Thirty years of resistance: Zig-zag through the plant immune system

1Plants are constantly challenged by diverse organisms, including viruses, bacteria, fungi, oomycetes, herbivores, and parasitic plants. Disease ensues when a plant is susceptible to any of these organisms. Plants carry powerful defense mechanisms. To cause disease, pathogens usually need to evade detection by the host and/or to suppress these immune responses. Cell-surface pattern recognition receptors (PRRs) in plants recognize conserved pathogen-/damage-/microbe-/herbivore-associated molecular patterns (PAMPs/DAMPs/MAMPs/HAMPs) and activate pattern-triggered immunity (PTI), which restricts pathogenicity. PRRs are plasma membrane (PM)-associated and are usually either receptor-like kinases (RLKs) or receptor-like proteins (RLPs) that lack a protein kinase domain. Pathogens have evolved to evade or suppress PTI through secreted effector molecules, which results in effector-triggered susceptibility (ETS). Plants, in turn, have evolved intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) to detect effectors, which are often encoded by Resistance (R) genes, and activate effector-triggered immunity (ETI) upon effector perception. Pathogens might then evolve or diversify or lose effectors to suppress or evade ETI. The interaction between PTI, ETS, and ETI was incorporated into the widely cited “zig-zag-zig” intellectual framework.

2PTI was originally an abbreviation for “PAMP-triggered immunity”, mediated by PRRs such as Arabidopsis thaliana Flagellin-Sensing 2 (FLS2). ETI is an acronym for “effector-triggered immunity,” which is mostly mediated by NLRs, but can also involve RLP-mediated detection of apoplastic effectors. While the terms PTI and ETI are frequently used in the literature, there are limitations to their use in describing specific immune responses. For example, the apoplastic effector Avr4 from the tomato (Solanum lycopersicum) leaf mold pathogen Cladosporium fulvum binds to fungal chitin to retard cell wall degradation by host chitinases and thus the release of N-acetyl glucosamine oligomers that activate defense. Avr4 is recognized by the tomato cell-surface RLP Cf-4. Thus, while immunity activated by some PRRs can be classified as PTI, others can be classified as ETI, since cell-surface receptors can recognize both PAMPs and apoplastic effectors. Other terms have been introduced to classify immune responses based on receptors, such as PRR-mediated immunity and NLR-mediated immunity. Immune responses are best defined by the location of recognition by the initiating protein, such as extracellularly triggered immunity and intracellularly triggered immunity, or surface-receptor-mediated immunity and intracellular-receptor-mediated immunity. Each of these terms has its own advantages and should be used with caution (Figure 1A). In this review, we try to minimize the overuse of these acronyms and emphasize immune responses triggered by the corresponding receptors.

3Plant PRR proteins are either RLPs or RLKs. RLKs consist of an extracellular domain, a transmembrane domain, and cytoplasmic kinase domain. RLPs lack a cytoplasmic kinase domain, and both require co-receptors to transduce immune signals. PRRs are localized to the PM via a transmembrane α-helix or a glycophosphatidylinositol (GPI) anchor. Both RLPs and RLKs perceive ligands via a range of extracellular domains. These include leucine-rich repeat (LRR), lectin, malectin, lysin motif (LysM), and epidermal growth factor-like domains.

4RLKs are found in Plasmodium, plants, and animals but not fungi. Conceivably, RLKs were present in the common ancestors of these organisms but were later lost in the fungi. Plant RLKs underwent remarkable expansion and constitute 60% of the kinases in the Arabidopsis genome. Arabidopsis RLKs can be classified into 44 subfamilies based on their kinase domains. The LRR–RLKs represent the largest subfamily of RLKs and are the best characterized RLKs in plants. A phylogenetic study of 33 plant species concluded that the average number of LRR–RLKs in angiosperms is approximately 250 per species (; Figure 1B). LRR–RLKs are further classified into 20 subgroups, with subgroup XII constituting genes involved in pathogen recognition, such as FLS2, EFR, and Xa21. Interestingly, the gene number in the LRR–RLK subgroup XII is highly variable across plant species, indicating that these genes underwent either expansion or contraction in particular lineages. Similarly, the LRR–RLPs represent the largest subfamily of RLPs in plants, and the size of this gene family is also highly variable across plant species (; Figure 1B).

5NLRs are grouped into three classes according to their N-terminal domains: coiled-coil (CC) NLRs (CNLs), Toll/Interleukin-1 receptor/Resistance (TIR) protein NLRs (TNLs), and RPW8-like CC domain (RPW8) NLRs (RNLs). Both CNLs and RNLs contain N-terminal CC-domains. Plant NLRs carry a nucleotide-binding (NB) domain shared by APAF-1, various plant R proteins and CED-4 (together, the NB-ARC domain), and LRR domains at their C-termini. These domains vary between NLRs, and additional noncanonical domains can be integrated into some NLRs (also known as NLR-integrated domains, or NLR-IDs; ). The functions of these domains also vary among NLRs. The LRR domain is involved in direct or indirect recognition of effectors. The NB-ARC domain exhibits ATP binding activity and acts as a switch for NLR activation (. The CC, TIR, and RPW8 domains function as signaling domains to downstream responses upon NLR activation. Some CC-domains are involved in effector recognition and interact directly with effectors (Avr-Pik) as well as a “guardee” protein (such as RIN4), which is a target of pathogen effectors. The α-helices in both the CC and RPW8 domains were recently shown to form cation channels required for defense signaling. TIR domains can also self-associate or associate with the TIR domains from paired TNLs, which is crucial for their activation. TIR domains, upon oligomerization, exhibit NADase activity, which leads to the production of variant-cyclic-ADP-ribose (v-cADPR;; ). TIR domains also exhibit 2′,3′-cAMP/cGMP synthetase activity. These small molecules produced by TIR domains likely function in signaling. The ID domain in NLR-IDs functions as a decoy, which enables the NLR to detect effectors targeting proteins with homology to the ID.

6NLR genes are present in the genomes of all land plants. CNLs, TNLs, and RNLs are present in basal angiosperm species such as Amborella and Nymphaea. However, TNLs are absent from most monocot genomes, indicating that gene loss likely occurred before monocots diverged from dicots. The loss of TNLs was also accompanied by the loss of TNL-signaling components, such as ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), PHYTOALEXIN DEFICIENT 4 (PAD4), and SENESCENCE-ASSOCIATED GENE 101 (SAG101;; ). The loss of these signaling components may have driven the contraction of TNLs in some angiosperm lineages, or vice versa. Similar to the LRR–RLK-XII and LRR–RLP, the number of NLRs (or NB-ARC containing proteins) is also highly variable across the angiosperms. Furthermore, the LRR–RLK-XII, LRR–RLP, and NLR gene families have undergone lineage-specific co-expansion or co-contraction (; Figure 1B). The cause of these concerted expansions and/or contractions is currently unclear but has been proposed to be linked to pathogen pressure and ecological specialization.

