Wound response is a crucial process for the survival of multicellular organisms and facilitates their adaptation to hostile environments. Plants, being sessile organisms, cannot escape from attack by insects or larger herbivores. Animals, although motile, are also exposed to mechanical damage and injuries inflicted by predators. Similarly, due to their absorptive nutrition mode and their immobility, multicellular (filamentous) fungi are prey to a variety of animal predators including fungivorous nematodes and insects. Nevertheless, the physiological response of fungi to wounding and its implications, if any, remains mostly unexplored.

The topic of damage signals and their perception in different organisms has been of recent interest since key mechanisms such as wound sealing and healing of the damaged tissue, as well as local or systemic responses to prevent infection, contain conserved elements. In highly regenerative animals, wounding can trigger regrowth of a missing body part, involving gene expression changes specific for tissue regeneration (Nacu and Tanaka, 2011; Lee and Gardiner, 2012; Wenemoser et al., 2012). Similarly, plant and moss cells can be reprogrammed to initiate tip growth in wounded tissues (Ishikawa et al., 2011). Unfortunately, our current knowledge of wound response in filamentous fungi is mostly limited to the well-characterized sealing of septal pores by Woronin bodies. This sealing reduces loss of cytoplasmic content to prevent cell death, and is followed by the formation of one or more hyphal tips at the plugged septum, resulting in reinitiation of growth and hyphal reconnection (Jedd, 2011). Despite our limited understanding of wound response in fungi, mechanical damage has been found to trigger entry into development in several species. One of the first reports refers to the formation of fruiting bodies in Schizophyllum commune in response to mycelial injury (Leonard and Dick, 1968). These authors suggested the participation of oxidative stress in the production of fruiting bodies in response to injury (Leonard and Dick, 1968). Later, Georgiou et al. (2006) reported the formation of sclerotia in response to oxidative stress in Sclerotium rolfsii, and increased reactive oxygen species (ROS) were found in damaged hyphae of the fungus Glomus intraradices (Fester and Hause, 2005).

Unfortunately, none of the mentioned reports provided mechanistic insights such that injury-signaling remains poorly understood. In other systems, reliable signals of tissue disruption are known to comprise fragments of the extracellular matrix, extracellular molecules such as ATP, adenosine, RNA or DNA, and certain proteins or protein fragments (Chen and Nuñez, 2010; Zeiser et al., 2011; Heil, 2012). These warning signals are known as damage-associated molecular patterns (DAMPs; Heil and Land, 2014).

The release of ATP is an important early danger signal in humans (Chen and Nuñez, 2010; Zeiser et al., 2011), fish (Kawate et al., 2009), algae (Torres et al., 2008), and plants (Chivasa et al., 2009; Heil et al., 2012). In humans, perception of extracellular ATP (eATP) by purinergic receptors is one of the main biological mechanisms responsible for epithelial intracellular calcium mobilization (Sherwood et al., 2011; Kurashima et al., 2012). In zebrafish, ATP released after an injury is sensed by a purinergic P2Y receptor, which in turn modulates NADPH-oxidase activity (Dual oxidase Duox1; de Oliveira et al., 2014). G-protein-coupled P2Y receptors can also recognize other nucleotides, such as adenosine diphosphate (ADP) and uridine triphosphate (UTP; Abbracchio et al., 2006; Covian-Nares et al., 2010). Purinergic receptors similar to those described in animal systems have not been found in plants. However, an extracellular lectin ATP receptor kinase (LecRK-I.9) was recently discovered in Arabidopsis (Choi et al., 2014).

One of the earliest events upon detection of a stress signal is the activation of mitogen-activated protein kinases (MAPKs). MAPKs are part of well-conserved eukaryotic signaling cascades. In Arabidopsis at least two MAPKs, MPK6, and MPK3, are rapidly activated by wounding and during plant-pathogen interactions. MPK6 directly phosphorylates the 1-aminocyclopropane-1-carboxylic acid synthases (ACS), ACS6, and ACS2 and thus stabilizes these enzymes, increasing the production of ethylene; while MPK3 activity increases after wounding (Ichimura et al., 2000; Liu and Zhang, 2004; Wang et al., 2008). In Nicotiana species, orthologs of MPK6 (SIPK) and MPK3 (WIPK) are activated by wounding (reviewed in Hettenhausen et al., 2014). Plants carrying a mutation in LecRK-I.9 that do not respond to ATP (DORN1), fail to trigger phosphorylation of MPK3 and MPK6 (Choi et al., 2014). Further, plant leaf extracts (which contain multiple DAMPs) can induce secretion of extrafloral nectar, an indirect defense response to damage (Heil et al., 2012).

