Like all multicellular organisms, plants require efficient mechanisms to detect injury. Specificity in the resistance to specialist herbivores and pathogens can be adaptive, because it allows for the synthesis of defensive compounds that are highly active against the current attacker. Specific responses can be triggered by the recognition of herbivore-associated molecular patterns (HAMPs; Wu and Baldwin 2010) or microbe- (or pathogen-) associated molecular patterns (MAMPs/PAMPs) in plants (Jones and Dangl, 2006). Common HAMPs are fatty acid-amino acid conjugates that stem from the saliva of insects or compounds in insect oviposition fluids (Wu and Baldwin, 2010). Common MAMPs are flagellin, chitin, and other molecules that are not produced by plants and that indicate the presence of enemies on or in a plant. Effector molecules that are secreted by adapted pathogens to overcome the plant resistance response can also be perceived as strain-specific PAMPs and then cause effector-triggered immunity (Jones and Dangl, 2006). Most of these molecules are recognized by pattern recognition receptors (PRRs), which induce a signaling cascade that normally consists of Ca²⁺-influxes and the corresponding membrane depolarization events, the generation of reactive oxygen species (ROS) such as the superoxide anion (O₂^-), hydrogen peroxide (H₂O₂) or the hydroxyl radical (⋅OH), and downstream signaling via mitogen-associated protein kinase (MAPK) cascades (Wu and Baldwin, 2010).

However, successfully dealing with injury requires multiple countermeasures that are independent of the detailed nature of the injury-causing agent. For example, wounds must be sealed to avoid desiccation and even so, they normally will be used by pathogens to enter the wounded tissue (Komarova et al., 2014). Moreover, induced plant responses that are completely based on the recognition of specific HAMPs or PAMPs would always be in danger to be overcome by co-evolving enemies, which change the molecular structure of the target molecules of plant receptors and thereby escape from recognition (Heil, 2012). Therefore, and in analogy to the immune system of mammals, which detects endogenous danger signal to perceive the “wounded self” (Gallucci and Matzinger, 2001; Matzinger, 2002; Land and Messmer, 2012), it has been hypothesized that plants are able to perceive fragmented or delocalised own molecules as damage-associated molecular patterns (DAMPs), which enable “damaged-self recognition” (Heil, 2009, 2012).

Indeed, the application of leaf homogenate frequently triggers plant resistance responses, which can be directed against herbivores, pathogens or both (Heil, 2009). Leaf homogenate contains fragments of plant macromolecules such as oligosaccharides, oligogalacturonides, and peptides and its application to a leaf also causes the presence in the extracellular space of molecules such as sucrose, Adenosintriphosphate (ATP), and nucleic acids, which in the intact tissue would be localized within the cell (Heil, 2009). Most of these molecules are produced or released passively as soon as tissue is being disrupted: macromolecules become exposed to (lytic) enzymes from which they are compartimentally separated in the intact cell, and small molecules are released into the extracellular space. In principle, such molecules can serve as DAMPs. Indeed, extracellular sucrose is an important signaling molecule in plants (Sheen et al., 1999; Rolland et al., 2006; Bolouri Moghaddam and Van den Ende, 2013), extracellular ATP (eATP) has immunmodulatory effects in mammals (Schwiebert and Zsembery, 2003; Chen and Nuñez, 2010; Ivison et al., 2011; Zeiser et al., 2011), insects (Moreno-Garcia et al., 2014), plants (Demidchik et al., 2003; Kim et al., 2006; Roux and Steinebrunner, 2007; Heil et al., 2012; Choi et al., 2014; Tanaka et al., 2014) and fungi (Medina-Castellanos et al., data not shown), methanol is released in wounded plant tissues from cell wall pectins by the action of pectin methylesterase (Dorokhov et al., 2012), and larger fragments of the plant cell wall matrix (Doares et al., 1995; Creelman and Mullet, 1997; Stennis et al., 1998; Bergey et al., 1999; Narváez-Vásquez et al., 2005) or of the extracellular matrix of mammals (Schaefer, 2010; Zeiser et al., 2011) serve as indicators of the ongoing injury and trigger resistance responses.

