Short title: Plant damage recognition · SAR depends on the immune signal 68 molecule salicylic acid (SA), whereas ISR relies on the signaling pathways activated by the plant 69 hormones

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Short title: Plant damage recognition 1

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Perception of damaged self in plants 3

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Qi Li, Chenggang Wang, and Zhonglin Mou* 5

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Department of Microbiology and Cell Science, University of Florida, P.O. Box 110700, 7

Gainesville, FL 32611, USA

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*Correspondence to: [email protected] 10

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One-sentence summary: Plants use specific receptor proteins on the cell surface to detect host-12

derived danger signals released in response to attacks by pathogens or herbivores and activate 13

immune responses against them. 14

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Author contributions: Q.L., C.W., and Z.M. conceived the content and wrote the article. 16

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Plant Physiology Preview. Published on January 6, 2020, as DOI:10.1104/pp.19.01242

Copyright 2020 by the American Society of Plant Biologists

www.plantphysiol.orgon June 9, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.

mailto:[email protected]://www.plantphysiol.org

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Multicellular eukaryotes including plants and animals have evolved highly complex, multi-20

layered immune systems to fight off microbial infections. How the immune systems function is a 21

fundamental question for immunologists. The animal immune system was originally thought to 22

function by distinguishing between “self “and “nonself ”(the Self-Nonself model) (Burnet, 23

1959), and later between “infectious-nonself” and “noninfectious-self” (the Infectious-Nonself 24

model) (Janeway, 1989, 1992). In 1994, Matzinger proposed that the immune system is more 25

concerned with “danger” than with “non-self” (the Danger model) (Matzinger, 1994, 2002, 26

2007). The Danger model suggests that the immune system is activated by danger/alarm signals 27

that are sent from both microbial pathogens and damaged host cells. In this model, it is assumed 28

that healthy cells or cells undergoing normal physiological death do not produce danger signals 29

(Matzinger, 2002). Over the years, the Danger model has been supported by the discovery of a 30

large number of endogenous danger signals (Tang et al., 2012; Pouwels et al., 2014; Schaefer, 31

2014; Hernandez et al., 2016; Yatim et al., 2017; Dinarello, 2018). 32

Danger signals consist of conserved pathogen-associated molecular patterns (PAMPs) 33

from the microbes and damage-associated molecular patterns (DAMPs) from injured host cells 34

(Matzinger, 2002). Although the term “DAMPs” originally referred to the hydrophobic portions 35

of biological molecules from dead and dying host and pathogen cells, which trigger immunity 36

when exposed (Seong and Matzinger, 2004), it is now generally used to describe danger signals 37

from damaged host cells (Martin, 2016; Yatim et al., 2017). Besides PAMPs and DAMPs, 38

pathogen-derived effectors, which are proteins expressed by pathogens to aid infection of their 39

hosts, and effector-caused perturbations on/in the host cells should also be considered as danger 40

signals, though they were not included in the original model (Matzinger, 2002; Boller and Felix, 41

2009). PAMPs/DAMPs and extracellular effectors or their disturbances are generally recognized 42

by germline-encoded cell-surface pattern recognition receptors (PRRs) (Takeuchi and Akira, 43

2010), whereas intracellular effectors or their interruptions are often sensed by cytoplasmic 44

nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) (Chen et al., 2009). 45

The plant immune system shares a similar conceptual logic with the animal immune 46

system, though plants lack adaptive immunity (Nurnberger et al., 2004; Haney et al., 2014). A 47

simple coevolutionary model called Zigzag model was proposed to describe the molecular events 48

in plant-microbe interactions (Jones and Dangl, 2006). Based on this model, plant cells employ 49

PRRs to detect PAMPs, activating PAMP-triggered immunity (PTI), while adapted pathogens 50


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utilize effectors to dampen PTI. Plants in turn exploit NLRs to sense the presence of effectors, 51

leading to effector-triggered immunity (ETI), which usually culminates in a hypersensitive cell 52

death response (HR) at the infection site. Natural selection constantly drives the arms race 53

between plants and pathogens, resulting in different levels of pathogen virulence and plant 54

resistance (Jones and Dangl, 2006). The Zigzag model has conceptually stimulated enormous 55

research in the plant-microbe interaction field; however, it did not encompass DAMPs. The 56

recent Invasion model included DAMPs and introduced a new term “invasion patterns”, which 57

essentially refers to the same type of molecules as “danger signals” (Cook et al., 2015). It was 58

suggested that adapting the Danger model for plants would allow the holistic concept of host 59

immunity to be better shared by the entire community of immunologists (Gust et al., 2017). 60

Nevertheless, neither the Zigzag model nor the Invasion model accommodates systemic 61

resistance, including systemic acquired resistance (SAR) and induced systemic resistance (ISR), 62

which are also essential parts of the plant immune system (Durrant and Dong, 2004; Pieterse et 63

al., 2014). SAR and ISR are two forms of induced systemic resistance wherein the plant immune 64

system is primed by a prior localized infection that results in resistance throughout the plant 65

against subsequent challenge by a broad spectrum of pathogens. However, induction of the two 66

forms of systemic resistance is mechanistically distinct. SAR depends on the immune signal 67

molecule salicylic acid (SA), whereas ISR relies on the signaling pathways activated by the plant 68

hormones jasmonic acid (JA) and ethylene (ET) (Durrant and Dong, 2004; Pieterse et al., 2014). 69

The SA, JA, and ET response pathways serve as the backbone of the plant immune signaling 70

network (Pieterse et al., 2012). 71

Compared to the large number of DAMPs that have been identified and characterized in 72

animals, research on DAMPs in plants has only just begun (Rubartelli and Lotze, 2007; Choi and 73

Klessig, 2016; Roh and Sohn, 2018). In the past several years, we have witnessed a marked 74

increase in the number of potential DAMPs in plants, and the number is still growing (Table 1) 75

(Duran-Flores and Heil, 2016; Gust et al., 2017; Hou et al., 2019). Moreover, potential receptors 76

for more than a dozen plant DAMPs have been identified (Gust et al., 2017; Hou et al., 2019). 77

Characterization of these receptors is expected to significantly boost DAMP research in plants. 78

While the DAMP field is blooming, the identity of DAMPs is under debate (Martin, 2016). It 79

was argued in animals that a canonical DAMP should (1) be only released from cells during 80

necrosis, (2) act through binding to cell-surface receptors, (3) be up-regulated, but not released, 81



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in response to PAMP detection or stress stimuli that are likely to pre-sage necrosis, (4) be 82

synergistic with PAMPs in activating robust immune responses, and (5) initiate relatively broad 83

acting responses in a manner similar to how pathogen components do (Martin, 2016). Based on 84

these characteristics, members of the extended interleukin-1 (IL-1) cytokine family (IL-1, IL-85

1, IL-18, IL-33, IL-36, IL-36, and IL-36) have been reasoned to be the canonical DAMPs 86

activating the immune system, whereas most other proposed DAMPs, e.g., ATP, uric acid, 87

calreticulin, HMGB1, HSPs, and DNA fragments, likely act through liberating IL-1 family 88

cytokines via promoting necrosis (Martin, 2016). 89

In plants, the identity of DAMPs has not been vigorously debated. Recently, 90

immunogenic plant factors were roughly divided into two categories, primary and secondary 91

DAMPs, which correspond to constitutive and inducible DAMPs proposed in animals (Gust et 92

al., 2017; Yatim et al., 2017). Primary/constitutive DAMPs are derived from pre-existing 93

structures or molecules, including breakdown products of extracellular matrix and passively 94

released intracellular molecules, while secondary/inducible DAMPs are actively processed and 95

released upon tissue damage and other stimuli (Gust et al., 2017). While this delineation of 96

primary DAMPs is aligned with the original definition of DAMPs (Matzinger, 2002; Seong and 97

Matzinger, 2004), it is worthwhile to compare the secondary DAMPs with the proposed 98

canonical DAMPs in animals (Martin, 2016). One central argument for members of the extended 99

IL-1 family being the canonical DAMPs in animals is that they do not possess N-terminal signal 100

sequences and are released during necrosis (Martin, 2016). In contrast, precursors of most of the 101

candidate peptide DAMPs in plants carry an N-terminal signal peptide (Table 1), suggesting 102

active release via the conventional secretion pathway. They would, nevertheless, also be 103

passively released upon cell damage during microbial infection and herbivore attack. Thus, 104

besides being DAMPs under pathological conditions, such molecules may function in normal 105

physiological processes. 106

In this review, we focus on several proposed plant primary and secondary DAMPs and 107

their receptors, which have been shown to physically bind each other. For a complete inventory 108

of potential DAMPs in plants, we refer interested readers to several recent excellent reviews and 109

references therein (Choi and Klessig, 2016; Duran-Flores and Heil, 2016; Gust et al., 2017; Hou 110

et al., 2019). A new item that was recently added to the inventory is the Arabidopsis 111

(Arabidopsis thaliana) SCOOP12 peptide, which is perceived by plants in a 112



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BRASSINOSTEROID INSENSITIVE1 (BRI1)-ASSOCIATED KINASE1 (BAK1) co-receptor-113

dependent manner (Table 1) (Gully et al., 2019). We explore potential roles of DAMPs in plant 114

immunity, particularly in SAR. Future perspectives of DAMPs in plants are also discussed. 115

