perception of damaged self in plants1[open]...insensitive1 (bri1)-associated kinase1 (bak1)...

Update on Perception of Damaged Self in Plants

Perception of Damaged Self in Plants1[OPEN]

Qi Li, Chenggang Wang, and Zhonglin Mou2,3

Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611

ORCID IDs: 0000-0002-4230-2651 (C.W.); 0000-0003-0243-4905 (Z.M.).

Multicellular eukaryotes including plants and ani-mals have evolved highly complex, multilayered im-mune systems to fight off microbial infections. How theimmune systems function is a fundamental questionfor immunologists. The animal immune system wasoriginally thought to function by distinguishing be-tween “self “and “nonself ”(the Self–Nonself model;Burnet, 1959), and later between “infectious-nonself”and “noninfectious-self” (the Infectious–Nonself model;Janeway, 1989, 1992). In 1994, Matzinger proposed thatthe immune system is more concerned with “danger”than with “non-self” (the Danger model; Matzinger,1994, 2002, 2007). The Danger model suggests that theimmune system is activated by danger/alarm signalsthat are sent from both microbial pathogens and dam-aged host cells. In this model, it is assumed that healthycells or cells undergoing normal physiological death donot produce danger signals (Matzinger, 2002). Overthe years, the Danger model has been supported bythe discovery of a large number of endogenous dan-ger signals (Tang et al., 2012; Pouwels et al., 2014;Schaefer, 2014; Hernandez et al., 2016; Yatim et al.,2017; Dinarello, 2018).Danger signals consist of conservedpathogen-associated

molecular patterns (PAMPs) from the microbes anddamage-associated molecular patterns (DAMPs) frominjured host cells (Matzinger, 2002). Although the term“DAMPs” originally referred to the hydrophobic por-tions of biological molecules from dead and dying hostand pathogen cells, which trigger immunity when ex-posed (Seong and Matzinger, 2004), it is now generallyused to describe danger signals from damaged hostcells (Martin, 2016; Yatim et al., 2017). Besides PAMPsand DAMPs, pathogen-derived effectors, which areproteins expressed by pathogens to aid infection of theirhosts, and effector-caused perturbations on/in the hostcells should also be considered as danger signals,though they were not included in the original model(Matzinger, 2002; Boller and Felix, 2009). PAMPs/DAMPs and extracellular effectors or their distur-bances are generally recognized by germline-encoded

cell-surface pattern recognition receptors (PRRs; Takeuchiand Akira, 2010), whereas intracellular effectors ortheir interruptions are often sensed by cytoplasmicnucleotide-binding oligomerization domain-like re-ceptors (Chen et al., 2009).The plant immune system shares a similar concep-

tual logic with the animal immune system, thoughplants lack adaptive immunity (Nürnberger et al., 2004;Haney et al., 2014). A simple coevolutionary modelcalled the “Zigzag” was proposed to describe the mo-lecular events in plant–microbe interactions (Jones andDangl, 2006). Based on this model, plant cells employPRRs to detect PAMPs, activating PAMP-triggeredimmunity (PTI), while adapted pathogens utilize ef-fectors to dampen PTI. Plants in turn exploit nucleotide-binding oligomerization domain-like receptors to sensethe presence of effectors, leading to effector-triggeredimmunity, which usually culminates in a hypersensi-tive cell death response at the infection site. Natural

1This work was supported by the National Science Foundation(ISO-1758932 to Z.M.).

2Author for contact: [email protected] author.Q.L., C.W., and Z.M. conceived the content and wrote the article.[OPEN]Articles can be viewed without a subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.19.01242

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https://orcid.org/0000-0002-4230-2651https://orcid.org/0000-0002-4230-2651https://orcid.org/0000-0003-0243-4905https://orcid.org/0000-0003-0243-4905https://orcid.org/0000-0002-4230-2651https://orcid.org/0000-0003-0243-4905http://crossmark.crossref.org/dialog/?doi=10.1104/pp.19.01242&domain=pdf&date_stamp=2020-03-25http://dx.doi.org/10.13039/100000001mailto:[email protected]://www.plantphysiol.org/cgi/doi/10.1104/pp.19.01242

selection constantly drives the arms race between plantsand pathogens, resulting in different levels of pathogenvirulence and plant resistance (Jones and Dangl, 2006).The Zigzag model has conceptually stimulated enor-mous research in the plant–microbe interaction field;however, it did not encompass DAMPs. The recent“Invasion” model included DAMPs and introduced anew term, “invasion patterns,” which essentially refersto the same type of molecules as “danger signals”(Cook et al., 2015). It was suggested that adapting theDanger model for plants would allow the holistic con-cept of host immunity to be better shared by the entirecommunity of immunologists (Gust et al., 2017). Nev-ertheless, neither the Zigzag model nor the Invasionmodel accommodates systemic resistance, includingsystemic acquired resistance (SAR) and induced sys-temic resistance (ISR), which are also essential parts ofthe plant immune system (Durrant and Dong, 2004;Pieterse et al., 2014). SAR and ISR are two forms of in-duced resistance wherein the plant immune system isprimed by a prior localized infection that results inresistance throughout the plant against subsequentchallenge by a broad spectrum of pathogens. How-ever, induction of the two forms of systemic resis-tance is mechanistically distinct. SAR depends on theimmune signal molecule salicylic acid (SA), whereasISR relies on the signaling pathways activated bythe plant hormones jasmonic acid (JA) and ethylene(ET; Durrant and Dong, 2004; Pieterse et al., 2014).The SA, JA, and ET response pathways serve as thebackbone of the plant immune signaling network(Pieterse et al., 2012).

Compared to the large number of DAMPs that havebeen identified and characterized in animals, researchonDAMPs in plants has only just begun (Rubartelli andLotze, 2007; Choi and Klessig, 2016; Roh and Sohn,2018). In the past several years, we have witnessed amarked increase in the number of potential DAMPs inplants, and the number is still growing (Table 1; Duran-Flores and Heil, 2016; Gust et al., 2017; Hou et al., 2019).Moreover, potential receptors for more than a dozenplant DAMPs have been identified (Gust et al., 2017;Hou et al., 2019). Characterization of these receptors isexpected to significantly boost DAMP research inplants. While the DAMP field is blooming, the identityof DAMPs is under debate (Martin, 2016). It was arguedin animals that a canonical DAMP should only be re-leased from cells during necrosis; act through bindingto cell-surface receptors; be upregulated, but not re-leased, in response to PAMP detection or stress stimulithat are likely to presage necrosis; be synergistic withPAMPs in activating robust immune responses; andinitiate relatively broad-acting responses in a mannersimilar to that shown by pathogen components (Martin,2016). Based on these characteristics, members of theextended IL-1 cytokine family (IL-1a, IL-1b, IL-18,IL-33, IL-36a, IL-36b, and IL-36g) have been reasonedto be the canonical DAMPs activating the immunesystem, whereas most other proposed DAMPs, e.g.ATP, uric acid, calreticulin, HMGB1, HSPs, and DNA

fragments, likely act through liberating IL-1 family cy-tokines via promoting necrosis (Martin, 2016).

In plants, the identity of DAMPs has not been vig-orously debated. Recently, immunogenic plant factorswere roughly divided into two categories, primary andsecondary DAMPs, which correspond to constitutiveand inducible DAMPs proposed in animals (Gust et al.,2017; Yatim et al., 2017). Primary/constitutive DAMPsare derived from pre-existing structures or molecules,including breakdown products of extracellular matrixand passively released intracellular molecules, whilesecondary/inducible DAMPs are actively processedand released upon tissue damage and other stimuli(Gust et al., 2017). Although this delineation of pri-mary DAMPs is aligned with the original definition ofDAMPs (Matzinger, 2002; Seong and Matzinger, 2004),it is worthwhile to compare the secondary DAMPswiththe proposed canonical DAMPs in animals (Martin,2016). One central argument for members of the ex-tended IL-1 family being the canonical DAMPs in ani-mals is that they do not possess N-terminal signalsequences and are released during necrosis (Martin,2016). In contrast, precursors of most of the candidatepeptide DAMPs in plants carry an N-terminal signalpeptide (Table 1), suggesting active release via theconventional secretion pathway. They would, never-theless, also be passively released upon cell damageduring microbial infection and herbivore attack. Thus,besides being DAMPs under pathological conditions,such molecules may function in normal physiologicalprocesses.

In this review, we focus on several proposed plantprimary and secondary DAMPs and their receptors,which have been shown to physically bind each other. Fora complete inventory of potential DAMPs in plants, werefer interested readers to several recent excellent reviewsand references therein (Choi and Klessig, 2016; Duran-Flores and Heil, 2016; Gust et al., 2017; Hou et al., 2019).Anew item thatwas recently added to the inventory is theArabidopsis (Arabidopsis thaliana) SCOOP12 peptide,which is perceived by plants in a BRASSINOSTEROIDINSENSITIVE1 (BRI1)-ASSOCIATED KINASE1 (BAK1)coreceptor-dependentmanner (Table 1; Gully et al., 2019).We explore potential roles of DAMPs in plant immunity,particularly in SAR. Future perspectives of DAMPs inplants are also discussed.

