Flagellin is the main protein component of bacterial flagella, that acts as a MAMP in both plants and animals. Plants have a notably sensitive perception system for the highly conserved domain in the N-terminus of eubacterial flagellin [13]. The perception thereof, and particularly the N-terminus flagellin peptides, flg22 and flg15 from Pseudomonas syringae pv. tabaci, lead to the alkalinisation of the culture medium in suspension-cultured cells of Lycopersicon peruvianum (a wild relative of tomato), A. thaliana, potato and tobacco respectively [13]. Flagellin from the rice-incompatible strain, N1141 of P. avenae, was also shown to induce the hypersensitive cell death and accumulation of EL2 mRNA (elicitor-responsive gene) in cultured rice cells [116]. As mentioned, FLS2 is a LRR-RLK involved in the perception of the bacterial flagellin and immune responses in A. thaliana [76,96]. Furthermore, the recognition of bacterial flg22 by Arabidopsis FLS2 induces a FLS2-BAK1 complex formation and triggers defence signalling [96,98]. By using chemical cross-linking and immunoprecipitation techniques, Chinchilla et al. [117] showed that the specificity of flagellin perception and immune responses is mediated by the binding of FLS2 to flg22 in A. thaliana. The fls2 mutants carrying T-DNA insertion in the flagellin receptor gene FLS2 were more susceptible to Pst DC3000 compared to the wildtype (WT) [118]. Upon flg22 treatment, FLS2 mutants (fls2-101), generated by the insertion of T-DNA at the third FLS2 exon, showed impaired binding of the flg22 and reduced seedling growth inhibition [81].

 

As stated, flg22 induces a FLS2-BAK1 complex in a ligand-dependent manner, and leads to defence responses in A. thaliana [96]. This study showed that although BAK1 mutants (bak1-3 and bak1-4 with T-DNA inserted in 5^th intron and 9^th exon, respectively) were defective in an oxidative burst generation, they did not affect the binding of flg22 to FLS2. Furthermore, flg22 induces recruitment of the U-box E3 ubiquitin ligases (PUB12 and PUB13), previously mentioned, to the FLS2-BAK1 complex and this causes FLS2 degradation and the concomitant attenuation of immune signalling in Arabidopsis [107]. Here, there was a decrease in FLS2 ubiquitination and Pst DC3000 infection in mutant plants (pub12-2, pub13 and pub12/13 double knockout), compared to WT controls. A recent study has furthermore shown that flagellin-sensing 3 (FLS3) directly and specifically binds flgII-28 (a second flagellin epitope distinct from flg22), which enhances immune responses in tomato [85]. Finally, it is also important to note that BAK1 is involved in the defence signalling activities of both FLS2 and FLS3 [85,96].

Elongation factor-thermo unstable (EF-Tu), a prokaryotic elongation factor involved in the synthesis of proteins, is a bacterial MAMP that is recognised by the LRR-RLK, EFR, in Arabidopsis [14]. N. benthamiana normally lacks EF-Tu responsiveness, but achieved the ability to recognise the MAMP when EFR was transiently expressed in the plant [14]. Kunze et al. [119] showed that EFR binds directly to the acetylated N-terminus epitope of elf18 (the first amino acids of EF-Tu) and elicits innate immunity in Arabidopsis and other Brassicaceae species. The first acetylated 12 N-terminal amino acids (elf12) were, however, not able to elicit an immune response, but rather acted as a specific antagonist of elf18 [119]. Interestingly, even though EF-Tu induces MTI in rice, the elf18 peptide failed to trigger an immune response. However, another epitope, EFa50 (50-amino acid from central region of EF-Tu comprising Lys176 to Gly222) induced MTI responses, although the related PRR is still unknown [120]. Rice leaves treated with EFa50 induced early defence responses such as increased H₂O₂ and callose deposition, and triggered resistance to coinfection with pathogenic bacteria. In this regard, Arabidopsis EFR T-DNA insertion mutants (efr-1 and efr-2), with both T-DNAs inserted in 1^st exon, were insensitive to EF-Tu and susceptible to the bacterium Agrobacterium tumefaciens [14] (Table 1). In fact, efr mutants did not show typical growth inhibition as for the WT, increased oxidative burst and ethylene biosynthesis or induced resistance to Pst DC3000 upon EF-Tu treatment. Jeworutzki et al. [121] reported involvement of EFR and FLS2 in the Ca²⁺-associated opening of plasma membrane anion channels during early bacterial flagellin and EF-Tu defence signalling in Arabidopsis mesophyll cells. Using electrophysiological approaches, they showed that the efr-1 was defective in the membrane potential depolarisation, which is indispensable for cytosolic calcium influx in response to elf18 treatment.