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PRIMARY/CONSTITUTIVE DAMP-RECEPTOR PAIRS 117

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OGs-WAK1 119

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Oligogalacturonides (OGs) are degradation products of the primary cell wall component pectin, a 121

complex polysaccharide comprising mainly esterified D-galacturonic acid residues in -(1-4)-122

chain (Cote and Hahn, 1994; Ferrari et al., 2013; Kohorn, 2016). Pectin is partially degraded by 123

pathogen- or plant-derived enzymes during pathogen infection or herbivore attack, resulting in 124

oligomers of D-galacturonic acids with varying degrees of polymerization (Bishop et al., 1981; 125

Cote and Hahn, 1994; Bergey et al., 1999; An et al., 2005). OGs with a degree of polymerization 126

between 10 and 15 are potent elicitors (Côté and Hahn, 1994; Moscatiello et al., 2006; Ferrari et 127

al., 2007; Denoux et al., 2008), able to induce reactive oxygen species (ROS) production, MAP 128

kinase activation, callose deposition, defense protein accumulation, and resistance to the 129

necrotrophic fungal pathogen Botrytis cinerea in multiple plant species (Hahn et al., 1981; Davis 130

and Hahlbrock, 1987; Broekaert and Peumans, 1988; Bellincampi et al., 2000; Aziz et al., 2004; 131

Denoux et al., 2008; Galletti et al., 2008; Rasul et al., 2012). Short OGs with a degree of 132

polymerization between two and six have also been shown to elicit immune responses, but the 133

effect of short OGs on the expression of immune-related genes appears to be not as strong as that 134

of long OGs (Moloshok et al., 1992; Davidsson et al., 2017). 135

WALL-ASSOCIATED KINASE (WAK) proteins are proposed receptors of OGs 136

(Kohorn and Kohorn, 2012; Ferrari et al., 2013). WAKs are receptor-like kinases, with an 137

extracellular domain containing epidermal growth factor motifs, a transmembrane domain, and 138

an intracellular Ser/Thr kinase domain (He et al., 1996; Anderson et al., 2001). There are five 139

WAK and 21 WAK-LIKE genes in Arabidopsis (Anderson et al., 2001; Verica and He, 2002). 140

Biochemical analyses suggested that WAK1 is tightly associated with pectin (He et al., 1996; 141

Wagner and Kohorn, 2001). The extracellular domains of WAK1 and WAK2 indeed bind pectin 142

in vitro (Kohorn et al., 2009). A recombinant peptide containing amino acids 67-254 of the 143



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extracellular domain of WAK1 (called WAK67-254) binds polygalacturonic acid (PGA), OGs, 144

pectins, and structurally-related alginates (Decreux and Messiaen, 2005). At least five specific 145

amino acids in the extracellular domain of WAK1 are involved in the interaction with PGA 146

(Decreux et al., 2006). Interestingly, binding of WAK67-254 to PGA, OGs, and alginates depends 147

on Ca2+

and ionic conditions that promote formation of Ca2+

bridges between oligomers or 148

polymers, resulting in a structure known as an egg-box dimer, which significantly enhances 149

binding to WAK1 and induces increased extracellular alkalinization when applied to Arabidopsis 150

cell suspensions (Decreux and Messiaen, 2005; Cabrera et al., 2008). 151

Multiple lines of genetic evidence strongly support that WAKs are OG receptors and 152

function in plant immune responses. First, a chimeric receptor with the extracellular domain of 153

WAK1 and the kinase domain of ELONGATION FACTOR Tu (EF-Tu) receptor (EFR) 154

responds to OGs and activates the kinase domain, and conversely, elf18, a polypeptide consisting 155

of the first 18 amino acids at the N-terminus of EF-Tu, activates a chimeric receptor formed by 156

the EFR ectodomain and the kinase domain of WAK1 and induces the typical responses 157

triggered by OGs (Brutus et al., 2010). Second, pectin- and OG-induced transcription of a 158

number of genes depends on WAK2 in Arabidopsis protoplasts (Kohorn et al., 2009; Kohorn et 159

al., 2012). Third, pathogen infection and SA treatment induce WAK1 gene expression and the 160

induction depends on NONEXPRESSOR OF PATHOGENESIS-RELATED (PR) GENES1 161

(NPR1), a key immune regulator (Cao et al., 1997; He et al., 1998). SA also induces the 162

expression of WAK2, WAK3, and WAK5 (He et al., 1999), and WAK1 and WAK2 are wound 163

inducible as well (Wagner and Kohorn, 2001). Fourth, overexpression of WAK1 enhances 164

tolerance to SA toxicity, and expression of an antisense allele of WAK1 reduces the level of PR1 165

gene expression induced by the biologically active analogue of SA, 2.6-dichloroisonicotinic acid 166

(He et al., 1998). Finally, a dominant gain-of-function WAK2 allele, WAK2cTAP, exhibits 167

autoimmune phenotypes including ROS accumulation and cell death (Kohorn et al., 2009; 168

Kohorn et al., 2012). Importantly, the stunted growth phenotype of WAK2cTAP is largely 169

suppressed by mutations in the key immune regulators, ENHANCED DISEASE 170

SUSCEPTIBILITY1, PHYTOALEXIN DEFICIENT4, and MAP KINASE6 (MPK6) genes (Kohorn 171

et al., 2012; Kohorn et al., 2014), which is reminiscent of autoimmune phenotypes (van Wersch 172

et al., 2016). 173

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eATP-DORN1 175

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Extracellular ATP (eATP) is one of the best-studied DAMPs in animals. As the energy currency, 177

cellular levels of ATP are normally maintained in the range of 1-10 mM. In animals, ATP is 178

constitutively released into the extracellular space through various mechanisms including ATP 179

binding cassette transporters, vesicular exocytosis, gap junctions, and pannexin hemichannels, as 180

well as the P2X7 receptor (Lazarowski et al., 2003; Spray et al., 2006; Suadicani et al., 2006; 181

Zhang et al., 2007). ATP also leaks into the extracellular milieu upon cell lysis or necrosis during 182

tissue damage and inflammation (la Sala et al., 2003). Once in the extracellular milieu, ATP 183

binds to either P2X ligand-gated channels or P2Y G-protein coupled receptors, triggering 184

outside-in signaling including changes in intracellular [Ca2+

], production of cytokines, and cell 185

death (Hattori and Gouaux, 2012; Jacobson et al., 2015). Depending on the tissue and cell types, 186

eATP signaling acts in both normal physiological and abnormal pathological processes in 187

animals (Trautmann, 2009). 188

In plants, research with exogenous ATP can be traced back to the 1960s (Jaffe and 189

Galston, 1966). However, it was unclear in the early studies whether the exogenously added ATP 190

functioned as a signal molecule, a precursor, or energy supply (Jaffe and Galston, 1966; 191

Williamson, 1975; Kamizyo and Tanaka, 1982; Nejidat et al., 1983). Recent studies with the 192

widely used stable ATP analog, adenosine 5’-[-thio]triphosphate (ATPS), suggested that eATP 193

might act as a signal molecule in the apoplast (Jeter et al., 2004; Song et al., 2006; Torres et al., 194

2008; Clark et al., 2010; Clark et al., 2011). The presence of eATP was proven by directly 195

measuring ATP accumulation in Arabidopsis leaves and roots (Thomas et al., 1999; Demidchik 196

et al., 2003; Deng et al., 2015), and active secretion of ATP in plants was confirmed by feeding 197

Arabidopsis cultures with [32

P]-H3PO4 and monitoring radiolabeled ATP in the extracellular 198

matrix (Chivasa et al., 2005). Furthermore, the distribution of eATP in plants was directly 199

visualized using luciferase reporters including a cellulose-binding domain-luciferase fusion, an 200

ecto-luciferase, and infiltration of a luciferase/luciferin mixture (Kim et al., 2006; Chivasa et al., 201

2009; Clark et al., 2011). These tools allowed discoveries of the dynamics of eATP accumulation 202

in roots, leaves, and around guard cells (Kim et al., 2006; Chivasa et al., 2009; Clark et al., 203

2011). 204



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The constitutive eATP appears to be essential for plant cell viability. Depletion of basal 205

eATP using the cell-impermeant traps glucose-hexokinase and apyrase triggers cell death in both 206

Arabidopsis cell cultures and whole plants (Chivasa et al., 2005). Competitive exclusion of eATP 207

from its binding sites with nonhydrolyzable ATP analog ,-methyleneadenosine 5’-triphosphate 208

also results in cell death in Arabidopsis, maize (Zea mays), bean (Phaseolus vulgaris), and 209

tobacco (Nicotiana tabacum) (Chivasa et al., 2005). Interestingly, the programmed cell death-210

eliciting mycotoxin fumonisin B1-induced cell death in Arabidopsis seems to be mediated by 211

depletion of eATP (Chivasa et al., 2005). Furthermore, environmental stresses induce ATP 212

release (Clark et al., 2011; Sun et al., 2012; Lim et al., 2014; Deng et al., 2015). Although the 213

biological relevance of the increases in endogenous eATP levels remains to be fully elucidated, 214

studies with exogenous ATP and/or ATPS have shown that eATP induces ROS and nitric oxide 215

production, Ca2+

influx, and H+ efflux in a G protein subunit and RESPIRATORY BURST 216

OXIDASE HOMOLOG (RBOH)-dependent manner (Jeter et al., 2004; Song et al., 2006; Foresi 217

et al., 2007; Wu et al., 2008; Wu and Wu, 2008; Demidchik et al., 2009; Clark et al., 2011; Hao 218

et al., 2012; Sun et al., 2012). Intriguingly, plants appear to respond to eATP in a dose-dependent 219

manner. Low doses of eATP induce stomatal opening, accelerate vesicular trafficking, and 220

stimulate cell elongation, whereas high doses of eATP trigger stomatal closure, inhibit vesicular 221

trafficking, and suppress cell elongation (Clark et al., 2010; Clark et al., 2011; Clark et al., 2013; 222

