*2.1.4. Perception of DAMPs*

oligomers [36]. In the absence of chitin, OsCEBiP exists as a homodimer [37]. On binding of chitin octamer, OsCEBiP associates with receptor-like kinase OsCERK1 and forms heterodim‐ ers [37,38]. This interaction activates the OsCERK1 kinase domain to become phosphorylated. Subsequently, active OsCERK1 phosphorylates a guanine nucleotide exchange factor OsRac‐ GEF, which activates a Rho-type small GTPase OsRac [39]. OsRac acts as a molecular switch in many plant signalling pathways and, among other things, regulates the production of ROS

The model dicot plant *Arabidopsis thaliana* harbours chitin elicitor receptor kinase 1/LysMcontaining receptor-like kinase1 (CERK1/LYK1), which perceives chitin (a polymer of Nacetyl-D-glucosamine, NAG) through its LysM motif [33]. Although the AtCERK1 extracellular domain contains three tandem LysMs, only LysM2 binds NAG5 [41]. This interaction, however, fails to trigger immune responses. Downstream signalling has been observed only on binding of chitin octamer, which acts as a bivalent ligand and induces CERK1 dimerization [41]. Another LysM-containing cell surface receptor, AtLYK5, has recently been proposed as the primary chitin receptor, due to a significantly higher binding affinity for NAG8 compared to AtCERK1 [42]. AtLYK5 exists as a homodimer in the absence of chitin. Binding of chitin to AtLYK5 homodimer promotes the association of AtLYK5 with AtCERK1. This leads to dimerization of AtCERK1 and activation of its kinase domain. The chitin signal is then transduced downstream to mitogen-activated protein kinases MPK3 and MPK6 [33].

Extensive research of the model plant *Arabidopsis thaliana* has elucidated molecular mecha‐ nisms triggered in response to recognition of bacterial flagellin by evolutionary conserved LRR RLK flagellin-sensitive 2 (FLS2) [31]. The extracellular domain of FLS2 contains 28 LRR and binds the 22-amino acid long flagellin epitope flg22 [43]. Immediately after, FLS2 associates with co-receptor brassinosteroid insensitive 1 (BRI1)-associated receptor kinase 1 (BAK1)/ somatic embryogenesis receptor-like kinase 3 (SERK3) to form a heterodimer [44–46]. BAK1 is a key regulatory LRR RLK coordinating growth–defence trade-offs [47], since it is required for early defence responses in PTI [48] but also implicated in brassinosteroid hormone signalling [49]. BAK1 phosphorylates receptor-like cytoplasmic kinase *Botrytis*-induced kinase 1 (BIK1), which interacts and forms a complex with both BAK1 and FLS2 [50]. BIK1 is subse‐ quently auto-phosphorylated at tyrosine and serine/threonine residues [51]. Activated BIK1 contributes to flg22-triggered calcium influx from apoplast [52] and phosphorylates NADPH

Peptidoglycan (PGN), a polymer of N-acetylglucosamine and N-acetylmuramic acid branched with a short peptide, is an essential component of a bacterial cell wall and is another wellstudied MAMP. In *Arabidopsis,* the PGN perception system is comprised of two GPI-anchored LysM domain RLPs, LYM1 and LYM3, which bind PGN, and a transmembrane RLK AtCERK1, which probably relays the PGN signal downstream [35]. Rice has a similar PGN detection

oxidase RbohD involved in reactive oxygen species (ROS) production [53].

by the NADPH oxidase OsRbohB [40].

414 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

*2.1.2. Perception of flagellin*

*2.1.3. Perception of peptidoglycan*

Some PRRs respond to damage-associated molecular patterns (DAMPs), endogenous mole‐ cules such as cell wall fragments or peptides released on pathogen attack or various abiotic stresses [25,56]. Partial degradation of pectic polysaccharide homogalacturonan (HGA) by pathogen or plant polygalacturonases produces oligogalacturonides (OGs), oligomers of α-1,4–linked galacturonic acid [57,58]. In *Arabidopsis,* OGs are perceived by wall-associated kinase 1 (WAK1) Ser/Thr RLK kinase with an extracytoplasmic domain that contains several EGF-like repeats [59]. The signal is then relayed to the MAP kinase signalling pathway, where AtMPK3 and AtMPK6 become phosphorylated and induce expression of several defence genes [60]. Additional OGs-triggered defence responses include activation of NADPH oxidase AtRbohD involved in the generation of ROS, production of NO and deposition of callose in the plant cell walls [57,58].