LPS is an amphipathic molecule found on the outer membrane of Gram-negative bacteria and protects the organism against antimicrobial compounds found in the environment [23,122]. This MAMP is involved in the bacterial adhesion and induction of defence-related responses in both mammals and plants [123]. In the former, LPS recognition is orchestrated by lipopolysaccharide binding protein (LBP), before recruitment into a complex comprising soluble myeloid differentiation protein 2 (MD-2), membrane attached cluster of differentiation 14 (CD14), and the transmembrane Toll-like receptor 4 (TLR4), thereby leading to mammalian defence activation against LPS [6,124]. LBP and bactericidal/permeability-increasing protein (BPI) thus play a vital role in regulation of immune responses against LPS in mammals [124,125]. As such, the mechanism by which LPS is recognised has been well-studied in animals, however, in plants, the mechanism of perception and recognised moiety/epitope(s) of LPS is still debateable.

 

In this regard, LPS, as well as its lipid A moiety from Burkholderia cepacia, Xanthomonas campestris and P. syringae, trigger the upregulation of genes involved in immunity and defence [126,127,128], with lipid A speculated to be the major elicitor in A. thaliana [129]. LPS has furthermore been shown to trigger defence responses, such as the generation of nitrogen oxide (NO), ROS, elevation of cytoplasmic Ca²⁺ concentration, stomatal closure and the expression of pathogenesis-related (PR) genes in Arabidopsis, tobacco and rice suspension cells [92,130,131,132,133]. Ranf et al. [92] earlier reported that LPS from Xanthomonas and Pseudomonas are sensed by the bulb-type (B-type) lectin S-domain (SD)-1 RLK lipooligosaccharide-specific reduced elicitation (LORE) and that LORE mutants showed defects in the LPS-induced elevation of cytosolic calcium, ROS and defence gene (AtFRK1) expression. Additionally, Shang-Guan et al. [129] reported the existence of two ROS production phases, characterised by a weak initial and second stronger ROS generation in A. thaliana. T-DNA insertion LORE mutants, however, showed little or no difference in second phase ROS production compared with WT, suggesting another LPS receptor(s). In contradiction to the original LORE study, a recent report implicated bacterial 3-hydroxy fatty acids in LORE-dependent induction of immune response in Arabidopsis [93]. Here, LORE could not sense LPS that was repurified to remove free 3-hydroxy fatty acids, indicating that LORE is not the receptor for LPS.

 

Using B. cepacia LPS-affinity capture strategies, Vilakazi et al. [134] and Baloyi et al. [135] identified BAK1 and other defence response proteins associated with the plasma membrane fraction in LPS-treated A. thaliana. Additionally, Arabidopsis LBP/BPI related-1 (AtLBR-1) and LBP/BPI related-2 (AtLBR-2) were shown to bind to both rough and smooth LPS, and regulate the expression of the pathogenesis-related 1 (PR1) gene [128]. AtLBR T-DNA single mutants (Atlbr-1, Atlbr-2-1, Atlbr2-2) and (Atlbr-DKO) double-knockouts, generated by crossing Atlbr-1 and Atlbr-2-1 plants, were defective in ROS generation and in upregulation of LPS-induced PR1 expression. A transcriptome analysis revealed that AtLBR-2 plays an indispensable role in the upregulation of 65 genes associated with defence responses upon Pseudomonas LPS treatment [136]. Furthermore, there was a defect in the upregulation of defence-related genes and salicylic acid (SA)-mediated signalling in the Atlbr-2-1 mutants, compared to the WT after Pseudomonas LPS treatment.

 

Lastly, a recent study has reported OsCERK1, the chitin co-receptor, as an LPS receptor/co-receptor in rice, but not in A. thaliana [137]. This indicates a significant difference between LPS perception in rice and Arabidopsis.

Rice chitin elicitor-binding protein (CEBiP) homologs in Arabidopsis, LYM1 and LYM3 RLPs, bind in a ligand-specific manner to PGNs, heteropolymers that are part of the building blocks of the cell walls of Gram-negative and Gram-positive bacteria [87]. There was a significant expression of the immune marker gene, flagellin-induced receptor kinase FRK1, upon treatment of Arabidopsis with PGNs from Gram-negative Pst DC3000 [87]. Homozygous T-DNA insertional mutants of LYM1 and LYM3 showed a strongly reduced PGN-inducible marker gene expression and were more susceptible to Pst DC3000 bacterial infection than the WT (Table 1). The same authors further reported the involvement of AtCERK1 in the LYM1/LYM3 perception of PGN and subsequent immune response in A. thaliana. Thus, AtCERK1 is the additional protein that provides the cytoplasmic kinase domain that is lacking in the LYM1/LYM3 RLPs needed for the downstream transphosphorylation of PGN signalling. The study suggests that Arabidopsis senses PGNs in a LYM1/LYM3 and AtCERK1-dependent manner, similar to the chitin perception system in rice that uses OsCEBiP and OsCERK1 [79]. Furthermore, the direct interaction of PGN with AtLYM1 and AtLYM3 has been reported, but not with AtCERK1 [87]. In rice, OsCERK1 associates with LysM motif-containing proteins (LYP4 and LYP6) in PGN-induced defence responses [80,91,138]. Importantly, the putative ability of CERK1 to participate in the recognition and signalling of more than one MAMP supports the hypothesis of the capability of one PRR/co-receptor to recognise more than one MAMP, which favours the generation of transgenic crop plants with enhanced/altered recognition capabilities [8].