Wang et al., 2014; Deng et al., 2015). Although depletion of eATP or exclusion of eATP from its 223

binding sites leads to cell death, high doses of eATP also reduce cell viability (Sun et al., 2012; 224

Deng et al., 2015). Currently, the molecular mechanisms underlying such biphasic responses are 225

unknown. 226

Identification of the eATP receptor DOES NOT RESPOND TO NUCLEOTIDES1 227

(DORN1) in Arabidopsis is a major breakthrough in eATP biology and provided a key to 228

addressing many questions about eATP (Choi et al., 2014a; Roux, 2014). DORNI is a L-type 229

(legume-like) lectin receptor kinase (LecRK), LecRK-I.9, which had previously been shown to 230

recognize RGD (arginine-glycine-aspartic acid) tripeptide motif-containing protein in mediating 231

plasma membrane-cell wall adhesions (Gouget et al., 2006). The extracellular domain of 232

DORN1 binds ATP with a dissociation constant (Kd) of ~46 nM (Choi et al., 2014a). A point 233

mutation in the DORN1 gene completely blocks exogenous ATP-induced transcriptional changes 234

in Arabidopsis seedlings, indicating that DORN1 is the major, if not the sole, receptor of eATP 235



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(Choi et al., 2014a). However, as eATP plays an important role in plant growth, development, 236

and cell viability (Tang et al., 2003; Chivasa et al., 2005; Clark and Roux, 2011; Liu et al., 2012; 237

Yang et al., 2015), but dorn1 mutants do not have obvious growth and developmental defects 238

(Choi et al., 2014a), it has been suggested that there might be other eATP receptors mainly 239

regulating plant growth signaling (Roux, 2014). 240

It was recently proposed that eATP functions as a DAMP in plants (Choi et al., 2014b; 241

Tanaka et al., 2014). Indeed, eATP levels at the wound sites reach ~40 M, well above the 242

concentration needed to induce ROS production and gene expression (Choi et al., 2014a), and 243

reducing eATP levels by overexpressing an apyrase suppresses wound responses (Song et al., 244

2006; Wang et al., 2019b). Furthermore, ~60% of the genes induced by exogenous ATP are also 245

induced by wounding (Choi et al., 2014a), and ATP mainly activates JA signaling through MYC 246

transcription factors (Tripathi et al., 2018; Jewell et al., 2019). Therefore, eATP clearly plays an 247

important role in wound responses. Furthermore, exogenous ATP induces resistance to the 248

necrotrophic fungal pathogen B. cinerea in Arabidopsis (Tripathi et al., 2018), suggesting a 249

potential role for eATP in immunity against fungal pathogens. Interestingly, although more than 250

a dozen ATP-induced genes depend on NPR1 (Jewell et al., 2019), eATP and SA antagonize 251

each other (Chivasa et al., 2009). Exogenous ATP reduces basal SA levels, whereas SA 252

treatment triggers collapse of eATP in tobacco leaves (Chivasa et al., 2009). In line with these 253

results, exogenous ATP does not induce apoplastic resistance to Pseudomonas syringae pv. 254

maculicola ES4326 (Psm) in Arabidopsis (Zhang and Mou, 2009). On the other hand, eATP 255

plays an important positive role in stomatal immunity. In Arabidopsis, bacterial infection induces 256

ATP release, particularly around guard cells, and exogenous ATP induces stomatal closure and 257

stomatal resistance against bacterial pathogens in a concentration-dependent manner (Chen et al., 258

2017). Importantly, exogenous ATP-induced stomatal movement and resistance depend on 259

DORN1 and RBOHD. It was proposed that eATP activates DORN1, which in turn 260

phosphorylates the N terminus of RBOHD, leading to ROS production that induces stomatal 261

closure (Chen et al., 2017). 262

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eNAD(P)-LecRK-I.8/VI.2 264

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It is well known that extracellular nicotinamide adenine dinucleotide (phosphate) [eNAD(P)] 266

plays a significant role in animal immune responses (Billington et al., 2006; Haag et al., 2007; 267

Adriouch et al., 2012). However, whether eNAD(P) is a DAMP in animals remains elusive (Roh 268

and Sohn, 2018). Under normal conditions, intracellular NAD+ levels are in the range of 0.2-0.5 269

mM (Canto et al., 2015), whereas eNAD levels, e.g., in mammalian serum, are around 0.1 M 270

(Zocchi et al., 1999; O'Reilly and Niven, 2003). Cell lysis during tissue damage and 271

inflammation presumably can lead to dramatic increases in eNAD(P) levels (Billington et al., 272

2006). At least three distinct mechanisms perceive eNAD(P) in animals. First, eNAD(P) can be 273

processed by a number of NAD(P)-metabolizing ectoenzymes such as CD38 and CD157, which 274

have ADP-ribosyl cyclase, cyclic ADP-ribose (cADPR)-hydrolase and NAD-hydrolase 275

activities, into Ca2+

-mobilizing second messengers cADPR and nicotinic acid adenine 276

dinucleotide phosphate (Ceni et al., 2003; Partida-Sanchez et al., 2003; De Flora et al., 2004; 277

Heidemann et al., 2005; Malavasi et al., 2006). Second, eNAD+ is a substrate of the 278

glycosylphosphatidylinositol (GPI)-anchored or secreted ectoenzymes known as mono(ADP-279

ribosyl)transferases in ADP-ribosylation of plasma membrane signaling proteins (Nemoto et al., 280

1996; Han et al., 2000; Bannas et al., 2005). Finally, eNAD(P) is a potential agonist of plasma 281

membrane receptors. It has previously been shown that NAD+ binds to rat brain synaptic 282

membranes and is a potential inhibitory neurotransmitter (Khalmuradov et al., 1983; Mutafova-283

Yambolieva et al., 2007). Recent studies have suggested that several purinergic P2X and P2Y 284

receptors function in eNAD(P)-triggered biological responses (Moreschi et al., 2006; Mutafova-285

Yambolieva et al., 2007; Grahnert et al., 2009; Klein et al., 2009). Nevertheless, binding between 286

NAD(P) and these receptors has not been reported. 287

In plants, intracellular NAD(P) levels are in the range of 1-2 mM (Noctor et al., 2006). 288

We found that, upon wounding and bacterial infection, NAD(P) concentrations in the 289

extracellular washing fluid are comparable to those from infiltration with ~0.7 and ~1.2 mM 290

NAD(P), respectively (Zhang and Mou, 2009). We also showed that treatment of Arabidopsis 291

and citrus plants with 0.2 mM NAD(P) significantly induces resistance to bacterial pathogens, 292

but not to the necrotrophic fungal pathogen B. cinerea (Zhang and Mou, 2009; Wang et al., 293

2016; Alferez et al., 2018). Importantly, exogenously applied NAD(P) does not change 294

intracellular NAD(P) homeostasis (Zhang and Mou, 2009), suggesting that it acts in the apoplast. 295

Furthermore, we found that transgenic expression of the human CD38 gene in Arabidopsis 296



11

reduces eNAD(P) concentrations and partially compromises SAR (Zhang and Mou, 2012). These 297

results together indicate that the eNAD(P) accumulated during pathogen infection is both 298

necessary and sufficient for activation of plant immune responses. In addition, exogenous 299

NAD(P) induces ROS production and changes in cytosolic [Ca2+

] (Pétriacq et al., 2016b; 300

Pétriacq et al., 2016a). Thus, eNAD(P) is a DAMP in plants. 301

Using a reverse genetic approach based on exogenous NAD+-induced transcriptome 302

changes in Arabidopsis, we have identified two potential eNAD(P) receptors, LecRK-I.8 and 303

LecRK-VI.2, both of which are L-type LecRKs (Singh et al., 2012; Wang et al., 2017a; Wang et 304

al., 2019a). The LecRK-I.8 and LecRK-VI.2 genes can be induced by exogenous NAD+, and both 305

LecRK-I.8 and LecRK-VI.2 are localized in the plasma membrane and have kinase activity (Xin 306

et al., 2009; Singh et al., 2013; Wang et al., 2017a). However, the two receptors are not alike. 307

LecRK-I.8 only binds NAD+ (Kd, ~437 nM), whereas LecRK-VI.2 binds both NAD

+ and 308

NADP+ with a slightly higher affinity for NADP

+ (Wang et al., 2017; Wang et al., 2019a). 309

LecRK-VI.2 binds 32

P-NAD+ with a Kd of ~787 nM, and the binding can be effectively 310

competed by unlabeled NAD+ (50% inhibition concentration, IC50, 1,887 nM) and NADP

+ (IC50, 311

945 nM) (Wang et al., 2019a). Consistently, mutations in LecRK-I.8 and LecRK-VI.2 suppress 312

NAD+- and NADP

+-induced immune responses, respectively (Wang et al., 2017; Wang et al., 313

2019a). Interestingly, the lecrk-I.8/VI.2 double mutant behaves like lecrk-I.8 for NAD+ responses 314

and like lecrk-VI.2 for NADP+ responses, indicating that the two receptors function in two 315

separate pathways (Wang et al., 2019a). Importantly, mutations in LecRK-I.8 and LecRK-VI.2 316

significantly compromise basal immunity and biological induction of SAR, respectively (Wang 317

et al., 2017a; Wang et al., 2019a), indicating that LecRK-I.8 primarily functions in basal 318

immunity, whereas LecRK-VI.2 plays a major role in SAR. 319

The leucine-rich repeat receptor kinase (LRR-RK) BAK1 is a co-receptor of a group of 320