Interestingly, authors who applied leaf homogenates obtained from other plants, or even from algae, frequently observed induced resistance to pathogens (Devaiah et al., 2009; Medeiros et al., 2009; Chandrashekhara et al., 2010; Akila et al., 2011; Ghazanfar et al., 2011; Jayaraman et al., 2011; Jithesh et al., 2012). By contrast, the application of conspecific leaf homogenates to leaves of cabbage (Brassica oleracea) or maize (Zea mays) plants caused the emission of herbivore-induced volatile organic compounds (HI-VOCs) (Turlings et al., 1993; Mattiacci et al., 1995). Similarly, applying conspecific leaf homogenate to wild lima bean (P. lunatus) induced the secretion of extrafloral nectar (EFN), enhanced the levels of endogenous jasmonic acid (JA), and caused an overall transcriptomic response that was very similar to the response to exogenous JA application (Heil et al., 2012). JA is the central hormone in the octadecanoid signaling pathway that controls multiple induced plant defense traits (including EFN and HI-VOCs) to chewing herbivores (Wasternack, 2007; Pieterse et al., 2009; Thaler et al., 2012). HI-VOCs play diverse roles in the resistance of plants, the most commonly reported one being the attraction of predators and parasitoids as a means of indirect defense against herbivores (Dicke and Baldwin, 2010). EFN is a taxonomically widespread, HI attractant of ants, wasps, and other predators and serves the indirect defense of plants against herbivores (Heil, 2008, 2011). Hence, it appears that leaf homogenates obtained from conspecific plants usually induce the resistance to herbivores, whereas homogenates from heterospecific plants more frequently induce the resistance to pathogens.

In the present study we used cultivated common bean (P. vulgaris, cultivar Negro San Luis, “NSL”; Fabaceae: Faboideae) to investigate whether leaf homogenates from species with different degrees of relatedness to the receiver plant (Figure 1) cause different responses. NSL-plants were treated with homogenate obtained from the same cultivar, the common bean cultivars, Pinto Villa and Negro Durango, a wild accession of common bean, and from Tecomari bean (P. coccineus), Lima bean (P. lunatus), Alfalfa (Medicago sativa; all: Fabaceae: Faboideae), an Acacia (Acacia farnesiana; Fabaceae: Magnolioidea), pumpkin (Cucurbita maxima; Cucurbitaceae), tomato (Solanum lycopersicum; Solanaceae), maize and sorghum (Sorghum spp.; both: Poaceae), a gymnosperm (Pinus arizonica) and a fern (Adiantum aleuticum). We also exposed leaves to the headspace of leaf homogenates to determine whether VOCs contribute to a putative induction event. The formation of ROS was quantified as an early marker of general resistance responses and EFN secretion was quantified as a late marker of the octadecanoid-dependent responses to herbivores. We also challenged the plants with the biotrophic bacterial pathogen Pseudomonas syringae, to quantify the induced resistance to pathogens. With these experiments we aimed to understand whether resistance induction after the application of leaf homogenate depends only on universal DAMPs such as extracellular sucrose or eATP, or whether a more specific blend of DAMPs allows for true “damaged-self” recognition.

The secretion of EFN responded significantly to the application of leaf homogenate from closely related (i.e., congeneric) species, but not from other plants (Figure 2). Homogenate from NSL-plants and Pinto Villa-plants as well as from the wild common bean accession caused the strongest response. Lower, but still significant, induction of EFN was caused by the application of homogenate obtained from the common bean cultivar Negro Durango or from the two congeneric species, P. coccineus or P. vulgaris. Although these extracts induced a significant response over control levels, they did not induce EFN secretion more strongly than MD inflicted by a needle. By contrast, none of the non-bean species resulted in EFN secretion rates that differed significantly from the rates observed on the control plants.

Very similar patterns were observed in the formation of ROS (Figure 3). Punching holes with a needle caused local induction of ROS production and related leaf homogenates caused a strong presence of ROS at 2 h after application (Figure 3A, supplementary Figure 4). The quantification of H₂O₂ revealed that homogenates obtained from NSL-plants, Pinto Villa-plants or the wild common bean accession significantly enhanced the concentration of ROS in the leaves (Figure 3B). Lower responses were observed after applying homogenates from the cultivar Negro Durango, P. coccineus or P. vulgaris, and no significant response was caused by any of the non-bean species (Figure 3B).

All homogenates tested (NSL, wild accession, P. coccineus, P. lunatus, A. farnesiana and maize) caused significant resistance to the bacterium, P. syringae, when they were applied 5 min before challenging the plant with the bacterium (Figure 4A). By contrast, no enhanced resistance to the bacterium could be observed when the leaf homogenate had been applied 24 h before the challenging treatment (Figure 4B). In a Petri dish assay, the leaf homogenates had no direct inhibitory effects on bacterial growth when the KB-medium was pre-treated with one of these homogenates 5 min before applying the bacterial suspension (Figure 5).