7PRRs recognize PAMPs/MAMPs/HAMPs from bacteria, fungi, oomycetes, parasitic plants, and herbivores. Some PRRs also recognize self-molecules, such as DAMPs and other plant endogenous peptides (phytocytokines; ). Some PRRs are not involved in direct ligand recognition but function as PRR co-receptors and negative regulators of immune signaling. There are more than 60 characterized immunity-related PRRs with known elicitors, and we attempt here to list those PRRs with known elicitors that are involved in pathogen recognition (Figure 2). Due to space limitations, some PRR gene names are abbreviated: the full gene names can be found in Supplemental Data Set 1.

8Plants perceive a range of PAMPs from bacteria, including peptides, lipids, peptidoglycans (PGs), and polysaccharides. Arabidopsis perceives the bacteria-derived peptides flg22, elf18, and xup25 via the LRR–RLKs AtFLS2, AtEFR, and AtXPS1 and the proteinaceous eMAX and translation initiation factor 1 via the LRR–RLPs AtRLP1 and AtRLP32, respectively. Other bacterial peptides such as RaxX21, flgII-28, and csp22 are perceived by rice (Oryza sativa) OsXa21, tomato SlFLS3, and SlCORE/NbCSPR (from tomato and Nicotiana benthamiana), respectively. The bacterial lipid 3-hydroxydecanoic acid is perceived through the lectin receptor kinase AtLORE. PGs from bacterial cell walls are perceived by the LysM-containing RLP AtLYM1/3 and rice OsLYP4/6. Bacterial exopolysaccharides are perceived by the LysM-containing RLK LjEPR3 from Lotus japonicus to control rhizobium infections (; Figure 2A).

9The fungal cell wall comprises chitin and oligo-galacturonides (OGs), which are perceived by multiple PRRs. Chitin is perceived by LysM-containing RLKs such as AtLYM2/4/5, OsLYP4/6, Medicago truncatula MtLYK4/9, grapevine (Vitis vinifera) VvLYK1-1/2, L. japonicus LjLYS6, and pea (Pisum sativum) PsLYK9. OGs are perceived by the cell wall-associated kinases AtWAK1/2. AtWAK1/2 also perceive pectin from the plant cell wall. The common wheat (Triticum aestivum) wall-associated kinase TaWAK perceives the protein SnTox1 from the necrotrophic fungal pathogen Parastagonospora nodorum and induces cell death. In addition to the fungal cell wall, apoplastic effectors from fungal pathogens are recognized by multiple LRR–RLPs. These include SlCf-2, SlCf-4, SlCf-5, SlCf-9, SlEIX2, SlVe1, SlHrc9-4E, SlI, SlI-3, and Brassica napus BnRLM2. A proteinaceous elicitor from the fungal pathogen Sclerotinia sclerotiorum, sclerotinia culture filtrate elicitor 1, is perceived by AtRLP30, and fungal endopolygalacturonases (endo-PGs) are perceived by the LRR–RLP AtRLP42 (,; Figure 2B).

10The oomycete cell wall is also composed of chitin, endo-PGs, and OGs. Thus, plants also perceive oomycetes via PRRs described in the previous section. In addition, some PRRs recognize specific PAMPs from oomycetes. For example, the glycoside hydrolase XEG1 from Phytophthora sojae is recognized by the LRR–RLP NbRXEG1. INF1 elicitin from Phytophthora infestans is recognized by the LRR–RLP SmELR from Solanum microdontum. Arabidopsis AtRLP23 recognizes a conserved peptide (nlp20) in necrosis and ethylene (ET)-inducing peptide 1-like protein (NLP) from multiple pathogens, including Phytophthora parasitica. The Arabidopsis lectin-receptor kinase AtRDA2 was recently shown to recognize 9-methyl sphingoid base, a PAMP derived from oomycete ceramide (; Figure 2C).

11Plants perceive DAMPs and phytocytokines from damaged or infected tissues to amplify and modulate immune responses against pathogens. Damage-induced cytosolic calcium influx activates metacaspases, which cleave the DAMP precursor PROPEPs into PEPs. PEPs are then secreted and perceived by the LRR–RLKs AtPEPR1/2. Multiple phytocytokines are upregulated during immunity. The stress-induced plant signaling peptides CTNIPs are upregulated during PTI and are perceived by the Arabidopsis LRR–RLK AtHSL3. Another defense-induced secreted peptide, PIP1, is recognized by AtRLK7. The Arabidopsis LRR–RLK AtMIK2 perceives the phytocytokine SCOOP peptides and SCOOP-like peptides from Fusarium spp.. Thus, AtMIK2 is involved in both self and fungal recognition during immunity. Plant PRRs also perceive a range of extracellular (e) self-molecules, such as eH2O2, eATP, and eNAD. These molecules are perceived by AtHPCA1 (also known as AtCARD1), AtDORN1, and AtLecRK-1.8, respectively. In tomato, the hormone peptide systemin is perceived by SlSYR1/2 to enhance resistance against herbivores (; Figure 2D).

12In addition to eH2O2, AtCARD1 has also been shown to perceive the self-derived quinone compound 2,6-dimethoxy-1,4-benzoquinone (DMBQ; ). Perception of DMBQ induces AtCARD1-dependent immune responses. On the other hand, the parasitic plant Phtheirospermum japonicum perceives DMBQ via AtCARD1 homologs PjCADL1/2/3, which leads to development of haustoria for parasitic infection. Thus, CARD1 is involved in both immunity (for nonparasitic plants) and parasitic plant infection. Plants also perceive PAMPs from parasitic plants to restrict infection. The tomato LRR–RLP SlCuRe1 perceives the peptide Crip21 from the parasitic plant Cuscuta spp.. Crip21 is derived from a Cuscuta glycine-rich cell wall protein. Activation of SlCuRe1 by Crip21 elicits cell death and defense responses in tomato (; Figure 2E).

13While some PRRs, such as AtNIK1, have been shown to be required for viral resistance, no PRR has been reported to directly perceive viral particles. However, the Arabidopsis PRR co-receptor bak1 loss-of-function mutant exhibits enhanced susceptibility to multiple viruses. In addition, exogenous application of double-stranded RNAs and viral coat protein (CP) elicits PTI responses in plants. Conceivably, some uncharacterized PRR(s) are involved in the recognition of viral PAMPs (Figure 2F).

14In addition to eNAD+, AtLecRK-1.8 and AtLecRK-1.1 are involved in the perception of Pieris brassicae (cabbage moth) eggs. The ligand from P. brassicae eggs that activates AtLecRK-1.8 remains to be identified and characterized. The Arabidopsis LRR–RLK AtNILR1 is involved in the perception of Heterodera schachtii (sugarbeet nematode) extracts, and nilr1 mutants are hypersusceptible to nematode infection. The cowpea (Vigna unguiculata) LRR–RLP VuINR was shown to perceive inceptin, a proteolytic fragment of chloroplastic ATP synthase from the oral secretions of Lepidopteran herbivores (a HAMP; ). Whether PRRs can perceive ligands directly from herbivores remains to be determined (Figure 2G).