The best studied peptides acting as DAMPs belong to the plant elicitor peptides (Peps) family. They are processed from precursor PROPEPs [61,62]. AtPep1, a 23 amino acid peptide released from the C-terminal of PROPEP1, was the first peptide elicitor isolated from *Arabidopsis* [61], but similar peptides were also later confirmed in other plants [63]. AtPep1 is recognized by two LRR RLK, PEPR1 and its paralog PEPR2 [64,65]. However, signalling is initiated only in complex with co-receptor LRR RLK BAK1 [66]. The active receptor complex consequently induces the expression of MAP kinase 3 (MPK3), WRKY transcription factors and defencerelated genes such as *PR-1* and *PDF1.2* (encoding defensin) [63]. Moreover, the cytosolic kinase domain of PEPR1 has guanylyl cyclase activity, which generates cGMP from GTP [67]. An increased local concentration of cGMP has been proposed to open cyclic nucleotide-gated channels (CNGC2) in the plasma membrane and activate cytosolic Ca2+ signalling [67,68].

#### **2.2. Intracellular immune receptors NLRs detect pathogen effectors**

In addition to PRRs, plants have evolved a second class of immune receptors that intercept effectors in different parts of the cell [8]. These intracellular receptors, so called NLRs, are characterized as multi-domain proteins that have a conserved central nucleotide-binding (NB) domain and variable C-terminal leucine-rich repeats (LRR) domain [28]. In terms of their distinct N-terminal domains, NLRs are broadly divided into two groups: TNLs that harbour a Toll–interleukin 1 receptor (TIR) domain and CNLs that contain a coiled-coil (CC) domain [69–71]. NLRs belong to signal transduction ATPases with numerous domains (STAND) that operate as molecular switches cycling between an inactive closed ADP-bound state and active open state with bound GTP [72–74]. In the resting state, N-terminal TIR or CC and C-terminal LRR domains sterically inhibit the NB domain from ADP–ATP exchange. On pathogen recognition, a series of conformational changes occur that expose the NB domain, promote ADP–ATP exchange and initiate signal transduction [8]. Effector recognition by NLRs often, but not always, leads to a form of programmed cell death termed as a hypersensitive response [75]. NLRs are, therefore, under precise control by accessory proteins. NLRs interact with conserved Hsp90-Sgt1-RAR1 protein complexes for proper folding, accumulation and regulation [76,77]. Moreover, Sgt1 interacts with the suppressor of *rps4-RLD* (SRFR1) nega‐ tively to regulate NLRs accumulation and prevent autoimmune activation [78].

Various strategies of effector recognition by NLRs exist and are represented in different models. In the gene-for-gene model, originally described by Flor in 1971, NLRs can recognize effectors directly (e.g. *Pita-AvrPita* [79], *Pto-AvrPto* [80] and many others) acting like receptor and ligand pairs that initiate a cascade of reactions leading to resistance [3]. When considering pathogen effector diversity, it is more likely that a single NLR recognizes multiple effectors from diverse pathogens in the presence of other host proteins. This hypothesis is explained by the guard model, in which the NLR protein is assigned the role of a sentinel that is activated indirectly by detecting an effector-modified host protein and induces a defence response [81,82]. An example of such mechanism is given by *Arabidopsis* CNL receptors, RPM1 and RPS2, which constantly monitor host protein RIN4 (a negative regulator of basal resistance) for interference with *Pseudomonas syringae* effectors AvrB, AvrRpm1 and AvrRpt2 [83]. Another indirect effector recognition strategy is proposed in the decoy model [84]. As guarded effector targets are evolutionarily unstable, it is likely that the targeted host gene has duplicated and evolved into decoy proteins. These serve as bait to trap effectors that target structurally related proteins involved in basal defence. For example, in the inactive state, *Arabidopsis* CNL RPS5 interacts with its N-terminal CC domain with protein kinase PBS1, which has no function in basal resistance [85]. Cleavage of PBS1 by *Ps. syringae* effector AvrPphB activates RPS5 [86]. However, several PBS1-like kinases (including Botrytis-induced kinase 1 (BIK1)) are also cleaved by AvrPphB [87]. PBS1, therefore, acts as a decoy that prevents cleavage of BIK1, which is an important component of PRR signalling [53] and the key AvrPphB target. An integrated decoy model has recently been proposed [5]. In this model, the effector-targeted plant protein is duplicated and fused to one member of the NLR pair to act as bait that, on effector binding, triggers defence signalling by the second NLR.