Chitin is a β(1→4)-linked polymer of N-acetylglucosamine, a major structural component in the exoskeleton of arthropods and cell walls of fungi [21]. Chitin and its fragments (chitooligosaccharides) are MAMPs recognised by plants PRRs, which elicit defence responses such as the oxidative burst, protein phosphorylation, transcriptional activation of defence-related genes and phytoalexin biosynthesis [21,78,139]. Kaku et al. [78] isolated and characterised a chitin elicitor binding protein (OsCEBiP), an RLP involved in the perception of chitin oligosaccharides in cultured rice cells. It was observed that OsCEBiP has two LysM motifs and a C-terminal transmembrane domain, however no intracellular kinase domain, suggesting that it requires additional protein-partner(s) to perform a signal transduction role. In rice, the LysM receptor, OsCEBiP, binds to the chitin oligosaccharide and forms a complex with OsCERK1 to trigger defence response [79]. Conversely, in A. thaliana, the LysM receptor, AtCERK1, directly binds to chitin, dimerises and triggers immune responses [140]. In the extracellular domain, OsCERK1 has one LysM motif, whereas AtCERK1 has three LysM motifs [15,79] that mediate the binding of chitin [141,142]. There are five genes encoding LysM receptor-like kinases (LYKs), which are comprised of LYK1 (CERK1) and LYK2 - LYK5, and three genes encoding LysM receptor-like proteins (LYPs) in the A. thaliana genome [143]. In this regard, LysM receptor-like kinase 1 (LysM RLK1) is involved in chitin signalling and fungal resistance in Arabidopsis [144]. A T-DNA insertion knockout mutant of LysM RLK1 blocked the induction of chitooligosaccharide-responsive genes (CRGs) by chitooligosaccharides and increased susceptibility to fungal pathogens, but not bacterial pathogens. This was reversed when the mutant was complemented with the WT LysM RLK1 gene using the cauliflower mosaic virus (CaMV) 35S promoter [15,144]. CERK1 KO mutants were unable to respond to chitin oligosaccharide elicitors when compared to the WT that exhibited rapid generation of ROS in Arabidopsis [15].

 

LYK4 is another LysM-RLK involved in chitin signalling and plant immunity in A. thaliana, and possibly in the chitin recognition receptor complex [89]. LYK4 mutants were defective in the activation of chitin-responsive genes and were also susceptible to bacterial and fungal pathogen infection. Cao et al. [90] showed that AtLYK5 binds chitin with higher affinity and forms a chitin-induced complex with AtCERK1, thereby triggering immunity in A. thaliana. The higher affinity of AtLYK5 for chitin, compared to AtCERK1, also suggests the former to be the major chitin binding protein in Arabidopsis. These authors [90] further reported involvement of AtLYK5 in chitin-induced AtCERK1 phosphorylation and homodimerisation [90]. While Atlyk5-2 was significantly impaired in chitin signal responses, Atlyk4/Atlyk5-2 double mutant resulted in a complete loss of chitin response, indicating an overlap of signal function between AtLYK5 and AtLYK4.

 

The LysM domain-containing glycosylphosphatidylinositol-anchored protein 2 (LYM2), one of the three CEBiP homologs in Arabidopsis, binds chitin and mediates a reduction in molecular flux via the plasmodesmata [88]. AtCERK1 is not involved in the chitin-mediated regulation of plasmodesmata flux, thereby suggesting the presence of an alternative novel disease resistance mechanism in Arabidopsis [88,145]. Shinya et al. [146] showed that the Arabidopsis LYM2 can recognise chitin oligosaccharides in a similar way as the rice OsCEBiP, but does not participate in chitin signalling. The KO mutant of LYM2 (lym2-1) was shown to be incapable of chitin-induced plasmodesmata flux, and susceptible to fungal pathogens (Botrytis cenerea and Alternaria brassicicola), but exhibited chitin-induced MAPK activation and an oxidative burst when compared to the WT [88,146]. LysM RLK1-interacting kinase 1 (LIK1) interacts with CERK1 and regulates chitin-induced MTI in Arabidopsis [147]. LIK1 mutants (lik1-1, lik1-2, lik1-3 and lik1-4), with T-DNA insertions located in intron 2, 13, and exon 18, respectively, showed an enhanced response to both chitin and flagellin elicitors. Furthermore, the mutants were defective in the expression of genes involved in jasmonic acid (JA) and ethylene (ET) signalling pathways, that have been shown to mediate resistance to necrotrophic pathogens [148]. In A. thaliana a powdery mildew-resistant kinase 1 (PMRK1), a RLK that is localised in the plasma membrane, is responsible for early chitin-induced defence signals against fungal pathogens [149]. PMRK1 KO mutant (pmrk1) was more susceptible to both Golovinomyces cichoracearum and Plectosphaerella cucumerina (Table 1). In fact, these numerous identified PPRs linked to chitin perception suggest the complexity of plant chitin defence signalling.