15Most, if not all, PRRs function with co-receptors to activate downstream immune responses. Multiple LRR–RLKs, such as FLS2, EFR, and PEPRs function with the co-receptors AtBAK1 and AtBKK1. LRR–RLPs function with the co-receptors SOBIR1 and BAK1, and the LysM-RLK LYKs and LysM-RLP LYMs function with the co-receptor CERK1. These co-receptors are highly conserved in land plants and are crucial for PRR-mediated immunity (Figure 2H).

16Sensor NLRs are involved in the recognition of effectors from viruses, bacteria, fungi, oomycetes, parasitic plants, and herbivores. Some NLRs act as helpers or co-receptors to transduce immune signals from sensor NLRs following effector recognition. Currently, there are more than 140 characterized NLRs with known recognized effectors. Here, we summarize a list of NLRs involved in effector recognition (Figure 3; Supplemental Data Set 2).

17Bacterial effectors have been selected that target PRR signaling components and suppress host immunity. Plants have evolved multiple NLRs to guard host immune components, which indirectly detect bacteria and induce ETI. For example, the Pseudomonas syringae effector AvrPto suppresses PTI by inhibiting host kinase activity. The tomato decoy kinase Pto is guarded by the CNL Prf, which detects the perturbation of Pto kinase activity by AvrPto and activates ETI. Since plants have evolved multiple NLRs to guard central immune signaling pathways, some effectors from P. syringae are recognized by multiple NLRs from different plant species. Examples include the following: AvrB is recognized by AtTAO1, AtRPM1, and Glycine max GmRPG1b. AvrRpm1 from P. syringae pv. maculicola (Pma) is recognized by AtRPM1, AtRPS2, and GmRPG1r. AvrPphB is recognized by AtRPS5, Hordeum vulgare HvPbr1.b and HvPbr1.c. AvrRpt2 from P. syringae and RipBN from Ralstonia pseudosolanacearum are recognized by the CNL Ptr1 from multiple Solanaceous species. In addition, AvrRpt2 is recognized by the CNL AtRPS2, and AvrRpt2_EA from Erwinia amylovora is recognized by FB_MR5 from Malus × robusta 5. HopA1 is recognized by AtRPS6, and HopAI1 is recognized by both AtSUMM2 and AtRPS6.

18On the other hand, central hubs of the immune system are targeted by multiple effectors. Correspondingly, NLRs, which guard central immune signaling components, can recognize multiple effectors. For example, the CNL AtZAR1 functions with the pseudokinase RKS1 to guard the receptor-like cytoplasmic kinase (RLCK) PBL2. By guarding RLCKs or decoy pseudokinases, AtZAR1 indirectly recognizes HopZ1a, HopF2, HopBA1, HopO1, HopX1, and AvrAC from P. syringae or Xanthomonas campestris, and potentially more effectors that target RLCKs. NbZAR1 is also required to recognize XopJ4 from Xanthomonas perforans via the pseudokinase JIM2. Other examples include the following: AvrRpm1Psa and AvrRpm1Psy from P. syringae pv. actinidiae biovar 3 (Psa) and P. syringae pv. syringae strain B728a (Psy) are recognized by Nicotiana tabacum NitabRPA1. AvrE and HopAA are both recognized by Arabidopsis CAR1. AvrRps4 from P. syringae pv. pisi and PopP2 from Ralstonia solanacearum are recognized by the paired-TNLs AtRRS1-R and AtRPS4. In addition, AvrRps4 can also be recognized by the paired-TNLs AtRRS1B and AtRPS4B. The TNL NbRoq1 recognizes HopQ1-1, XopQ, and RipB from P. syringae, Xanthomonas, and R. solanacearum, respectively. Multiple TRANSCRIPTION ACTIVATOR-LIKE (TAL) effectors from Xanthomonas oryzae are recognized by the CNLs OsXo1 and OsXa1.The tomato TNL SlBs4 also recognizes multiple Xanthomonas effectors. AvrRxo1-ORF1 from X. oryzae and Burkholderia andropogonis are recognized by the CNL ZmRxo1 from maize (Zea mays;; Figure 3A).

19Plant NLRs recognize multiple effectors and molecules from fungal pathogens. Victorin, a secondary metabolite from Cochliobolus victoriae, is recognized by LOV1 from Arabidopsis and Phaseolus vulgaris. AvrFom2 from Fusarium oxysporum is recognized by the CNL CmFom-2 from Cucumis melo. Hordeum vulgare RESISTANCE LOCUS A NLRs recognize a range of effectors from Blumeria graminis (;; and can even recognize races of wheat stripe rust. Multiple TNLs from Linum usitatissimum recognize effectors from Melampsora lini. Effectors from the rice blast fungus Magnaporthe oryzae are recognized by multiple CNLs from O. sativa (;,;,;;;;;;;;. Effectors from B. graminis, P. nodorum, Pyrenophora tritici-repentis, and Puccinia graminis are recognized by multiple CNLs from Triticum species. AvrSr50 from P. graminis is recognized by ScSr50 from Secale cereale. Avr2 from F. oxysporum is recognized by the CNL SlI2, and Pc-toxin from Periconia circinata is recognized by the CNL SbPc from Sorghum bicolor (;;; Figure 3B).

20Multiple effectors from Hyaloperonospora arabidopsidis (Hpa) are recognized by Arabidopsis NLRs. ATR1, ATR4, ATR5, ATR13, and ATR39 are recognized by AtRPP1, AtRPP4, AtRPP5, AtRPP13, and AtRPP39, respectively. CX2CX5G effector-like proteins (CCG effectors) from Albugo candida are recognized by AtWRR4A and AtWRR4B.

21The oomycete genus Phytophthora carries multiple phytopathogenic species that cause enormous crop losses worldwide. Identification of NLRs that recognize Phytophthora effectors provides resources for crop resistance. The P. sojae effectors Avr1k and Avr1b-1 are recognized by GmRps1-k. Effectors from P. infestans are also recognized by NLRs from multiple Solanaceae species. For example, the effectors Avramr1 and Avramr3, with homologs in many Phytophthora species, are recognized by Rpi-amr1 (from Solanum americanum) and Rpi-amr3, respectively. Avrblb1 is recognized by Rpi-blb1 (from Solanum bulbocastanum), Rpi-pta1, and Rpi-sto1 (from Solanum stoloniferum;; ). Avrblb2 is recognized by Rpi-blb2 and R9a (from S. bulbocastanum and Solanum demissum, respectively;; ). PexRD12 is recognized by Rpi-chc1 (from Solanum chacoense;; ). Avr1, Avr3b, and Avr8 are recognized by R1, R3b, and R8, respectively. PiAvr2 is recognized by multiple NLRs from Solanaceae. Avr3a is recognized by Rpi-sto2 and R3a (from Solanum tuberosum;;;; ). Avrvnt1 is recognized by Rpi-vnt1 from Solanum venturi (;; Figure 3C).

22In mammals, DAMPs can be indirectly recognized the intracellular NOD-, LRR-, and pyrin domain-containing protein 3-inflammasome in macrophages. However, no plant NLRs have been reported to detect self-molecules so far (Figure 3D).

