopoletin assay, respectively. Each bars represent the standard error (n = 6). The one-way analysis of variance (ANOVA) was performed (p = 0.05), and letters above the data points indicate the significance and data points with the same letters are not significantly different. (c) Visualization of bacterial growth in methyl viologen (MV) treated N. benthamiana leaves infected with Pseudomonas syringae pv. tabaci. Plants were sprayed with ROS inducer, MV, followed by infection with GFPuv expressing P. syringae pv. tabaci. Photographs were taken under UV light at 4 dpi.

Figure 5

Increased expression of plant defense genes in drought acclimated plants. The relative mRNA expression levels of PR-5 (a) and PDF1.2 (b) were quantified by qRT-PCR in drought acclimated and control plants (100% FC). The fold change values were calculated using the 2^−ΔΔCT method and represented as changes in mRNA levels relative to 100% FC. NbActin was used as an internal control to normalize gene expression levels. The data are averages of two biologically independent experiments each consisting of three technical replicates.