PRIMARY/CONSTITUTIVE DAMP-RECEPTOR PAIRS

Oligogalacturonides—WALL-ASSOCIATED KINASE1

Oligogalacturonides (OGs) are degradation productsof the primary cell wall component pectin, a complexpolysaccharide comprising mainly esterified D-GalUAresidues in a-(1-4)-chain (Côté and Hahn, 1994; Ferrariet al., 2013; Kohorn, 2016). Pectin is partially degradedby pathogen- or plant-derived enzymes during patho-gen infection or herbivore attack, resulting in oligo-mers of D-galacturonic acids with varying degrees of

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Table 1. Putative DAMPs proposed in plants

Abbreviations: CAPE1, CYS-RICH SECRETORY PROTEINS, ANTIGEN5, AND PATHOGENESIS-RELATED1 PROTEINS (CAP)-DERIVED PEPTIDE1;eDNA, extracellular DNA; GmSubPep, G. max Subtilase Peptide; HMGB3, HIGH MOBILITY GROUP BOX3; HypSys, Hyp-rich Systemin; IDLp,INFLORESCENCE DEFICIENT IN ABSCISSION (IDA)-LIKE peptide; INR, INCEPTIN RECEPTOR; N/A, Not Applicable; RGS1, REGULATOR OFG-PROTEIN SIGNALING1; SCOOP12, SER-RICH ENDOGENOUS PEPTIDE12; Zip1, Z. mays immune signaling peptide1.

DAMP Precursor

N-Terminal

Signal

Peptide

Degree of

Polymerization,

Amino Acids,

or Bps

Receptor Plant References

Primary/Constitutive DAMPsOGs Pectin N/A 10–15 WAK1 Arabidopsis Côté and Hahn, 1994; He

et al., 1996; Brutus et al.,2010

eATP N/A N/A N/A DORN1 Arabidopsis Choi et al., 2014a; Roux,2014

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

Arabidopsis Zhang and Mou, 2009; Wanget al., 2017a, 2019a

Amino acids andglutathione

N/A N/A N/A GLR3.3/3.6

Arabidopsis Qi et al., 2006; Stephenset al., 2008; Li et al., 2013;Toyota et al., 2018

Extracellularsugars

N/A N/A N/A RGS1 Arabidopsis Johnston et al., 2007; BolouriMoghaddam and van denEnde, 2012

HMGB3 N/A No N/A Unknown Arabidopsis Choi et al., 2016Cutin monomers Cuticle N/A N/A Unknown Arabidopsis, tomato Fauth et al., 1998; Buxdorf

et al., 2014Cellooligomers Cellulose N/A 2–7 Unknown Arabidopsis Souza et al., 2017; Johnson

et al., 2018Xyloglucans Hemicellulose N/A 7–9 Unknown Common grape vine,

ArabidopsisClaverie et al., 2018

Methanol Pectin N/A N/A Unknown Arabidopsis Dixit et al., 2013; Hann et al.,2014; Tran et al., 2018

eDNA fragments DNA N/A ,700 Unknown Pea (Pisum sativum),lima bean (Phaseoluslunatus), maize,common bean

Wen et al., 2009; Barberoet al., 2016; Duran-Floresand Heil, 2018

Secondary/Inducible DAMPsSystemin Prosystemin No 18 SYR1/2 Some Solanaceae

speciesPearce et al., 1991; Wang

et al., 2018bHypSys ProHypSys Yes 18–20 Unknown Some Solanaceae

speciesPearce et al., 2001a; Pearce,

2011Peps PROPEPs No 23 PEPR1/2 Arabidopsis, maize Huffaker et al., 2006, 2011,

2013; Yamaguchi et al.,2006, 2010

RALFs RALFpreproproteins

Yes 49 FER Tobacco, Arabidopsis Pearce et al., 2001b; Harutaet al., 2008, 2014;Stegmann et al., 2017

PSKs PSK precursors Yes 5 PSKR1/2 Asparagus, rice,Arabidopsis

Matsubayashi and Sakagami,1996; Yang et al., 1999,2001; Matsubayashi et al.,2002; Amano et al., 2007

PIP1/2 PrePIP1/2 Yes 13/15 RLK7 Arabidopsis Hou et al., 2014IDLp IDLs Yes 14 HAE/HSL2 Arabidopsis Stenvik et al., 2008; Butenko

et al., 2014; Patharkaret al., 2017; Vie et al.,2017; Wang et al., 2017b

GRIp GRI Yes 11 PRK5 Arabidopsis Wrzaczek et al., 2009, 2015CAPE1 PR1 Yes 11 Unknown Tomato, Arabidopsis Chen et al., 2014Zip1 PROZIP1 No 17 Unknown Maize Ziemann et al., 2018Inceptins ATP synthase

g-subunitproteins

No 11 INR Cowpea (Vignaunguiculata), maize

Schmelz et al., 2006;Steinbrenner et al., 2019

GmSubPep Subtilase Yes 12 Unknown Soybean Pearce et al., 2010a(Table continues on following page.)

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polymerization (Bishop et al., 1981; Côté and Hahn,1994; Bergey et al., 1999; An et al., 2005). OGs with adegree of polymerization between 10 and 15 are potentelicitors (Côté and Hahn, 1994; Moscatiello et al., 2006;Ferrari et al., 2007; Denoux et al., 2008), able to inducereactive oxygen species (ROS) production, MAP ki-nase activation, callose deposition, defense proteinaccumulation, and resistance to the necrotrophic fun-gal pathogen Botrytis cinerea in multiple plant spe-cies (Hahn et al., 1981; Davis and Hahlbrock, 1987;Broekaert and Peumans, 1988; Bellincampi et al., 2000;Aziz et al., 2004; Denoux et al., 2008; Galletti et al.,2008; Rasul et al., 2012). Short OGs with a degree ofpolymerization between two and six have also beenshown to elicit immune responses, but the effect of shortOGs on the expression of immune-related genes appearsto be not as strong as that of long OGs (Moloshok et al.,1992; Davidsson et al., 2017).

WALL-ASSOCIATED KINASE (WAK) proteins areproposed receptors of OGs (Kohorn and Kohorn, 2012;Ferrari et al., 2013). WAKs are receptor-like kinases(RLKs), with an extracellular domain containing epi-dermal growth factormotifs, a transmembrane domain,and an intracellular Ser/Thr kinase domain (He et al.,1996; Anderson et al., 2001). There are five WAK and21 WAK-LIKE genes in Arabidopsis (Anderson et al.,2001; Verica and He, 2002). Biochemical analyses sug-gested that WAK1 is tightly associated with pectin(He et al., 1996; Wagner and Kohorn, 2001). The extra-cellular domains of WAK1 and WAK2 indeed bindpectin in vitro (Kohorn et al., 2009). A recombinantpeptide containing amino acids 67 to 254 of the extra-cellular domain of WAK1 (called “WAK67–254”) bindspolygalacturonic acid (PGA), OGs, pectins, andstructurally related alginates (Decreux and Messiaen,2005). At least five specific amino acids in the extra-cellular domain of WAK1 are involved in the interac-tion with PGA (Decreux et al., 2006). Interestingly,binding of WAK67–254 to PGA, OGs, and alginatesdepends on Ca21 and ionic conditions that promoteformation of Ca21 bridges between oligomers or pol-ymers, resulting in a structure known as an “egg-boxdimer,” which significantly enhances binding toWAK1 and induces increased extracellular alkalini-zation when applied to Arabidopsis cell suspensions(Decreux and Messiaen, 2005; Cabrera et al., 2008).

Multiple lines of genetic evidence strongly supportthat WAKs are OG receptors and function in plantimmune responses. First, a chimeric receptor with theextracellular domain of WAK1 and the kinase domain

of ELONGATION FACTOR Tu receptor (EFR) re-sponds to OGs and activates the kinase domain, andconversely, elf18, a polypeptide consisting of the first 18amino acids at the N terminus of ELONGATIONFACTOR Tu, activates a chimeric receptor formed bythe EFR ectodomain and the kinase domain of WAK1and induces the typical responses triggered by OGs(Brutus et al., 2010). Second, pectin- and OG-inducedtranscription of a number of genes depends on WAK2in Arabidopsis protoplasts (Kohorn et al., 2009, 2012).Third, pathogen infection and SA treatment induceWAK1 gene expression and the induction depends onNONEXPRESSOR OF PATHOGENESIS-RELATED(PR) GENES1 (NPR1), a key immune regulator (Caoet al., 1997; He et al., 1998). SA also induces the ex-pression of WAK2, WAK3, and WAK5 (He et al., 1999),and WAK1 and WAK2 are wound-inducible as well(Wagner and Kohorn, 2001). Fourth, overexpression ofWAK1 enhances tolerance to SA toxicity, and expres-sion of an antisense allele of WAK1 reduces the level ofPR1 gene expression induced by the biologically activeanalog of SA, 2.6-dichloroisonicotinic acid (He et al.,1998). Fifth, a dominant gain-of-function WAK2 allele,WAK2cTAP, exhibits autoimmune phenotypes includ-ing ROS accumulation and cell death (Kohorn et al.,2009, 2012). Importantly, the stunted growth pheno-type of WAK2cTAP is largely suppressed by mutationsin the key immune regulators, ENHANCED DISEASESUSCEPTIBILITY1, PHYTOALEXIN DEFICIENT4, andMAP KINASE6 (MPK6) genes (Kohorn et al., 2012,2014), which is reminiscent of autoimmune phenotypes(van Wersch et al., 2016).