Activated NLRs trigger a variety of immune responses, from the generation of ROS, elevation of intracellular Ca2+, activation of MAPK cascades, transcriptional reprogramming to produc‐ tion of phytohormones [8]. Although effector-triggered responses are qualitatively similar to immune responses elicited by MAMPs/DAMPs, there are quantitative differences in the strength and duration of pathways, which result in different resistance responses and signal‐ ling networks [17].

#### **2.3. Signal transduction cascades**

Perception of MAMPs/DAMPs by their cognate receptors triggers an array of immune responses, comprising changes in intracellular calcium levels [Ca2+]i , membrane potential depolarization, extracellular alkalinization, production of ROS, NO and phosphatidic acid, activation of kinases (mitogen-activated protein kinases (MAPKs) or Ca2+-dependent protein kinases (CDPKs)), transcriptional reprogramming and changes in plant hormone concentra‐ tions (e.g. ethylene, salicylic and jasmonic acid) [88,89].

#### *2.3.1. Calcium and ROS signalling interconnection*

Ca2+ is a ubiquitous second messenger released in response to various stresses and develop‐ mental processes. In *Arabidopsis,* various MAMPs/DAMPs induce distinct and sustained elevations of intracellular calcium concentration ([Ca2+]i ), which differ in the lag phase and amplitude of response [89,90]. Moreover, changes in [Ca2+]i are organ-specific and correlate with the expression patterns of the corresponding MAMP/DAMP receptors. Chitin octamer and Pep1 induce similar responses in seedling shoots and roots, while roots are insensitive to elf18 and show only a minor response to flg22 [90]. Furthermore, Ca2+ fluxes are generated from different sources; flg22/FLS2 signalling involves the release of Ca2+ from intracellular stores (e.g. endoplasmic reticulum and/or tonoplast) and inositol phosphate signalling, whereas Pep/PEPR signalling requires an influx of Ca2+ from the apoplast [68]. The identity of plant Ca2+ channels and pumps involved in the generation of Ca2+ signals is largely unknown, although some candidates (e.g. ionotropic glutamate receptor (iGluR)-like channels, cyclic nucleotide gated channels (CNGCs) and annexins in plasma membrane and two-pore-channel 1 (TPC1) in the tonoplast membrane) have been investigated [88,91]. Elevated [Ca2+]i is detected by Ca2+sensor proteins such as calmodulins (CaMs), calcium-dependent protein kinases (CDPKs), calcineurin B-like (CBL) proteins and CBL-interacting protein kinases (CIPKs) [88,91]. CaMs are highly conserved eukaryotic proteins that bind free Ca2+ with four EF-hand motifs and regulate the function of their interacting proteins, such as CaM-binding transcrip‐ tion factors [88]. CDPKs are unique proteins acting as sensors and decoders of Ca2+ signals and are suited for rapid responses to stimuli. Binding of Ca2+ via four EF-hand domain motifs in the C-terminus activates the CDPKs' N-terminal kinase domain and promotes transmission of a Ca2+ signal by phosphorylating different target proteins [91]. The functional specificity of CDPKs is achieved by targeting distinct membrane subdomains and involves specific lipid modifications (e.g. N-terminal myristoylation, S-acylation) [91]. In contrast to CDPKs, CBLs are Ca2+ sensors without enzymatic activity. They bind Ca2+ with four EF hands and then associate with CIPKs through the NAF motif in the kinase C-terminal regulatory domain. This interaction liberates kinase from auto-inhibition and enables conversion of the Ca2+ signal into phosphorylation events [91]. Like CDPKs, CBLs have different lipid modifications (e.g. Nterminal myristoylation, S-acylation) that determine their localization and, consequently, the site of action of CBL–CIPK complexes [91].

regulation [76,77]. Moreover, Sgt1 interacts with the suppressor of *rps4-RLD* (SRFR1) nega‐

