**3.1. General perception of MAMPs in roots**

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)

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 ET-

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

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

[161,162].

stabilized TF EIN3 [165].

responses in plants [115].

**2.5. Inactivation of immune signalling pathways**

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

Despite the fact that roots are subjected to a rich microbial community, the perception of MAMPs and immune responses in roots are poorly understood. Millet et al. [172] studied immune responses in *Arabidopsis* roots after exposure to flg22, PGN and chitin. Flg22 and PGN initiated signalling only in association with LRR RLK BAK1. Furthermore, bacterial flg22 and PGN triggered a localized response in the elongation zone of the root tip, while chitin induced a response only in the mature zones of roots. It is thus likely plants have evolved tissue-specific MAMP-triggered immune responses, depending on the nature of the attacker [172,173]. While fungi and nematodes can directly penetrate the epidermal layer of roots, bacteria cannot and therefore exploit the weakest part of the roots as infection site. This hypothesis has been confirmed by recent FLS2 expression studies [174], which indicated that basal FLS2 promotor activity is restricted to the vascular cylinder and outgrowing lateral roots. Moreover, the FLS2 receptor system in roots is functional, since flg22 treatment induced rapid calcium influx and caused phosphorylation of MAPK [174]. Whole transcriptome expression analysis of flg22 elicited roots also revealed a set of genes specifically upregulated in roots, with functions in hormone and stress signalling, root and lateral root development, signalling and defence [174].

#### **3.2. Perception of vascular wilt pathogens**

At early stages of infection, vascular wilt pathogens are faced with preformed physical and chemical root defences and MAMP-induced immune responses that hinder their invasion [20]. Once they breach the rigid secondary xylem walls and enter the xylem vessels, vascular wilt pathogens are presumably recognized by specific extracellular receptors in the parenchyma cells surrounding the xylem vessels [21].

#### *3.2.1. Perception of Verticillium spp.*

In tomato, extracellular LRR RLP Ve1 [24,175,176] plays a role in xylem defence and provides resistance against race 1 strains of *V. dahliae* and *V. albo-atrum* [177,178]. In recent years, several other homologue genes have been reported in *Gossypium*, *Solanum* and *Mentha*. A functional *Ve1* orthologue has also been discovered in *Nicotiana glutinosa* [179]. Ve1 recognizes a small effector protein, Ave1, with a similarity to plant natriuretic peptides involved in regulation of water and ion homeostasis [180]. Phylogenetic analysis has indicated hundreds of Ave1 homologues in plants but only a few in fungi, suggesting *Verticillium* spp. acquired Ave1 through horizontal gene transfer [180].

Ve1 forms heterodimers with a tomato orthologue of the *Arabidopsis* RLK suppressor of BIR1-1/ evershed (SOBIR1/EVR) in the absence of Ave1 [181]. However, Ve1-mediated signalling also requires other critical signalling components, such as SERK1 and SERK3/BAK1, to establish *Verticillium* resistance in tomato and *Arabidopsis* [177,178,182]. Additionally, Ve1-mediated signalling depends on ER-QC-assisted folding mediated by ER-resident chaperones HSP70 binding proteins (BiPs) and lectin-type calreticulins (CRTs) [183].