Extracellular ATP-DOES NOT RESPONDTO NUCLEOTIDES1

Extracellular ATP (eATP) is one of the best-studiedDAMPs in animals. As the energy currency, cellularlevels of ATP are normally maintained in the range of1 to 10 mM. In animals, ATP is constitutively releasedinto the extracellular space through various mecha-nisms including ATP binding cassette transporters,vesicular exocytosis, gap junctions, and pannexinhemichannels, as well as the P2X7 receptor (Lazarowskiet al., 2003; Spray et al., 2006; Suadicani et al., 2006;Zhang et al., 2007). ATP also leaks into the extracellularmilieu upon cell lysis or necrosis during tissue damageand inflammation (la Sala et al., 2003). Once in the ex-tracellular milieu, ATP binds to either P2X ligand-gated

Table 1. (Continued from previous page.)

DAMP Precursor

N-Terminal

Signal

Peptide

Degree of

Polymerization,

Amino Acids,

or Bps

Receptor Plant References

GmPep914/890 GmPROPEP914/890

No 8 Unknown Soybean Yamaguchi et al., 2011

SCOOP12 PROSCOOP12 Yes 13 Unknown Arabidopsis Gully et al., 2019


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channels or P2Y G-protein coupled receptors, trigger-ing outside–in signaling including changes in intra-cellular [Ca21], production of cytokines, and cell death(Hattori and Gouaux, 2012; Jacobson et al., 2015).Depending on the tissue and cell types, eATP signalingacts in both normal physiological and abnormalpathological processes in animals (Trautmann, 2009).In plants, research with exogenous ATP can be traced

back to the 1960s (Jaffe and Galston, 1966). However, itwas unclear in the early studies whether the exoge-nously added ATP functioned as a signal molecule, aprecursor, or an energy supply (Jaffe and Galston, 1966;Williamson, 1975; Kamizyo and Tanaka, 1982; Nejidatet al., 1983). Recent studies with the widely used stableATP analog, adenosine 59-[g-thio]triphosphate, sug-gested that eATP might act as a signal molecule in theapoplast (Jeter et al., 2004; Song et al., 2006; Torres et al.,2008; Clark et al., 2010, 2011). The presence of eATPwasproven by directly measuring ATP accumulation inArabidopsis leaves and roots (Thomas et al., 1999;Demidchik et al., 2003; Deng et al., 2015), and activesecretion of ATP in plants was confirmed by feedingArabidopsis cultures with [32P]H3PO4 and monitoringradiolabeled ATP in the extracellular matrix (Chivasaet al., 2005). Furthermore, the distribution of eATP inplants was directly visualized using luciferase reportersincluding a cellulose-binding domain-luciferase fusion,an ecto-luciferase, and the infiltration of a luciferase/luciferin mixture (Kim et al., 2006; Chivasa et al., 2009;Clark et al., 2011). These tools allowed discoveries of thedynamics of eATP accumulation in roots, leaves, andaround guard cells (Kim et al., 2006; Chivasa et al., 2009;Clark et al., 2011).The constitutive eATP appears to be essential for

plant cell viability. Depletion of basal eATP using thecell-impermeant traps Glc-hexokinase and apyrasetriggers cell death in both Arabidopsis cell culturesand whole plants (Chivasa et al., 2005). Competitiveexclusion of eATP from its binding sites with non-hydrolyzable ATP analog b,g-methyleneadenosine 59-triphosphate also results in cell death in Arabidopsis,maize (Zea mays), bean (Phaseolus vulgaris), and tobacco(Nicotiana tabacum; Chivasa et al., 2005). Interestingly,the programmed cell death-eliciting mycotoxin fumo-nisin B1-induced cell death in Arabidopsis seems to bemediated by depletion of eATP (Chivasa et al., 2005).Furthermore, environmental stresses induce ATP re-lease (Clark et al., 2011; Sun et al., 2012; Lim et al., 2014;Deng et al., 2015). Although the biological relevance ofthe increases in endogenous eATP levels remains to befully elucidated, studies with exogenous ATP and/oradenosine 59-[g-thio]triphosphate have shown thateATP induces ROS and nitric oxide production, Ca21influx, and H1 efflux in a G protein a-subunit and RES-PIRATORY BURST OXIDASE HOMOLOG (RBOH)-dependent manner (Jeter et al., 2004; Song et al., 2006;Foresi et al., 2007; Wu et al., 2008; Wu and Wu, 2008;Demidchik et al., 2009; Clark et al., 2011; Hao et al.,2012; Sun et al., 2012). Intriguingly, plants appear torespond to eATP in a dose-dependent manner. Low

doses of eATP induce stomatal opening, acceleratevesicular trafficking, and stimulate cell elongation,whereas high doses of eATP trigger stomatal closure,inhibit vesicular trafficking, and suppress cell elon-gation (Clark et al., 2010, 2011, 2013; Wang et al., 2014;Deng et al., 2015). Although depletion of eATP or ex-clusion of eATP from its binding sites leads to celldeath, high doses of eATP also reduce cell viability(Sun et al., 2012; Deng et al., 2015). Currently, themolecular mechanisms underlying such biphasic re-sponses are unknown.Identification of the eATP receptor DOES NOT RE-

SPOND TONUCLEOTIDES1 (DORN1) in Arabidopsisis a major breakthrough in eATP biology and provideda key to addressing many questions about eATP (Choiet al., 2014a; Roux, 2014). DORN1 is a legume-typelectin receptor kinase (LecRK), LecRK-I.9, which hadpreviously been shown to recognize RGD (Arg-Gly-Asp) tripeptide motif-containing protein in mediatingplasma membrane-cell wall adhesions (Gouget et al.,2006). The extracellular domain of DORN1 binds ATPwith a dissociation constant (Kd) of ;46 nM (Choi et al.,2014a). A point mutation in the DORN1 gene com-pletely blocks exogenous ATP-induced transcriptionalchanges in Arabidopsis seedlings, indicating thatDORN1 is the major, if not the sole, receptor of eATP(Choi et al., 2014a). However, as eATP plays an im-portant role in plant growth, development, and cellviability (Tang et al., 2003; Chivasa et al., 2005; Clarkand Roux, 2011; Liu et al., 2012; Yang et al., 2015), butdorn1 mutants do not have obvious growth and de-velopmental defects (Choi et al., 2014a), it has beensuggested that there might be other eATP receptorsmainly regulating plant growth signaling (Roux, 2014).It was recently proposed that eATP functions as a

DAMP in plants (Choi et al., 2014b; Tanaka et al., 2014).Indeed, eATP levels at the wound sites reach ;40 mM,well above the concentration needed to induce ROSproduction and gene expression (Choi et al., 2014a),and reducing eATP levels by overexpressing an apy-rase suppresses wound responses (Song et al., 2006;Wang et al., 2019b). Furthermore, ;60% of the genesinduced by exogenous ATP are also induced bywounding (Choi et al., 2014a), and ATP mainly acti-vates JA signaling through MYC transcription factors(Tripathi et al., 2018; Jewell et al., 2019). Therefore,eATP clearly plays an important role in wound re-sponses. Furthermore, exogenous ATP induces resis-tance to the necrotrophic fungal pathogen B. cinereain Arabidopsis (Tripathi et al., 2018), suggesting apotential role for eATP in immunity against fungalpathogens. Interestingly, although more than a dozenATP-induced genes depend on NPR1 (Jewell et al.,2019), eATP and SA antagonize each other (Chivasaet al., 2009). Exogenous ATP reduces basal SA levels,whereas SA treatment triggers collapse of eATP in to-bacco leaves (Chivasa et al., 2009). In line with theseresults, exogenous ATP does not induce apoplastic re-sistance to Pseudomonas syringae pv. maculicola strainES4326 in Arabidopsis (Zhang and Mou, 2009). On the



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other hand, eATP plays an important positive role instomatal immunity. In Arabidopsis, bacterial infec-tion induces ATP release, particularly around guardcells, and exogenous ATP induces stomatal closureand stomatal resistance against bacterial pathogensin a concentration-dependent manner (Chen et al.,2017). Importantly, exogenous ATP-induced stoma-tal movement and resistance depend on DORN1 andRBOHD. It was proposed that eATP activates DORN1,which in turn phosphorylates theN terminus of RBOHD,leading to ROS production that induces stomatal closure(Chen et al., 2017).

Extracellular NAD(P)—LecRK-I.8/LecRK-VI.2

It is well known that extracellular NAD (eNAD) andNADP (eNADP) play a significant role in animalimmune responses (Billington et al., 2006; Haaget al., 2007; Adriouch et al., 2012). However, whethereNAD(P) is a DAMP in animals remains elusive (Rohand Sohn, 2018). Under normal conditions, intracellularNAD1 levels are in the range of 0.2 to 0.5 mM (Cantóet al., 2015), whereas eNAD levels, e.g. in mammalianserum, are ;0.1 mM (Zocchi et al., 1999; O’Reilly andNiven, 2003). Cell lysis during tissue damage and in-flammation presumably can lead to dramatic increasesin eNAD(P) levels (Billington et al., 2006). At leastthree distinct mechanisms perceive eNAD(P) in ani-mals. First, eNAD(P) can be processed by a number ofNAD(P)-metabolizing ectoenzymes such as CD38 andCD157, which have ADP-ribosyl cyclase, cADP-ribosehydrolase and NAD-hydrolase activities, into Ca21-mobilizing second messengers cADP-ribose and nic-otinic acid adenine dinucleotide phosphate (Ceni et al.,2003; Partida-Sánchez et al., 2003; de Flora et al., 2004;Heidemann et al., 2005; Malavasi et al., 2006). Second,eNAD1 is a substrate of the GPI-anchored or secretedectoenzymes known as mono(ADP-ribosyl)transfer-ases in ADP-ribosylation of plasma membrane sig-naling proteins (Nemoto et al., 1996; Han et al., 2000;Bannas et al., 2005). Finally, eNAD(P) is a potential ag-onist of plasma membrane receptors. It has previouslybeen shown that NAD1 binds to rat brain synapticmembranes and is a potential inhibitory neurotrans-mitter (Khalmuradov et al., 1983; Mutafova-Yambolievaet al., 2007). Recent studies have suggested that severalpurinergic P2X and P2Y receptors function in eNAD(P)-triggered biological responses (Moreschi et al., 2006;Mutafova-Yambolieva et al., 2007; Grahnert et al.,2009; Klein et al., 2009). Nevertheless, binding be-tweenNAD(P) and these receptors has not been reported.