LRR-RK receptors including BRI1, PRR FLAGELLIN-SENSITIVE2 (FLS2), EFR, and PEP 321

RECEPTOR1 (PEPR1)/2 (Li et al., 2002a; Nam and Li, 2002; Chinchilla et al., 2007; Heese et 322

al., 2007; Postel et al., 2010; Schulze et al., 2010; Roux et al., 2011). BAK1 is also required for 323

signaling triggered by several other potential DAMPs including the Arabidopsis HMGB3 protein 324

and the SCOOP12 peptide (Choi et al., 2016; Gully et al., 2019). BAK1 and LecRK-VI.2 form a 325

complex in vivo and function in eNAD(P) signaling and SAR (Wang et al., 2019). The 326

interaction between BAK1 and LecRK-VI.2 appears to be constitutive and independent of 327



12

eNAD(P), which is different from the inducible associations between BAK1 and LRR-RK 328

receptors. Moreover, the bak1-5 mutation has been shown to impair signaling mediated by the 329

non-RD kinases FLS and EFR, but not that mediated by the RD kinase BRI1 (Schwessinger et 330

al., 2011). Interestingly, although LecRK-VI.2 is an RD kinase, eNAD(P) signaling is 331

significantly inhibited in bak1-5 (Wang et al., 2019). In addition, it has been shown that C-332

terminal tags on BAK1 have limited effects on several BR responses, but strongly impact PTI 333

signaling (Ntoukakis et al., 2011). Surprisingly, a BAK1-GFP fusion protein is able to 334

complement the defects of bak1-5 in NADP+-induced immune responses and biological 335

induction of SAR (Wang et al., 2019). Since C-terminally tagged BAK1 fusion proteins are not 336

phosphorylated at S612 upon PAMP treatment (Perraki et al., 2018), it would be interesting to 337

test whether S612 phosphorylation in BAK1 is required for eNAD(P) signaling and SAR. 338

Interestingly, exogenously added NAD+ moves systemically and induces systemic 339

resistance (Wang et al., 2019), suggesting that eNAD(P) might be an SAR mobile signal. 340

Consistently, high levels of exogenous NAD(P) induces SA accumulation and NADPH oxidase-341

independent ROS production (Zhang and Mou, 2009; Pétriacq et al., 2016b). Surprisingly, 342

exogenous NAD(P)-induced systemic resistance does not depend on the putative SAR mobile 343

signals pipecolic acid (Pip), N-hydroxy-Pip (NHP), azelaic acid (AzA), and glycerol-3-phosphate 344

(G3P), but requires an intact SA signaling pathway (Wang et al., 2019). Although DEFECTIVE 345

IN INDUCED RESISTANCE1 (DIR1) and ROS have not been tested for systemic resistance, 346

exogenous NAD(P)-induced local resistance and PR gene expression is independent of DIR1 and 347

NADPH oxidase, respectively (Zhang and Mou, 2009; Wang et al., 2019). It appears that 348

eNAD(P) functions either downstream or independently of the putative SAR mobile signals Pip, 349

NHP, AzA, G3P, DIR1, and ROS in both local and systemic resistance. Furthermore, although 350

exogenous eNAD(P) requires SA signaling for immune response activation, SA induces the 351

expression of LecRK-VI.2 in an NPR1-dependent manner (Wang et al., 2019). In addition, since 352

Pip, ROS, AzA, and G3P form a signaling amplification loop (Wang et al., 2018a), it is possible 353

that ROS produced in the amplification loop causes reversible or irreversible damages to the 354

plasma membrane (Cwiklik and Jungwirth, 2010; Tero et al., 2016), leading to leakage of 355

cellular NAD(P) into the apoplast. Thus, the interplay between eNAD(P) and SA as well as other 356

SAR signal molecules is complicated and deserves further investigation. 357

358



13

Glutamate-GLR3.3/3.6 359

360

Glutamate is the most prominent neurotransmitter in the brain and excites postsynaptic neural 361

cells through different types of receptors including ionotropic and metabotropic glutamate 362

receptors (Brassai et al., 2015). Ionotropic glutamate receptors (iGluRs) are ligand-gated 363

channels that are activated upon glutamate binding (Krieger et al., 2019). The Arabidopsis 364

genome encodes 20 GLUTAMATE-RECEPTORs (GLRs) that are homologous to iGluRs (Chiu 365

et al., 2002). GLRs carry the same signature domains as animal iGluRs, including the ‘three-366

plus-one’ transmembrane domains and the extracellular ligand-binding domains (Lam et al., 367

1998; Chiu et al., 1999; Lacombe et al., 2001). Upon herbivore and mechanical damage, 368

glutamate is released into the apoplast where it activates GLR3.3 and GLR3.6, triggering long-369

distance electric and Ca2+

signaling as well as JA accumulation and defense gene expression in 370

undamaged leaves (Mousavi et al., 2013; Toyota et al., 2018). At least six amino acids (glutamic 371

acid, glycine, alanine, serine, asparagine, and cysteine) and the tripeptide glutathione can also 372

serve as agonists of GLR3.3 and induce membrane depolarization and cytosolic [Ca2+

] elevation 373

in a GLR3.3-dependent manner (Qi et al., 2006; Stephens et al., 2008; Li et al., 2013). Moreover, 374

seven out of the 20 standard amino acids (methionine, tryptophan, phenylalanine, leucine, 375

tyrosine, asparagine, and threonine) activate GLR1.4 transiently expressed in Xenopus oocytes to 376

various extents, and methionine-induced membrane depolarization in Arabidopsis leaves 377

depends on GLR1.4 (Tapken et al., 2013). 378

Interestingly, several amino acids have been shown to induce disease resistance in plants. 379

For instance, histidine induces ET biosynthesis and ET-related defense gene expression as well 380

as resistance to the soil-borne bacterial pathogen Ralstonia solanacearum and the fungal 381

pathogen B. cinerea partially in an ET-dependent manner in tomato (Solanum lycopersicum) and 382

Arabidopsis (Seo et al., 2016). Glutamate induces several genes of the SA signaling pathway in 383

rice (Oryza sativa) and tomato fruit, and enhances resistance to Magnaporthe oryzae and 384

Alternaria alternata in rice and tomato fruit, respectively (Kadotani et al., 2016; Yang et al., 385

2017). Surprisingly, other amino acids except tryptophan and tyrosine also improve rice 386

resistance to M. oryzae to various degrees (Kadotani et al., 2016). Furthermore, cysteine, aspartic 387

acid, and GSH enhance resistance to P. syringae pv. tomato (Pst) DC3000 in Arabidopsis (Li et 388

al., 2013). Importantly, cysteine- and GSH-induced disease resistance depends on GLR3.3, and 389



14

mutations of the GLR3.3 gene compromise resistance to Pst DC3000 and Hyaloperonospora 390

arabidopsidis in Arabidopsis (Li et al., 2013; Manzoor et al., 2013), suggesting that GLR3.3 is a 391

potential receptor for cysteine and GSH released into the apoplast during pathogen infection. 392

393

SECONDARY/ INDUCIBLE DAMP-RECEPTOR PAIRS 394

395

Systemin-SYR1/2 396

397

Systemin is the first reported extracellular peptide that induces defense signaling in plants. It was 398

purified from tomato leaf extracts using high-performance liquid chromatography based on its 399

proteinase inhibitor gene-inducing activity (Pearce et al., 1991). Systemin is an 18-amino acid 400

(aa) peptide processed from a 200-aa precursor named prosystemin (Pearce et al., 1991; 401

Beloshistov et al., 2018). Genes encoding well-conserved prosystemins were identified in the 402

Solanaceae species tomato, potato, bell pepper, and nightshade, but not in tobacco (McGurl et 403

al., 1992; Constabel et al., 1998). The tomato prosystemin gene is constitutively expressed 404

throughout the plant except in the roots, and is further induced by wounding (McGurl et al., 405

1992). The prosystemin protein accumulates in the cytosol and nucleus of vascular parenchyma 406

cells in response to wounding and methyl JA (MeJA) treatment (Narvaez-Vasquez and Ryan, 407

2004). Prosystemin does not carry an N-terminal signal sequence and, upon cell damage, is 408

expected to passively leak into the apoplast where it is processed by phytaspases and possibly 409

leucine aminopeptidase A (Ryan and Pearce, 1998; Beloshistov et al., 2018). Systemin is highly 410

active. When supplied to the cut stems of young tomato plants, ~40 fmol of systemin per plant is 411

sufficient to induce half maximal accumulation of two wound-inducible proteinase inhibitors that 412

break the activity of digestive enzymes in the insect midgut (Green and Ryan, 1972; Pearce et 413

al., 1991). Overexpression of the presystemin gene leads to constitutive synthesis of the 414

proteinase inhibitors (McGurl et al., 1994). 415

Although exogenously supplied systemin moves systemically, systemin may not be the 416

mobile signal mediating systemic wound responses. Grafting experiments with tomato JA 417

biosynthesis and recognition mutants indicated that systemic wound signaling requires both 418

biosynthesis of JA at the wound site and recognition of a JA signal in remote tissues, suggesting 419

that JA controls the production of or acts as the mobile wound signal (Li et al., 2002b). It was 420



