Self/nonself perception in plants in innate immunity and defense

Abstract

The ability to distinguish ‘self’ from ‘nonself’ is the most fundamental aspect of any immune system. The evolutionary solution in plants to the problems of perceiving and responding to pathogens involves surveillance of nonself, damaged-self and altered-self as danger signals. This is reflected in basal resistance or non-host resistance, which is the innate immune response that protects plants against the majority of pathogens. In the case of surveillance of nonself, plants utilize receptor-like proteins or -kinases (RLP/Ks) as pattern recognition receptors (PRRs), which can detect conserved pathogen/microbe-associated molecular pattern (P/MAMP) molecules. P/MAMP detection serves as an early warning system for the presence of a wide range of potential pathogens and the timely activation of plant defense mechanisms. However, adapted microbes express a suite of effector proteins that often interfere or act as suppressors of these defenses. In response, plants have evolved a second line of defense that includes intracellular nucleotide binding leucine-rich repeat (NB-LRR)-containing resistance proteins, which recognize isolate-specific pathogen effectors once the cell wall has been compromised. This host-immunity acts within the species level and is controlled by polymorphic host genes, where resistance protein-mediated activation of defense is based on an ‘altered-self’ recognition mechanism.

Introduction: The Age Old Question of “What is Self?”

The ability to distinguish self from nonself is the most fundamental aspect of an immune system. Although expressing an apparent passivity associated with their sedentary lifestyle and, being simultaneously exposed to evolving pathogens as well as environmental stresses, plants have evolved a unique metabolic plasticity that allows them to perceive pathogens and unleash effective defense strategies.¹ Since a plant's entire immune response is not based on an adaptive/acquired system as seen in mammals, it would appear to be an evolutionary ancient defense mechanism able to genetically distinguish ‘self’ from ‘nonself’ and result in downstream cascades to counter pathogen attack or eliminate the pathogen. The question therefore arises how such a system could perceive so many diverse pathogen-derived signals when the mechanism originated before the evolving variables in potential invaders and when plants are unable to acquire or adapt specifically, whilst simultaneously being exposed to environmental stresses? In this context it is important to define ‘self’ and ‘nonself’ during a defense response and to focus on how plants distinguish between self and nonself/damaged-self/altered-self using the perception and signal transduction mechanisms available.

Self.

The philosophical debate regarding self appears to be mute when considering plant cells that are surrounded by a cell wall. In this biological context everything originating from within the wall is self and all molecules of foreign origin outside the cell wall are nonself. This definition applies to the self recognition (i.e., the identification of the same genetic homology) that is observed in the specific example of pollen recognition in the same species, from the same plant during self incompatibility (SI).

The established system of self/nonself recognition in SI systems utilizes receptor-ligand type interactions to perceive, recognize and reject incompatible pollen when the same S-haplotype is expressed by both pollen and pistil. Thus, SI prevents self-pollination. Although SI responses are generally comprised of a self/nonself recognition process, SI systems have evolved independently (as exemplified in the Solanaceae, Papaveraceae and Brassicaceae) and do not utilize one molecular mechanism exclusively. Rather, SI encompasses a collection of divergent cellular responses leading to pollen rejection.^2–4 In Brassicaceae, the recognition of self and self-incompatibility are components of a receptor-ligand based mechanism that utilizes an S receptor kinase (SRK) to perceive and reject self-pollen. SRK is an S-domain RLK, which in turn is part of the RLK family, some members of which represent PRRs involved in the detection of P/MAMPs (discussed below). S-domain RLKs also occur in species that do not exhibit self-incompatibility and exhibit upregulated gene expression profiles in response to wounding and pathogen recognition, suggesting that they may fulfill a role in perception and/or defense. Although evolution may have driven expansion of certain RLK families to serve roles in particular physiological processes, this may not exclude these receptor types from functioning in different programs.¹ The evolutionary origins of plant SI, centered on the hypothesis that SI evolved from a defense pathway, was discussed by Nasrallah.⁵ Parallels exist where plant SI and plant immunity have similar outcomes, such as the elimination of undesirable cells or organisms. Interestingly, the process of pollen rejection is closely associated with rapid and effective proteolytic events, including the ubiquitin-proteasome pathway and the vacuolar sorting pathways; processes that are also of great importance in plant defense. SI is not further discussed and the reader is referred to Zhang et al.⁴ for a recent review on the subject and to Sanabria et al.¹ for the conceptual and mechanistic links between microbial recognition and self-incompatibility.

Nonself.

The definition for self, as described above, is not entirely sufficient. However, with regards to immune responses, it appears to be more constructive to define what plants perceive as nonself. In the same biological context, nonself should be redefined as a biological molecule/organism that the plant perceives to be (I) of different origin, e.g., pathogenic species recognized during a defense response (discussed in detail below) or (II) of different state, e.g., altered or damaged cellular components recognized during routine ‘house-keeping’ maintenance and regulatory metabolism (discussed in detail below).

The Constant Battle between Self and Nonself: Principles of Immunity

During co-evolution with pathogens, plants have evolved systems to distinguish self and nonself based on the detection of P/MAMPs. PAMPs may be described as invariant epitopes within molecules that are fundamental to the pathogens' fitness. They are widely distributed among different microbes, absent from the host and recognized by a wide array of potential hosts (Table 1). PAMP-triggered immunity (PTI), constitutes the first line of inducible defense against infectious disease.^10,11 In response, many Gram-negative bacteria inject effector proteins, previously termed avirulence (Avr) proteins, into the host cells, through type III secretion systems, which suppress the P/MAMP-mediated immune responses.^12–15 As a counter move, plants have co-evolved specific resistance (R) proteins to recognize the effector proteins.^10,13,14 This then leads to effector-triggered immunity (ETI) and/or the hypersensitive response (HR) representing a form of programmed cell death.^14,16 Moreover, host inhibition of bacterial virulence effectors can trigger immunity to infection.¹⁷ And so the cycle continues, (Figure 1), thus perpetuating the constant battle between pathogens and plants, described as an “arms race between pattern recognition receptors in plants and effectors in microbial pathogens”.¹⁸

Table 1.

Summary of selected PAMPs^* recognized by plants (adapted from Nürnberger and Lipka, Schwessinger and Zipfel)⁶,⁷