Based on these precedents we hypothesized that fungi could regulate wound response via MAPK pathways, triggering the activation of developmental processes such as cell growth and differentiation. In most filamentous fungi, there are three MAPK pathways that are involved in several processes, such as asexual and sexual reproduction, general stress response, spore germination, cell fusion, secondary metabolism, and mycoparasitism (Mendoza-Mendoza et al., 2003; Delgado-Jarana et al., 2006; Reithner et al., 2007; Fleissner et al., 2009; Kumar et al., 2010; Lara-Rojas et al., 2011; Bayram et al., 2012; Lichius et al., 2012). Defective sexual and asexual development resulting from MAP kinase mutations have been reported in Magnaporthe grisea (Xu et al., 1998), Fusarium graminearum (Hou et al., 2002), Neurospora crassa (Lichius et al., 2012), and Aspergillus nidulans (Wei et al., 2003).

Trichoderma species are often a predominant component of the mycoflora in native and agricultural soils, and are considered effective biocontrol agents due to their ability to parasitize phytopathogenic fungi (Herrera-Estrella and Chet, 2003). These ascomycete fungi reproduce asexually forming conidia, as a mechanism for survival and dispersal. Accordingly, the switch from vegetative to reproductive growth is triggered by several types of stress such as light, nutrient deprivation, and acid environments (for a review see Carreras-Villasenor et al., 2012). Recently, Hernández-Oñate et al. (2012) showed that Trichoderma atroviride responds to mechanical damage with a morphogenetic change initiated by the activation of regenerative processes, and entry into asexual reproduction (conidiation). Their transcriptome analysis revealed over nine hundred injury-responsive genes, including subsets involved in cell cycle control, oxidative stress, and calcium signaling and transport. The injury response depended on activation of the NADPH-oxidase complex formed by the catalytic (Nox1) and the regulatory (NoxR) subunits, which resulted in ROS production at the hyphal tip within the first minutes after injury. Both Nox1 and NoxR are essential for conidiation since Δnox1 and ΔnoxR mutants do not conidiate in response to this stimulus.

Trichoderma species have three MAPKs that belong to the so-called mycoparasitism/filamentous growth, cell wall integrity, and osmotic stress response pathways. In T. atroviride they are named Tmk1, Tmk2, and Tmk3, and their corresponding orthologs in yeast are Kss1/Fus3, Slt2, and Hog1 (Mendoza-Mendoza et al., 2003; Delgado-Jarana et al., 2006; Reithner et al., 2007; Zeilinger and Omann, 2007). None of the reports on the role of Trichoderma’s MAPKs establishes a connection between them and signaling molecules of the injury response.

Here, we show that eATP serves as a cue that signals tissue disruption to as yet intact hyphae (and, thus, acts as a DAMP) and that Ca²⁺ also plays an important role in asexual reproduction triggered by wounding in T. atroviride, likely as a downstream second messenger. We further demonstrate that transduction of eATP signaling takes place through the activation of MAPK cascades involving Tmk1 and Tmk3. Activation of Tmk3 depends on this NoxR/Nox1 complex, whereas that of Tmk1 is independent of the complex. By contrast, calcium signaling appears to take place through a MAPK-independent pathway. In conclusion, DAMPs are involved in the fungal wound response and trigger downstream signaling events that are similar to those known in other organisms.

Wound detection and healing represent important processes for the survival of any multicellular organism, involving conserved mechanisms across species. Multicellular fungi respond to wounding by sealing the septa of the cell adjacent to the damaged one by rapidly mobilizing proteins and forming Woronin bodies (Jedd, 2011). Interestingly, several fungi respond also by initiating a morphogenetic change that leads to the formation of different structures, such as sexual fruiting bodies, and conidiophores (Leonard and Dick, 1968; Hernández-Oñate et al., 2012). Unfortunately, there is scarce information about the perception of potential danger signaling molecules and their transduction. To the best of our knowledge, the present report is the first that contributes to understanding the signaling pathways involved in the wound response in filamentous fungi.