15

proposed that systemin promotes systemic wound signaling by augmenting JA biosynthesis in 421

the vascular tissues (Schilmiller and Howe, 2005). 422

Identification of the receptor of systemin was a daunting task. A 160-kDa systemin-423

binding protein named SR160 was initially purified from plasma membranes of tomato 424

suspension cells using a photoaffinity analog of systemin (Scheer and Ryan, 2002). SR160 425

turned out to be the tomato homolog of the steroid hormone brassinolide receptor BRI1 (Scheer 426

and Ryan, 2002; Scheer et al., 2003). Later studies indicated that, although SR160 increases 427

binding of systemin to tobacco plasma membranes, it does not mediate systemin-triggered 428

defense responses (Holton et al., 2007; Lanfermeijer et al., 2008; Malinowski et al., 2009). Two 429

distinct LRR-RKs termed SYR1 and SYR2 were recently identified as the bona fide systemin 430

receptors (Wang et al., 2018b). Tobacco leaves expressing SYR1 and SYR2 respond with an 431

EC50 of ~0.03 and >30 nM systemin based on systemin-induced ROS production, respectively 432

(Wang et al., 2018b). Importantly, systemin is unable to induce production of ET and expression 433

of the proteinase inhibitor gene PIN1 in tomato mutant lines lacking functional SYR1 and SYR2 434

(Wang et al., 2018b). Surprisingly, mechanical wounding still induces local and systemic 435

expression of the PIN1 gene, though tomato plants expressing a prosystemin antisense gene 436

accumulate less than 40% of the wild-type level of proteinase inhibitor I (McGurl et al., 1992; 437

Wang et al., 2018b). Nevertheless, both the prosystemin antisense lines and the receptor mutant 438

line support significantly better herbivore larval growth than wild type (McGurl et al., 1992; 439

Wang et al., 2018b), demonstrating that systemin signaling contributes to resistance against 440

insect herbivores in tomato. 441

442

Peps-PEPR1/2 443

444

The first plant elicitor peptide (Pep), Pep1, was isolated as a 23-aa peptide from extracts of 445

Arabidopsis leaves, which is derived from the C-terminus of a 92-aa precursor protein encoded 446

by the PROPEP1 gene (Huffaker et al., 2006). The PROPEP1 protein does not carry an N-447

terminal signal peptide (Huffaker et al., 2006). It has been shown that PROPEP1 is processed by 448

Ca2+

-dependent type-II metacaspases in Arabidopsis (Hander et al., 2019; Shen et al., 2019). The 449

Arabidopsis genome carries eight PROPEP genes, PROPEP1-8 (Huffaker et al., 2006; Bartels et 450

al., 2013). PROPEP1, 2, 3, 5, and 8 are expressed in the roots and slightly in the leaf vasculature, 451



16

and are inducible by wounding, and expression of PROPEP4 and 7 is restricted to the root tip 452

and is not inducible by wounding (Bartels et al., 2013). Expression of PROPEP1, 2, and 4 is 453

inducible by MeJA, whereas that of PROPEP2 and 3 is inducible by methyl SA (MeSA) 454

(Huffaker and Ryan, 2007). PROPEP2 and 3 are also inducible by pathogen attacks and elicitors 455

derived from pathogens (Huffaker et al., 2006). Furthermore, expression of PROPEP1 is 456

strongly induced by Pep1-3, PROPEP2 and 3 are strongly induced by Pep1-6, PROPEPE 4 and 457

5 are weakly inducible, and PROPEP6 is not inducible by the peptides (Huffaker et al., 2006; 458

Huffaker and Ryan, 2007; Yamaguchi et al., 2010). Interestingly, while PROPEP3-YFP is 459

localized in the cytoplasm, PROPEP1-YFP and PROPEP6-YFP are associated with the tonoplast 460

(Bartels et al., 2013). The different gene expression patterns and localization suggest non-461

redundant roles among the members of the PROPEP family. Based on the responses of PROPEP 462

gene promoters to various stimuli, PROPEP genes were classified into four groups, with 463

PROPEP1 in the first group, PROPEP2 and 3 in the second group, PROPEP4, 7, and 8 in the 464

third group, and PROPEP5 in the fourth group (Safaeizadeh and Boller, 2019). Nevertheless, all 465

Peps, when applied exogenously, activate MPK3 and MPK6, induce ethylene production, and 466

inhibit seedling growth (Bartels et al., 2013). Exogenous Peps also induces expression of several 467

defense genes including PDF1.2, MPK3, and WRKY33, production of ROS, elevation of 468

cytosolic [Ca2+

], and resistance to the bacterial pathogen Pst DC3000 (Huffaker et al., 2006; Qi 469

et al., 2010; Yamaguchi et al., 2010). Pep1 also induces resistance against B. cinerea (Liu et al., 470

2013). Overexpression of PROPEP1 and PROPEP2 in Arabidopsis results in constitutive 471

PDF1.2 expression and/or resistance against a root oomycete pathogen Pythium irregulare 472

(Huffaker et al., 2006). 473

The first Pep receptor, a LRR-RK called PEP RECEPTOR1 (PEPR1), was purified from 474

Arabidopsis suspension cells using a photoaffinity analog of Pep1, 125

I1-Tyr-Pep1 (Yamaguchi et 475

al., 2006). 125

I1-Tyr-Pep1 is as active as Pep1 and binds to Arabidopsis suspension cells with a 476

Kd of ~0.25 nM (Yamaguchi et al., 2006). The second Pep receptor, PEPR2, was identified by 477

phylogenetic analysis and searching for the most closely related gene to PEPR1 (Yamaguchi et 478

al., 2010). Transgenic tobacco cells expressing PEPR1 and PEPR2 bind 125

I1-Tyr-Pep1 with Kd’s 479

of 0.56 and 1.25 nM, respectively. PEPR1 and PEPR2 also bind Pep2-6 and Pep2, respectively 480

(Yamaguchi et al., 2010). Both PEPRs carry a guanylyl cyclase (GC) catalytic domain with 481

residues for catalysis being conserved (Qi et al., 2010; Yamaguchi et al., 2010), and the GC 482



17

activity of PEPR1 has been experimentally demonstrated (Qi et al., 2010). It has been shown that 483

Pep1 induces rapid formation of a heterocomplex containing de novo phosphorylated BAK1 and 484

a ~160 kDa polypeptide that is expected to be PEPR1 (Schulze et al., 2010), while Pep2 induces 485

PEPR1 association with BAK1, BAK1-LIKE1, SOMATIC EMBRYOGENESIS RECEPTOR-486

LIKE KINASE1 (SERK1), and SERK2 in N. benthamiana (Yamada et al., 2016). Consistently, 487

the kinase domains of PEPR1 and PERP2 interact with that of BAK1 in yeast (Postel et al., 488

2010), and disruption of BAK1 sensitizes PEPR signaling (Yamada et al., 2016). The kinase 489

domain of PEPR1 also interacts with and directly phosphorylates the receptor-like cytoplasmic 490

kinase BOTRYTIS-INDUCED KINASE1 (BIK1) and BIK is required for Pep1-induced 491

resistance against B. cinerea (Liu et al., 2013). 492

Expression of PEPR1 and PEPR2 is inducible by wounding, MeJA, most Peps, and 493

PAMPs such as flg22 (a 22-amino acid peptide corresponding to the N terminus of bacterial 494

flagellin) and elf18 (Yamaguchi et al., 2010). It appears that PEPR1 is inducible in different 495

parts of the plant, whereas PEPR2 induction is restricted to the root (Safaeizadeh and Boller, 496

2019). Pep-induced expression of defense genes including MPK3 and WRKY33 is partially 497

suppressed in the pepr1 and pepr2 single mutants, and completely blocked in the pepr1 pepr2 498

double mutant (Yamaguchi et al., 2010). Pep1-induced expression of PR1 and PDF1.2 as well as 499

resistance against Pst DC3000 are also compromised in the double mutant (Yamaguchi et al., 500

2010). Interestingly, ET-induced expression of defense genes and resistance to B. cinerea are 501

also compromised in the pepr1 pepr2 double mutant (Liu et al., 2013). Furthermore, local 502

application of Pep2 activates both JA and SA signaling pathways and resistance to 503

Colletotrichum higginsianum path-29 strain in systemic leaves, although Pep2 may not be a 504

mobile signal (Ross et al., 2014). In agreement with this result, biological induction of SAR is 505

compromised in the pepr1 pepr2 mutant (Ross et al., 2014). 506

507

RALFs-FER 508

509

RAPID ALKALINIZATION FACTOR (RALF) peptides were first isolated from tobacco, 510

tomato, and alfalfa leaves based on their activity in alkalinating the medium of tobacco 511

suspension cells (Pearce et al., 2001b), and later from sugarcane leaves using a similar approach 512

(Mingossi et al., 2010). The tobacco RALF is a 49-aa peptide located at the C terminus of a 115-513



18

aa preproprotein. The preproprotein carries an N-terminal signal peptide and the derived RALF 514

peptide contains four cysteines that form two disulfide bridges important for its activity (Pearce 515

et al., 2001b). Later studies indicated that many, but not all, RALF preproproteins are cleaved at 516

a conserved dibasic site RRXL by plant subtilisin-like serine proteases such as the Arabidopsis 517

SITE-1 PROTEASE (S1P)/SBT6.1 (Matos et al., 2008; Srivastava et al., 2009; Stegmann et al., 518