PAMP	Plant	Pathogen(s)	Active epitope
β-glucans	Rice Legume	Fungi (Pyricularia oryzae), Oomycetes (Phytophthora spp.,), Brown algae	Tetraglucosyl glucitol, branced hepta-β-glucoside, linear oligo-β-glucosides
Cerebrosides A, C	Rice	Fungi (Magnaporthe spp.,)	Sphingoid base
Chitin/Chitosan	Arabidopsis, rice, tomato and wheat	All fungi	Chitin oligosaccharides (degree of polymerization >3)
Cold shock protein	Solanaceae	Gram-negative bacteria, Gram-positive bacteria	RNP-1 motif (amino terminal fragment of cold shock protein)
Elongation factor (EF-Tu)	Brassicaceae	Gram-negative bacteria	elf18 (N-acetylated amino terminal fragment of EF-Tu)
Ergosterol	Tomato	All fungi
Flagellin	Most plants (except rice)	Gram-negative bacteria	flg22 (amino terminal fragment of flagellin)
Harpin (HrpZ)	Various plants	Gram-negative bacteria (Pseudomonads, Erwinia)	Undefined
Invertase	Tomato	Yeast	N-mannosylated peptide (fragment of the peptide)
Lipid-transfer proteins (elicitins)	Tobacco	Oomycetes (Phytophthora spp., Pythium spp.,)	Undefined
LPS	Arabidopsis, pepper and tobacco	Gram-negative bacteria (Xanthomonads, Pseudomonads, Burkholderia spp.)	Lipid A/inner core/Glucosamine backbone/Combinations of motifs?
Necrosis-inducing protein	Many dicot plants	Bacteria (Bacillus spp.,), Fungi (Fusarium spp., Verticillium spp.,) Oomycetes (Phytophthora spp., Pythium spp.)	Undefined
Peptidoglycan	Arabidopsis and tobacco	Gram-positive bacteria	Muramyl dipeptide
Rhamnolipids**	Grapevine	Pseudomonas species	Mono-/dirhamnolipids
Siderophores***	Tobacco	Undefined	Pseudomonas fluorenscens
Sulphated fucans	Tobacco	Brown algae	Fucan oligosaccharide
Transglutaminase	Parsley and potato	Oomycetes (Phytophthora spp.)	Pep-13 motif (surface-exposed epitope of the transglutaminase)
Xylanase	Tobacco and tomato	Fungi (Trichoderma spp.)	TKLGE pentapeptide (suface-exposed epitope of xylanase)

^*PAMPs may be described as invariant epitopes within molecules that are fundamental to the pathogens' fitness; They are widely distributed among different microbes, absent from the host and recognized by a wide array of potential hosts

^**varnier et al.;⁸

^***van Loon et al.⁹

Figure 1.

Perception systems for damaged-self, altered-self and nonself signals in plants. Pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), and pathogen-derived effectors are perceived as nonself danger signals. Extracellular P/MAMPs originating from prototypical microbes and DAMPs generated by their enzymes, are recognized via pattern recognition receptors (PRRs). Pathogen effectors injected into the cell are detected, directly or indirectly, by intracellular resistance (R) proteins and associated proteins. The domain organization of typical extracellular and intracellular receptors in plants is shown.²¹,¹⁴¹ Successful pathogens have acquired the ability to interfere or suppress generated signals and to circumvent plant defenses (e.g., AvrPto, AvrProB, AvrRpt2, HopAl1, HopU1, HopM1, Hopi1).¹⁴⁶ Six possible targets for interference or suppression include: (1) MAMP perception through PRRs, (2) the MAPK cascade, (3) RNA metabolism, (4) vesicle trafficking, (5) regulation of ETI/PTI and (6) chloroplast function (adapted from Boller and He,¹⁸ Boller and Felix,¹⁹ as well as Tör et al.¹⁴⁷).

Innate immunity.

Plants possess an innate immune system consisting of PTI and ETI that detects and defends against potentially dangerous microbes.^14,16,18,19 It draws its origins from a phylogenetically ancient form of immunity that is common to all Metazoa and Viridiplantae,²⁰ which precedes SI.^1,21 The innate immune system in plants is unable to acquire or specifically adapt like the animal adaptive immune system.²² Rather, it relies on a spectrum of predetermined receptors expressed in non-mobile cells. These receptors may be proteins with similar morphologies or proteins that are able to multi-task between different functions in order to compensate for the inability to acquire antibodies.

Basal resistance, non-host and host immunity.

Non-host immunity refers to an evolutionary ancient, multilayered resistance mechanism consisting of constitutive and inducible components.²³ Non-host immunity remains operative even in susceptible plants to limit pathogen growth and is associated with the release of molecules (ligands or elicitors) derived from the pathogen.²⁴ In addition, it is also associated with peptides or oligouronides originating from hydrolytic events during the interaction between plants and pathogens and acting as endogenous elicitors. These are analogous to the ‘danger signals’ of the vertebrate immune system, such as heat-shock proteins, nucleotides, reactive oxygen intermediates, extracellular-matrix breakdown products, neuromediators and cytokines.²⁵ Basal resistance is the innate immune response that protects plants against the majority of pathogens. In addition, another, more recently evolved form of immunity is operative in plants. Host-immunity acts within the species level and is controlled by polymorphic host genes, such as the R genes, the products of which interact, directly or indirectly, with secreted avr proteins or effectors of the pathogen.^20,26

Current models of plant immunity; PAMP- vs. Effector-triggered immunity.

Two branches to the plant immune system are now recognized: PTI and ETI, associated with different perception mechanisms in the host.^14,16

PTI. PTI refers to the inducible responses activated upon recognition of conserved P/MAMPs such as lipopolysaccharides (LPS), peptidoglycan and flagellin of bacteria, or chitin and glucan of fungi etc. It has been reported that P/MAMPs can interact, either directly or indirectly, with each other as well as the cell wall matrix of the host. This interaction would influence the speed, magnitude, versatility as well as the organization of the defense response.²⁷ Recent evidence indicates that some identified PRRs are members of the RLK family, e.g., the flagellin receptor (FLS2) and elongation factor Tu (EF-Tu)-receptor (EFR).^28,29 The work regarding flagellin and EF-Tu, indicates that there must be a requirement for numerous such signal perception and transduction systems in plants able to recognize all potential invaders.^29,30 Indeed, sequencing of the Arabidopsis thaliana genome has revealed the presence of >400 RLK sequences with various receptor configurations, of which those containing a leucine-rich repeat (LRR) in the extra-cellular domain constitute the largest group with 216 members.^30,32 The diversity and large number of plant RLKs suggest that they may be involved in the perception of a wide range of stimuli. Other PRRs are also found amongst non-RLK proteins such as Glycine max β-glucan elicitor binding protein (GmGBP), Lycopersicon esculentum ethylene-inducing xylanase (LeEIX2) and chitin elicitor-binding protein (CeBIP) for perception of β-glucans (soybean), xylanase (tomato) and chitin fragments (rice) respectively.^33–35

ETI. The second branch of the plant immune system, ETI, in contrast acts mostly inside the cell, using polymorphic resistance proteins encoded by R genes, reviewed by Liu et al.³⁶ as well as Tameling and Takken.³⁷ R gene-mediated resistance is a form of host-immunity activated upon recognition of an avirulence factor, a pathogen effector protein that elicits resistance (via direct recognition of the effector by the plant, or via their action on targeted host molecules, i.e., indirectly). Since R genes act in a race-specific manner, there are few that confer broad-spectrum resistance.

‘Zigzag’ model and ETS. A ‘zigzag’ model to illustrate the quantitative output of the plant immune system as well as to illustrate the evolutionary relationship between PTI and ETI was recently proposed.¹⁶ In phase 1, P/MAMPs are recognized by PRRs, resulting in PTI that can stop further colonization. In phase 2, successful pathogens deploy effectors that contribute towards pathogen virulence. When effectors suppress or interfere with PTI, it results in effector-triggered susceptibility (ETS).^15,38 In phase 3, an effector is specifically recognized by an R protein, which results in ETI. ETI is regarded as an accelerated and amplified PTI response, which results in disease resistance and may lead to an HR at the infection site. In phase 4, natural selection drives pathogens to avoid ETI. This is achieved by either shedding or diversifying the recognized effector gene, or by acquiring effectors that suppress ETI. Thereafter, natural selection results in the evolution of new R specificities leading to ETI being triggered again.