When plants were exposed to leaf homogenate without any direct contact between homogenate and leaf surface, we could not detect any induction of EFN secretion. By contrast, the same homogenates caused an effect when they were directly applied on the leaves. Moreover, similar responses were observed after the use of fresh or lyophilized homogenates (Figure 6). The application of ATP induced EFN secretion only at a concentration of 1 mM but not at lower concentrations (0.5, 0.1, or 0.04 mM, see Figure 7).

During method establishment we had detected that the homogenates required at least 1 h resting time before application to elicit maximum responses (supplementary Figure 3) and that adding 0.05% Tween20®; enhanced the response of plants to a given homogenate (Figure 8, supplementary Figures 1 and 6), likely because Tween20®; facilitates the uptake of lipophilic compounds into the leaf tissue. However, Tween20®; enhanced the EFN-inducing effect of a homogenate even further when it was added to the homogenate before a resting time of 2 h (Figure 8). By contrast, no induction of EFN secretion was observed when the homogenate was heated for 3 min to 100°C before the 2 h of resting time, independent of whether or not Tween20®; had been added, whereas boiling the homogenate immediately before its application on the leaf did not diminish its EFN-inducing effect (Figure 8).

Applying leaf homogenates obtained from bean plants on the leaves of common bean induced the formation of ROS after 2 h and the secretion of EFN over the following 24 h, and decreased the infection by the bacterial pathogen, P. syringae, over the following 2 days. No insects, pathogens, HAMPs, or MAMPs/PAMPS were used in any of the induction treatments. Thus, our results show that leaf homogenates contain DAMPs that can trigger both early and late resistance-related responses in intact, non-infected leaves. eATP induced EFN secretion in this and an earlier study (Heil, 2012) and in fact, eATP represents a universal DAMP that is active in organisms across the tree of life (Heil and Land, 2014). However, we observed a strong phylogenetic signal in the intensity of the formation of ROS and the induction of EFN by different homogenates: homogenates produced from leaves of the own cultivar and two genetically very similar common bean accessions (one cultivar and one wild accession) elicited the strongest response, a less related common bean cultivar and two other species of Phaseolus (P. coccineus and P. lunatus) elicited significant, but lower responses, and none of the non-bean species caused a significant induction of ROS formation or EFN secretion. We conclude that both responses are induced by a blend of DAMPs, rather than a single DAMP of wide taxonomic distribution, and that the entire blend of DAMPs that is released from the destroyed own cells allows plants to specifically detect the “damaged-self,” rather than allowing only for a general, diffuse “damaged plant” recognition. No phylogenetic effects were observed in the induced resistance to the pathogen: all homogenates reduced the rate of infection rate by P. syringae when they had been applied 5 min before inoculation. Because none of the leaf homogenates tested caused any detectable direct inhibitory effect on the growth of P. syringae, we conclude that the short-term inhibitory effect on P. syringae that we observed in homogenate-treated bean plants indicates an enhancement of pathogen resistance that was likely to be caused by a few, conserved DAMPs. In this context, the microflora that was associated to the plants from which the homogenates had been produced might have interfered with the observed resistance phenotype. However, no fungi or bacteria could be detected in the control Petri dishes that received only leaf homogenate but no P. syringae suspension, and no endophytic fungi or bacteria could be cultivated on standard media from bean plants that were cultivated under the same experimental conditions as used in the present study (data not shown). Thus, we assume that the inhibitory effects on P. syringae that we observed in the in planta assays were more likely to represent a transient induction of resistance in the receiver plant, rather than direct inhibitory effects of components (of plant or microbial origin) of the homogenate. However, putative resistance-inducing effects of endosymbiotic microorganisms cannot be excluded by now and require further investigation that will also have to consider non-culturable and, perhaps, intracellular endosymbionts.

The detailed nature of all DAMPs that are involved in this highly specific “damaged-self recognition” in plants will form the goal of future research efforts. We observed that heating the homogenates to 100°C for 3 min before the 2 h resting time strongly reduced the induction effect, which indicates that enzymatic activities and/or intact peptides or proteins are likely to be involved in the formation of the DAMPs. Since mixing the homogenate with Tween20®; 2 h before the application resulted in stronger induction effects than mixing the homogenate with Tween20®; directly before the application, we conclude that enzymatic activity during the 2 h of resting time might have helped with the formation of the full blend of DAMPs and that the disintegration of membranes by the detergent further contributed to DAMP formation, likely by exposing the endogenous enzymes to their substrates. Lyophilized homogenates were as effective as fresh homogenates, whereas no induction effect could be observed when leaves were only exposed to the headspace of the homogenates. Thus, it appears that VOCs did not play a significant role in the induction effects that we observed here, although they are well-known to induce or prime resistance responses in several plants, including bean (Engelberth et al., 2004; Heil and Kost, 2006; Kessler et al., 2006; Frost et al., 2007; Heil and Silva Bueno, 2007; Ton et al., 2007; Yi et al., 2009).