2017). A photoaffinity analog of the tomato RALF peptide, 125

I-azido-LeRALF, which has 519

biological activity similar to the native LeRALF, binds to tomato suspension cells with a Kd of 520

0.8 nM (Scheer et al., 2005). A highly conserved YISY motif located at positions 5 through 8 521

from the N terminus is essential for RALF activity, presumably being required for productive 522

binding to its putative receptor (Pearce et al., 2010b). 523

RALF proteins have been identified in a large number of plant species that represent a 524

variety of land plant lineages (Cao and Shi, 2012; Murphy and De Smet, 2014). The Arabidopsis 525

genome carries 39 RALF genes (Sharma et al., 2016). Comprehensive analysis of the identified 526

795 RALF proteins from various plant species revealed four major clades. Clades I, II, and III 527

carry the features important for RALF activity, including the RRXL cleavage site and the YISY 528

motif important for receptor binding, whereas clade IV is highly diverged and lacks these 529

features (Campbell and Turner, 2017). While the mean length of the RALF proteins in clades I, 530

II, and III is 125 amino acids, the clade IV RALFs have an average length of only 88 amino 531

acids, suggesting that the members in clade IV may not be true RALFs (Campbell and Turner, 532

2017). 533

RALF peptides were initially found to suppress root growth of tomato and Arabidopsis 534

seedlings as well as tomato pollen tube growth (Pearce et al., 2001b; Covey et al., 2010). In line 535

with these results, silencing of the tobacco RALF gene leads to increased root growth and 536

abnormal root hair development (Wu et al., 2007), whereas transgenic overexpression of the 537

Arabidopsis RALF1 and RALF23 genes results in dwarf phenotypes (Matos et al., 2008; 538

Srivastava et al., 2009). Moreover, RALF genes are highly expressed in roots, shoots, and 539

flowers (Zhang et al., 2010; Cao and Shi, 2012; Campbell and Turner, 2017). Collectively, these 540

results support a role for RALF peptides in plant growth and development. On the other hand, 541

the fungal pathogen Fusarium oxysporum f. sp. ciceri (Race 1)-induced expression of a RALF-542

related EST is 5-fold higher in resistant chickpea plants than in a susceptible variety (Gupta et 543

al., 2010). In Arabidopsis, RALF8 is induced by a combination of water deficit and nematode 544



19

stress, and overexpression of RALF8 confers susceptibility to drought stress and nematode 545

infection (Atkinson et al., 2013). Moreover, synthetic RALF17 peptide increases resistance to 546

Pst DC3000, while RALF23 reduces resistance to Pst DC3000 (Stegmann et al., 2017). 547

Consistently, overexpression of RALF23 inhibits resistance to Pst DC3000 coronatine-minus 548

(COR-), whereas loss of RALF23 enhances resistance to Pst DC3000 COR

- (Stegmann et al., 549

2017). Interestingly, genomes of 26 species of phytopathogenic fungi encode RALF homologs, 550

and the predicted F. oxysporum RALF appears to contribute to the virulence of the pathogen in 551

tomato plants (Masachis et al., 2016; Thynne et al., 2017). These data together suggest potential 552

involvement of RALFs in plant immunity. 553

The first RALF receptor FERONIA (FER), a Catharanthus roseus receptor-like kinase 1-554

like (CrRLK1L) receptor, was identified by quantitative phosphoproteomic profiling of RALF1-555

treated Arabidopsis seedlings (Haruta et al., 2014). The finding that the abundance of FER 556

phosphopeptides increased in RALF1-treated samples led to the hypothesis that FER might be 557

the receptor of RALF1. This hypothesis was supported by reduced RALF1 sensitivity of fer 558

mutants and binding of RALF1 to FER (Haruta et al., 2014). Recent studies have shown that 559

RALF4 and RALF19 bind to other CrRLK1L receptors including ANXUR1 (ANX1), ANX2, 560

Buddha’s Paper Seal1 (BUPS1), and BUPS2, as well as LEUCINE-RICH REPEAT EXTENSIN 561

proteins in regulating pollen tube integrity and sperm release in Arabidopsis (Mecchia et al., 562

2017; Ge et al., 2017). FER is also a receptor of RALF23 and perhaps RALF33 as well 563

(Stegmann et al., 2017). Interestingly, FER constitutively associates with both FLS2 and BAK1 564

to act as scaffolds for ligand-induced FLS2-BAK1 complex formation. The constitutive 565

association between BAK1 and FER can be strongly enhanced upon treatment with flg22, 566

whereas binding of RALF23 to FER inhibits flg22/elf18-induced complex formation between 567

FLS2/EFR and BAK1, leading to attenuation of FLS2/EFR-mediated PTI signaling (Stegmann et 568

al., 2017). Furthermore, the GPI-anchored protein (GPI-AP) LORELEI (LRE)-like GPI-AP1 569

(LLG1) constitutively associates with both FER and FLS2 and is required for PTI signaling (Li 570

et al., 2015; Shen et al., 2017). LLG1 and the related LLG2 directly bind RALF23 to nucleate the 571

assembly of a RALF23-LLG1/2-FER heterocomplex (Xiao et al., 2019), suggesting that RALFs 572

may be perceived by distinct CrRLK1L receptor kinase-LLG/LRE heterocomplexes in regulating 573

various biological processes including plant immunity. 574

575



20

PSKs-PSKR1/2 576

577

Phytosulfokines (PSKs) are sulfated tyrosine-containing pentapeptides with mitogenic activity in 578

vitro. The first PSK was purified from conditioned medium of rapidly growing asparagus 579

(Asparagus officinalis) cell cultures by following its mitogenic activity (Matsubayashi and 580

Sakagami, 1996). Based on the amino acid sequence of the asparagus PSK, rice (Oryza sativa) 581

and Arabidopsis PSK genes were subsequently identified (Yang et al., 1999, 2001; Matsubayashi 582

et al., 2006). PSKs are derived from ~77-89-aa prepropeptide precursors through tyrosylprotein 583

sulfotransferase-mediated tyrosine sulfation and subtilisin-like serine protease-catalyzed 584

proteolytic cleavage (Srivastava et al., 2008; Komori et al., 2009). The PSK precursors carry N-585

terminal signal sequences and are sulfated in the Golgi apparatus, secreted, and cleaved in the 586

extracellular milieu (Yang et al., 1999, 2001; Srivastava et al., 2008; Komori et al., 2009). 587

PSK binds to plasma membrane-enriched fractions with both high and low affinities (Kd 588

values ranging from 1 to 100 nM) (Matsubayashi et al., 1997; Matsubayashi and Sakagami, 589

1999). Photoaffinity cross-linking analysis indicated that the putative receptors for PSK in rice 590

are 120- and 160-kDa glycosylated proteins (Matsubayashi and Sakagami, 2000). The first PSK 591

receptor, a LRR-RK, was purified from microsomal fractions of carrot suspension cells using 592

ligand-based affinity chromatography, and the carrot PSK receptor gene encodes both 120- and 593

150-kDa proteins (Matsubayashi et al., 2002). Amino acid homology search revealed that the 594

Arabidopsis genome encodes two PSK receptors, PSKR1 and PSKR2 (Matsubayashi et al., 595

2006; Amano et al., 2007). Structure analysis indicated that PSK interacts with and stabilizes an 596

island domain of PSKR, which enhances PSKR heterodimerization with a SERK co-receptor 597

(Wang et al., 2015). The cytoplasmic domain of PSKR1 has not only kinase activity but also GC 598

activity. Both exogenous PSK treatment and overexpression of PSKR1 increase cGMP levels in 599

protoplasts (Kwezi et al., 2011). Moreover, PSKR1, BAK1, CNGC17, and H+-ATPAses AHA1 600

and AHA2 form a complex in mediating PSK-triggered signaling (Ladwig et al., 2015). 601

PSK was initially shown to induce the proliferation of asparagus suspension cells 602

(Matsubayashi and Sakagami, 1996; Matsubayashi et al., 1997). PSK precursors are 603

constitutively secreted by suspension cells, and overexpression and silencing of PSK genes led to 604

increased and reduced PSK levels in conditioned media of rice transgenic cells, respectively 605

(Yang et al., 1999, 2001). PSK genes are stably expressed not only in suspension cells but also in 606



21

intact plants (Yang et al., 1999, 2001). Overexpression of PSK genes resulted in enlarged 607

transgenic calli (Yang et al., 2001; Matsubayashi et al., 2006). Similarly, transgenic carrot cells 608

expressing high levels of sense mRNA of the PSK receptor exhibited accelerated proliferation, 609

whereas those expressing antisense showed substantially reduced callus growth (Matsubayashi et 610

al., 2002). Individual cells of the Arabidopsis pskr1-1 mutant gradually lose their potential to 611

form calli as the tissues mature, while PSKR1-overexpressing plants exhibit significantly greater 612

callus-forming potential than wild type (Matsubayashi et al., 2006). 613

Genes encoding PSK precursors, processing enzymes, and/or receptors are inducible by 614

wounding, elf18, flg22, and B. cinerea (Srivastava et al., 2008; Igarashi et al., 2012; Hou et al., 615