Downstream signaling. Similar to PTI, R-protein triggered immunity is also linked to reactive oxygen intermediate accumulation and activation of defense genes, but the two responses differ quantitatively and kinetically. The outcome of ETI can lead to programmed cell death of the host cell in the form of the HR in order to limit the spread of the infection. It results in local induced/acquired resistance (LAR), acting at the site of infection to contain the invader, and systemic acquired resistance (SAR), which induces defenses in distal, non-infected parts of plants after activation of local resistance. Due to the fact that PTI and ETI have similar output responses, it is possible that the downstream signaling pathways converge.³⁹ It should be noted that SAR has also been demonstrated to be induced by recognition of PAMPs like LPS40 and that certain PAMPs like flagellin and flg22 can cause an HR.⁴¹ Both salicylic acid (SA) and jasmonic acid (JA) are required for P/MAMP-induced defense responses.⁴² P/MAMP, as well as effector-triggered processes are linked to SA pathways,⁴³ therefore, SA-mediated responses may be an important part of R gene-mediated defense.

Biochemistry of Perception and Recognition: Nonself Detection

Perception of pathogens by plants.

The ability to monitor microbial presence at the cell surface is essential for plant defense mechanisms. Plant innate immunity is activated either upon perception of P/MAMPs by PRRs or upon recognition of pathogen race-specific effector molecules by R processes (Fig. 1). Recognition of potential pathogen-derived molecules or pathogen activity in planta, results in signal initiation and signal transduction, culminating in the activation or de-repression of defense-associated genes. The recognition specificities in the different kingdoms probably arose independently in order to recognize highly conserved molecules.^14,16

The plant cell wall as a sensor of integrity.

The structure of the plant cell wall distinguishes it from all other eukaryotic cells. It represents the first barrier to an invading pathogen. If the invasion is halted, cellular damage is minimized and no other defensive actions are required. In this context the plant cell wall is not only a rigid or static structure used for mechanical support; it exists as a highly dynamic and responsive structure in a relationship with the plasma membrane and cytoskeleton, where the external and internal environments are joined, and where information from external stimuli is relayed.⁴⁴ The plant cell is able to perceive changes to the cell wall, be responsive and adapt with regards to growth and development, as well as stresses, e.g., wounding and pathogen attack, which was reviewed by Humphrey et al.⁴⁴

Pathogen attack may lead to cell damage and influence the cell wall integrity. When the cell wall responds to stress or change, it may be due to the recognition of its ‘damaged-self,’ through damage-associated molecular pattern molecules (DAMPs). The stress or change is perceived by a sensor or sentinels and the plant responds to the change in a defensive manner. An example of recognition of the plant’s ‘damaged-self’ is when pathogen-secreted or endogenous plant polygalacturonases or pectate lyases cause enzymatic degradation of pectin in the plant cell wall, i.e., an altered/damaged state of self. Here, the polygalacturonase-inhibiting protein, an extracellular LRR R protein, interacts to generate oligogalacturonides, which are perceived by a sentinel in order to generate a signal, triggering defense related responses.^45–47 Other potential DAMPs include cellodextrins and cutin monomers, originating as degradation products from the plant cell wall cellulose and cutin layers. Plants can perceive modifications of the cuticle and activate a multi-factorial defense response.⁴⁸ However, sentinels that alert the plant to activate defense responses in response to DAMPs have only recently been explored.

In addition to signals generated due to cell wall degradation, conditions that lead to a decrease in cellulose content (e.g., due to loss of function mutations in cellulose synthase (CESA) or chemical inhibition of cellulose synthase/cell wall synthesis) are associated with a corresponding increase in defense-associated cell wall strengthening through lignin and callose synthesis.^49–52 This implies a feedback mechanism involving sensors of wall integrity. In addition, these conditions cause constitutive expression of genes associated with JA or ethylene signaling.⁵³ The synthesis of these hormones is usually associated with responses to pathogens, wounding and drought.^54–56

The protein components in the cell wall probably play a determining role in perception and these include a variety of potential sensors. Arabinogalactan proteins (AGPs) are regarded as potential sensors of wall integrity.⁴⁴ AGPs are glycosylphos-phatidylinositol (GPI)-anchored proteins (GAPs). GAPs may play a role in cell surface signaling, adhesion, matrix remodeling and pathogen response.⁵⁷ In addition, leucine-rich extensin (LRX) proteins that bind to the cellulose microfibrils, have also been identified as potential cell wall sensors. The wall-associated kinases (WAKs) are the best characterized of the potential cell wall receptors and are ideally situated for sensing and signaling from the cell wall.⁵⁸ WAK expression can be induced in response to pathogen attack and, being able to bind pectin fragments and oligogalacturonides,⁵⁹ may serve as potential sensors of damaged-self. Other RLKs found associated with the cell wall include lectin receptor kinases, a subset of which are found in plasma membrane-cell wall adhesions and proline-rich extensin-like receptor kinases (PERKs) involved in sensing of cell wall damage due to wounding by pathogens.⁶⁰ THESEUS1, a receptor kinase, is a new candidate for sensing cell wall integrity, but has not been proven to play a role in defense.⁴⁴ Furthermore, various plasma membrane proteins with extracellular domains, such as RLPs and RLKs important in relaying information from external stimuli (discussed below), interact with and within the wall matrix.

Sentinels of nonself: pattern recognition receptors.

Plants have evolved a large range of potential immune receptors (refer to Tables 2–531 y) that recognize P/MAMPs as determinants of nonself, or mediate effector perception in the form of ‘sentinels.’ Sentinels may contain pattern recognition domains combined with accessory domains that participate in signal relay. The diversity of P/MAMPs and the identification of the corresponding PRRs, a term describing a functional category, were recently reviewed.¹⁹ Receptors, a term describing a molecular category, which detect microbial patterns can either be surface based or intracellular receptors (Fig. 1). Surface receptors are known to detect primarily microbe-derived elicitors (including P/MAMPs, if the molecule contains a conserved ‘pattern’), as well, in certain cases, avirulence effectors such as Xa21, in which case they are regarded as R gene products. The surface receptors include RLKs, RLPs and extracellular binding proteins that may form part of multi-component recognition complexes.⁶¹ The few P/MAMP receptors identified thus far in plants are all surface receptors that physically interact with their cognate ligands.⁶¹ In contrast, interaction between effectors and the intracellular R proteins (which can contain a LRR domain) probably occurs indirectly through a multi-member complex.⁶²

Table 2.