23Virus-induced silencing of the CNL VuRSG3-301 from V. unguiculata leads to enhanced susceptibility to the parasitic plant Striga gesnerioides race 3. The effector recognized by VuRSG3-301 has not yet been identified (Figure 3E).

24The CPs from different viruses are recognized by pepper (Capsicum annuum) CaL1, CaL1a, Capsicum baccatum CbL2b, Capsicum chacoense CchaL4, Capsicum chinense CchiL1c, CchiL3, Capsicum frutescens CfL2, Nicotiana sylvestris NsN′, Solanum acaule Rx2, S. stoloniferum Rysto, and potato (S. tuberosum) Rx. Viral movement proteins are recognized by Tm2, SlTm22, and SlSw5-b. The RNA-dependent RNA Polymerase (NIb) of potyviruses is recognized by the Ca Pvr4. The RNA silencing suppressor protein NSs from tomato spotted wilt virus is recognized by CchiTsw. P3 cistrons from soybean mosaic virus are recognized by Gm3gG2. The helicase domain of the tobacco mosaic virus replicase (p50) is recognized by Nicotiana glutinosa N. Cucumber mosaic virus 2a protein is recognized by P. vulgaris PvRT4-4. To summarize, multiple components involved in the process of viral infection are recognized by NLRs (Figure 3F).

25Multiple NLRs were shown to be involved in resistance against herbivores. NLRs involved in nematode resistance include the TIR–NB–LRR pair AtDSC1 and AtWRKY19, Aegilops tauschii AtaCD3.1, CaMi, Prunus cerasifera PcMa, SlHero, StGpa-2, and StGro1-4. In addition, the tomato Mi gene confers resistance to multiple herbivores, such as nematodes, aphids, and whiteflies. Other NLRs have been shown to confer resistance against the arthropod Nilaparvata lugens (brown planthopper). These include the rice OsBph1/9 and OsBph14. While multiple NLRs are involved in herbivore resistance, more work is needed to identify the recognized effectors (Figure 3G).

26While some sensor NLRs do not require helper NLRs, many NLRs function with helper NLRs to transduce immune signals. In Arabidopsis, some CNLs and/or most TNLs require the RNLs ACTIVATED DISEASE RESISTANCE 1 (collectively known as ADR1s, which includes AtADR1, AtADR1-L1, and AtADR1-L2) and/or N REQUIREMENT GENE 1 (collectively known as NRG1s, which includes AtNRG1A and AtNRG1B;;;; ). In Arabidopsis accession Col-0, the four RPW8 homologs, AtHR1, AtHR2, AtHR3, and AtHR4, also contribute to resistance against bacterial and fungal pathogens. In Solanaceous plants, the CNLs NB-LRR REQUIRED FOR HR-ASSOCIATED CELL DEATH-2 (NRC2), NRC3, and NRC4 function as helper NLRs for multiple sensor NLRs (; Figure 3H). The contribution of the NRC network to the functions of sensor NLRs has been extensively discussed.

27The extracellular domains of plant PRRs perceive diverse ligands. Binding of ligands leads to heterodimeric receptor complex formation between PRRs and their co-receptors, such as BAK1 and CERK1. On the other hand, RLPs constitutively interact with SOBIR1 and recruit BAK1 upon ligand recognition. In Arabidopsis, the bacterial flagellin peptide flg22 is perceived by the LRR–RLK FLS2. Flg22 acts as a “molecular glue” and interacts with and brings together the extracellular LRR domains of FLS2 and BAK1. Heterodimeric complex formation between the LRR domains of FLS2 and BAK1 brings their cytoplasmic kinase domains into close proximity, which leads to a series of auto- and trans-phosphorylation events. This activated receptor complex then phosphorylates RLCKs. RLCK subfamily VII members (collectively known as RLCK-VIIs) were first shown to be important for surface receptor-mediated immunity in tomato and tobacco and to be required for Cf-4 and Cf-9 to confer fungal resistance (. In Arabidopsis, RLCKs play particularly important roles during PRR-mediated immunity. BAK1 associates with and phosphorylates the RLCK-VII BIK1 at the Try243 and Try250 residues.

28The activation of RLCK-VIIs promotes the phosphorylation of multiple signaling components, including the calcium channels CNGC2/4 and OSCA1.3, the NADPH oxidase respiratory burst oxidase protein D (RbohD), and the mitogen-activated protein kinase kinase kinase (MAPKKK5). The activation of multiple calcium channels by BIK1 leads to cytosolic calcium influx, which activates calcium-dependent protein kinases (CPKs). In Arabidopsis, CPK4/5/6/11, together with BIK1, phosphorylate and activate RbohD, which leads to reactive oxygen species (ROS) production. The phosphorylation of multiple ion channels by RLCKs also leads to stomatal closure in response to PAMPs. In parallel, MAPKKK3 and MAPKKK5 phosphorylate the MAPKKs MKK4 and MKK5, which then phosphorylate the MAPKs MPK3 and MPK6 in Arabidopsis. In parallel, MKK1/MKK2 also phosphorylate MPK4. RLCK-VIIs, CPKs, and MPKs phosphorylate and activate multiple defense-related transcription factors, such as WRKY transcription factors, resulting in the upregulation of defense-related genes. PTI-induced transcriptional reprogramming leads to the biosynthesis of antimicrobial compounds and defense-related hormones, such as ET and salicylic acid (SA;;;; ). Hydrogen peroxide (a type of ROS) promotes protein and phenolic cross-linking, which result in callose deposition and restricts fungal and oomycete infection (;; Figure 4A).

29NLR-mediated immunity is triggered by the detection of effectors through intracellular NLRs. NLRs detect effectors either via direct interactions with effectors, guarding effector targets, or guarding decoy proteins. In Arabidopsis, CNLs and TNLs act as sensor NLRs that recognize effectors, while RNLs act as helper NLRs to transduce immune signals. While the majority of sensor NLRs in Arabidopsis require helper NLRs to mediate immunity, some CNLs mediate immune responses alone. These are known as singleton NLRs, such as ZAR1 and RPM1. ZAR1 recognizes a range of effectors by monitoring pseudokinases such as RKS1 and PBL2, which mimic authentic RLCK targets of effectors. The bacterial effector AvrAC from X. campestris uridylylates the RLCK PBL2. The ZAR1/RKS1 heterodimer associates with uridylylated PBL2 (PBL2UMP), which leads to conformational changes in the heterodimer. ADP in the NB-ARC domain in ZAR1 is ejected and replaced by ATP (. This results in the oligomerization of ZAR1/RKS1/PBL2UMP oligomers into pentameric resistosomes that localize to the PM to trigger downstream immune responses.