2014; Zhang et al., 2018), suggesting a potential involvement of PSK-PSKR signaling in plant 616

immunity. Indeed, elf18-triggerred immune responses are enhanced in the Arabidopsis pskr1-3 617

mutant (Igarashi et al., 2012). Mutations of the PSKR1 and TYROSYLPROTEIN 618

SULFOTRANSFERASE (TPST) genes enhance resistance to Pst DC3000 and increase 619

susceptibility to A. brassicicola, whereas overexpression of PSK2, PSK4, and PSKR1 leads to 620

opposite effects (Mosher et al., 2013). However, overexpression of the rice PSKR1 gene 621

activates SA signaling and enhances resistance to the bacterial pathogen Xanthomonas oryzae 622

pv. oryzicola (Yang et al., 2019). Furthermore, exogenous application of PSK enhances Pst 623

DC3000 growth in the Arabidopsis tpst-1 mutant (Mosher et al., 2013), and increase resistance to 624

B. cinerae in tomato (Zhang et al., 2018). In addition, silencing of the tomato PSKR1 gene 625

enhances susceptibility to B. cinerae (Zhang et al., 2018). Binding of PSK to tomato PSKR1 626

elevates cytosolic [Ca2+

], which enhances interaction between calmodulins and auxin 627

biosynthetic YUCCAs, resulting in auxin-dependent immunity against B. cinerae (Zhang et al., 628

2018). 629

630

GRIp-PRK5 631

632

GRIM REAPER (GRI) belongs to a small family with six members in Arabidopsis. Its C-633

terminal cysteine-rich domain is highly homologous to STIGMA-SPECIFIC PROTEIN1 634

(STIG1) that functions in regulation of exudate secretion in the pistils and promotion of pollen 635

tube growth (Verhoeven et al., 2005; Huang et al., 2014). The GRI protein is 169-aa long, carries 636

a predicted N-terminal signal peptide (amino acids 1-30), and is secreted into the apoplast 637



22

(Wrzaczek et al., 2009). As the GRI gene expression in flowers is 1,000-fold higher than in 638

leaves (Wrzaczek et al., 2009), GRI likely plays a role in reproduction. Indeed, a gain-of-639

function gri mutant and GRI-overexpressing plants exhibit reduced seed content in the siliques 640

(Wrzaczek et al., 2009). Interestingly, the low basal GRI expression in leaves is inducible by 641

ozone exposure and both gri and GRI-overexpressing plants are sensitive to ozone (Wrzaczek et 642

al., 2009). The gri mutant is also resistant to the virulent bacterial pathogen Pst DC3000 643

(Wrzaczek et al., 2009). These gri phenotypes are likely caused by accumulation of a GRI 644

peptide (GRIp) corresponding to the N-terminal variable region after the signal peptide (amino 645

acids 31-96) (Wrzaczek et al., 2015). Exogenous GRIp31-96

induces superoxide- and SA-646

dependent ion leakage, an indicator of cell death. GRI is cleaved by an apoplast-localized type II 647

metacaspase METACASPASE9 (MC9), releasing an 11 amino acid peptide, GRIp68-78

, which is 648

sufficient for induction of ion leakage (Wrzaczek et al., 2015). GRIp-induced ion leakage 649

depends on the atypical LRR-RK, POLLEN-SPECIFIC RECEPTOR-LIKE KINSASE5 (PRK5) 650

(Wrzaczek et al., 2015). Full-length GRI without the signal peptide and GRIp31-96

interact with 651

the extracellular domain of PRK5 in vitro. A radiolabeled GRIp, 125

I-Y-GRIp68-78

, which is 652

active for ion leakage induction, binds to Arabidopsis membrane extracts with a Kd of 1.9 nM. 653

Binding of 125

I-Y-GRIp68-78

to membrane extracts is reduced to background levels in prk5 654

mutants (Wrzaczek et al., 2015). These results support that PRK5 is a receptor of GRIp. 655

However, since the prk5 and mc9 mutations have no significant effects on extracellular 656

superoxide-induced ion leakage and resistance to Pst DC3000 (Wrzaczek et al., 2015), whether 657

GRIp is a bona fide DAMP requires further investigation. 658

659

PIP1-RLK7 660

661

Genes encoding PAMP-INDUCED SECRETED PEPTIDE (PIP) precursors named prePIP1, 662

prePIP2, and prePIP3 were identified by searching flg22- and elf18-induced transcription data 663

(Hou et al., 2014). Eleven prePIP homologs were identified in Arabidopsis based on the highly 664

conserved C-terminal sequences. All of the prePIP family members carry a N-terminal signal 665

peptide (Hou et al., 2014; Vie et al., 2015). Orthologs of prePIPs were also identified in multiple 666

other plant species such as soybean, grape, maize, and rice (Hou et al., 2014). The prePIP1 gene 667

is induced not only by PAMPs but also by MeSA, Pst DC3000, and the fungal pathogen 668



23

Fusarium oxysporum f. sp. conglutinans strain 699 (Foc 699) (Hou et al., 2014). Overexpression 669

of prePIP1 and prePIP2 inhibits root growth and enhances resistance to Foc 699. Synthetic PIP1 670

and PIP2 comprising the conserved C terminus also inhibit root growth and induce immune 671

responses similar to PTI (Hou et al., 2014). Interestingly, PIP1- and PIP2-mediated root growth 672

inhibition and immune responses are compromised in T-DNA insertion mutants of the 673

RECEPTOR-LIKE KINASE7 (RLK7) gene, which encodes a class XI LRR-RK, suggesting that 674

RLK7 is a potential receptor of these PIPs (Hou et al., 2014). Indeed, RLK7-HA was pulled 675

down with PIP1-biotin-associated streptavidin beads from membrane extracts of transgenic 676

Arabidopsis plants expressing RLK7-HA, and specific binding of radiolabeled 125

I-Y-PIP1 was 677

detected in homogenates of tobacco leaves transiently expressing RLK7-HA in photoaffinity 678

labeling assays, indicating that PIP1 directly binds to RLK7 (Hou et al., 2014). Moreover, PIP1-679

induced root growth inhibition and/or ROS production are reduced in the bak1-4 mutant but not 680

in the bik1 mutant, indicating that PIP1-RLK7 signaling is partially dependent on BAK1, but 681

independent of BIK1 (Hou et al., 2014). Finally, both PIP1 and PEP1 induce the expression of 682

PrePIP1, ProPEP1, RLK7, PEPR1, and FLS2, suggesting that PIP1 and PEP1 function 683

cooperatively in amplification of FLS2-initiated immune signaling (Hou et al., 2014). 684

685

IDL6p-HAE/HSL2 686

687

INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) and IDA-LIKE (IDL) proteins are 688

precursors of peptides that induce floral abscission (Butenko et al., 2003; Stenvik et al., 2008). 689

The Arabidopsis IDA family has nine members (IDA and IDL1-8) characterized by an N-690

terminal signal peptide, a variable region, and a C-terminal conserved region where the PIP motif 691

is located (Butenko et al., 2003; Stenvik et al., 2008; Vie et al., 2015). Genetic studies suggested 692

that two LRR-RKs, HAESA (HAE) and HAE-LIKE2 (HSL2), are receptors of IDA/IDL-derived 693

peptides (Stenvik et al., 2008). A chemiluminescent acridinium labeled VPIPPo (PIP with a 694

valine residue at the N terminus and hydroxylation of the conserved proline at position 7) termed 695

acri-PIPPo binds to leaf materials of N. benthamiana expressing HSL2KD with a Kd of ~20 696

nM (Butenko et al., 2014), demonstrating that HSL2 is a bona fide receptor of IDA/IDL 697

peptides. The IDA and IDL6 genes are up-regulated by PAMPs and IDL6 is also induced by Pst 698

DC3000 (Hou et al., 2014; Wang et al., 2017b). Synthetic IDL6 and IDL7 extended PIP peptides 699



24

down-regulate the expression of a broad range of stress-responsive genes (Vie et al., 2017). 700

Moreover, overexpression of IDL6 enhances susceptibility to Pst DC3000, whereas silencing of 701

IDL6 increases resistance to the bacterial pathogen (Wang et al., 2017b). IDL6 elevates the 702

transcription of Arabidopsis DEHISCENCE ZONE POLYGALACTURONASE2 (ADPG2), which 703

encodes an active polygalacturonase that promotes pectin degradation to facilitate Pst DC3000 704

infection. Consistent with HAE and HSL2 being receptors of IDL6, IDL6-mediated ADPG2 705

expression and Pst DC3000 susceptibility are completely suppressed in the hae hsl2 double 706

mutant (Wang et al., 2017b). Interestingly, the IDA-HEA/HSL2 ligand-receptor pair is required 707

for P. syringae type III effector-triggered leaf abscission, which likely represents a new form of 708

plant immunity (Patharkar et al., 2017). 709

710

CONCLUSIONS AND FUTURE PERSPECTIVES 711

712

A large and compelling body of evidence has accumulated in recent years, which supports an 713

important role for DAMPs in plant immune responses (Figure 1). Nevertheless, the identity of 714

DAMPs in plants remains to be unambiguously defined. The Danger model postulates that 715

healthy cells or cells undergoing normal physiological death do not generate danger signals 716

(Matzinger, 1994, 2002). It was recently further argued in animals that a canonical DAMP can be 717

up-regulated, but not released, in response to PAMP detection or stress stimuli that presumably 718

leads to necrosis (Martin, 2016). In plants, however, it seems that some DAMPs are actively 719

released upon PAMP detection or environmental stresses (Deng et al., 2015; Chen et al., 2017). 720

Release of DAMPs in the absence of cell death appears to be inconsistent with the Danger 721

model. However, before we arrive at such a conclusion, we must consider the following 722

possibilities. First, some DAMPs may play dual functions in plants. For instance, as in animals 723