Plant LRR-RLKs associated with plant-microbe interactions, innate immunity and defense

Gene	Plant	Class	Type	Function	Perception	Putative ligands	References
CURL3	Solanum esculentum	LRR	RD-kinase	Putatively systemin perception during wounding; possibly brassinosteroid perception *Orthologue of Sr160	Nonself recognition: altered self	Systemin BL	107
DIPM1 to 4	Malus x domestica	LRR	Non-RD-kinase	Disease resistance and plant-pathogen interaction signaling	Nonself recognition: different origins	DspA/E	108
EFR	Arabidopsis thaliana	LRR	Non-RD-kinase	Disease resistance and plant-pathogen interaction signaling	Nonself recognition: different origins	EF-Tu (elf18)	29
ERECTA	Arabidopsis thaliana	LRR	RD-kinase	Resistance to Ralstonia solanacearum	Nonself recognition: different origins		109
FLS2	Arabidopsis thaliana	LRR	Non-RD-kinase	Flagellin perception	Nonself recognition: different origins	flg22	28
HAR1	Lotus japonicas	LRR	RD-kinase	Nodule development during nitrogen fixation symbiosis *Orthologue of NARK	Symbiotic relationship, recognition of different origins		110
LRPKm1	Malus x domestica	LRR	RD-kinase	Disease resistance and plant-pathogen interaction signaling. Highly induced expression during incompatible reactions and lower induction during compatible interactions with Venturia inaequalis	Nonself recognition: different origins		111
Nork	Medicago sativa	LRR	Non-RD-kinase	Bacterial symbiosis. root nodule and mycorrhiza formation *Orthologue of SYMRK	Symbiotic relationship, recognition of different origins		112
PEPR1	Arabidopsis thaliana	LRR	RD-kinase	Activation of defense and possible positive feedback loop mechanism to amplify PAMP-induced responses.	Nonself recognition: altered self	AtPep1	113
SIRK	Arabidopsis thaliana	LRR	RD-kinase	Upregulated during senescence and pathogen challenge	Nonself recognition: different origins		114
SR160	Lycopersicon esculentum	LRR	RD-kinase	Putatively systemin perception during wounding; possibly brassinosteroid perception *Orthologue of CURL3	Nonself recognition: altered self	Systemin BL	115
SYMRK	Lotus japonicus	LRR	RD-kinase	Bacterial symbiosis. root nodule and mycorrhiza formation *Orthologue of Nork	Symbiotic relationship, recognition of different origins		116
Xa21 and Xa26	Oryza sativa	LRR	Non-RD-kinase	Specific disease resistance to Xanthomonas oryzae pv oryzae	Nonself recognition: different origins	AvrXa21 elicitor	117, 118

Table 5.

Dual functioning in plant signaling

Component	Dual Function	Reference
BAK1	Is associated with developmental regulation through the plant hormone receptor BRI 1, but also has a functional role in PRR-dependant signaling which initiates innate immunity	105, 106
ERECTA	Affects development of aerial organs by controlling organ size and shape, and is also involved in disease resistance	142, 143
HAESE/RLK5	Functions in both developmental processes (abscission) and defense (hypersensitive cell death)	100
LTP1	In wheat, it binds putative receptors for elicitins.	144
	In tobacco, it binds jasmonic acid providing protection against Phytophthora parasitica similar to that invoked by elicitin
LysM type receptors	*CEBiP is a chitin oligosaccharide elicitor binding protein with two LysM motifs with a proposed role in chitin signaling and transcriptional regulation	35
	*NRF1 and NRF5 are two LysM receptor kinases found in Lotus japonicus, which are putative receptors for lipochitooligosaccharide Nod-factors	127
	*LYK3, found in Medicago truncatula, is involved in specific recognition during later stages of bacterial infection	145
Mi gene product	Confers resistance to nematodes as well as aphids	85
PERK	Upregulated by wounding and infection in Brassica, also plays a role in regulation of growth in Arabidopsis	146, 147
Plant gp91PHOX NADPH oxidase	Although involved in the oxidative burst, it also functions in a variety of developmental and physiological processes	103
RIN4	Guardee—interacts with two different R genes, RPM1 and RPS2	92
RPM1	Able to recognize two different Avr effectors	85
RPP8/HRT	Recognizes both viral and oomycete pathogens	85
Rx/Gpa gene	Confer both viral and nematode resistance	85
WAK1	Involved with an epidermal growth factor EGF-like motif linked to plant growth, also up regulated in response to pathogen infection and exogenous salicylic acid	139

Extracellular sentinels—receptor-like proteins/kinases (RLP/Ks). According to the zig-zag model, plants will first be exposed to pathogen-derived elicitors or PAMPs, which upon perception will trigger PTI. Plants are therefore dependent on the initial local recognition of the invader to activate defenses, and this is where perception by RLP/Ks can play a determining role.

In Arabidopsis, 610 RLK and 56 RLP have been identified, but only a limited number have been functionally characterized and even fewer are reported to act as immune receptors.^63,64 Many Arabidopsis genes encoding RLK and RLP were found to be induced upon the amino-terminal fragment of flagellin (flg22) or EF-Tu treatment, suggesting that they may function as immune receptors.⁶⁵ Indeed, 27 out of a total 216 LRR-RLK in Arabidopsis were found to be transcriptionally induced upon treatment with flg22 or EF-Tu.^61,65 In addition to FLS2, the EFR was identified as a novel PRR and is a LRR-RLK.^28,29,66 Among the upregulated genes, there also were three encoding RLK containing lysine motifs (LysM) in their extracellular domains, which potentially could recognize carbohydrate microbial structures containing N-acetyl glucosamine (GlcNAc). It appears that P/MAMPs also trigger enhanced expression of their own cognate receptors, as reported for flg22, EF-Tu and chitin.^29,35,65

The receptor protein kinases (RPKs) can be classified according to different substrate specificities (tyrosine, serine/threonine or histidine) in the kinase domains.^67,68 RLKs form part of the receptor serine/threonine kinase (RSTK) family, also known as the interleukin-1 receptor-associated kinase (IRAK/Pelle) family. The RLKs can be subdivided into transmembrane receptor kinases (TMRKs) and receptor-like cytoplasmic kinases (RLCKs) if an extracellular domain is absent.⁶³ RLKs are transmembrane proteins with versatile N-terminal extracellular domains and a C-terminal intracellular kinase domain related to the Drosophila Pelle kinase.^21,69 They are classified according to their extracellular domains, except for those who do not have a signal peptide and/or a transmembrane region, referred to as RLCKs. TMRKs can be further grouped into arginine and aspartate (RD)-kinases, non-RD kinases and RD-minus kinases.⁶⁸ Although only a few RLKs have been shown to play a role in either development, plant defense or even symbiotic interactions, their large number and diversity suggest that they may be able to recognize and respond to a variety of stimuli. Despite the small number of non-RD kinases (10% of the Arabidopsis kinome), kinases known or predicted to function in PRR signaling fall into the non-RD class. The reader is referred to Dardick and Ronald,⁷⁰ for a review on receptor signaling through non-RD kinases, predicted to function in PRR signaling and thought to be involved in pathogen recognition and innate immunity. RLPs differ from RLKs in that they contain an extracellular domain and a membrane-spanning domain, but they lack an intracellular activation domain. Therefore, they require interaction with adaptor molecule(s) or RLCKs for signal transduction.

The proposed evolutionary relationships between receptor kinase family members arose from an ancient duplication event leading to the divergence of RLKs/Pelle from receptor tyrosine kinases (RTKs)/Raf.^21,64 In the case of PTI, the evolutionary history of the plant RLKs indicate that the kinase domains were recruited numerous times by fusion with different extracellular domains to form the subfamilies found in Arabidopsis. Subfamilies are assigned based on kinase phylogeny and are shown according to the domain organization of the majority of members in a given subfamily.^21,69

Diverse sequence motifs are present in the extracellular domains of RLKs and these motifs are potentially responsible for interactions with other proteins, carbohydrates or lipids.⁶⁹ The data indicates that RLKs involved in resistance or defense responses may have been duplicated or retained at higher rates in a lineage-specific fashion.³¹ The preferential expansion of defense/resistance-related RLKs could be the consequence of strong selection pressure for recognizing pathogens.³¹ The large family of plant RLK proteins, therefore, contain distinct protein kinases where each might play a unique role in cellular signaling.⁷¹ These probably comprise receptors for further P/MAMP recognition.²⁹ In addition, in certain cases, plant defense mechanisms seem to exhibit ‘multi-tasking,’ i.e., the use and application of pre-existing biochemical modules or systems to compensate for evolving variables in potential invaders (discussed below).