Various strategies of effector recognition by NLRs exist and are represented in different models. In the gene-for-gene model, originally described by Flor in 1971, NLRs can recognize effectors directly (e.g. *Pita-AvrPita* [79], *Pto-AvrPto* [80] and many others) acting like receptor and ligand pairs that initiate a cascade of reactions leading to resistance [3]. When considering pathogen effector diversity, it is more likely that a single NLR recognizes multiple effectors from diverse pathogens in the presence of other host proteins. This hypothesis is explained by the guard model, in which the NLR protein is assigned the role of a sentinel that is activated indirectly by detecting an effector-modified host protein and induces a defence response [81,82]. An example of such mechanism is given by *Arabidopsis* CNL receptors, RPM1 and RPS2, which constantly monitor host protein RIN4 (a negative regulator of basal resistance) for interference with *Pseudomonas syringae* effectors AvrB, AvrRpm1 and AvrRpt2 [83]. Another indirect effector recognition strategy is proposed in the decoy model [84]. As guarded effector targets are evolutionarily unstable, it is likely that the targeted host gene has duplicated and evolved into decoy proteins. These serve as bait to trap effectors that target structurally related proteins involved in basal defence. For example, in the inactive state, *Arabidopsis* CNL RPS5 interacts with its N-terminal CC domain with protein kinase PBS1, which has no function in basal resistance [85]. Cleavage of PBS1 by *Ps. syringae* effector AvrPphB activates RPS5 [86]. However, several PBS1-like kinases (including Botrytis-induced kinase 1 (BIK1)) are also cleaved by AvrPphB [87]. PBS1, therefore, acts as a decoy that prevents cleavage of BIK1, which is an important component of PRR signalling [53] and the key AvrPphB target. An integrated decoy model has recently been proposed [5]. In this model, the effector-targeted plant protein is duplicated and fused to one member of the NLR pair to act as bait that, on effector binding,

Activated NLRs trigger a variety of immune responses, from the generation of ROS, elevation of intracellular Ca2+, activation of MAPK cascades, transcriptional reprogramming to produc‐ tion of phytohormones [8]. Although effector-triggered responses are qualitatively similar to immune responses elicited by MAMPs/DAMPs, there are quantitative differences in the strength and duration of pathways, which result in different resistance responses and signal‐

Perception of MAMPs/DAMPs by their cognate receptors triggers an array of immune

depolarization, extracellular alkalinization, production of ROS, NO and phosphatidic acid, activation of kinases (mitogen-activated protein kinases (MAPKs) or Ca2+-dependent protein kinases (CDPKs)), transcriptional reprogramming and changes in plant hormone concentra‐

Ca2+ is a ubiquitous second messenger released in response to various stresses and develop‐ mental processes. In *Arabidopsis,* various MAMPs/DAMPs induce distinct and sustained

, membrane potential

responses, comprising changes in intracellular calcium levels [Ca2+]i

tions (e.g. ethylene, salicylic and jasmonic acid) [88,89].

*2.3.1. Calcium and ROS signalling interconnection*

tively to regulate NLRs accumulation and prevent autoimmune activation [78].

416 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

triggers defence signalling by the second NLR.

ling networks [17].

**2.3. Signal transduction cascades**

Rapid production of reactive oxygen species (ROS) in response to MAMPs/DAMPs depends primarily on respiratory burst oxidase homologues (RBOHs) [92,93]. These NADPH oxidases are integral membrane proteins that generate superoxide anions (O2 − ), which are rapidly converted into hydrogen peroxide (H2O2). RBOHs have cytosolic FAD- and NADPH-binding domains in the C-terminal region, six membrane-spanning domains and a cytosolic N-terminal extension that harbours two EF-hand motifs and multiple phosphorylation sites [94]. Recent studies in *Arabidopsis* have revealed different regulation mechanisms of RBOHD and RBOHFdependent ROS production. RBOHF regulation involves direct binding of Ca2+ to its EF-hands and Ca2+-dependent phosphorylation by CBL1/9–CIPK26 complexes [95]. Direct binding of Ca2+to EF-hand motifs on MAMP-induced elevation of [Ca2+]i causes conformational changes and activation of RBOHD [96]. Additionally, RBOHD is activated by protein phosphorylation at multiple sites via calcium-dependent protein kinase 5 (CPK5)[97,98] and MAMP-receptorassociated Botrytis-induced kinase 1 (BIK1) [53]. In addition to local defences, Ca2+ and RBOHD-dependent ROS production is implicated in the systemic signal propagation required for long-distance signalling [98–100]. In accordance with the current model [98], the perception of MAMPs triggers a rapid rise of [Ca2+]i, causing the activation of CPK5 and subsequent phosphorylation of RBOHD and other CPK5 substrates. Apoplastic H2O2, generated after dismutation of the O2 − produced by the RBOHD, probably represents the cell-permeable signal, which serves as the stimulus for further reiterations of calcium-dependent CPK5 activation and RBOHD phosphorylation, resulting in rapid propagation of the MAMP signal throughout the plant.