Hernández-Oñate et al. (2012) recently showed that mycelial injury in T. atroviride results in the formation of conidiophores that are produced exclusively from the newly regenerated hyphae. This observation suggests that signal molecules are released to the extracellular matrix during injury and that adjacent cells recognize these molecules. In plants, eATP is considered a damage signaling molecule, included in a group of molecules known as DAMPs, produced during herbivory, mechanical damage, or pathogen attack (Roux and Steinebrunner, 2007; Chivasa et al., 2009; Heil, 2012; Heil et al., 2012). Interestingly, our data show that eATP induces conidiation in response to injury and suggest that eATP is an important signal molecule that is released from damaged hyphae. Thus, eATP can also be considered a damage signaling molecule in fungi, which plays a similar role in wound signaling to that reported in triggering immune responses in mammals (Chen and Nuñez, 2010; Zeiser et al., 2011; Cordeiro and Jacinto, 2013), fish (Kawate et al., 2009), insects (Moreno-García et al., 2014), algae (Torres et al., 2008), and plants (Chivasa et al., 2009). Elevations in the concentration of intracellular calcium have also been observed in Arabidopsis upon application of eATP (Tanaka et al., 2010). Further, the induction of conidiation by eATP displayed a dose-dependent trend. This observation suggests that the fungus senses the concentration of eATP, which is probably correlated with the level or extension of the injury, and, thus produces more conidia to warrant survival when it suffers from particularly strong damage.

In accordance with reports of plants and animals, where Ca²⁺ influxes into the cytosol follow the perception of DAMPs (Heil and Land, 2014), our observation that chelating extracellular calcium blocked injury-induced conidiation would suggest that calcium plays a key role as second messenger of wounding. The early wound signaling response in animals, including humans (Shabir and Southgate, 2008; Covian-Nares et al., 2010), and plants (Arimura and Maffei, 2010; Beneloujaephajri et al., 2013) includes an increase in intracellular calcium and the activation of the calcium signaling machinery. Interestingly, calcium induces the formation of conidia in submerged cultures of T. viride (Simkovic et al., 2008). Furthermore, transcriptomic analysis of the response to injury in T. atroviride suggested the participation of a calcium signaling pathway, since several genes related to calcium signaling, including calcium transporters, phospholipase C, and a Ca²⁺/calmodulin-dependent kinase-1 (CAMK-1) were induced (Hernández-Oñate et al., 2012). In agreement with these observations, Nelson et al. (2004) showed that hypo-osmotic shock and external calcium treatment induce transient increase in intracellular calcium in Neurospora crassa, Aspergillus niger, and Aspergillus awamori. Our results strongly indicate that eATP is a damage signal, and that calcium acts downstream of this DAMP.

The recognition of nucleotides through purinergic receptors (a family of receptors initially classified according to the relative potency of purine nucleotides to stimulate them), which exhibit different affinities for different nucleotides, is well known in animal systems. Efforts to identify plant ATP receptors through homology of their genomic sequence to animal purinergic receptors failed to find any suitable candidates, but recently the lectin receptor kinase-I.9 (LecRK-I.9) has been shown to perceive eATP (Choi et al., 2014). In contrast, no single nucleotide receptor has been reported in fungi to date. However, our results indicate that there must be a nucleotide receptor, with higher affinity for ATP than for other nucleotides.

Here we show that T. atroviride also responds to extracellular CTP by forming conidia, consistent with evidence in Arabidopsis, where a significant elevation in cytosolic Ca²⁺ could be elicited by the application of exogenous ATP or CTP but not by ITP, TTP, or UTP ( Tanaka et al., 2010; Choi et al., 2014). In addition GTP, CTP, and UTP (as well as ATP) were found to induce superoxide production in Arabidopsis leaves (Song et al., 2006).

Although a BLAST based search for homologues of lectin receptor kinases in the T. atroviride genome database failed to find any match, the kinase domain of the lectin receptor presents high similarity with a MAPKKK orthologous to yeast Bck1, which participates in the protein kinase C signaling pathway that controls cell wall integrity (Lee and Levin, 1992; Lee et al., 1993). In this sense, one of the earliest signaling events after wound in animals and plants is the activation of MAPKs (Wu and Baldwin, 2010; Suzuki and Mittler, 2012). The first report of the involvement of MAPKs in plant–herbivore interactions showed that transcription and activity of wound-induced protein kinase (WIPK), a member of MAPK subfamily A, increased 1 min after wounding (Seo et al., 1995). In filamentous fungi, MAPKs play a central role in development and sexual/asexual reproduction (Xu et al., 1998; Hou et al., 2002; Wei et al., 2003; Lara-Rojas et al., 2011; Lichius et al., 2012). In agreement, Tvk1, the T. virens ortholog of Tmk1, is involved in conidiation and the activation of genes encoding cell wall proteins (Mendoza-Mendoza et al., 2007), and the ortholog of Tmk3 in T. harzianum plays an important role in the oxidative and osmotic stress response (Delgado-Jarana et al., 2006). Nevertheless, according to these reports there was no evidence of their involvement during wound response.