In plants, intracellular NAD(P) levels are in the rangeof 1 to 2 mM (Noctor et al., 2006). We found that, uponwounding and bacterial infection, NAD(P) concentra-tions in the extracellular washing fluid are comparableto those from infiltration with ;0.7 and ;1.2 mM ofNAD(P), respectively (Zhang and Mou, 2009). We alsoshowed that treatment of Arabidopsis and citrus plantswith 0.2 mM of NAD(P) significantly induces resistance

to bacterial pathogens, but not to the necrotrophicfungal pathogen B. cinerea (Zhang and Mou, 2009;Wang et al., 2016; Alferez et al., 2018). Importantly,exogenously applied NAD(P) does not change intra-cellular NAD(P) homeostasis (Zhang and Mou, 2009),suggesting that it acts in the apoplast. Furthermore,we found that transgenic expression of the humanCD38 gene in Arabidopsis reduces eNAD(P) concen-trations and partially compromises SAR (Zhang andMou, 2012). These results together indicate that theeNAD(P) accumulated during pathogen infection isboth necessary and sufficient for activation of plantimmune responses. In addition, exogenous NAD(P)induces ROS production and changes in cytosolic[Ca21] (Pétriacq et al., 2016a, 2016b). Thus, eNAD(P) isa DAMP in plants.

Using a reverse genetic approach based on exoge-nous NAD1-induced transcriptome changes in Arabi-dopsis, we have identified two potential eNAD(P)receptors, LecRK-I.8 and LecRK-VI.2, both of which arelegume-type LecRKs (Singh et al., 2012; Wang et al.,2017a, 2019a). The LecRK-I.8 and LecRK-VI.2 genescan be induced by exogenous NAD1, and both LecRK-I.8 and LecRK-VI.2 are localized in the plasma mem-brane and have kinase activity (Xin et al., 2009; Singhet al., 2013; Wang et al., 2017a). However, the tworeceptors are not alike. LecRK-I.8 only binds NAD1(Kd, ;437 nM), whereas LecRK-VI.2 binds both NAD1and NADP1 with a slightly higher affinity for NADP1(Wang et al., 2017a, 2019a). LecRK-VI.2 binds 32P-NAD1with a Kd of;787 nM, and the binding can be effectivelycompeted by unlabeled NAD1 (50% inhibition concen-tration, IC50, 1,887 nM) and NADP1 (IC50, 945 nM; Wanget al., 2019a). Consistently, mutations in LecRK-I.8 andLecRK-VI.2 suppress NAD1- and NADP1-induced im-mune responses, respectively (Wang et al., 2017a, 2019a).Interestingly, the lecrk-I.8/VI.2 double mutant behaveslike lecrk-I.8 for NAD1 responses and like lecrk-VI.2 forNADP1 responses, indicating that the two receptorsfunction in two separate pathways (Wang et al., 2019a).Importantly, mutations in LecRK-I.8 and LecRK-VI.2significantly compromise basal immunity and bio-logical induction of SAR, respectively (Wang et al.,2017a, 2019a), indicating that LecRK-I.8 primarilyfunctions in basal immunity, whereas LecRK-VI.2 playsa major role in SAR.

The Leu-rich repeat receptor kinase (LRR-RK) BAK1is a coreceptor of a group of LRR-RK receptors includ-ing BRI1, FLAGELLIN-SENSITIVE2 (FLS2), EFR, andPEP RECEPTOR1 (PEPR1)/PEPR2 (Li et al., 2002a;Nam and Li, 2002; Chinchilla et al., 2007; Heese et al.,2007; Postel et al., 2010; Schulze et al., 2010; Roux et al.,2011). BAK1 is also required for signaling triggered byseveral other potential DAMPs including the Arabi-dopsis HMGB3 protein and the SCOOP12 peptide(Choi et al., 2016; Gully et al., 2019). BAK1 and LecRK-VI.2 form a complex in vivo and function in eNAD(P)signaling and SAR (Wang et al., 2019a). The interactionbetween BAK1 and LecRK-VI.2 appears to be consti-tutive and independent of eNAD(P), which is different


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from the inducible associations between BAK1 andLRR-RK receptors. Moreover, the bak1-5 mutation hasbeen shown to impair signaling mediated by the non-RD kinases FLS and EFR, but not that mediated by theRD kinase BRI1 (Schwessinger et al., 2011). Interestingly,although LecRK-VI.2 is an RD kinase, eNAD(P) signal-ing is significantly inhibited in bak1-5 (Wang et al.,2019a). In addition, it has been shown that C-terminaltags on BAK1 have limited effects on several BR re-sponses, but strongly impact PTI signaling (Ntoukakiset al., 2011). Surprisingly, a BAK1-GFP fusion protein isable to complement the defects of bak1-5 in NADP1-induced immune responses and biological inductionof SAR (Wang et al., 2019a). Because C-terminallytagged BAK1 fusion proteins are not phosphory-lated at S612 upon PAMP treatment (Perraki et al.,2018), it would be interesting to test whether S612phosphorylation in BAK1 is required for eNAD(P)signaling and SAR.Interestingly, exogenously added NAD1 moves

systemically and induces systemic resistance (Wanget al., 2019a), suggesting that eNAD(P) might be anSAR mobile signal. Consistently, high levels of exoge-nous NAD(P) induces SA accumulation and NADPHoxidase-independent ROS production (Zhang andMou, 2009; Pétriacq et al., 2016b). Surprisingly, exoge-nous NAD(P)-induced systemic immunity does notdepend on the putative SAR mobile signals pipecolicacid (Pip), N-hydroxy-Pip, azelaic acid (AzA), andglycerol-3-phosphate (G3P), but requires an intact SAsignaling pathway (Wang et al., 2019a). Althoughthe role of DEFECTIVE IN INDUCED RESISTANCE1(DIR1) and ROS in NAD(P)-induced systemic immu-nity has not been tested, exogenous NAD(P)-inducedlocal resistance and PR gene expression is independentof DIR1 and NADPH oxidase, respectively (Zhang andMou, 2009;Wang et al., 2019a). It appears that eNAD(P)functions either downstream or independently of theputative SAR mobile signals Pip, N-hydroxy-Pip, AzA,G3P, DIR1, and ROS in both local and systemic resis-tance. Furthermore, although exogenous eNAD(P) re-quires SA signaling for immune response activation,SA induces the expression of LecRK-VI.2 in an NPR1-dependent manner (Wang et al., 2019a). In addition,because Pip, ROS, AzA, and G3P form a signalingamplification loop (Wang et al., 2018a), it is possiblethat ROS produced in the amplification loop causesreversible or irreversible damages to the plasma mem-brane (Cwiklik and Jungwirth, 2010; Tero et al., 2016),leading to leakage of cellular NAD(P) into the apoplast.Thus, the interplay between eNAD(P) and SA aswell asother SAR signal molecules is complicated and de-serves further investigation.

Glu—GLU-RECEPTOR3.3/GLU-RECEPTOR3.6

Glu is the most prominent neurotransmitter in thebrain and excites postsynaptic neural cells throughdifferent types of receptors including ionotropic and

metabotropic Glu receptors (Brassai et al., 2015). Iono-tropic Glu receptors (iGluRs) are ligand-gated channelsthat are activated upon Glu binding (Krieger et al.,2019). The Arabidopsis genome encodes 20 GLU-RECEPTORs (GLRs) that are homologous to iGluRs(Chiu et al., 2002). GLRs carry the same signature do-mains as animal iGluRs, including the “three-plus-one”transmembrane domains and the extracellular ligand-binding domains (Lam et al., 1998; Chiu et al., 1999;Lacombe et al., 2001). Upon herbivore and mechanicaldamage, Glu is released into the apoplast where it ac-tivates GLR3.3 and GLR3.6, triggering long-distanceelectric and Ca21 signaling as well as JA accumulationand defense gene expression in undamaged leaves(Mousavi et al., 2013; Toyota et al., 2018). At least sixamino acids (Glu, Gly, Ala, Ser, Asn, and Cys) and thetripeptide glutathione can also serve as agonists ofGLR3.3 and induce membrane depolarization and cy-tosolic [Ca21] elevation in a GLR3.3-dependent man-ner (Qi et al., 2006; Stephens et al., 2008; Li et al., 2013).Moreover, seven out of the 20 standard amino acids(Met, Trp, Phe, Leu, Tyr, Asn, and Thr) activateGLR1.4 transiently expressed in Xenopus oocytes tovarious extents, and Met-induced membrane depo-larization in Arabidopsis leaves depends on GLR1.4(Tapken et al., 2013).Interestingly, several amino acids have been shown

to induce disease resistance in plants. For instance, Hisinduces ET biosynthesis and ET-related defense geneexpression as well as resistance to the soil-borne bac-terial pathogen Ralstonia solanacearum and the fungalpathogen B. cinerea partially in an ET-dependent man-ner in tomato (Solanum lycopersicum) and Arabidopsis(Seo et al., 2016). Glu induces several genes of the SAsignaling pathway in rice (Oryza sativa) and tomatofruit, and enhances resistance toMagnaporthe oryzae andAlternaria alternata in rice and tomato fruit, respectively(Kadotani et al., 2016; Yang et al., 2017). Surprisingly,other amino acids except Trp and Tyr also improve riceresistance to M. oryzae to various degrees (Kadotaniet al., 2016). Furthermore, Cys, Asp, and GSH en-hance resistance to P seudomonas syringae pv. tomato(Pst) DC3000 in Arabidopsis (Li et al., 2013). Impor-tantly, Cys- and GSH-induced disease resistance de-pends on GLR3.3, and mutations of the GLR3.3 genecompromise resistance to Pst DC3000 and Hyaloper-onospora arabidopsidis in Arabidopsis (Li et al., 2013;Manzoor et al., 2013), suggesting that GLR3.3 is a po-tential receptor for Cys and GSH released into theapoplast during pathogen infection.