### *2.3.2. MAPK signalling*

Plant mitogen-activated protein kinase (MAPK) cascades generally comprise MAPKK kinases (or MEKKs), which receive signals from receptors/sensors and phosphorylate downstream MAPK kinases (or MKKs) and which subsequently activate MAPKs (or MPKs) that control the activities and synthesis of a plethora of transcription factors (TFs), enzymes, hormones, peptides and antimicrobial chemicals [101,102]. In *Arabidopsis*, two kinase cascades, MKK4/ MKK5–MPK3/MPK6 [103] and MEKK1–MKK1/MKK2–-MPK4, [104] are activated after perception of MAMPs/DAMPs. The activation of MEKK1–MKK1/MKK2–MPK4 negatively regulates ROS and salicylic acid (SA) production [105,106], as well as repressing cell death and immune responses [107]. MKK4/MKK5–MPK3/MPK6 cascade positively regulates the expression of several defence–related genes [60,103] and promotes accumulation of camalexin via transcription factor WRKY33 [108,109]. Moreover, activation of MPK3/MPK6 is required for full priming of stress responses [110] and increases ethylene production via ACC synthases ACS2/ACS6 [111]. Given the essential nature of the MKK4/MKK5–MPK3/MPK6 cascade, its activation has to be precisely controlled, since inappropriate activation (e.g. constitutively activated MKK4/MKK5 [112] or over-expression of MPK3 [113]) may promote hypersensitive response (HR)-like cell death or be lethal to plants. MPK3 has also recently been indicated to be a negative regulator of defence gene expression, flg22-triggered SA accumulation and disease resistance to *Pseudomonas syringae*[114]. Another negative regulator of MAPK activities is MAPK phosphatase 2 (MKP2), which interacts with and dephosphorylates MPK3 and MPK6 [115]. Additionally, a Raf-like MAPKK kinase (EDR1) has been proposed to negatively regulate the MKK4/MKK5–MPK3/MPK6 cascade by physically interacting with MKK4 and MKK5 via its N-terminal domain [113].

#### **2.4. Transcriptional reprogramming converges with complex phytohormone signalling networks**

Transcription factors (TFs) involved in plant immunity reside in transcriptional complexes and, together with co-regulatory proteins, directly or indirectly recruit RNA polymerase II to the target promoters or release it from them [116]. TFs vital for plant immunity comprise members of the AP2/ERF, bHLH, bZIP, MYB, NAC and WRKY TF families and perform diverse roles [9]. For instance, certain members of apetala2/ethylene-response element binding factor (AP2/ERF) participate in the regulation of genes related to the jasmonic acid (JA) and ethylene hormone signalling pathways [117]. *At*MYC2/JAI1/JIN1 and closely related proteins *At*MYC3 and *At*MYC4 belong to basic-helix-loop-helix (bHLH) TFs and coordinate JAmediated defence responses with other phytohormones (salicylic acid (SA), abscisic acid (ABA), gibberellins (GA) and auxin) [118]. TGA/basic domain leucine zipper (bZIP) family members are central players in SA-mediated resistance to biotrophic pathogens. Moreover, *At*TGA2, 5 and 6 TF have central roles in establishing systemic acquired resistance (SAR), regulate host detoxification pathways and are essential activators of certain ethylene-induced defence responses [119].

RBOHD-dependent ROS production is implicated in the systemic signal propagation required for long-distance signalling [98–100]. In accordance with the current model [98], the perception of MAMPs triggers a rapid rise of [Ca2+]i, causing the activation of CPK5 and subsequent phosphorylation of RBOHD and other CPK5 substrates. Apoplastic H2O2, generated after

which serves as the stimulus for further reiterations of calcium-dependent CPK5 activation and RBOHD phosphorylation, resulting in rapid propagation of the MAMP signal throughout

