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.