SECONDARY/ INDUCIBLE DAMP-RECEPTOR PAIRS

Systemin—Systemin Receptor1/Systemin Receptor2

Systemin is the first reported extracellular peptidethat induces defense signaling in plants. It was puri-fied from tomato leaf extracts using HPLC based onits proteinase inhibitor (PIN) gene-inducing activity



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(Pearce et al., 1991). Systemin is an 18-amino acidpeptide processed from a 200-amino acid precursornamed “prosystemin” (Pearce et al., 1991; Beloshistovet al., 2018). Genes encoding well-conserved prosyste-mins were identified in the Solanaceae species tomato,potato (Solanum tuberosum), bell pepper (Capsicumannuum), and black nightshade (Solanum nigrum), butnot in tobacco (McGurl et al., 1992; Constabel et al.,1998). The tomato prosystemin gene is constitutivelyexpressed throughout the plant except in the roots, andis further induced by wounding (McGurl et al., 1992).The prosystemin protein accumulates in the cytosol andnucleus of vascular parenchyma cells in response towounding and methyl JA (MeJA) treatment (Narvíez-Vásquez and Ryan, 2004). Prosystemin does not carryan N-terminal signal sequence and, upon cell damage,is expected to passively leak into the apoplast where itis processed by phytaspases and possibly Leu amino-peptidase A (Ryan and Pearce, 1998; Beloshistov et al.,2018). Systemin is highly active. When supplied to thecut stems of young tomato plants,;40 fmol of systeminper plant is sufficient to induce half-maximal accumu-lation of two wound-inducible PINs that break the ac-tivity of digestive enzymes in the insect midgut (Greenand Ryan, 1972; Pearce et al., 1991). Overexpression ofthe prosystemin gene leads to constitutive synthesis ofthe PINs (McGurl et al., 1994).

Although exogenously supplied systemin movessystemically, systemin may not be the mobile signalmediating systemic wound responses. Grafting exper-iments with tomato JA biosynthesis and recognitionmutants indicated that systemic wound signaling re-quires both biosynthesis of JA at the wound site andrecognition of a JA signal in remote tissues, suggestingthat JA controls the production of or acts as the mobilewound signal (Li et al., 2002b). It was proposed thatsystemin promotes systemic wound signaling byaugmenting JA biosynthesis in the vascular tissues(Schilmiller and Howe, 2005).

Identification of the receptor of systemin was a daunt-ing task. A 160-kD systemin-binding protein namedSR160 was initially purified from plasma membranes oftomato suspension cells using a photoaffinity analog ofsystemin (Scheer and Ryan, 2002). SR160 turned out tobe the tomato homolog of the steroid hormone brassi-nolide receptor BRI1 (Scheer and Ryan, 2002; Scheeret al., 2003). Later studies indicated that, althoughSR160 increases binding of systemin to tobacco plasmamembranes, it does not mediate systemin-triggered de-fense responses (Holton et al., 2007; Lanfermeijer et al.,2008; Malinowski et al., 2009). Two distinct LRR-RKstermed SYSTEMIN RECEPTOR1 (SYR1) and SYR2were recently identified as the bona fide systemin re-ceptors (Wang et al., 2018b). Tobacco leaves expressingSYR1 and SYR2 respond with an EC50 of ;0.03 and.30 nM systemin based on systemin-induced ROS pro-duction, respectively (Wang et al., 2018b). Importantly,systemin is unable to induce production of ET and ex-pression of the PIN gene PIN1 in tomato mutant lineslacking functional SYR1 and SYR2 (Wang et al., 2018b).

Surprisingly, mechanical wounding still induces localand systemic expression of the PIN1 gene, even thoughtomato plants expressing a prosystemin antisense geneaccumulate,40% of the wild-type level of PIN1 (McGurlet al., 1992; Wang et al., 2018b). Nevertheless, both theprosystemin antisense lines and the receptor mutant linesupport significantly better herbivore larval growth thanwild type (McGurl et al., 1992; Wang et al., 2018b),demonstrating that systemin signaling contributes toresistance against insect herbivores in tomato.

Plant Elicitor Peptides—Pep Receptor1/Pep Receptor2

The first plant elicitor peptide, Pep1, was isolated as a23-amino acid peptide from extracts of Arabidopsisleaves, which is derived from the C terminus of a 92-amino acid precursor protein encoded by the PROPEP1gene (Huffaker et al., 2006). The PROPEP1 protein doesnot carry an N-terminal signal peptide (Huffaker et al.,2006). It has been shown that PROPEP1 is processed byCa21-dependent type-II metacaspases in Arabidopsis(Hander et al., 2019; Shen et al., 2019). The Arabidopsisgenome carries eight PROPEP genes, PROPEP1 toPROPEP8 (Huffaker et al., 2006; Bartels et al., 2013).PROPEP1, PROPEP2, PROPEP3, PROPEP5, and PRO-PEP8 are expressed in the roots and slightly in the leafvasculature, and are inducible by wounding, and ex-pression of PROPEP4 and PROPEP7 is restricted to theroot tip and is not inducible by wounding (Bartels et al.,2013). Expression of PROPEP1, PROPEP2, and PRO-PEP4 is inducible by MeJA, whereas that of PROPEP2and PROPEP3 is inducible by methyl SA (Huffaker andRyan, 2007). PROPEP2 and PROPEP3 are also inducibleby pathogen attacks and elicitors derived from patho-gens (Huffaker et al., 2006). Furthermore, expression ofPROPEP1 is strongly induced by Pep1 to Pep3, PRO-PEP2 and PROPEP3 are strongly induced by Pep1 toPep6, PROPEP4 and PROPEP5 are weakly inducible,and PROPEP6 is not inducible by the peptides (Huffakeret al., 2006; Huffaker and Ryan, 2007; Yamaguchi et al.,2010). Interestingly, while PROPEP3-YFP is localized inthe cytoplasm, PROPEP1-YFP and PROPEP6-YFP areassociated with the tonoplast (Bartels et al., 2013). Thedifferent gene expression patterns and localization sug-gest nonredundant roles among the members of thePROPEP family. Based on the responses of PROPEPgene promoters to various stimuli, PROPEP genes wereclassified into four groups, with PROPEP1 in the firstgroup, PROPEP2 and PROPEP3 in the second group,PROPEP4, PROPEP7, and PROPEP8 in the third group,and PROPEP5 in the fourth group (Safaeizadeh andBoller, 2019). Nevertheless, all Peps, when applied ex-ogenously, activate MPK3 and MPK6, induce ET pro-duction, and inhibit seedling growth (Bartels et al., 2013).Exogenous Peps also induce expression of several de-fense genes including PDF1.2, MPK3, and WRKY33,production of ROS, elevation of cytosolic [Ca21], andresistance to the bacterial pathogenPstDC3000 (Huffakeret al., 2006; Qi et al., 2010; Yamaguchi et al., 2010). Pep1