The members of the RLK family are divided into classes. The S-class/domain RLKs share homology with the self-incompatibility-locus glycoproteins (SLG) from Brassica.⁷² The extracellular domain has 12 characteristic conserved cysteines, CX₅ CX₅ CX₇ CXCX_N CX₇CX_N CX₃ CX₃ CXCX_N C. Usually, 10 cysteines are absolutely conserved.⁷³ A conserved PTDT box was observed in seven different S-domain RLKs.⁷¹ The LRR class contains conserved repeats of leucines, LX₂ LX₂ LX₂ LXLX₂ X_N XLXGXIPX₂ and the regions are also surrounded by paired cysteines.^74,75 The lectin-like class has an extracellular domain that shares homology with lectin proteins.⁶³ Some RLKs bind to plant cell-wall components. The extracellular domains of cell wall-associated kinase (WAK)-type RLKs are associated with pectin, a structural component in the middle lamella and primary cell wall.^21,76 WAKs contain an extracellular domain with similarity to epidermal growth factor (EGF)-like domains.⁴⁴ The tumor necrosis factor receptor (TNFR) class has a repeat motif that resembles the extracellular domain of the mammalian tumor necrosis factor receptor.⁷⁷ The pathogenesis-related (PR) class contains all 16 cysteines in the extracellular domain that are conserved in PR5 antimicrobial proteins.⁷⁸ The chitinase-like class has an extracellular domain homologous to both class V tobacco and Bacillus WL-12 A chitinases.⁷⁹ The cysteine-rich repeat (CRR) class has one or more repeats of the C-X₈-C-X₂-C motif.⁸⁰ RLKs containing lysine motifs in their extracellular domains are characterized as the LysM class. The ‘miscellaneous’ or ‘other’ types of RLKs include those with extracellular domains that do not share homology with other known proteins, contain unique motifs, and therefore cannot be grouped into the above mentioned classes.

Receptor proteins have also been identified that lack the characteristic RLK kinase domain (i.e., RLPs), or proteins that are functional kinases that lack extracellular ligand binding domains (RLCKs). In some cases the proteins have an intracellular kinase domain, as well as a transmembrane region, but only have a short extracellular domain. Tables 2 and 3 serve to summarize which RLKs are involved with disease resistance and/or associated with plant-microbe interactions. The role and regulation of RLKs that have been identified in elicitor-initiated defense responses and as incompatibility-locus glycoproteins (SLG) from Brassica.⁷² The extracellular domain has 12 characteristic conserved cysteines, CX5 CX5 CX7 CXCXN CX7CXN CX3 CX3 CXCXN C. Usually, 10 cysteines are absolutely conserved.⁷³ A conserved PTDT box was observed in seven different S-domain RLKs.⁷¹ The LRR class contains conserved repeats of leucines, LX2 LX2 LX2 LXLX2 XN XLXGXIPX2 and the regions are also surrounded by paired cysteines.^74,75 The lectin-like class has an extracellular domain that shares homology with lectin proteins.⁶³ Some RLKs bind to plant cell-wall components. The extracellular domains of cell wall-associated kinase (WAK)-type RLKs are associated with pectin, a structural component in the middle lamella and primary cell wall.^21,76 WAKs contain an extracellular domain with similarity to epidermal growth factor (EGF)-like domains.⁴⁴ The tumor necrosis factor receptor (TNFR) class has a repeat motif that resembles the extracellular domain of the mammalian tumor necrosis factor receptor.⁷⁷ The pathogenesis-related (PR) class contains all 16 cysteines in the extracellular domain that are conserved in PR5 antimicrobial proteins.⁷⁸ The chitinase-like class has an extracellular domain homologous to both class V tobacco and Bacillus WL-12 A chitinases.⁷⁹ The cysteine-rich repeat (CRR) class has one or more repeats of the C-X8-C-X2-C motif.⁸⁰ RLKs containing lysine motifs in their extracellular domains are characterized as the LysM class. The ‘miscellaneous’ or ‘other’ types of RLKs include those with extracellular domains that do not share homology with other known proteins, contain unique motifs, and therefore cannot be grouped into the above mentioned classes. Receptor proteins have also been identified that lack the characteristic RLK kinase domain (i.e., RLPs), or proteins that are functional kinases that lack extracellular ligand binding domains (RLCKs). In some cases the proteins have an intracellular kinase domain, as well as a transmembrane region, but only have a short extracellular domain. Tables 2 and 3 serve to summarize which RLKs are involved with disease resistance and/or associated with plant-microbe interactions. The role and regulation of RLKs that have been identified in elicitor-initiated defense responses and as dominant R genes in race-specific pathogen defense, was recently reviewed.⁸¹

Table 3.

Non-LRR RLKs and RLPs associated with plant-microbe interactions, innate immunity and defense