Plant mitogen-activated protein kinase (MAPK) cascades generally comprise MAPKK kinases (or MEKKs), which receive signals from receptors/sensors and phosphorylate downstream MAPK kinases (or MKKs) and which subsequently activate MAPKs (or MPKs) that control the activities and synthesis of a plethora of transcription factors (TFs), enzymes, hormones, peptides and antimicrobial chemicals [101,102]. In *Arabidopsis*, two kinase cascades, MKK4/ MKK5–MPK3/MPK6 [103] and MEKK1–MKK1/MKK2–-MPK4, [104] are activated after perception of MAMPs/DAMPs. The activation of MEKK1–MKK1/MKK2–MPK4 negatively regulates ROS and salicylic acid (SA) production [105,106], as well as repressing cell death and immune responses [107]. MKK4/MKK5–MPK3/MPK6 cascade positively regulates the expression of several defence–related genes [60,103] and promotes accumulation of camalexin via transcription factor WRKY33 [108,109]. Moreover, activation of MPK3/MPK6 is required for full priming of stress responses [110] and increases ethylene production via ACC synthases ACS2/ACS6 [111]. Given the essential nature of the MKK4/MKK5–MPK3/MPK6 cascade, its activation has to be precisely controlled, since inappropriate activation (e.g. constitutively activated MKK4/MKK5 [112] or over-expression of MPK3 [113]) may promote hypersensitive response (HR)-like cell death or be lethal to plants. MPK3 has also recently been indicated to be a negative regulator of defence gene expression, flg22-triggered SA accumulation and disease resistance to *Pseudomonas syringae*[114]. Another negative regulator of MAPK activities is MAPK phosphatase 2 (MKP2), which interacts with and dephosphorylates MPK3 and MPK6 [115]. Additionally, a Raf-like MAPKK kinase (EDR1) has been proposed to negatively regulate the MKK4/MKK5–MPK3/MPK6 cascade by physically interacting with MKK4 and MKK5 via

**2.4. Transcriptional reprogramming converges with complex phytohormone signalling**

Transcription factors (TFs) involved in plant immunity reside in transcriptional complexes and, together with co-regulatory proteins, directly or indirectly recruit RNA polymerase II to the target promoters or release it from them [116]. TFs vital for plant immunity comprise members of the AP2/ERF, bHLH, bZIP, MYB, NAC and WRKY TF families and perform diverse roles [9]. For instance, certain members of apetala2/ethylene-response element binding factor (AP2/ERF) participate in the regulation of genes related to the jasmonic acid (JA) and

produced by the RBOHD, probably represents the cell-permeable signal,

dismutation of the O2

*2.3.2. MAPK signalling*

its N-terminal domain [113].

**networks**

the plant.

−

418 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

TF expression and activities are regulated in multiple ways. Certain nucleotide-binding site/ leucine-rich repeat (NLR) proteins directly regulate transcription by physically interacting with TFs [120–123]. Several TFs are controlled by phosphorylation as downstream targets of activated MAPK cascades [109,124–127]. Another mechanism of TFs activation is carried out by Ca2+sensors such as CaMs and CDPKs [128–133]. Additional factors (e.g. components of mediator complex [134,135], chromatin modifications [136–138]) and levels of regulation (e.g. ubiquitination [139], sumoylation [140], alternative mRNA splicing [141]) also contribute to the complexity of transcriptional networks and fine-tuning of immune responses.

Coordination of diverse stress responses and growth is resolved within complex phytohor‐ mone signalling networks, in which salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) perform central roles, while other hormones merely modulate their responses [142]. SA is synthesized in chloroplasts from chorismate by isochorismate synthase [143] and exported to the cytosol [144]. In response to pathogens and various abiotic stresses, SA mediates expression of pathogenesis-related (PR) genes and the synthesis of antimicrobial compounds to provide basal defence and systemic acquired resistance (SAR) [145]. SA activates these defence responses through transcription cofactor nonexpresser of PR genes 1 (NPR1) [146] and transcription factors TGA2, TGA5 and TGA6 [119]. In the absence of SA, NPR1 is sequestered in the cytosol and forms oligomeric complexes stabilized by intermolecular disulphide bonds [147]. In response to activation of SA pathway, thioredoxins reduce these disulphide bonds, causing the release of NPR1 in monomeric form, which can translocate to the nucleus via a nuclear translocation signal (NLS) [148]. In addition, NPR1 protein levels oscillate through CUL3NPR3- and CUL3NPR4-mediated degradation in the nucleus, which is required for finetuning of immune responses [147,149,150].