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also induces resistance against B. cinerea (Liu et al.,2013). Overexpression of PROPEP1 and PROPEP2 inArabidopsis results in constitutive PDF1.2 expressionand/or resistance against a root oomycete pathogenPythium irregulare (Huffaker et al., 2006).The first PEPR, an LRR-RK called “PEPR1,” was pu-

rified from Arabidopsis suspension cells using a photo-affinity analog of Pep1, 125I1-Tyr-Pep1 (Yamaguchi et al.,2006). 125I1-Tyr-Pep1 is as active as Pep1 and binds toArabidopsis suspension cells with a Kd of ;0.25 nM(Yamaguchi et al., 2006). The second PEPR, PEPR2, wasidentified by phylogenetic analysis and searching for themost closely related gene to PEPR1 (Yamaguchi et al.,2010). Transgenic tobacco cells expressing PEPR1 andPEPR2 bind 125I1-Tyr-Pep1 with Kd values of 0.56 and1.25 nM, respectively. PEPR1 and PEPR2 also bind Pep2to Pep6 and Pep2, respectively (Yamaguchi et al., 2010).Both PEPRs carry a guanylyl cyclase (GC) catalyticdomain with residues for catalysis being conserved(Qi et al., 2010; Yamaguchi et al., 2010), and the GCactivity of PEPR1 has been experimentally demon-strated (Qi et al., 2010). It has been shown that Pep1induces rapid formation of a heterocomplex con-taining de novo phosphorylated BAK1 and an;160-kD polypeptide that is expected to be PEPR1(Schulze et al., 2010), while Pep2 induces PEPR1 as-sociation with BAK1, BAK1-LIKE1, SOMATIC EM-BRYOGENESIS RECEPTOR-LIKE KINASE1 (SERK1),and SERK2 in Nicotiana benthamiana (Yamada et al.,2016). Consistently, the kinase domains of PEPR1and PERP2 interact with that of BAK1 in yeast (Postelet al., 2010), and disruption of BAK1 sensitizes PEPRsignaling (Yamada et al., 2016). The kinase domain ofPEPR1 also interacts with and directly phosphory-lates the receptor-like cytoplasmic kinase BOTRYTIS-INDUCED KINASE1 (BIK1) and BIK is requiredfor Pep1-induced resistance against B. cinerea (Liuet al., 2013).Expression of PEPR1 and PEPR2 is inducible by

wounding,MeJA,most Peps, and PAMPs such as flg22(a 22-amino acid peptide corresponding to the N ter-minus of bacterial flagellin) and elf18 (Yamaguchiet al., 2010). It appears that PEPR1 is inducible in dif-ferent parts of the plant, whereas PEPR2 inductionis restricted to the root (Safaeizadeh and Boller, 2019).Pep-induced expression of defense genes includingMPK3 andWRKY33 is partially suppressed in the pepr1and pepr2 single mutants, and completely blocked inthe pepr1 pepr2 double mutant (Yamaguchi et al., 2010).Pep1-induced expression of PR1 and PDF1.2 as well asresistance against Pst DC3000 are also compromised inthe double mutant (Yamaguchi et al., 2010). Interest-ingly, ET-induced expression of defense genes and re-sistance to B. cinerea are also compromised in the pepr1pepr2 double mutant (Liu et al., 2013). Furthermore,local application of Pep2 activates both JA and SA sig-naling pathways and resistance to Colletotrichum hig-ginsianum path-29 strain in systemic leaves, althoughPep2 may not be a mobile signal (Ross et al., 2014).In agreement with this result, biological induction of

SAR is compromised in the pepr1 pepr2 mutant (Rosset al., 2014).

RAPID ALKALINIZATION FACTORs—FERONIA

RAPID ALKALINIZATION FACTOR (RALF) pep-tideswere first isolated from tobacco, tomato, and alfalfa(Medicago sativa) leaves based on their activity in alka-linating the medium of tobacco suspension cells (Pearceet al., 2001b), and later from sugarcane (Saccharum)leaves using a similar approach (Mingossi et al., 2010).The tobacco RALF is a 49-amino acid peptide located atthe C terminus of a 115-amino acid preproprotein. Thepreproprotein carries an N-terminal signal peptide andthe derived RALF peptide contains four cysteines thatform two disulfide bridges important for its activity(Pearce et al., 2001b). Later studies indicated that many,but not all, RALF preproproteins are cleaved at a con-served dibasic site RRXL by plant subtilisin-like Ser pro-teases such as theArabidopsis SITE-1 PROTEASE/SBT6.1(Matos et al., 2008; Srivastava et al., 2009; Stegmann et al.,2017). Aphotoaffinity analog of the tomatoRALFpeptide,125I-azido-LeRALF,which has biological activity similar tothe native LeRALF, binds to tomato suspension cellswith a Kd of 0.8 nM (Scheer et al., 2005). A highly con-served YISYmotif located at positions 5 through 8 fromthe N terminus is essential for RALF activity, presum-ably being required for productive binding to its pu-tative receptor (Pearce et al., 2010b).RALF proteins have been identified in a large number

of plant species that represent a variety of land plant lin-eages (Cao and Shi, 2012; Murphy and de Smet, 2014).The Arabidopsis genome carries 39 RALF genes (Sharmaet al., 2016). Comprehensive analysis of the identified 795RALF proteins from various plant species revealed fourmajor clades. Clades I, II, and III carry the features im-portant for RALF activity, including the RRXL cleavagesite and the YISY motif important for receptor binding,whereas clade IV is highly diverged and lacks these fea-tures (Campbell andTurner, 2017).While themean lengthof the RALF proteins in clades I, II, and III is 125 aminoacids, the clade-IV RALFs have an average length of only88 amino acids, suggesting that the members in clade IVmay not be true RALFs (Campbell and Turner, 2017).RALF peptides were initially found to suppress root

growth of tomato and Arabidopsis seedlings as well astomato pollen tube growth (Pearce et al., 2001b; Coveyet al., 2010). In line with these results, silencing of thetobacco RALF gene leads to increased root growth andabnormal root hair development (Wu et al., 2007),whereas transgenic overexpression of the ArabidopsisRALF1 and RALF23 genes results in dwarf phenotypes(Matos et al., 2008; Srivastava et al., 2009). Moreover,RALF genes are highly expressed in roots, shoots, andflowers (Zhang et al., 2010; Cao and Shi, 2012; Campbelland Turner, 2017). Collectively, these results support arole for RALF peptides in plant growth and develop-ment. On the other hand, the fungal pathogen Fusariumoxysporum f. sp. ciceri (Race 1)-induced expression of a



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RALF-related EST is 5-fold higher in resistant chickpea(Cicer arietinum) plants than in a susceptible variety(Gupta et al., 2010). In Arabidopsis, RALF8 is inducedby a combination of water deficit and nematode stress,and overexpression of RALF8 confers susceptibility todrought stress and nematode infection (Atkinson et al.,2013). Moreover, synthetic RALF17 peptide increasesresistance to Pst DC3000, while RALF23 reduces resis-tance to Pst DC3000 (Stegmann et al., 2017). Consis-tently, overexpression of RALF23 inhibits resistance toPst DC3000 coronatine-minus, whereas loss of RALF23enhances resistance to Pst DC3000 coronatine-minus(Stegmann et al., 2017). Interestingly, genomes of 26species of phytopathogenic fungi encode RALF homo-logs, and the predicted F. oxysporum RALF appears tocontribute to the virulence of the pathogen in tomatoplants (Masachis et al., 2016; Thynne et al., 2017). Thesedata together suggest potential involvement of RALFsin plant immunity.

The first RALF receptor FERONIA (FER), a Cathar-anthus roseus receptor-like kinase1-like (CrRLK1L) recep-tor, was identified by quantitative phosphoproteomicprofiling of RALF1-treatedArabidopsis seedlings (Harutaet al., 2014). The finding that the abundance of FERphosphopeptides increased inRALF1-treated samples ledto the hypothesis that FER might be the receptor ofRALF1. This hypothesis was supported by reducedRALF1 sensitivity of fermutants and binding of RALF1 toFER (Haruta et al., 2014). Recent studies have shown thatRALF4 and RALF19 bind to other CrRLK1L receptorsincluding ANXUR1, ANXUR2, Buddha’s Paper Seal1,and Buddha’s Paper Seal2, aswell as LEU-RICHREPEATEXTENSIN proteins in regulating pollen tube integrityand sperm release inArabidopsis (Mecchia et al., 2017; Geet al., 2017). FER is also a receptor of RALF23 and perhapsRALF33 aswell (Stegmann et al., 2017). Interestingly, FERconstitutively associates with both FLS2 and BAK1 to actas scaffolds for ligand-induced FLS2-BAK1 complex for-mation. The constitutive association between BAK1 andFER can be strongly enhanced upon treatment with flg22,whereas binding of RALF23 to FER inhibits flg22/elf18-induced complex formation between FLS2/EFR andBAK1, leading to attenuation of FLS2/EFR-mediatedPTI signaling (Stegmann et al., 2017). Furthermore, theGPI-anchored protein LORELEI-like GPI-AP1 (LLG1)constitutively associates with both FER and FLS2 and isrequired for PTI signaling (Li et al., 2015; Shen et al., 2017).LLG1 and the related LLG2 directly bind RALF23 to nu-cleate the assembly of a RALF23–LLG1/2–FER hetero-complex (Xiao et al., 2019), suggesting that RALFsmay beperceived bydistinct CrRLK1L receptor kinase-LLG/LREheterocomplexes in regulating various biological pro-cesses including plant immunity.

Phytosulfokines—Phytosulfokine Receptor1/Phytosulfokine Receptor2

Phytosulfokines (PSKs) are sulfated Tyr-containingpentapeptides with mitogenic activity in vitro. The first

PSK was purified from conditioned medium of rapidlygrowing asparagus (Asparagus officinalis) cell cultures byfollowing its mitogenic activity (Matsubayashi andSakagami, 1996). Based on the amino acid sequence ofthe asparagus PSK, rice and Arabidopsis PSK geneswere subsequently identified (Yang et al., 1999, 2001;Matsubayashi et al., 2006). PSKs are derived from;77-to 89-amino acid prepropeptide precursors throughtyrosylprotein sulfotransferase (TPST)-mediated Tyrsulfation and subtilisin-like Ser protease-catalyzedproteolytic cleavage (Srivastava et al., 2008; Komoriet al., 2009). The PSK precursors carry N-terminalsignal sequences and are sulfated in the Golgi appa-ratus, secreted, and cleaved in the extracellular milieu(Yang et al., 1999, 2001; Srivastava et al., 2008; Komoriet al., 2009).