Gene	Plant	Class	Type	Function	Perception	Putative ligands	References
At-RLK3	Arabidopsis thaliana	DUF-26	RD-kinase	Expressed under oxidative stress and pathogen attack	Nonself recognition: altered self		119
CERK1	Arabidopsis thaliana	LysM	RD-kinase	Chitin elicitor signaling *Homologue of LysM RLK1	Nonself recognition: different origins	chitin	120
CHRK1	Nicotiana tabacum	Chitinase	RD-kinase	Induced by tobacco mosaic virus, Phytophthora parastitica	Nonself recognition: different origins		79
LecRK-a1	Arabidopsis thaliana	Lectin	RLP	Expressed during wounding	Nonself recognition: altered self		121
LRK10	Triticum aestivum	‘Other’/S-domain	Non-RD-kinase	Specific resistance to wheat rust fungi	Nonself recognition: different origins		122, 123
LYK3	Medicago truncatula	LysM	RD-kinase	Involved in early events during nitrogen fixation symbiosis (symbiotic interactions) *Orthologue of NFR1	Symbiotic relationship, recognition of different origins	Nod factors	124
LYK4	Medicago truncatula	LysM	RD-kinase	Involved in early events during nitrogen fixation symbiosis (symbiotic interactions)	Symbiotic relationship, recognition of different origins	Nod factors	124
LysM RLK1	Arabidopsis thaliana	LysM	RD-kinase	Chitin elicitor signalling *Homologue of CerK1	Nonself recognition: different origins	chitin	125
NARK	Glycine max		RD-kinase	Nodule development during nitrogen fixation symbiosis *Orthologue of HAR1	Symbiotic relationship, recognition of different origins		126
NFR1	Lotus japonicas	LysM	RD-kinase	Involved in early events during nitrogen fixation symbiosis (symbiotic interactions) *Orthologue of LYK3	Symbiotic relationship, recognition of different origins	Nod factors	127
NFR5	Lotus japonicas	LysM	RD-kinase	Involved in early events during nitrogen fixation symbiosis (symbiotic interactions) *Orthologue of SYM10	Symbiotic relationship, recognition of different origins	Nod factors	128
PBS1	Arabidopsis thaliana	RLCK	RD-kinase	Specific resistance to Pseudomonas syringae pv phaseolicola	Nonself recognition: different origins		129
PERK1	Brassica napus	RLK	RD-kinase	Sensing of cell wall damage due to wounding by pathogens	Nonself recognition: altered self		60
Pi-d2	Oryza sativa	LecrK/S-domain	Non-RD-kinase	R gene that confers resistance to blast disease	Nonself recognition: different origins		130
PnLPK	Populus nigra var. Italic (Lombardy poplar)	Lectin-like	RD-kinase	Expressed during wounding	Nonself recognition: altered self		131
PR5K	Arabidopsis thaliana	PR5	Non-RD-kinase	Disease/stress response	Nonself recognition: altered self		78
PvRK20-1	Phaseolus vulgaris	DUF-26	RD-kinase	Expressed during wounding and plant-microbe interactions	Nonself recognition: altered self		132
RFO1	Arabidopsis thaliana	WAK/EGF	RD-kinase	Involved in resistance response to Fusarium oxysporum	Nonself recognition: different origins		133
RKC1	Arabidopsis thaliana	DUF-26	RD-kinase	Salicylic acid inducible	Nonself recognition: altered self		100
RKS1, 2	Arabidopsis thaliana	S-domain	RD-kinase	Salicylic acid inducible	Nonself recognition: altered self		100
RLK1	Arabidopsis thaliana	S-domain	RLP	Salicylic acid inducible	Nonself recognition: altered self		134
RLK4	Arabidopsis thaliana	DUF26	RD-kinase	Salicylic acid inducible	Nonself recognition: altered self		135
SFR1, 2	Brassica oleracea	S-domain	RD-kinase	Defense response signaling, wounding, pathogenic (Xanthomonas campestris, Ralstonia solanacearum) and non-pathogenic (Escherichia coli; E. coli) bacterial infection	Nonself recognition: different origins & altered self		136
SYM10	Pisum sativum	LysM	RD-kinase	Involved in early events during nitrogen fixation symbiosis (symbiotic interactions) *Orthologue of NFR5	Symbiotic relationship, recognition of different origins	Nod factors	128
WAK1 to 4	Arabidopsis thaliana	WAK/EGF	RD-kinase	Cell expansion and disease response, Pseudomonas syringae (compatible), repressed by wounding	Nonself recognition: altered self		137–140

Intracellular sentinels—R proteins. A second class of immune receptors encoded by R genes, which occur mainly intracellularly, has the capacity to perceive isolate-specific pathogen effectors encoded by AVR genes of the pathogen. This perception can occur either directly or indirectly by sensing the host proteins upon which effectors have acted.^37,61,82,83 Some R proteins structurally resemble RLK and RLP receptors and probably evolved from PAMP receptors through the loss of a kinase domain.⁷⁰ However, as exceptions, some intracellular R proteins can consist of one (Pto and Fen) or even two kinase domains (RPG1). Similar to RLKs, R genes subfamilies have evolutionary expansion patterns that show lineage-specific expansions marked by tandem duplicates.^64,70 Some R genes are more rapidly evolving as components of the plant immune system, compared to the evolution of P/MAMP receptors.⁸⁴

It has been recognized that plants might not possess enough R genes to intercept all potential avirulence determinants, due to the diversity of pathogens and their associated effectors. According to Dangl and Jones,⁸⁵ the likelihood of the known R genes linked to defense being able to recognize all the possible effector signals, where ‘a surprisingly small number of genes mediate recognition of all possible pathogen-encoded effectors’ is questionable. For this reason, bacterial effector recognition has likely evolved as an indirect mechanism (within a complex),⁶² with a limited repertoire of plant resistance receptors.¹⁴

The mechanisms of R gene-mediated immunity may be explained by the ‘gene-for-gene’ genetic model or the ‘guard hypothesis’ molecular model. A ‘guard’ can refer to a typical R protein, whereas the ‘guardee’ represents a target of pathogen effectors.⁸⁵ Many plant R proteins might be activated indirectly by pathogen-encoded effectors, and not by direct recognition.⁸⁵ This form of ‘guard hypothesis’ implies that R proteins are able to indirectly recognize pathogen effectors by monitoring the structural integrity of the host cell targets following effector action. The R proteins in question are, thus, activated as sensors or sentinels of ‘pathogen-induced altered-self’ molecular patterns and can thereby potentially perceive the presence of more than one effector protein. Plants are able to sense an ‘infectious-self,’ where the host molecules that are normally not available for recognition (but rather are released following microbe detection, wounding or during infection), are recognized as an altered-self.⁸⁶ This ‘altered/nonself’ concept can explain how plants can recognize a diverse set of pathogens and pathogen-specific molecules, using a relatively limited number of pathogen receptors. A recent modification to the model describes ‘decoys’ that mimic effector targets in the plant in order to trap the pathogen in a recognition event.⁸⁷

Much information has been gained from molecular studies of R genes about their organization within genomes and their functional domains. Although polymorphic, and divided into several classes, common structural modules found in intracellular plant R proteins are a C-terminal LRR domain, which is believed to sense microbe-derived signals, and a central nucleotide-binding (NB) domain (Table 4). The NB domain is part of a larger NB-ARC domain (due to its occurrence in plant R proteins, the apoptotic protease-activating factor, APAF-1, and its Caenorhabditis elegans homolog CED-4).⁸⁸ These NB-ARC domain proteins belong to the family of STAND (signal transduction ATPases with numerous domains) NTPases. STAND ATPases are modular proteins and display a wide range of fusions to domains involved in protein-protein or protein-DNA interactions, small molecule-binding domains, as well as catalytic domains involved in signal transduction.

Table 4.

Some plant R proteins associated with plant-microbe interactions and innate immunity (adapted from Dickinson)¹⁴¹