30ZAR1 resistosomes were recently shown to exhibit cation channel activity. The N-terminal α-helices in ZAR1 form a funnel-shaped structure with a negatively charged carboxylate ring, which allows cations to pass through into the cytosol. Co-expression of ZAR1 with RKS1, PBL2, and AvrAC in plant protoplasts results in cytosolic calcium influx, ROS accumulation, and the perturbation of chloroplasts and vacuoles. Robust ROS accumulation during ZAR1 activation is likely caused by the activation of multiple downstream signaling components, such as the NADPH oxidases, since the CPKs are activated by cytosolic calcium influx. In addition, multiple CPKs and RbohD have been shown to be phosphorylated during RPS2 activation. Defense-related transcription factors are also likely activated by cytosolic calcium influx. The perturbation of chloroplasts and vacuoles is quickly followed by the loss of PM integrity and cellular rupture (; Figure 4B). How these processes are regulated by immune signaling components and their relationships to transcriptional reprogramming are currently unclear.

31The majority of sensor NLRs requires helper NLRs to mediate immunity. In solanaceous plants, the NB-LRR REQUIRED FOR HR-ASSOCIATED CELL DEATH proteins (collectively known as NRCs) are required for immunity and hypersensitive cell death response (HR) mediated by multiple sensor NLRs. Interestingly, the N-terminal CC domain in ZAR1 contains a “MADA motif” that is also present in NRCs. This suggests that perhaps NRCs also form cation channels with α-helices following activation. In Arabidopsis, ADR1s and NRG1s are required for resistance and HR mediated by some CNLs and many TNLs. Following effector recognition, TNLs also oligomerize into resistosomes to mediate resistance. The Arabidopsis RPP1 recognizes the Hpa effector ATR1, and N. benthamiana ROQ1 recognizes the Xanthomonas effector XopQ. These effectors are recognized by the LRR and post-LRR domain, which likely leads to conformational changes and oligomerization of these TNLs into tetrameric resistosomes.

32The TIR domains of TNLs are brought into close proximity following oligomerization, activating NADase activity and producing v-cADPR. TIR domains also exhibit 2′,3′-cAMP/cGMP synthetase activity by hydrolyzing RNA or DNA. v-cADPR and 2′,3′-cAMP/cGMP are proposed to be signaling molecules that activate downstream signaling components. Following the activation of TNLs, the EP-domain containing proteins (EP-proteins) SAG101 and EDS1 associate with NRG1. Similarly, the activation of TNLs also leads to the association of the EP-proteins PAD4 and EDS1 with ADR1. These associations lead to the activation of these signaling components, which in turn activate downstream immune responses, such as defense-related gene expression and HR. The RNLs ADR1 and NRG1 were also recently shown to function as calcium channels to activate immunity. It is conceivable that the association and activation of helper RNLs and EP-proteins induces calcium influx and triggers downstream immune responses (Figure 4C).

33Following ligand perception, the PRR co-receptor BAK1 and the RLCK BIK1 are phosphorylated. This leads to the phosphorylation and activation of multiple signaling components. The activation of multiple calcium channels and NADPH oxidases leads to calcium influx, stomatal closure, ROS production, and callose deposition. The activation of CPKs and MAPKs leads to transcriptional reprograming and the biosynthesis of defense-related hormones. In Arabidopsis, MPK3/MPK6 activate 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE (ACS) isoforms ACS2 and ACS6, which are involved in ET biosynthesis. The transcription factors SYSTEMIC-ACQUIRED RESISTANCE DEFICIENT 1 (SARD1) and CALMODULIN-BINDING PROTEIN 60 G (CBP60g) are required for PTI-induced upregulation of SA biosynthesis genes, such as ISOCHORISMATE SYNTHASE 1 (ICS1), EDS5, and AVRPPHB SUSCEPTIBLE 3 (PBS3;; ). SARD1 and CBP60g are also required for the upregulation of pipecolic acid (N-hydroxyl-pipecolic acid [NHP])-biosynthesis genes, such as FLAVIN-CONTAINING MONOOXYGENASE 1 (;; Figure 5).

34Similar to RLKs, RLPs also require PRR co-receptors, RLCKs, CPKs, and MAPKs to transduce immune signals. In Arabidopsis, nlp20-induced immune responses mediated by RLP23 require the co-receptors BAK1, SOBIR1, and multiple RLCKs such as PBL19/20/30/31/32 (;;. The activation of RLP23 leads to changes in PM potential, an ROS burst, the phosphorylation of BIK1 and MAPKs, callose deposition, and SA and ET production, similar to the activation of FLS2. In addition, flg22 and nlp20 induce highly overlapping transcriptional reprogramming in Arabidopsis. Thus, RLKs and RLPs induce overlapping responses due to the activation of similar downstream signaling components. However, the individual activation of multiple RLPs, such as SlCf-4, SlCf-9, and AtRLP23, leads to the HR, perhaps due to the prolonged activation of downstream signaling components. PAD4, EDS1, and ADR1 are required for both RLK- and RLP-mediated immunity (;. Thus, EP-proteins and helper NLRs might also be activated during some PTI signaling, although it remains to be established whether EP proteins play a primary or secondary role in defense signaling (Figure 5).

35Activation of the Arabidopsis CNL RPS2 in the absence of PTI leads to the phosphorylation of RbohD (in Ser343/347), CPKs, and MAPKs. RPS2-induced RbohD phosphorylation and ROS production are dependent on BAK1/BKK1 and BIK1. However, it is currently unclear whether BAK1/BKK1 and BIK1 are directly or indirectly activated by CNLs. While the ZAR1 resistosome directly triggers calcium influx, other calcium channels may also be activated by CNLs. The activation of RPM1, RPS2, and RPS5 leads to MAPK activation and the HR. In addition, the activation of many CNLs leads to the upregulation of SA- and NHP-biosynthesis genes. Thus, ET, SA, and NHP are likely to be produced during CNL activation (Figure 5).

36Activation of the Arabidopsis TNL RRS1/RPS4 does not lead to the phosphorylation of BIK1, RbohD (in Ser39/343/347), MAPKs, calcium influx, ROS accumulation, or the HR. Thus, RLCKs, NADPH oxidases, calcium channels, or CPKs are unlikely to be activated by RRS1/RPS4 alone. Activation of RRS1/RPS4 induces weak callose deposition, perhaps via SA accumulation. Activation of TNLs leads to the association of EP-proteins with helper NLRs, which induces transcriptional reprogramming. Similar to CNLs, the activation of TNLs leads to the upregulation of SA- and NHP-biosynthesis genes. Thus, SA and NHP are likely to be produced during TNL activation (Figure 5).

37Co-activation of PRRs and NLRs (“PTI + ETI”) leads to the robust activation of BIK1, RbohD, and MPK3. This results in stronger calcium influx, ROS accumulation, and callose deposition compared to PTI or ETI alone. In addition, “PTI + ETI” leads to stronger accumulation of SA and NHP compared to PTI alone, which is likely due to the stronger expression of SA- and NHP-biosynthesis genes during ETI (;;;; Figure 5).

38The PRR-signaling pathway is tightly regulated as the excessive activation of PRRs leads to autoimmunity and growth inhibition.