(Trautmann, 2009), eATP in plants not only acts as a DAMP in wound response, but also plays a 724

major role in growth control (Choi et al., 2014b; Roux, 2014). The constitutive eATP and 725

actively released ATP may be crucial for cell viability and growth changes (Chivasa et al., 2005; 726

Liu et al., 2012; Deng et al., 2015). Second, the amount of DAMPs actively released may not be 727

sufficient for immune activation. For example, in response to cold stress (4C for 7 days), the 728

concentration of eATP in the extracellular root medium of seven-day-old Arabidopsis seedlings 729

is ~8 nM, whereas that in the fluid released at the sites of physical wounding is ~40 M (Choi et 730



25

al., 2014; Deng et al., 2015). The eATP concentration under cold stress is likely too low to 731

activate the eATP receptor DORN1 (Kd, ~46 nM) for wound response (Choi et al., 2014a). 732

These results suggest that DAMPs may induce immune responses in a concentration-dependent 733

manner, or there may be a threshold below which DAMPs do not activate immune response. And 734

third, since plants lack specialized immune cells and adaptive immunity, cell-autonomous 735

immunity may play a more important role in plants than in animals (Randow et al., 2013). Plants 736

might have thus evolved mechanisms to actively release high amounts of DAMPs for activation 737

of cell-autonomous immunity. Clearly, further investigations are required to determine whether 738

sufficient DAMPs can be released in the absence of cell death for immune activation in plants. 739

Regardless, even though the Danger model may need some modifications for the plant immune 740

system, the general principles should be applicable. 741

It is expected that multiple DAMPs would be released upon any type of cell damage. 742

However, the combinations of DAMPs following different types of cell damages may be 743

different. For instance, besides primary DAMPs, mechanical damage leads to release of 744

wounding-induced secondary DAMPs such as systemin (Pearce, 2011), whereas pathogen attack 745

results in release of pathogen-induced secondary DAMPs including Peps and PIPs (Huffaker et 746

al., 2006; Hou et al., 2014). Moreover, DAMPs may be released at various times during plant-747

microbe interaction due to their different subcellular localizations. In this regard, DAMPs 748

derived from the cell wall would be released early, followed by those from the cytoplasm, and 749

finally from the nucleus. Additionally, the half-lives and apoplastic mobility of DAMPs as well 750

as the activities of receptors for DAMPs may differ significantly (Adriouch et al., 2012). Thus, 751

DAMPs should function cooperatively with each other, as well as with PAMPs in a temporal, 752

spatial, and stress-specific manner to generate a peculiar immune response. 753

Determining the role of DAMPs in plant immune responses is an important but 754

challenging task (see Outstanding Questions). Several studies have investigated the interplays 755

between DAMPs and PAMPs (such as flg22 and elf18) as well as between different DAMPs. 756

Both synergism and antagonism between DAMPs and PAMPs/DAMPs have been observed 757

(Fauth et al., 1998; Stennis et al., 1998; Aslam et al., 2009; Ma et al., 2012; Flury et al., 2013; 758

Tintor et al., 2013; Stegmann et al., 2017). However, since DAMPs are released during pathogen 759

infection or herbivore attack, the context is extremely complex. It would be difficult to sort out 760

the contribution of individual DAMPs to the final specific immune phenotype. Perhaps 761



26

something similar to the recently proposed PAMP/DAMP combination-based ‘inflammatory 762

code’ could help solve this puzzle (Escamilla-Tilch et al., 2013). Moreover, although evidence 763

supporting a role for DAMPs in ETI is accumulating (Ma et al., 2012; Zhang and Mou, 2012), 764

in-depth investigations are warranted. In addition, several DAMPs have been implicated in 765

systemic responses including SAR (Pearce et al., 1991; Ross et al., 2014; Toyota et al., 2018; 766

Wang et al., 2019a), suggesting that DAMP signaling is an integral component of biological 767

induction of systemic responses. Future research should investigate whether DAMPs move 768

systemically or act through other signal molecules similarly to systemin (Li et al., 2002b; 769

Schilmiller and Howe, 2005), and how the DAMP signal is transduced into the nucleus. 770

It is worth mentioning that what we currently know about DAMPs is just the tip of the 771

iceberg. Among the countless number of intracellular molecules, many could potentially become 772

DAMPs if released into the apoplast. Furthermore, a recent study using a bioinformatics 773

approach identified more than 1,000 putative secreted peptides in Arabidopsis (Lease and 774

Walker, 2006), not to mention other plant species with larger genomes than Arabidopsis. Many 775

of the putative peptides could potentially function as DAMPs. Identification of potential new 776

DAMPs as well as the processing enzymes and/or receptors for the candidate DAMPs would 777

greatly improve our understanding of plant DAMP signaling and the plant immune system as a 778

whole. It is expected that a deeper understanding of plant DAMPs and the plant immune system 779

could significantly help design new strategies to breed crop varieties with increased resistance 780

against pathogens and/or herbivores. 781

782



27

783

Acknowledgments 784

785

We apologize to researchers whose relevant studies were not cited in this review due to page 786

limitations, and would like to thank Fiona M. Harris for careful reading of the manuscript. 787

788

Footnotes 789

790

This work was supported by NSF grant (ISO-1758932) awarded to Z.M. 791

792

Figure Legends 793

794

Figure 1. Putative DAMP-receptor pairs and their functions in plant immunity 795

796

The cell surface receptor cartoons depict the putative DAMP receptors with their co-receptors or 797

associated proteins. The cartoon for the glutamate receptors GLR3.3/3.6 is based on an animal 798

ionotropic glutamate receptor, a ligand-gated ion channel formed by four subunits. Each subunit 799

has four domain layers: the extracellular N-terminal domain and ligand-binding domain, the 800

transmembrane (TM) domain, and an intracellular C-terminal domain. For the sake of clarity, 801

only two subunits are shown in the cartoon for GLR3.3/3.6. Moreover, although several RALFs 802

including RALF17, 23, 33, and 34 are potential DAMPs that positively or negatively regulate 803

immunity, RALF23 has been shown to bind LLG1/2 and FER to nucleate the assembly of 804

RALF23-LLG1/2-FER heterocomplexes. Thus, only RALF23-LLG1/2-FER-mediated inhibition 805

of PTI is presented here. Note that both LLG1 and FER are required for PTI signaling. In 806

addition, although PIP1-induced ROS production and root growth inhibition partially depend on 807

BAK1, whether the PIP1 receptor RLK7 interacts with BAK1 has not been reported. A question 808

mark (?) is thus included in the RLK7/BAK1 cartoon to illustrate the uncertainty. Finally, dashed 809

arrows are used to indicate the immune responses that are induced either by exogenously added 810

DAMPs or by overexpression of the receptors; however, whether these immune responses are 811

induced by the DAMPs through their receptors is unclear. By contrast, solid arrows represent 812

immune responses that are activated by the DAMPs through their receptors. Abbreviations: 813



28

DAMPs: damage-associated molecular patterns; OGs: oligogalacturonides; WAK1: WALL-814

ASSOCIATED KINASE1; eATP: extracellular ATP; DORN1: DOES NOT RESPOND TO 815

NUCLEOTIDES1; eNAD(P): extracellular NAD(P); LecRK-I.8/VI.2: Lectin RECEPTOR 816

KINASE-I.8/VI.2; GLR3.3/3.6: GLUTAMATE-RECEPTOR3.3/3.6; SYR1/2: SYSTEMIN 817

RECEPTOR1/2; Pep: Plant elicitor peptide; PEPR1/2: Pep RECEPTOR1/2; RALF23: RAPID 818

ALKALINIZATION FACTOR23; FER: FERONIA; PSK: phytosulfokine; PSKR1/2: PSK 819

RECEPTOR1/2; GRIp: GRIM REAPER peptide; PRK5: POLLEN-SPECIFIC RECEPTOR-820

LIKE KINSASE5; PIP1: pathogen-associated molecular pattern (PAMP)-INDUCED 821

SECRETED PEPTIDE1: RLK7: RECEPTOR-LIKE KINASE7; IDL6p: INFLORESCENCE 822

DEFICIENT IN ABSCISSION-LIKE6 peptide; HAE/HSL2: HAESA/HAE-LIKE2; INR: 823

INCEPTIN RECEPTOR; SERKs: SOMATIC EMBRYOGENESIS RECEPTOR-LIKE 824

KINASEs; LLG1/2: LORELEI-LIKE GLYCOSYLPHOSPHATIDYLINOSITOL (GPI)-825

ANCHORED PROTEIN1/2; BAK1: BRASSINOSTEROID INSENSITIVE1-ASSOCIATED 826

KINASE1; SOBIR1: SUPPRESSOR OF BIR1-1; EGF: epidermal growth factor; ROS: reactive 827

oxygen species; MPKs: mitogen-activated protein kinases; JA: jasmonic acid; SA: salicylic acid; 828

SAR: systemic acquired resistance; PIN1: PROTEINASE INHIBITOR1; PR1: PATHOGENESIS-829

RELATED GENE1; FDF1.2: PLANT DEFENSIN1.2; PTI: PAMP-triggered immunity; ADPG2: 830

DEHISCENCE ZONE POLYGALACTURONASE2. 831

832

833



29

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Documents

Short title: Plant damage recognition · SAR depends on the immune signal 68 molecule salicylic acid (SA), whereas ISR relies on the signaling pathways activated by the plant 69 hormones