Here we describe, for the first time in a filamentous fungus, the activation of MAPKs upon wounding, as well as the phosphorylation of two of them (Tmk1 and Tmk3) in response to eATP. Interestingly, we observed that Tmk1 protein levels decreased in the tmk3 mutant background. In this regard, in Saccharomyces cerevisiae Hog1 (the ortholog of Tmk3) induces changes in RNA Pol II localization, with a shift toward stress-responsive genes (Nadal-Ribelles et al., 2012). Therefore, it is tempting to speculate that lack of tmk3 could result in a modified expression profile, including decreased expression of tmk1. Remarkably, Tmk2 appears to also be induced by injury and eATP.

During tissue regeneration and healing in Drosophila melanogaster, Grainy head, a transcription factor responsible for epidermal barrier formation and repair, is phosphorylated by ERK1 (Rämet et al., 2002; Kim and McGinnis, 2011), and the activation of ERK is required in mammalian cells for both restoration of damaged tubular epithelial cells and inhibition of fibrosis progression following injury (Jang et al., 2013). Consistently, Tmk1, the T. atroviride ortholog of mammalian ERK1/2 and plant MAP2K1, as well as Tmk3, the ortholog of mammalian MAPK p38 and plant MAPK3, were also activated by wounding (for a review see Taj et al., 2010). We further showed that eATP induces Nox1-dependent ROS production, and that this activates exclusively the Tmk3 pathway. The Tmk1 pathway is likely activated by small GTPases, as proposed in previous reports (Schmoll, 2008). Similarly, Hernández-Oñate et al. (2012) showed in T. atroviride that wounding promotes ROS production through Nox1.

Interestingly, a recent report in the fungus Ganoderma lucidum revealed that Nox-generated ROS elevated cytosolic Ca²⁺ levels by activating a plasma membrane Ca²⁺ influx pathway, thereby regulating ganoderic acid biosynthesis and hyphal branching (Mu et al., 2013). ROS play essential roles in sexual development in Aspergillus nidulans and N. crassa (Lara-Ortíz et al., 2003; Cano-Domínguez et al., 2008), as well as cell signaling roles (Aguirre et al., 2005). In agreement with these observations, we showed that T. atroviride responds to eATP by developing asexual structures. In animals, production of ROS by Nox1/NoxR or Dual oxidases is crucial for the inflammatory response or activation of systemic defense after wounding (de Oliveira et al., 2014). In plants and animals, Ca²⁺ stimulates Nox1/NoxR and Dual oxidase activity through their EF-hand calcium-binding domain, a domain not found in fungal Nox1/NoxR. These enzymes in turn produce ROS, which provoke liberation of intracellular Ca²⁺, likely causing feed back regulation (Niethammer et al., 2009; Wu and Baldwin, 2010; Razzell et al., 2013). The precise role of Ca²⁺ in the damage response of Trichoderma remains to be proven, since calcium released from a broken cell could be detected by neighboring cells as a signal molecule, but it could also serve as a second messenger liberated from intracellular pools or transported across the plasma membrane upon detection of DAMPs. Our results suggest that eATP induces conidiation in a Ca-independent manner, as phosphorylation of Tmk1 and Tmk3 occurred also in the presence of the Ca-chelating agent, EGTA. On the other hand, increasing the extracellular concentration of Ca²⁺ was sufficient to trigger conidiation. Thus, we hypothesize that more than one signaling pathway may converge in triggering the expression of genes that are required for the wound-induced formation of conidia.

In summary, we have shown that eATP can serve as DAMP that activates ROS production by Nox1. Tmk3 is activated in a NoxR–Nox1 dependent fashion, whereas Tmk1 is independently activated. In parallel, Ca²⁺ signaling, which appears to be essential for the wound response, likely activates CAMK or the PKC pathway, to finally trigger a transcriptional response that turns on genes related to cellular stress, regeneration, and conidiation. Figure 6 shows a model of the cellular response to damage of T. atroviride, where we propose eATP as a damage molecule released from wounded hyphae that promotes Nox1-dependent ROS production and in turn activates Tmk3 phosphorylation. Activation of Tmk1 appears to be independent of ROS and extracellular Ca²⁺ but important for injury-induced conidiation. Nevertheless, we can not exclude the possible activation of Tmk1 by Ca²⁺ mobilized from intracellular pools in response to damage. In conclusion, wound signaling in T. atroviride is an evolutionarily conserved process displaying multiple similarities to processes described in other higher eukaryotes. The relative simplicity of T. atroviride makes it an excellent model for the study of wound response and regeneration in multicellular organisms.

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.