PSK binds to plasma membrane-enriched frac-tions with both high and low affinities (Kd valuesranging from 1 to 100 nM; Matsubayashi et al., 1997;Matsubayashi and Sakagami, 1999). Photoaffinitycross-linking analysis indicated that the putative re-ceptors for PSK in rice are 120- and 160-kD glycosylatedproteins (Matsubayashi and Sakagami, 2000). The firstPSK receptor, an LRR-RK, was purified from micro-somal fractions of carrot suspension cells using ligand-based affinity chromatography, and the carrot PSKreceptor (PSKr) gene encodes both 120- and 150-kDproteins (Matsubayashi et al., 2002). Amino acid ho-mology search revealed that the Arabidopsis genomeencodes two PSKRs, PSKR1 and PSKR2 (Matsubayashiet al., 2006; Amano et al., 2007). Structure analysisindicated that PSK interacts with and stabilizes anisland domain of PSKR, which enhances PSKR het-erodimerization with a SERK coreceptor (Wang et al.,2015). The cytoplasmic domain of PSKR1 has not onlykinase activity but also GC activity. Both exogenousPSK treatment and overexpression of PSKR1 increasecGMP levels in protoplasts (Kwezi et al., 2011).Moreover, PSKR1, BAK1, CNGC17, and H1-ATPasesAHA1 and AHA2 form a complex in mediating PSK-triggered signaling (Ladwig et al., 2015).

PSK was initially shown to induce the prolifera-tion of asparagus suspension cells (Matsubayashi andSakagami, 1996; Matsubayashi et al., 1997). PSK pre-cursors are constitutively secreted by suspension cells,and overexpression and silencing of PSK genes led toincreased and reduced PSK levels in conditionedmediaof rice transgenic cells, respectively (Yang et al., 1999,2001). PSK genes are stably expressed not only in sus-pension cells but also in intact plants (Yang et al., 1999,2001). Overexpression of PSK genes resulted in en-larged transgenic calli (Yang et al., 2001; Matsubayashiet al., 2006). Similarly, transgenic carrot cells express-ing high levels of sense mRNA of the PSK recep-tor exhibited accelerated proliferation, whereas thoseexpressing antisense showed substantially reducedcallus growth (Matsubayashi et al., 2002). Individualcells of the Arabidopsis pskr1-1 mutant gradually losetheir potential to form calli as the tissues mature, whilePSKR1-overexpressing plants exhibit significantly greater


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callus-forming potential than wild type (Matsubayashiet al., 2006).Genes encoding PSK precursors, processing en-

zymes, and/or receptors are inducible by wounding,elf18, flg22, and B. cinerea (Srivastava et al., 2008;Igarashi et al., 2012; Hou et al., 2014; Zhang et al.,2018), suggesting a potential involvement of PSK-PSKR signaling in plant immunity. Indeed, elf18-triggered immune responses are enhanced in theArabidopsis pskr1–3 mutant (Igarashi et al., 2012).Mutations of the PSKR1 and TPST genes enhanceresistance to Pst DC3000 and increase susceptibilityto A. brassicicola, whereas overexpression of PSK2,PSK4, and PSKR1 leads to opposite effects (Mosheret al., 2013). However, overexpression of the ricePSKR1 gene activates SA signaling and enhances re-sistance to the bacterial pathogen Xanthomonas oryzaepv. oryzicola (Yang et al., 2019). Furthermore, exoge-nous application of PSK enhances PstDC3000 growthin the Arabidopsis tpst-1mutant (Mosher et al., 2013),and increases resistance to Botrytis cinerae in tomato(Zhang et al., 2018). In addition, silencing of the to-mato PSKR1 gene enhances susceptibility to B. cinerae(Zhang et al., 2018). Binding of PSK to tomato PSKR1elevates cytosolic [Ca21], which enhances interac-tion between calmodulins and auxin biosyntheticYUCCAs, resulting in auxin-dependent immunityagainst B. cinerae (Zhang et al., 2018).

GRIM REAPER Peptide—POLLEN-SPECIFIC RLK5

GRIM REAPER (GRI) belongs to a small family withsix members in Arabidopsis. Its C-terminal Cys-richdomain is highly homologous to STIGMA-SPECIFICPROTEIN1 that functions in regulation of exudate se-cretion in the pistils and promotion of pollen tubegrowth (Verhoeven et al., 2005; Huang et al., 2014). TheGRI protein is 169-amino acids long, carries a predictedN-terminal signal peptide (amino acids 1–30), and issecreted into the apoplast (Wrzaczek et al., 2009). As theGRI gene expression in flowers is 1,000-fold higher thanin leaves (Wrzaczek et al., 2009), GRI likely plays a rolein reproduction. Indeed, a gain-of-function gri mu-tant and GRI-overexpressing plants exhibit reducedseed content in the siliques (Wrzaczek et al., 2009).Interestingly, the low basal GRI expression inleaves is inducible by ozone exposure and both griand GRI-overexpressing plants are sensitive to ozone(Wrzaczek et al., 2009). The gri mutant is also resis-tant to the virulent bacterial pathogen Pst DC3000(Wrzaczek et al., 2009). These gri phenotypes arelikely caused by accumulation of a GRI peptide(GRIp) corresponding to the N-terminal variable re-gion after the signal peptide (amino acids 31–96;Wrzaczek et al., 2015). Exogenous GRIp31–96 inducessuperoxide- and SA-dependent ion leakage, an indica-tor of cell death. GRI is cleaved by an apoplast-localizedtype II metacaspase METACASPASE9, releasing an11-amino acid peptide, GRIp68–78, which is sufficient

for induction of ion leakage (Wrzaczek et al., 2015).GRIp-induced ion leakage depends on the atypicalLRR-RK, POLLEN-SPECIFIC RECEPTOR-LIKE KI-NASE5 (PRK5; Wrzaczek et al., 2015). Full-length GRIwithout the signal peptide and GRIp31–96 interact withthe extracellular domain of PRK5 in vitro. A radiola-beled GRIp, 125I-Y-GRIp68–78, which is active for ionleakage induction, binds to Arabidopsis membraneextracts with a Kd of 1.9 nM. Binding of 125I-Y-GRIp68–78to membrane extracts is reduced to background levelsin prk5 mutants (Wrzaczek et al., 2015). These resultssupport that PRK5 is a receptor of GRIp. However,because the prk5 andmc9mutations have no significanteffects on extracellular superoxide-induced ion leakageand resistance to Pst DC3000 (Wrzaczek et al., 2015),whether GRIp is a bona fide DAMP requires furtherinvestigation.

PAMP-INDUCED SECRETED PEPTIDE1—RLK7

Genes encoding PAMP-INDUCED SECRETEDPEPTIDE (PIP) precursors named prePIP1, prePIP2,and prePIP3 were identified by searching flg22- andelf18-induced transcription data (Hou et al., 2014).Eleven prePIP homologs were identified inArabidopsisbased on the highly conserved C-terminal sequences.All of the prePIP family members carry a N-terminalsignal peptide (Hou et al., 2014; Vie et al., 2015).Orthologs of prePIPs were also identified in multipleother plant species such as soybean (Glycine max), grape(Vitis vinifera), maize, and rice (Hou et al., 2014). TheprePIP1 gene is induced not only by PAMPs but alsoby methyl SA, Pst DC3000, and the fungal pathogenF. oxysporum f. sp. conglutinans strain 699 (Foc 699; Houet al., 2014). Overexpression of prePIP1 and prePIP2inhibits root growth and enhances resistance to Foc 699.Synthetic PIP1 and PIP2 comprising the conservedC terminus also inhibit root growth and induce im-mune responses similar to PTI (Hou et al., 2014). In-terestingly, PIP1- and PIP2-mediated root growthinhibition and immune responses are compromised intransferred DNA insertion mutants of the RLK7 gene,which encodes a class XI LRR-RK, suggesting thatRLK7 is a potential receptor of these PIPs (Hou et al.,2014). Indeed, RLK7-HA was pulled down with PIP1-biotin–associated streptavidin beads from membraneextracts of transgenic Arabidopsis plants expressingRLK7-HA, and specific binding of radiolabeled 125I-Y-PIP1 was detected in homogenates of tobacco leavestransiently expressing RLK7-HA in photoaffinity la-beling assays, indicating that PIP1 directly binds toRLK7 (Hou et al., 2014). Moreover, PIP1-induced rootgrowth inhibition and/or ROS production are reducedin the bak1-4 mutant but not in the bik1 mutant, indi-cating that PIP1-RLK7 signaling is partially dependenton BAK1, but independent of BIK1 (Hou et al., 2014).Finally, both PIP1 and PEP1 induce the expressionof PrePIP1, ProPEP1, RLK7, PEPR1, and FLS2, sug-gesting that PIP1 and PEP1 function cooperatively