Class	Protein Structure	Gene	Plant	Function	Perception	Putative ligands
1		EILP	Nicotiana tabacum	Non-host disease resistance, induced by hyphal wall component elicitor, Pseudomonas syringae pv. glycinae, Pseudomonas syringae pv. tabaci	Nonself recognition: different origins and altered self
		L		Resistance against Melampsora lini	Nonself recognition: altered self	AvrL567
		M	Linum usitatissimum	Resistance against Melampsora lini	Nonself recognition: altered self	AM
	TIR-NBS-LRR	P		Resistance against Melampsora lini	Nonself recognition: altered self	AvrP4, AvrP123
		N	Nicotiana tabacum	Resistance against Tobacco mosaic virus	Nonself recognition: altered self	TMV replicase
		RPP1		Resistance against Peronospora parasitica	Nonself recognition: altered self	ATR1
		RPP5	Arabidopsis thaliana	Resistance against Peronospora parasitica	Nonself recognition: altered self	AvrRPP5
		RPS4		Resistance against Pseudomonas syringae	Nonself recognition: altered self	AvrRps4
		Prf	Solanum esculentum	Resistance against Pseudomonas syringae	Nonself recognition: altered self	AvrPto
		MI		Resistance against Melodogyne incognita	Nonself recognition: altered self
		Gpa2/Rx1	Solanum tuberosum	Resistance against Globodera pallida and Potato virus X	Nonself recognition: altered self
	CC-NBS-LRR	RPS2	Arabidopsis thaliana	Resistance against Pseudomonas syringae	Nonself recognition: altered self	AvrRpt2
		RPS5		Resistance against Pseudomonas syringae	Nonself recognition: altered self	AvrPphB
		RPM1		Resistance against Pseudomonas syringae	Nonself recognition: altered self	AvrRpm1, AvrB
		RPP8/HRT		Resistance against Peronospora and Turnip crinkle virus	Nonself recognition: altered self	AvrRPP8
	NBS-LRR	Bs2	Capsicum chacoense	Resistance against Xanthomonas campestris	Nonself recognition: altered self	AvrBs2
		Dm3	Lactuca serriola	Resistance against Bremia lactuca	Nonself recognition: altered self	Avr3
		I2	Solanum esculentum	Resistance against Fusarium oxysporum	Nonself recognition: altered self
		Cre3	Triticum aestivum	Resistance against Heterodera avenae	Nonself recognition: altered self
		Xa1		Resistance against Xanthomonas oryzae	Nonself recognition: altered self	AvrXa1
		Pib	Oryza sativa	Resistance against Magnaporthe grisea	Nonself recognition: altered self
		Pi-ta		Resistance against Magnaporthe grisea	Nonself recognition: altered self	AvrPita
		Rp1	Zea mays	Resistance against Puccinia sorghi	Nonself recognition: altered self
		Mla	Hordeum vulgare	Resistance against Blumeris graminis	Nonself recognition: altered self	AvrMla
	TIR-NBS-LRR-NLS-WRKY	RRS1-R	Arabidopsis thaliana	Resistance against Ralstonia solanacearum	Nonself recognition: altered self	PopP2
2	LRR-TM (RLP)	Cf-2, Cf-4, Cf-5, Cf-9	Solanum esculentum	Resistance against Cladosporium fulvum	Nonself recognition: different origins	Avr2, Avr4, Avr5, Avr9
		Ve1, Ve2		Resistance against Verticillium albo-atrum and V. dahliae	Nonself recognition: different origins
3	Kinase	Pto		Specific resistance to Pseudomonas syringae pv tomato	Nonself recognition: altered self	AvrPto
		PtI1		Specific resistance to Pseudomonas syringae pv tomato	Nonself recognition: altered self
		PBS1	Arabidopsis thaliana	Resistance against Pseudomonas syringae	Nonself recognition: altered self	AvrPphB
	Kinase-Kinase	Rpg1	Hordeum vulgare	Resistance against Puccinia graminis	Nonself recognition: altered self	AvrB
4	LRR-TM-Kinase (RLK)	Xa21	Oryza sativa	Resistance against Xanthamonas oryzae	Nonself recognition: different origins
	FLS2	Arabidopsis thaliana	Resistance against	Nonself recognition: different origins
5	Unique	HS1 pro-1	Beta vulgaris	Resistance against Heterodera schachtii	Nonself recognition: altered self
6	Membrane protein	RPW8	Arabidopsis thaliana	Resistance against Erysiphe	Nonself recognition: altered self
		mlo	Hordeum vulgare	Resistance against Blumeria graminis	Nonself recognition: altered self
7	Toxin reductase	Hm1	Zea mays	Resistance against Cochliobolus carbonum	Nonself recognition: altered self

These immune sensor proteins are considered to act as regulatory signal transduction switches where the regulatory switch, scaffolding and occasionally, sensory as well as signal-generating moieties are integrated into a single multidomain protein.^89,90 In addition, a structurally diverse range of domains was co-opted during evolution and is found on the N-terminal side of the NB domain. These include the coiled coil (CC, formerly referred to as leucine zipper) or TOLL/interleukin-1 receptor (TIR) domains (Fig. 1). In the case of RRS1, a WRKY DNA-binding domain is located at the C-terminus.

Current data points to the existence of the R proteins in auto-repressed conformations in the absence of a cognate pathogen effector. Direct or indirect recognition of effectors by the polymorphic LRR regions initiates conformational changes and ADP/ATP exchange that renders the respective N-terminal effector domains accessible for interactions with downstream targets.⁹¹

Various studies have shown that the C-terminal part of the LRR domain provides pathogen recognition specificity. Hence, the LRR domain has a dual function; it provides auto-inhibition and it translates pathogen recognition into activation. How exactly the LRR domains recognize a pathogen or pathogen action is unclear. Whereas some R proteins bind effectors directly, others require an intermediary host factor(s). This factor often interacts with the N-terminal domain of the R protein and could represent either the virulence target (thereby acting as a guardee) or a target mimic (thereby acting as a decoy).^87,92 In this situation, the LRR domain is likely involved in sensing the effector-induced perturbations/altered-self of the target.

Many R genes contain nuclear localization signals.^93,94 Recent data indicate that members of the TIR- and CC-type of R protein families function inside the nucleus with nucleocytoplasmic partitioning occurring upon activation. Inside the nucleus, the N-terminal domains of the activated receptor can act as signal relays to transcription factors of the WRKY class.⁹⁴ The subgroup of R proteins that have co-opted a WRKY-domain, may exhibit direct DNA-binding capacity. Members of the WRKY transcription factors bind to cis-acting regulatory elements called W-boxes and can act as repressors of PAMP-triggered immune responses whilst others act as positive regulators.^95,96

De-repression of defense genes could thus amplify PAMP-triggered responses and integrate signals generated by defenseassociated RLKs and R proteins.³⁸ It is considered likely that, in addition to interference with WRKY repressors, other potential convergence points between P/MAMP- and R protein-triggered signaling pathways exist.⁹⁴ P/MAMP-triggered and mitogen-activated protein kinase (MAPK)-dependent phosphorylation of R proteins can modulate effector-triggered receptor-activity and/or nucleo-cytoplasmic receptor partitioning.³⁸ This offers an explanation of how perception of nonself structures by RLP/Ks and R proteins can lead to transcriptional activation of defense-responsive genes, thereby linking receptor function to transcriptional reprogramming of the host cells for pathogen defense.³⁸

Upregulation of Surveillance and a Primed State

Perception of general elicitors such as LPS and flagellin from bacteria by plants, resembles recognition based on PAMPs in animals.^97,98 As all types of plant immunity may be considered innate, the response to PAMPs should be considered as an expression of basal resistance. Genes expressed in Arabidopsis in response to elicitation by flg22,⁹⁹ indicate that a considerable number of the upregulated genes can be classified as being involved in signal perception (RLK and R genes) and signal transduction. This indicates a positive feedback regulation operating in innate immunity with transcriptional activation of the components involved in the perception and signaling.⁶⁵ Similar results were found in a transcriptional microarray analysis of genes expressed in Arabidopsis in response to elicitation by LPS (TAIR accession expression set 100808727, Nürnberger T 2006).

