Introduction
Plants respond to invading organisms triggering fast signalling processes leading to the activation of diverse defence mechanisms. They have adapted their immune system to rely on an early molecular recognition of the potential aggressor, crucial for an efficient defence reaction (Jones and Dangl, 2006). Immune responses are controlled by pattern recognition receptors, and defence signalling starts with the perception of conserved molecules associated to the damaging organism, such as pathogen (or microbe)-associated molecular patterns (PAMPs or MAMPs). Moreover, they can also recognize self-molecules associated to damage, the so called damage-associated molecular patterns (DAMPs) (Zipfel et al., 2017).
DAMPs are damaged-self molecules released from host tissue disruption that act as endogenous danger signals in both animals and plants (Heil and Land 2014). DAMPs comprise a mixture of molecules from diverse origin such as extracellular ATP, extracellular DNA, inducible proteins and fragments of the cell wall (Heil and Land 2014). In plants, DAMPs are released from disintegrated plant cells and are sensed by the pattern recognition receptors of adjacent cells. After the recognition, plants go into an “alarm state” activating signalling cascades and triggering defence responses not only locally, at the damaged tissue, but also in distal tissues that will then be prepared to respond more efficiently to a potential upcoming aggression (Orozco-Cardenas and Ryan 1999). Local responses to DAMPs involve the generation of H2O2, MAPKs activation, increased flux of calcium, production of phenylpropanoids and hypomethylation in CpG sites (Barbero et al., 2016, Duran-Flores et al., 2017, Vega-Muñoz et al., 2018). Damage perception also involves cell-to-cell communication to prime distal parts of the plant. Consequently, plants activate a myriad of mobile signals that transmit the alarm state and activate defence responses over long distances. It has been reported that jasmonic acid (JA) signalling mediates some of the systemic responses in tomato plants after DAMPs perception (Sun et al., 2011). Generation of hydrogen peroxide, accumulation of proteinase inhibitors and other defence-related proteins are produced in distal leaves upon wounding or application of the peptidic, wound-related hormone systemin in tomato (Orozco-Cardenas and Ryan 1999, Sun et al., 2011).
Oligogalacturonides (OGs) are among the best characterized plant DAMPs. They are pectin fragments hydrolysed from the cell wall that act as danger signals, triggering a signalling cascade that activates plant immunity (Ferrari et al., 2013, Savatin et al., 2014; De Lorenzo et al.,2018). OGs are oligomers of α-1,4-galacturonic acid that are released to the extracellular cell space through the action of polygalacturonases, usually generated during pathogens or insects attack (Benedetti et al., 2015). Exogenous application of OGs induces defence responses in plants when they have a degree of polymerization between 10 and 15 and they have acquired an egg-box conformational state dependent on calcium and sodium (Benedetti et al., 2015, Cabrera et al., 2008). Short oligomers have been also shown to trigger plant defences, although to a lesser extent than long OGs (Davidsson et al., 2017).
It has been demonstrated that OGs perception stimulates antioxidant systems in plants (Camejo et al., 2012) and the biosynthesis of different antimicrobial enzymes through responses regulated by the main defence related phytohormones: JA, salicylic acid (SA) and ethylene (ET) (Bishop et al., 1984, Doares et al., 1995, Ferrari et al., 2007; Denoux et al., 2008; Gravino et al., 2015). These hormonal signalling pathways play a key regulatory function in the interaction of plants with potential aggressors as pathogens and herbivores (Pieterse et al., 2014). Therefore, the modulation of these pathways by OGs would likely have a relevant impact in these biotic interactions.
The ability of OGs to induce defence responses in plants stimulated the scientific community to study the potential of OGs for plant protection. In grape, pre-incubation of excised leaves with OGs leads to protection against the necrotrophic pathogen Botrytis cinerea (Aziz et al., 2004), and protection was also achieved in Arabidopsis by spray-application of OGs (Ferrari et al., 2007; Galletti et al., 2008). Moreover, in-vivo production of bioactive OGs oligomers in Arabidopsis boosts plant defences and induces resistance to necrotrophic and biotrophic pathogens (Benedetti et al., 2015). Some research efforts have been devoted to analyse the plant responses to OGs that mediate this locally induced enhanced resistance. In Arabidopsis, OG-induced resistance against B. cinerea does not require JA and SA signalling, nor the oxidative burst generated in plants by OG perception (Aziz et al., 2004, Ferrari et al., 2007, Galletti et al., 2008; Galletti et al. 2011; Gravino et al., 2015). Instead, it requires a functional PAD3, which encodes the last step of camalexin biosynthesis (Ferrari et al., 2007). In addition, Botrytis success in colonizing the host plant depends, in part, on the methylesterification degree of the pectin that is tightly regulated by OGs (Lionetti et al., 2017).
Despite this knowledge on OG-mediated local responses in the model plant Arabidopsis, little is known in other plant species. Even more unexplored are the responses induced by OGs at the systemic level, despite the well-established relevance of systemic defence responses in plants (Hilleary and Gilroy, 2018). The induction of systemic resistance to pathogens upon OG treatment has been so far reported only in Arabidopsis (Ferrari et al., 2007), but the molecular mechanisms behind this response are unexplored. Moreover, responses to OGs in roots have been mostly studied in relation to morphogenesis but not to defence (Hernandez Mata et al., 2010; Bellincampi et al., 1993, Savatin et al. 2011).
Tomato was one of the model plants for the pioneer studies addressing systemic wound responses (ODonell et al., 1996; Birkenmeier and Ryan, 1998; Schilmiller and Howe, 2005). In fact, the first observations of the biological activity of OGs as proteinase inhibitor-inducing factors and their ability to depolarize membranes, regulate protein phosphorylation and hormone biosynthesis were obtained in tomato (Bishop et al., 1981, Sympson et al., 1998; Thain et al., 1995; Reymond et al., 1995). However, while several reports support a regulatory role of JA, SA, ET and abscisic acid (ABA) in the tomato immune responses againstB. cinerea (El Oirdi et al., 2011, Díaz et al., 2002, Achuo et al., 2002, Asselbergh et al., 2007, Curvers et al., 2010), the role of OGs in the process remains unexplored.
In this study we examined how t plant coordinate local and systemic responses to OG perception in different organs. In addition, we addressed the biological relevance of these responses by testing their efficacy in enhancing plant resistance against B. cinerea , a common and polyphagous necrotrophic pathogen. We show that changes in hormone levels induced by OGs are fast and transient at the local level and more sustained at the systemic level, and notably, that OGs have a stronger impact in roots than in leaves, regardless of the application site. Untargeted metabolomic analysis highlights the differential response to OGs in local and systemic tissues, supporting the notion of a precise fine-tuning of plant defences in response to this class of DAMPs, and uncovers the major pathways targeted by OG signalling. Finally, we show that root or leaf treatment with OGs induces systemic resistance against B. cinerea in tomato plants. The results highlight the differences among local and systemic responses and their dependence on the site of signal perception.