39Both the transcript and protein levels of PRRs are regulated by multiple mechanisms. For example, the expression of FLS2 is regulated by the microRNA miR172b. The expression of FLS2 is also upregulated by ET. U-BOX DOMAIN-CONTAINING PROTEIN 12 (PUB12) and PUB13 mediate the polyubiquitination of FLS2, which leads to the endocytosis and degradation of this protein. Cf-4 also undergoes endocytosis upon Avr4 recognition. The activation of PRRs and their co-receptors must also be regulated. BAK1-INTERACTING RECEPTOR (BIR)-LIKE KINASE 1 is an RLK that associates with and sequesters BAK1 to prevent the auto-activation of BAK1-associated PRRs. Following PAMP perception, the peptide RAPID ALKALINIZATION FACTOR 23 (RALF23) is perceived by a PRR complex composed of the CrRLK1L FERONIA (FER) and the LORELEI-LIKE-GPI ANCHORED PROTEIN 1. The perception of RALF23 by FER negatively regulates the formation of the FLS2–BAK1 complex. FER regulates PM nanodomain organization to modulate PRR signaling. In addition, the phosphorylation status of PRRs is regulated by multiple protein phosphatases. In Arabidopsis, POLTERGEIST-LIKE 4 (PLL4) and PLL5 associate with EFR and negatively regulate elf18-induced responses. PROTEIN PHOSPHATASE 2A negatively regulates the phosphorylation status of BAK1 (; Figure 6).

40In addition to PRRs, downstream signaling components are also regulated to prevent prolonged activation. As a central signaling component in the PRR-signaling pathway, the Arabidopsis RLCK BIK1 is regulated by multiple mechanisms. EXTRA-LARGE G PROTEIN 2 (XLG2) functions with other heterotrimeric G proteins to attenuate proteasome-mediated degradation of BIK1. The turnover of BIK1 is regulated by CPK28, PUB4/25/26, and the E3 ubiquitin ligases RING-H2 FINGER A3A/B. The phosphorylation status of BIK1 is also negatively regulated by the protein phosphatase PP2C38. In addition to RLCKs, other PRR-signaling components must also be regulated. RbohD is ubiquitinated by the E3 ubiquitin ligase PIRE (PBL13 interacting RING domain E3 ligase), which leads to proteasome-mediated degradation. PHAGOCYTOSIS OXIDASE/ BEM1P (PB1) DOMAIN-CONTAINING PROTEIN negatively regulates ROS production by controlling the localization of RbohD. The PP2C phosphatases PP2C5 and AP2C1 negatively regulate the phosphorylation of MPK3 and MPK6 (; Figure 6).

41Similar to PRRs, the prolonged activation of NLRs also leads to autoimmunity. Thus, the regulation of both NLRs and downstream signaling components is important to prevent autoimmunity.

42The expression of NLRs is regulated at multiple levels. The transcription of NLRs is regulated by chromatin-remodeling proteins such as DECREASE IN DNA METHYLATION 1, SWI/SNF CHROMATIN REMODELER SYD, and multiple WRKY transcription factors. NLR transcript stability is also regulated by microRNAs and NONSENSE-MEDIATED mRNA DECAY factors, such as UP-FRAMESHIFT1/2/3. NLR transcripts also undergo alternative splicing, which is regulated by some MODIFIER OF SUPPRESSOR OF NPR1-1 (SNC1; MOS) proteins such as MOS4/12/14.

43REQUIRED FOR MLA12 RESISTANCE 1 (RAR1), SUPPRESSOR OF THE G2 ALLELE OF SKP1 (SGT1), and HEAT SHOCK PROTEIN 90 (HSP90) function together as protein chaperones to regulate the folding, localization, and turnover of NLRs. In addition, NLR protein turnover is regulated by the SGT1-interacting protein SUPPRESSORS OF RPS4-RLD, multiple MUTANT SNC1-ENHANCING proteins, and the E3 ligases SNIPER1 and SNIPER2.

44The localization of the ZAR1 resistosome to the PM is required for ZAR1-mediated resistance. In addition, the Arabidopsis importin-α nuclear transport receptor protein IMP-α3/MOS6 is required for SUPPRESSOR OF SNC1-mediated immunity. Thus, the localization of NLRs is important and is likely regulated by proteins involved in trafficking (Figure 6).

45The correct localization of helper NLRs is likely important for signaling. For example, the helper NLR NRC4 accumulates at the extra-haustorial membrane following P. infestans infection. In addition, the balanced activity of both cytosolic- and nuclear-EDS1 is required for full immunity. Thus, the localization of helper NLRs and NLR-signaling components is important for defense. The activity of NLR signaling components is also negatively regulated. The Arabidopsis RNL NRG1C functions as a negative regulator in NLR-mediated immunity; overexpressing NRG1C compromised TNL-mediated HR and resistance. In addition, an atypical member of the NRC family, NRCX, negatively regulates other NRC members to modulate immunity. Posttranslational modifications (PTMs) are important for the functions of both PRRs and NLRs. For example, the phosphorylation of the C-terminus of the TNL RRS1-R is crucial for its recognition of the effector PopP2. It is currently unclear whether PTMs are important for the activation and/or stability of NLR-signaling components. Perhaps, EP-proteins and helper NLRs must also undergo PTMs in order to function properly. The additional regulation of NLR-signaling components pre-NLR activation and postNLR activation remains to be investigated (Figure 6).

46Multiple effectors have been shown to target both the PRR- and NLR-signaling pathways. Here, we summarize our knowledge of effectors reported to target PTI or ETI. Unless specified, the effectors mentioned in this section are from various P. syringae strains. AvrPtoB is an E3 ubiquitin ligase that induces the degradation of FLS2. HopB1 specifically degrades activated BAK1. AvrPto targets SOBIR1 and the FLS2–BAK1 complex by inhibiting their kinase activities. Similarly, the conserved Colletotrichum effector NIS1 also targets receptor kinase complexes. The tyrosine phosphatase HopAO1 directly dephosphorylates EFR. As RLCKs are central immune regulators, they are targeted by multiple effectors. AvrAC from X. campestris uridylylates BIK1 and PBL2. HopZ1a acetylates RLCKs, and AvrPphB is a cysteine protease that degrades RLCKs such as BIK1, PBS1, and PBL1. Other downstream PRR signaling components are also targeted by effectors. The ADP-ribosyltransferase HopF2 targets both BAK1 and MKK5 to suppress PTI signaling. HopAI1 inactivates MPK3, MPK4, and MPK6 via its phosphothreonine lyase activity. AvrRpt2 suppresses MPK4/11 activation. Interestingly, many parallel mechanisms are employed to suppress the same PRR-signaling node in different hosts by different pathogens (Figure 6).