Many receptors are transcriptionally activated upon perception of their ligands as well as SA, an effector of SAR.¹⁰⁰ Wang et al.¹⁰¹ provided evidence for a model where the RPW8 resistance gene from Arabidopsis could be induced by invasion of powdery mildew isolates and amplified by a SA-feedback circuit, leading to activation of defense responses, via a conserved basal resistance pathway in a non-race-specific manner. In the case of LRR-RLK genes, 49 were found to be upregulated upon either PAMP elicitation and/or pathogen infection.⁸³

Recent data indicate the existence of similar and complementary, but independent perception systems, for different PAMPs (e.g., flg22 and elf18), where perception of one PAMP at a binding site induces higher amounts of binding sites for a second PAMP, and vice versa. Interestingly, the genes for the RLKs FLS2 and EFR, are also induced by LPS and other PAMPs.²⁹ Signaling cascades generated by these independent receptors converge to lead to the activation of plant innate immunity systems.²⁹ If that is generally applicable, plants seem to induce the gene products recognizing the attacking pathogen, thus activating the plant's surveillance system and thereby sensitize or ‘prime’ the rest of the plant to control the spread of the pathogen. It would also imply sensitization of the innate immune system to perceive and respond to the attacking pathogen, analogous to what constitutes local and systemic induced resistance.¹⁰² The upregulated expression of RLK and R genes presumably leads to an enhanced sensitivity of the plant to further stimuli, sensing the presence of invading microorganisms with other PAMPs or effector signals, i.e., a primed or sensitized state.¹

Dual Functioning in Plant Signaling

There is currently no conclusive evidence for evolutionary conservation of an ancient P/MAMP detection system,^32,85,103 and independent recruitment of components during evolution is equally plausible. Moreover, there are also various examples where a specific type of biochemical module or protein appears to be used to fulfill a requirement in more than one process, i.e., dual functioning or ‘multi-tasking.’ Since the pre-existing mechanisms of innate immunity must be specifically utilized to distinguish plant from pathogen, the question arises if it is possible that there might be a sharing of receptors between similar signal molecules, such as a general receptor and/or co-receptor complex for PAMPs with common molecular architectures.

The re-use of highly evolved processes for diverse functions was recently pointed out.¹⁰³ It was concluded that a form of the ‘guard hypothesis’ best explains how plants can potentially recognize a diverse set of pathogens and pathogen-specific molecules, using a relatively limited number of pathogen receptors, but emphasizes that (in addition to PTI) the evolutionary solution in plants to identify pathogens involves surveillance of ‘self vs. altered-self,’ whereas the evolutionary solution in the adaptive immune response in vertebrates involves detection of foreign antigens.

The work regarding innate immunity, with specific reference to flagellin, has lead to the perception of a one-to-one specific recognition of pathogen (ligand) by the host (receptor) in plants, as also observed in animal and insect adaptive (and innate) immune responses.^29,30,99 However, it is not necessarily a specific one-to-one recognition system (such as the Avr-R model). Rather, plant defense mechanisms may follow an adaptation of the guard hypothesis, such as ‘one post, multiple guards’.⁹² In addition, many R proteins (guards) may perceive the presence of more than one effector protein, whether that protein comes from pathogens with similar or different lifestyles.⁸⁵

An example of independent recruitment of biochemical components for different functions is the LRR motif. LRR domains are found in transmembrane proteins, -kinases and intracellular R proteins. Collectively, LRRs appear to be involved in a range of processes from development to intercellular communication and to disease resistance.^14,104 A number of LRR transmembrane and intracellular proteins act as integral components of ligand perception complexes during ETI.⁸⁵ In addition, the LRR motif also plays an important role in PRRs in the evolutionary older PTI.⁸³

Is it possible that the same type of receptor could perceive different signals, for example both PAMP signals for defense and MAMP rhizobial signals for symbiosis; although the downstream signaling and the outcome of the plant-microbe interactions are different?¹⁰ An important recent discovery is the role that co-receptors might play in receptor-ligand interactions, and it has been suggested that co-receptors might modulate receptor specificity.¹⁰⁵ The brassinosteroid receptor BRI1-associated kinase (BAK1), may be upregulated and seems to be a crucial component of plant disease resistance and a positive regulator/general signaling adaptor/signal amplifier in signaling, and exerts this activity independent of brassinosteroids.^86,106 In addition to BAK1 interacting with FLS2 in a stimulus-dependent manner, it may also have a common role as an adaptor or co-receptor for the regulation of various other receptors. BAK1 is thus not only associated with developmental regulation through the hormone receptor BRI1, but also has a functional role in PRR-dependent signaling which initiates innate immunity.^11,105 Other examples of multifunctional proteins, in addition to BAK1, are compiled in Table 5, and include multiple applications of specific motifs and the re-use of highly evolved processes for diverse functions.

Conclusion

The concept of innate immunity centers on the recognition of ‘nonself‗ components, which is accomplished by sentinels. Plants have evolved a unique metabolic plasticity that allows them to perceive pathogens and unleash effective defense strategies, but how the plant can distinguish between itself and pathogens during a defense response has only recently been explored. This highly evolved surveillance system in plants is able to detect a broad range of signals originating from microbes or damaged plant tissues, initiating sophisticated molecular mechanisms that result in defense. Microbe/pathogen-associated molecular pattern molecules, damage-associated molecular pattern molecules, virulence factors, secreted proteins and processed peptides can be recognized directly or indirectly by this surveillance system. Together, receptor-like kinases or receptor like proteins, as membrane bound signaling molecules with an extracellular receptor domain and intracellular nucleotide binding-leucine-rich repeat proteins as receptors of pathogen-secreted effector proteins, provide an early warning system for the presence of potential pathogens and activate protective immune signaling in plants. Much remains to be discovered, e.g., how different perception mechanisms in plants, based on self, damaged-self, altered-self and nonself, are employed for different threats, and how those signals are transduced within the inter-connected relay system observed during defense responses.

Abbreviations

Avr

avirulence

AGP

arabinogalactan protein

BAK

BRI1-associated kinase

BRI

brassinosteroid insensitive (receptor)

CC

coiled coil

CRR

cysteine-rich repeat

DAMP

damage-associated molecular pattern

EFR

elongation factor Tu receptor

EGF

epidermal growth factor

ETI

effector-triggered immunity

ETS

effector-triggered susceptibility

FLS

flagellin (sensing) binding receptor

GAP

GPI-associated protein

GlcNac

N-acetyl glucosamine

GPI

glycosylphospatidylinositol

HR

hypersensitive response

IRAK

interleukin-1 receptor-associated kinase

JA

jasmonic acid

LAR

local induced/acquired resistance

LRR

leucine-rich repeat

LRX

leucine-rich extensin

LPS

lipopolysaccharides

LysM

lysine motif

MAPK

mitogenactivated protein kinase

NB

nucleotide-binding

P/MAMP

pathogen/microbe-associated molecular pattern

PR

pathogenesisrelated

PRR

pattern recognition receptor

PTI

pathogen-triggered immunity

R

resistance

RD

arginine-aspartate

TIR

toll/interleukin-1 receptor

RPK

receptor protein kinase

RLCK

receptor-like cytoplasmic kinase

RLK

receptor-like kinase

RLP

receptor-like protein

RSTK

receptor serine/threonine kinase

RTK

receptor tyrosine kinase

SA

salicylic acid

SAR

systemic induced resistance

SI

self incompatibility

SLG

self incompatibility locus glycoprotein

SRK

S receptor kinase

TMRK

trans membrane receptor kinase

TNFR

tumor necrosis factor receptor

WAK

wall-associated kinase

WRKY

trp-arg-lys-tyr

PERK

proline-rich extensin-like receptor kinase