**2. Plant immune signalling initiation**

such threats. Plants lack specialized immune cells and their survival relies upon a highly sophisticated innate immune system, in which each plant cell responds autonomously [2–5]. The first line of defence is a basal resistance response called pattern-triggered immunity (PTI). It is induced by recognition of exogenous microbe or pathogen-associated molecular patterns (MAMPs or PAMPs) or endogenous molecules released on pathogen perception or pathogeninduced cell damage (damage-associated molecular patterns, DAMPs) via pattern-recognition receptors (PRRs) in the plasma membrane [4]. Successful pathogens overcome PTI by secreting effectors, hydrolytic enzymes or toxins, which suppress or interfere with host defence molecules [6]. In an evolutionary arms race, plants have evolved a robust defence response network termed effector-triggered immunity (ETI) to intercept pathogen effectors through intracellular receptors, such as nucleotide-binding site/leucine-rich repeat (NLR) proteins [5,7,8]. An intricate network of signalling pathways transduces these incoming signals into a diverse array of immune responses activating reactive oxygen species (ROS) generation, MAP kinases, Ca2+ signalling, the production of phytohormones and extensive transcriptional

In the past, comprehensive research has been dedicated to understanding plant physiolog‐ ical and molecular responses to individual abiotic and biotic stresses under controlled laboratory conditions. Recent studies of plant responses to concurrent abiotic and biotic stress conditions [10–16] have demonstrated that plants perceive and respond to com‐ bined stresses in a specific and unique manner. Moreover, the underlying signalling pathways are carefully modulated [14,17,18] and coordinated to ensure that plant growth

Vascular wilt pathogens are soil-borne bacteria, fungi and oomycetes that employ various infection strategies to invade plant roots at different infection sites [20]. They subsequently advance inter- or intracellularly through the root cortex and enter the xylem vessels, where they proliferate and spread passively with xylem sap to aerial plant parts [21]. The character‐ istic wilt symptoms develop as a consequence of obstructed transportation of water and minerals, either due to the physical blockage of vessels by the pathogen or indirectly due to the activation of plant physical defence responses (e.g. formation of tyloses, accumulation of pectin-rich gels and gums) that confine the further spread of the pathogen [22]. In addition to wilting, other disease symptoms include vein clearing, leaf epinasty, chlorosis, vascular

Primarily due to the specific lifestyle of vascular wilt pathogens, relatively little is known about their interactions with host plants and root-specific defence responses on molecular and biochemical levels compared to foliar pathogens. This chapter, therefore, summarizes the currently available molecular, cellular and systems biology data gathered from studies of signalling networks in model plants and crops challenged by bacterial or fungal pathogens and applies this general knowledge to advance understanding of vascular wilt pathogenesis and implement all these findings into the design of new strategies for the protection of crops

reprogramming [9].

and forest ecosystems.

and fitness are not significantly retarded [19].

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

browning, stunting, necrosis and eventually plant death [21–24].

Several factors contribute to the complex regulatory mechanisms in the initiation of plant immune signalling: (i) as sessile organisms, plants need to respond promptly to danger signals, (ii) each plant cell reacts autonomously to different stimuli, but the response needs to be integrated at a higher organizational level to ensure the plant's survival and (iii) immune reactions are energy- and resource-demanding processes requiring the proper timing and amplitude of response [4]. Typically, immune responses occur on recognition of conserved microbe-, pathogen- or damage-associated molecular patterns or after perception of effector molecules that are species-, race-, or strain-specific and contribute to pathogen virulence [25]. However, not all microbial elicitors conform to the common distinction between PAMPs and effectors, and so Thomma et al. [26] proposed that plant immunity should be considered as a continuum, instead of a two-branched system composed of PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI). An alternative perspective of plant innate immunity as a system that evolves to detect invasion has recently been extended into a so-called invasion model [27]. In this model, invasion patterns (IPs), externally encoded or modified-self ligands that signify invasion, are perceived by plant invasion pattern receptors (IPRs) and induce IPtriggered responses that do not result in immunity by default.

### **2.1. Plant PRRs convey danger signals to the intracellular immune signalling pathways**

Recently emerging structural biology data on plant–pathogen interactions, [28] together with data obtained primarily from genetic and biochemical studies on the leaves of model plants and crops, have revealed that fine-tuning and coordination of immune responses are achieved within large protein complexes at the plasma membrane, where plant PRRs reside [4]. Plant PRRs are either receptor-like kinases (RLKs) or receptor-like proteins (RLPs) [4,6,25,29]. RLKs are modular proteins comprised of an extracellular domain involved in recognition of MAMPs/ DAMPs, a single-pass transmembrane domain and a cytosolic serine/threonine kinase domain that transmits a signal to downstream signalling components. RLPs have a similar architecture, but their short cytoplasmic tail lacks kinase activity. RLPs, therefore, probably form hetero‐ meric complexes with RLKs or other cytosolic kinases to relay downstream signalling. Extracellular domains of PRRs contain various motifs involved in recognition and binding of ligands. Leucine-rich repeat (LRR) motifs are widespread and serve as a scaffold for proteinor peptide–protein interactions [30]. Well-characterized examples of LRR–RLKs are *Arabidop‐ sis* FLS2 (flagellin-sensitive 2) and EFR (elongation factor Tu receptor), which bind flagellin fragment flg22 and EF-Tu peptide elf18, respectively [31,32]. Lysine motifs (LysMs), lectin and epidermal growth factor (EGF)-like domains are found in PRRs that recognize carbohydrate moieties, such as fungal chitin [33,34] or bacterial peptidoglycans [35].

#### *2.1.1. Perception of chitin*

The chitin-responsive PRR system has been thoroughly investigated in both dicots and monocots. The perception of chitin in monocot plants is best described in rice (*Oryza sativa*). Chitin elicitor binding protein (OsCEBiP) is a receptor-like protein that specifically binds chitin 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 by the NADPH oxidase OsRbohB [40].

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].

#### *2.1.2. Perception of flagellin*

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

#### *2.1.3. Perception of peptidoglycan*

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 system, involving LysM RLK OsCERK1 [54] and two LysM-containing proteins, OsLYP4 and OsLYP6, which not only bind PGN but also associate with chitin oligomers [55].