Plant resistance responses to biological enemies are orchestrated by two main signaling pathways, of which the salicylic acid (SA) pathway mainly controls responses to biotrophic pathogens, whereas ethylene/JA signaling mainly controls the resistance to chewing herbivores and necrotrophic pathogens (Pieterse and Dicke, 2007; Pieterse et al., 2009). In most plant species, these two pathways are subject to a negative crosstalk (Thaler et al., 2012). In our study, the formation of ROS and the secretion of EFN were positively correlated with each other, and both responded strongest to the same treatment. The formation of ROS is a widespread early response in plants and mammals to abiotic stress, invasion by pathogens or exposure to DAMPs (see references in Heil and Land, 2014), whereas the secretion of EFN serves the indirect defense of plants against insect herbivores (Heil, 2008). Thus, our results appear to contradict a major paradigm in induced plant defenses. However, the formation of ROS as an early event also been reported after herbivory in Arabidopsis thaliana (Beneloujaephajri et al., 2013) and sweet potato (Rajendran et al., 2014) or after sterile mechanical wounding in multiple plant species (Orozco-Cardenas and Ryan, 1999), and enhanced concentrations of ROS were observed after MD in the fungus, Trichoderma atroviride (Medina-Castellanos et al., data not shown). Thus, ROS might represent early signals in the response to injury in all multicellular organisms. Finally, responses that can be observed after mechanical injury in plants such as the release of green leaf volatiles (Heil and Karban, 2010; Dorokhov et al., 2012; Scala et al., 2013) or methanol (Dorokhov et al., 2012) and the synthesis of terpenoids such as limonene (Byun-McKay et al., 2006) all are directed against both pathogens and herbivores. Even serine proteinase inhibitors, which represent the first group of wound-inducible defensive proteins that have been discovered in plants (Green and Ryan, 1972), act against endopeptidases in both animals and microorganisms (Turra and Lorito, 2011).

Like all multicellular organisms, plants rely on an intact surface to prevent infection and desiccation. In the words of Komarova et al. (2014), “plant wounding is one of the conditions for pathogen entry.” Thus, as soon as a plant is damaged, it must seal the wound and prepare locally for infection. Applying leaf homogenates 5 min (but not 24 h) before challenging with P. syringae significantly decreased the levels of infection by this bacterium, and this effect depended much less on the detailed taxonomic nature of the homogenate than the induction of ROS development or EFN secretion. Because we observed no direct inhibitory effect of the homogenates on the bacterium in in vitro assays, we conclude that the application of leaf homogenate caused an unspecific and transient induction of the resistance in bean to pathogens, but no longer-lasting priming or induction of pathogen resistance, as it has been observed in VOC-exposed plants (Yi et al., 2009; Scala et al., 2013).

In summary, leaf homogenates contain a complex blend of DAMPs, which trigger early responses in plant tissues that are exposed to these DAMPs. These responses can be directed against both, pathogens and herbivores. An emerging general pattern appears to be that homogenates obtained from the same species trigger mainly responses against herbivores, whereas heterospecific leaf homogenates can enhance the resistance to pathogens. Many human “danger signals” play a dual role in resistance induction and act both as antimicrobial agents and as signals that enhance immunity-related responses in intact cells (Gallucci and Matzinger, 2001). Similarly, green leaf volatiles (Dorokhov et al., 2012; Scala et al., 2013), MeOH (Dorokhov et al., 2012) and wound-induced terpenoids such as limonene (Byun-McKay et al., 2006) have direct antimicrobial activity and also act as signals that induce the expression of defense-related genes in the surrounding tissues, distant parts of the same plant, and even in neighboring plants. The formation of ROS appears to play a central role in the responses in plants, fungi, and mammals to biotic and biotic stress and increasing evidence indicates their direct involvement in the perception of DAMPs in all these organisms. Future studies will have to identify the DAMPs are used by plants to perceive the “damaged-self” and to investigate which of these astonishing similarities and parallels between the ways in which plants and animals respond to injury represent the product of parallel evolution and which ones represent homologies.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.