Jasmonates (JAs) are plant hormones with essential roles in plant defence and development [118]. JAs are derived from *α*-linolenic acid liberated from membrane phospholipids by the action of phospholipase A and enzymatically converted in a series of steps in chloroplasts and peroxisomes, to be finally transformed into bioactive molecule JA-isoleucine (JA-Ile) in the cytosol [151]. JA-signalling is activated after repressor removal [152,153]. In unstimulated cells, jasmonate ZIM domain (JAZ) proteins repress transcription of JA signalling components, such as the basic-helix–loop-helix (bHLH) master transcription factor MYC2 and its close homo‐ logues MYC3 and MYC4 [154]. On JA signal perception by coronatine insensitive 1 (COI1), a component of the Skp1-Cul-F-box protein (SCF) E3 ligase complex, JAZ repressor proteins are targeted for proteasome-mediated degradation and MYC2 activates the transcription of several JA-responsive genes [154,155].

Ethylene (ET) is a gaseous hormone that often works synergistically with JA [156]. Important steps in ET biosynthesis are the conversion of *S-*AdoMet to 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase and oxidation of ACC by ACC oxidase to form ET [157]. In *Arabidopsis*, MPK3 and MPK6 phosphorylate ACS2 and ACS6 proteins to prevent rapid degradation of ACS2/ACS6 by the 26S proteasome pathway and enhance ET production in response to MAMP perception [111]. MPK3 and MPK6 also phosphorylate TF WRKY33, which subsequently binds to ACS promoters and regulates ET production [158]. Generated ET binds to its receptors, such as ethylene response 1 (ETR1) in the ER membrane. When ET is absent, active receptors ETR1 negatively regulate ethylene insensitive2 (EIN2) through phosphoryla‐ tion via Raf-like protein kinase constitutive triple response1 (CTR1) [159]. At the same time, TFs ethylene insensitive3 (EIN3) and EIN3-like1 (EIL1) are recruited by two F-box proteins, EBF1 and EBF2, to 26S proteasomal degradation. On ET signal perception, the ETR1 receptors are inactivated and CTR1 repressed. Subsequently, the C-terminal part of EIN2 is cleaved and translocated to the nucleus [160]. This induces degradation of EBF1 and EBF2 and stabilizes EIN3 and EIL1, which regulate expression of ET-responsive genes (e.g. TF ERF1 and ORA59) [161,162].

Plant hormonal crosstalk is extensive and occurs in several combinations [163]. The molecular mechanism underlying SA-mediated reprogramming of the JA transcriptional network points to immune signalling antagonism and the involvement of transcriptional regulators NPR1, TGA, WRKY and ORA59 as signal integrators [164]. Phytohormones JA and ET synergistically regulate plant defence responses to necrotrophic fungi via JA-induced EIN3 and EIL1 activa‐ tion and ET-induced EIN3 and EIL1 stabilization. In addition, antagonistic effects observed in JA and ET signalling are mediated by the interaction of JA-activated MYC2 TF and ETstabilized TF EIN3 [165].

#### **2.5. Inactivation of immune signalling pathways**

Various negative regulation mechanisms ensure immune signalling activation is switched off when there is no danger. In the absence of ligand, several phosphatases interact with PRRs and their associated kinases to keep immune complexes inactive through dephosphorylation. For instance, Ser/Thr phosphatase type 2A (PP2A) constitutively associates with BRI1 associated kinase1 (BAK1) and negatively controls BAK1 activation in PRR immune receptor complexes [166]. Negative regulation exerted by downstream phosphatases is illustrated by *Arabidopsis* MAPK phosphatase 1 (MKP1) operating as a negative regulator of MPK6-mediated MAMP responses [167] and also observed with MAPK phosphatase 2 (MKP2), which acts as the key regulator of MPK3 and MPK6 networks controlling both abiotic and specific pathogen responses in plants [115].

Ubiquitination and proteasomal degradation are other mechanisms by which plant immune responses are attenuated. For example, two U-box E3 ubiquitin ligases, PUB12 and PUB13, are recruited to flagellin-induced FLS2/BAK1 receptor complex and phosphorylated by BAK1 to polyubiquitinate FLS2 and promote its degradation [168]. Downregulation of immune signalling can also be achieved by ligand-induced endocytosis and degradation [169]. This has been reported in localization studies of flg22-induced FLS2 receptors in *Arabidopsis* [170] and was recently proposed as a mechanism for desensitizing host cells to flg22 stimulus and in turning over ligand-bound FLS2 [171].