47Phosphorylation of SGT1 by MAPKs is required for NLR activation, implying that NLRs are regulated by SGT1 following PTI-induced MAPK activation. The R. solanacearum effector RipAC prevents MAPK-mediated phosphorylation of SGT1, which suppresses NLR-mediated immunity. Two effectors were recently shown to suppress NRC-mediated HR. The P. infestans effector AVRcap1b and the cyst nematode effector SPRYSEC15 can suppress autoimmunity induced by autoactive alleles of NRC2 and NRC3. Suppression of NRC2 and NRC3 by AVRcap1b is dependent on the membrane trafficking-associated protein TARGET OF MYB 1-LIKE PROTEIN 9A (NbTOL9a; ). AVRcap1b suppresses NRC2 and NRC3 by directly interacting with their NB-ARC domains. Another Phytophthora effector (from Phytophthora capsici), PcAvh103, suppresses immunity by promoting the disassociation of the EDS1–PAD4 complex. More studies are needed to identify pathogen effectors that target the NLR signaling pathway.

48In Arabidopsis, the transcription factors CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR 1/2/3 (CAMTA1/2/3) and CBP60a negatively regulate defense-induced transcriptional reprogramming. Pathogens also target defense-related transcription factors to suppress immunity. For example, the R. solanacearum effector PopP2 acetylates and inhibits WRKY transcription factors to suppress immunity. In addition, the Verticillium dahliae effector VdSCP41 inhibits SARD1 and CBP60g to facilitate its proliferation (; Figure 6).

49While PRR- and NLR-mediated immunity has been extensively studied for the last 20 years, it has not been clear how or if these defense mechanisms interact. NLR-mediated immunity is mostly activated in the presence of microbes or PAMPs. Most studies on NLR-mediated immunity have involved transient expression-based comparisons between PTI and “PTI + ETI.” The activation of NLRs in the absence of PTI has not been extensively studied until recently. There have been multiple reports on the different interactions between these two immune systems. Here, we describe three situations in which PTI and ETI interact with each other.

50Many effectors target the PRR-signaling pathway. Plants have evolved multiple NLRs to detect these effectors via the guarding of PRR-signaling components or decoys. As a result, many PRRs and PRR-signaling loss-of-function mutants, such as the Arabidopsis mutants bak1-4 bkk1-1, bik1, cngc2/4, rbohd/f, mekk1, mkk1/2, mpk4, and camta3, exhibit autoimmune phenotypes (;;;;;;;. The autoimmunity observed in some of these mutants is caused by the activation of multiple NLRs. The TNL CONSTITUTIVE SHADE-AVOIDANCE 1 guards both BIR3 and BAK1. In addition, bak1-3 bkk-1-autoimmunity and HopB1-triggered immunity are dependent on ADR1s. RLCKs are targeted by multiple effectors. The CNL ZAR1 together with the RLCK RKS1 monitor PBL2, and the CNL RPS5 monitors PBS1, to reverse ETS. The CNL SUMM2 guards and senses the disruption of the MEKK1–MKK1/2–MPK4 kinase cascade via CALMODULIN-BINDING RECEPTOR-LIKE CYTOPLASMIC KINASE 3, a substrate protein of MPK4. SUMM2 also detects the P. syringae effector HopAI1, which inhibits MPK4 kinase activity. The TNL RPS6 also contributes to HopAI1-triggered immunity. Whether the autoimmunity in bik1, cgnc2/4, and rbohd/f is dependent on NLRs remains unclear. Other NLRs that guard the PRR-signaling pathway remain to be identified (Figure 7A).

51PRR co-receptors, RLCKs, NADPH oxidases, calcium channels, CPKs, and MAPKs are considered to be canonical PRR-signaling components, while EP proteins and helper NLRs are considered to be canonical NLR-signaling components. However, recent studies indicated that PRR-mediated resistance is dependent on canonical NLR-signaling components and vice versa (;;;; Figure 7B). As mentioned, flg22- and nlp20-induced resistance is partially dependent on EDS1, PAD4, SAG101, ADR1s, and NRG1s (;. proposed that EP-proteins and helper NLRs are activated by RLPs through interactions between RLP co-receptors (SOBIR1), EP-proteins, and helper NLRs, although it remains to be determined whether EP-proteins play a primary or secondary role in RLP defense signaling. Another report, however, suggested that the activation of PRRs leads to increased expression of multiple NLRs and other TIR-domain-containing proteins, promoting downstream signaling (. These two hypotheses are not mutually exclusive, and the exact mechanisms by which PRR-mediated immunity involves NLR-signaling components remain to be determined.

52NLR-mediated immunity is also dependent on PRRs and multiple PRR-signaling components. In Arabidopsis, RPS2-, RPS5-, and RRS1/RPS4-mediated resistance is dependent on BAK1 and BKK1. RPS2-mediated resistance is also dependent on BIK1 and RbohD. Both RPM1- and RPS2-mediated resistance and the HR are dependent on CPK1/2/5/6. The activation of MPK3 and MPK6 is also required for the HR and resistance mediated by multiple NLRs including RPM1, RPS2, RPS5, and RRS1/RPS4. One of the proposed key mechanisms by which ETI halts pathogen infection is to potentiate and restore PTI from turnover and the action of pathogen effectors. As a result, PRRs and PRR-signaling components are required for NLR-mediated resistance. The molecular mechanisms by which ETI potentiates PTI will be discussed in the next section.

53Activation of the TNLs RRS1/RPS4 and RPP4 using an estradiol-inducible recognized effector (ETI without PTI) did not trigger the HR. The presence of PAMPs/MAMPs restored the HR induced by these TNLs. Similarly, the HR induced by the CNLs RPM1, RPS2, and RPS5 was also potentiated by the activation of PRRs. In addition, the HR and resistance induced by RPS2 are compromised in PRR mutants. There are a few possible mechanisms by which PRRs potentiate NLR-induced immunity. First, the activation of PRRs could induce the expression of NLRs and NLR-signaling components. A recent transcriptomics study suggested that the activation of different PRRs induces highly overlapping transcriptional changes. Indeed, the activation of six distinct PRRs led to the upregulation of genes encoding most TNLs, CNLs, EP-proteins, and helper NLRs in Arabidopsis (; Figure 7C; Supplemental Data Set 3). The increased abundance of these proteins might therefore “prime” the activation of NLRs upon effector recognition. Second, the activation of PRRs might prime NLR-mediated immunity via PTMs. Upon PAMP perception, SGT1 is phosphorylated by MAPKs, which is important for the stability of NLRs. In addition, nonsense-mediated decay of NLR transcripts is inhibited upon PAMP recognition. Thus, the stability of NLRs can be affected by both transcriptional and posttranscriptional modifications activated by PTI. Conceivably, EP proteins and helper NLRs might also be primed via PTMs induced by PTI. Flg22 treatment led to reduced polyubiquitination levels of EDS1. Whether and how PTI primes NLR-signaling components remain to be investigated.

