Damaged-self recognition in common bean (Phaseolus vulgaris) shows taxonomic specificity and triggers signaling via reactive oxygen species (ROS)

Abstract

Plants require reliable mechanisms to detect injury. Danger signals or "damage-associated molecular patterns" (DAMPs) are released from stressed host cells and allow injury detection independently of enemy-derived molecules. We studied the response of common bean (Phaseolus vulgaris) to the application of leaf homogenate as a source of DAMPs and measured the production of reactive oxygen species (ROS) as an early response and the secretion of extrafloral nectar (EFN) as a jasmonic acid (JA)-dependent late response. We observed a strong taxonomic signal in the response to different leaf homogenates. ROS formation and EFN secretion were highly correlated and responded most strongly to leaf homogenates produced using the same cultivar or closely related accessions, less to a distantly related cultivar of common bean or each of the two congeneric species, P. lunatus and P. coccineus, and not at all to homogenates prepared from species in different genera, not even when using other Fabaceae. Interestingly, leaf homogenates also reduced the infection by the bacterial pathogen, Pseudomonas syringae, when they were applied directly before challenging, although the same homogenates exhibited no direct in vitro inhibitory effect in the bacterium. We conclude that ROS signaling is associated to the induction of EFN secretion and that the specific blend of DAMPs that are released from damaged cells allows the plant to distinguish the "damaged-self" from the damaged "non-self." The very early responses of plants to DAMPs can trigger resistance to both, herbivores and pathogens, which should be adaptive because injury facilitates infection, independently of its causal reason.

Keywords: Pseudomonas syringae; damage-associated molecular pattern; danger signal; extrafloral nectar; induced defense; induced resistance; plant pathogen; wound response.

FIGURE 1

Phylogenetic relations to the receiver, Phaseolus vulgaris cultivar “Negro San Luis” (NSL), of the plant species used for the preparation of leaf homogenates. The relative phylogenetic distances among the different accessions within the species P. vulgaris were assessed using AFLPs (data not shown) whereas relations among species are depicted according to public information (URL http://www.ncbi.nlm.nih.gov/taxonomy/).

FIGURE 2

Induction of extrafloral nectar (EFN) by leaf homogenates depends on phylogenetic distance. The EFN secretion after applying different leaf homogenates on intact leaves is depicted as means ± SE of mg soluble solids per gram of dry leaf mass as quantified 24 h after treatment of n = 6 biological replicates per homogenate. Control plants were treated with water (light gray bars), Tween20 (black bars) or mechanically damaged (dark gray bars). The intensity of green of the bars indicates the relatedness among the receiver plant and the plants from which the homogenates were prepared. Different letters above bars indicate significant differences among treatments (univariate ANOVA and post hoc Tukey test: p < 0.05).

FIGURE 3

Formation of hydrogen peroxide (H₂O₂) after application of leaf homogenates depends on phylogenetic distance. (A) The presence of H₂O₂ was detected by staining with diaminobenzidine (DAB) 2 h after application of leaf homogenates. (B) Concentrations of H₂O₂ in nanomol per gram leaf fresh mass. The intensity of blue of the bars indicates the relatedness among the receiver plant and the plants from which the homogenates were prepared. Bars depict means ± SE of n = 3 biological replicates per homogenate and different letters above bars indicate significant differences among treatments (univariate ANOVA and post hoc Tukey test: p < 0.05).

FIGURE 4

Resistance to Pseudomonas syringae in plants treated with different leaf homogenates does not depend on phylogenetic distance. We depict the number of colony forming units (CFUs) of P. syringae in leaves inoculated with the pathogen 5 min (A) or 24 h (B) after application of leaf homogenates. Bars indicate means ± SE of n = 6 biological replicates and different letters indicate significant differences among treatments (univariate ANOVA and post hoc Tukey test: p < 0.05).

FIGURE 5

Leaf homogenates do not directly inhibit P. syringae. We depict numbers of CFUs 2 days after inoculating (I) the bacterium on Petri dishes that 5 min before had been prepared with sterile distilled water, Tween20^® or the indicated leaf homogenates. Bars indicate means ± SE of n = 4 repetitions and different letters indicate significant difference among treatments (univariate ANOVA and post hoc Tukey test: p < 0.05). Negative controls (C) were not inoculated with P. syringae to ensure that the leaf homogenates themselves did not carry bacteria.

FIGURE 6

Volatile organic compounds (VOCs) released from leaf homogenate do not induce EFN secretion. EFN secretion (mg soluble solids per gram of dry leaf mass as quantified 24 h after treatment) was quantified on plants treated with mechanical damage (MD) and leaf homogenate of NSL plants obtained from fresh or lyophilized leaves. Leaves were directly treated with the homogenate (D) or had no direct contact, that is, they were only exposed to the headspace (H) of the homogenates. Controls were treated with water (W). Bars indicate the mean ± SE of n = 6 biological replicates and different letters indicate significant differences among treatments (univariate ANOVA and post hoc Tukey test: p < 0.05).

FIGURE 7

Exogenous ATP at high concentrations induces EFN secretion. EFN secretion (mg soluble solids per gram of dry leaf mass as quantified 24 h after treatment) was quantified on plants treated with different concentrations of exogenous ATP and compared to plants treated with NSL homogenate or MD as positive controls or with Tween20^® (T) or water (W) as negative controls. Bars indicate the mean ± SE of n = 7 biological replicates and different letters indicate significant differences among treatments (univariate ANOVA and post hoc Tukey test: p < 0.05).

FIGURE 8

Heat treatment of the homogenate before resting diminishes its effects on EFN secretion. EFN secretion (mg soluble solids per gram of dry leaf mass as quantified 24 h after treatment) was quantified on plants treated with water, Tween20^®, boiled Tween20^®, or NSL leaf homogenate that was subjected to different temporal combinations of Tween20^® application, boiling (3 min at 100°C) and resting: (A) Tween20^® added to homogenate, then 2 h resting time; (B) Tween20^® added to homogenate, then 2 h resting time, then boiled; (C) Tween20^® added to homogenate, then boiled, then 2 h resting time; (D) homogenate with water only, then 2 h resting time, then Tween20^® added directly before application; (E) homogenate with water only, then 2 h resting time, then boiled, then Tween20^® added directly before application. Bars indicate the mean ± SE of n = 7 biological replicates and different letters above bars indicate significant differences among treatments (univariate ANOVA and post hoc Tukey test: p < 0.05).