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

IDA-LIKE6 Peptide—HAESA/HAE-LIKE2

INFLORESCENCE DEFICIENT IN ABSCISSION(IDA) and IDA-LIKE (IDL) proteins are precursorsof peptides that induce floral abscission (Butenkoet al., 2003; Stenvik et al., 2008). The ArabidopsisIDA family has nine members (IDA and IDL1–8)characterized by an N-terminal signal peptide, avariable region, and a C-terminal conserved regionwhere the PIP motif is located (Butenko et al., 2003;Stenvik et al., 2008; Vie et al., 2015). Genetic studiessuggested that two LRR-RKs, HAESA (HAE) andHAE-LIKE2 (HSL2), are receptors of IDA/IDL-de-rived peptides (Stenvik et al., 2008). A chemilumi-nescent acridinium-labeled PIP with a Val residue atthe N terminus and hydroxylation of the conservedPro at position 7 termed “acri-PIPPo” binds to leafmaterials of N. benthamiana expressing HSL2DKDwith a Kd of ;20 nM (Butenko et al., 2014), demon-strating that HSL2 is a bona fide receptor of IDA/IDLpeptides. The IDA and IDL6 genes are upregulated byPAMPs, and IDL6 is also induced by Pst DC3000(Hou et al., 2014; Wang et al., 2017b). Synthetic IDL6and IDL7 extended PIP peptides downregulate theexpression of a broad range of stress-responsivegenes (Vie et al., 2017). Moreover, overexpression ofIDL6 enhances susceptibility to Pst DC3000, whereassilencing of IDL6 increases resistance to the bacterialpathogen (Wang et al., 2017b). IDL6 elevates thetranscription of Arabidopsis DEHISCENCE ZONEPOLYGALACTURONASE2 (ADPG2), which encodesan active polygalacturonase that promotes pectindegradation to facilitate Pst DC3000 infection. Con-sistent with HAE and HSL2 being receptors of IDL6,IDL6-mediated ADPG2 expression and Pst DC3000susceptibility are completely suppressed in the haehsl2 double mutant (Wang et al., 2017b). Interest-ingly, the IDA-HEA/HSL2 ligand-receptor pair isrequired for P. syringae type III effector-triggered leaf

abscission, which likely represents a new form ofplant immunity (Patharkar et al., 2017).

CONCLUSIONS AND FUTURE PERSPECTIVES

A large and compelling body of evidence has accu-mulated in recent years, which supports an importantrole for DAMPs in plant immune responses (Fig. 1).Nevertheless, the identity of DAMPs in plants remainsto be unambiguously defined. The Danger model pos-tulates that healthy cells or cells undergoing normalphysiological death do not generate danger signals(Matzinger, 1994, 2002). It was recently further arguedin animals that a canonical DAMP can be upregulated,but not released, in response to PAMP detection orstress stimuli that presumably lead to necrosis (Martin,2016). In plants, however, it seems that some DAMPsare actively released upon PAMP detection or envi-ronmental stresses (Deng et al., 2015; Chen et al., 2017).Release of DAMPs in the absence of cell death appearsto be inconsistent with the Danger model. However,before we arrive at such a conclusion, we must considerthe following possibilities. First, some DAMPs mayplay dual functions in plants. For instance, as in animals(Trautmann, 2009), eATP in plants not only acts as aDAMP in wound response, but also plays a major rolein growth control (Choi et al., 2014b; Roux, 2014). Theconstitutive eATP and actively released ATP may becrucial for cell viability and growth changes (Chivasaet al., 2005; Liu et al., 2012; Deng et al., 2015). Second,the amount of DAMPs actively released may not besufficient for immune activation. For example, in re-sponse to cold stress (4°C for 7 d), the concentration ofeATP in the extracellular root medium of 7-d–oldArabidopsis seedlings is ;8 nM, whereas that in thefluid released at the sites of physical wounding is;40 mM (Choi et al., 2014a; Deng et al., 2015). The eATPconcentration under cold stress is likely too low to ac-tivate the eATP receptor DORN1 (Kd ;46 n M) forwound response (Choi et al., 2014a). These resultssuggest that DAMPs may induce immune responses ina concentration-dependent manner, or there may be a

Figure 1. (Continued.)Putative DAMP-receptor pairs and their functions in plant immunity. The cell surface receptor cartoons depict the putative DAMPreceptors with their coreceptors or associated proteins. The cartoon for the Glu receptors GLR3.3/GLR 3.6 is based on an animaliGluR, a ligand-gated ion channel formed by four subunits. Each subunit has four domain layers: the extracellular N-terminaldomain and ligand-binding domain, the transmembrane domain, and an intracellular C-terminal domain. For the sake of clarity,only two subunits are shown in the cartoon for GLR3.3/3.6. Moreover, although several RALFs including RALF17, RALF23,RALF33, and RALF34 are potential DAMPs that positively or negatively regulate immunity, RALF23 has been shown to bind LLG1/2 and FER to nucleate the assembly of RALF23-LLG1/2-FER heterocomplexes. Thus, only RALF23-LLG1/2-FER–mediated inhi-bition of PTI is presented here. Note that both LLG1 and FER are required for PTI signaling. In addition, although PIP1-induced–ROS production and root-growth inhibition partially depend on BAK1, whether the PIP1 receptor RLK7 interactswith BAK1 has not been reported. A question mark (?) is thus included in the RLK7/BAK1 cartoon to illustrate the uncertainty.Finally, dashed arrows are used to indicate the immune responses that are induced either by exogenously added DAMPs or byoverexpression of the receptors; however, whether these immune responses are induced by the DAMPs through their receptors isunclear. By contrast, solid arrows represent immune responses that are activated by the DAMPs through their receptors. EGF,epidermal growth factor; IDL6p, INFLORESCENCE DEFICIENT IN ABSCISSION-LIKE6 peptide; INR, INCEPTIN RECEPTOR;SOBIR1, SUPPRESSOR OF BIR1-1; TM, transmembrane.



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threshold belowwhichDAMPs do not activate immuneresponse. Third, because plants lack specialized im-mune cells and adaptive immunity, cell-autonomousimmunity may play a more important role in plantsthan in animals (Randow et al., 2013). Plantsmight havethus evolved mechanisms to actively release highamounts of DAMPs for activation of cell-autonomousimmunity. Clearly, further investigations are requiredto determine whether sufficient DAMPs can be releasedin the absence of cell death for immune activation inplants. Regardless, even though the Danger model mayneed some modifications for the plant immune system,the general principles should be applicable.

It is expected that multiple DAMPs would be re-leased upon any type of cell damage. However, thecombinations of DAMPs following different types ofcell damages may be different. For instance, besidesprimary DAMPs, mechanical damage leads to releaseof wounding-induced secondary DAMPs such as sys-temin (Pearce, 2011), whereas pathogen attack resultsin release of pathogen-induced secondary DAMPs in-cluding Peps and PIPs (Huffaker et al., 2006; Houet al., 2014). Moreover, DAMPs may be released atvarious times during plant-microbe interaction dueto their different subcellular localizations. In thisregard, DAMPs derived from the cell wall would bereleased early, followed by those from the cytoplasm,and finally from the nucleus. Additionally, the half-lives and apoplastic mobility of DAMPs as well as theactivities of receptors for DAMPs may differ signifi-cantly (Adriouch et al., 2012). Thus, DAMPs should

function cooperatively with each other, as well as withPAMPs in a temporal, spatial, and stress-specific man-ner to generate a peculiar immune response.

Determining the role of DAMPs in plant immuneresponses is an important but challenging task (seeOutstanding Questions). Several studies have investi-gated the interplays between DAMPs and PAMPs(such as flg22 and elf18) as well as between differentDAMPs. Both synergism and antagonism betweenDAMPs and PAMPs/DAMPs have been observed(Fauth et al., 1998; Stennis et al., 1998; Aslam et al., 2009;Ma et al., 2012; Flury et al., 2013; Tintor et al., 2013;Stegmann et al., 2017). However, because DAMPs arereleased during pathogen infection or herbivore attack,the context is extremely complex. It would be diffi-cult to sort out the contribution of individual DAMPs tothe final specific immune phenotype. Perhaps some-thing similar to the recently proposed PAMP/DAMPcombination-based “inflammatory code” could helpsolve this puzzle (Escamilla-Tilch et al., 2013). More-over, although evidence supporting a role for DAMPsin effector-triggered immunity is accumulating (Maet al., 2012; Zhang and Mou, 2012), in-depth investi-gations are warranted. In addition, several DAMPshave been implicated in systemic responses includingSAR (Pearce et al., 1991; Ross et al., 2014; Toyota et al.,2018; Wang et al., 2019a), suggesting that DAMP sig-naling is an integral component of biological inductionof systemic responses. Future research should investi-gate whether DAMPsmove systemically or act throughother signal molecules similarly to systemin (Li et al.,2002b; Schilmiller and Howe, 2005), and how theDAMP signal is transduced into the nucleus.

It is worth mentioning that what we currently knowabout DAMPs is just the tip of the iceberg. Among thecountless numbers of intracellular molecules, manycould potentially become DAMPs if released into theapoplast. Furthermore, a recent study using a bio-informatics approach identified .1,000 putative se-creted peptides in Arabidopsis (Lease and Walker,2006), not to mention other plant species with largergenomes than Arabidopsis. Many of the putative pep-tides could potentially function as DAMPs. Identifica-tion of potential new DAMPs as well as the processingenzymes and/or receptors for the candidate DAMPswould greatly improve our understanding of plantDAMP signaling and the plant immune system as awhole. It is expected that a deeper understanding ofplant DAMPs and the plant immune system couldsignificantly help design new strategies to breed cropvarieties with increased resistance against pathogensand/or herbivores.

ACKNOWLEDGMENTS

We apologize to researchers whose relevant studies were not cited in thisreview due to page limitations, and would like to thank Fiona M. Harris forcareful reading of the article.

Received October 7, 2019; accepted December 16, 2019; published January 6,2020.


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