54The activation of NLRs potentiates PAMP-induced cellular responses, such as ROS production, callose deposition, and defense-related gene expression. The activation of multiple PRR signaling components, such as BIK1, RbohD, and MPK3, is also potentiated by ETI. ETI induces the transcript and protein accumulation of SOBIR1, BAK1, BIK1, RbohD, and MPK3. Transcriptomic analysis confirmed that multiple PRR signaling components are also upregulated upon the activation of RRS1/RPS4. These include CPK1/2/5/6, XLG2, and the calcium channels OSCA1.3, CNGC19/20, GLR2.7/2.8/2.9 (; Figure 7C; Supplemental Data Set 4). Interestingly, the transcript levels of BIK1, MPK3, and RbohD are only transiently upregulated during ETI. However, the protein levels of these genes remain upregulated for an extensive period of time. This implies that PTMs or other posttranscriptional mechanisms might also influence the stability of PRR-signaling components during ETI. The protein abundance of PRR signaling components, such as BAK1, BIK1, and RbohD, is tightly regulated by multiple processes (Figure 6). How ETI regulates or affects these processes remains unclear. In addition, calcium influx induced by NLRs might contribute to the potentiation of PTI through CPKs. To summarize, PTI and ETI mutually potentiate each other through multiple mechanisms to induce robust immunity against pathogens (Figure 7C).

55Researchers identified the first PRR-encoding gene, Cf-9, back in 1994. Multiple PRR genes, such as Xa21, Cf-2, Cf-4, FLS2, EFR, and RLP23, were subsequently identified and used as models to study PTI. Researchers then explored PRR-induced physiological responses and identified multiple signaling components. The activation of MAPKs by cell-surface receptors were reported back in 1997 and was verified for Cf- genes 2 years later. In tobacco (N. tabacum), the perception of PAMPs leads to the activation of wounding-induced protein kinase (WIPK) and SA-induced protein kinase (SIPK;; ). WIPKs and SIPKs are orthologs of the subsequently identified Arabidopsis MPK3 and MPK6, respectively. Accumulation of ROS and callose deposition during infection were also reported in 1997, and for Cf-initiated responses. Researchers identified the human Rbohs in Arabidopsis and showed that two of these (RbohD and RbohF) are required for ROS production during infection. It was unclear how these signaling components were activated by PRRs until the identification of the PRR co-receptors and RLCKs. BAK1 was identified as a co-receptor essential for FLS2-mediated resistance in 2007. In the same year, CERK1 was also shown to be essential for chitin-mediated immunity. In 2013, SOBIR1 was identified as a co-receptor of RLPs, and the structure of the FLS2/BAK1 receptor complex was also defined. In 2018, a genome-wide analysis of Arabidopsis LRR–RLKs interactions was reported, further supporting the theory that PRRs interact with each other to modulate and transduce signals. Tomato ACIK1 was the first RLCK shown to be an essential signaling component in PRR-mediated immunity (. The Arabidopsis ortholog BIK1 was subsequently shown to be a central PRR-signaling component. RbohD, MAPKKKs, and multiple calcium channels were shown to be phosphorylated by RLCKs, which leads to downstream immune responses (;;;;;;; Figure 8A).

56More than 60 immunity-related PRRs with known ligands have now been identified. Arabidopsis EFR has been introduced into multiple plant species, such as tomato, rice, orange, and apple, providing broadspectrum resistance to many bacteria. Therefore, the identification of novel PRRs that recognize PAMPs or other elicitors would provide resources to engineer disease-resistant crops. Other challenges in PRR biology include trying to understand how PRRs activate downstream signaling components and physiological responses, how these processes are regulated and suppressed by effectors, and how resistance against pathogens is achieved (Figure 8B).

57Arabidopsis RPS2 and the tobacco N gene were the first reported NLR genes. Multiple NLRs, including RPM1 and L6, were subsequently identified. Understanding how NLRs detect effectors has led to multiple models. The guard hypothesis was proposed to explain how the protein kinase Pto confers Prf-dependent recognition of AvrPto. Many other examples have emerged that are consistent with this hypothesis, such as the requirement of the protease Rcr3 for Cf-2-mediated resistance. The decoy model was then proposed, which is further supported by the discovery of integrated decoy domains in NLRs. The discovery of NRCs led to the concept of NLR networks. Following the identification of multiple NLRs, researchers identified multiple genetic components required for NLR-mediated immunity. These include EDS1, NDR1, PAD4, RPW8, SGT1, RAR1, HSP90, SAG101, NRG1s, and ADR1s. EDS1 was later shown to co-function with SAG101 and PAD4 to mediate HR and resistance during ETI. Similarly, ADR1 and NRG1 have been shown to function downstream of multiple sensor NLRs to mediate the HR and resistance. How sensor NLRs activate these signaling components is currently under investigation. v-cADPR produced by TIR domains might contribute to the activation of EP-proteins and helper NLRs. NLRs were shown to oligomerize and trigger cytosolic calcium influx following effector recognition. The discovery of the structures of multiple NLR resistosomes proved that the oligomerization of NLRs is required for resistance, likely through the formation of cation channels. However, oligomerization of TIR domains imposed by an NLRC4 scaffold is sufficient to activate defense (; Figure 8A).

58More than 140 NLRs with known recognized effectors have been identified. Cross-species transfer of NLR “stacks” provides durable resistance against pathogens. Identification of novel NLRs will provide resources to engineer crop resistance against multiple pathogens. Current challenges in NLR biology include understanding how NLRs activate downstream signaling components, how these signaling components then trigger immune responses, how these processes are regulated and suppressed by effectors, and how NLRs and PRRs co-function to achieve resistance against pathogens (Figure 8B).

59Plants respond to pathogens using a two-tier innate immune system activated by both cell-surface and intracellular immune receptors. The perception of PAMPs/MAMPs/DAMPs/HAMPs on the cell surface leads to PRR-mediated immunity, and the recognition of effectors leads to intracellular NLR-mediated immunity. The first plant Resistance (R) gene, Hm1, was cloned back in 1992. Many immune receptors have been identified since 1994, when the first PRR and NLRs were identified. Tremendous efforts have been made to understand the PRR- and NLR-signaling pathways. PRRs and NLRs utilize some overlapping but also unique signaling components to activate each of their downstream physiological responses, which thwart pathogen proliferation. Both signaling pathways are tightly regulated to prevent autoimmunity, while being suppressed by pathogen effectors. Recent studies have shown that PRR- and NLR-mediated immunity can be mutually potentiated and are dependent on each other. Great opportunities for novel discoveries remain in addressing the following challenges in the research of plant immunity: (1) identifying novel immune receptors; (2) understanding the signaling pathways and physiological responses triggered by both cell-surface and intracellular immune receptors; (3) understanding how immunity is intrinsically regulated and manipulated by external biotic and/or abiotic factors; (4) understanding the vastly diverse mechanisms by which plants resist pathogen infections; and (5) understanding how different immune systems function synergistically during infections. These challenges overlap with some of the “top 10 unanswered questions in molecular plant-microbe interactions” and will shape our understanding of plant immunity in the coming decades (Figure 8B).

60The following materials are available in the online version of this article.

61Supplemental Data Set 1. PRRs involved in plant immunity.

62Supplemental Data Set 2. NLRs involved in plant immunity.

63Supplemental Data Set 3. Expression of ETI-related genes during PTI.

64Supplemental Data Set 4. Expression of PTI-related genes during ETI.