Characteristics, Roles and Applications of Proteinaceous Elicitors from Pathogens in Plant Immunity

In interactions between pathogens and plants, pathogens secrete many molecules that facilitate plant infection, and some of these compounds are recognized by plant pattern recognition receptors (PRRs), which induce immune responses. Molecules in both pathogens and plants that trigger immune responses in plants are termed elicitors. On the basis of their chemical content, elicitors can be classified into carbohydrates, lipopeptides, proteinaceous compounds and other types. Although many studies have focused on the involvement of elicitors in plants, especially on pathophysiological changes induced by elicitors in plants and the mechanisms mediating these changes, there is a lack of up-to-date reviews on the characteristics and functions of proteinaceous elicitors. In this mini-review, we provide an overview of the up-to-date knowledge on several important families of pathogenic proteinaceous elicitors (i.e., harpins, necrosis- and ethylene-inducing peptide 1 (nep1)-like proteins (NLPs) and elicitins), focusing mainly on their structures, characteristics and effects on plants, specifically on their roles in plant immune responses. A solid understanding of elicitors may be helpful to decrease the use of agrochemicals in agriculture and gardening, generate more resistant germplasms and increase crop yields.


Introduction
Plants, the main nutrient sources for humans, are attacked by many pathogens that can consume them, devastating yields and causing economic losses, which make pathogen infection among the most formidable challenges to global food security. To reduce crop loss, many chemical bactericides and fungicides have been used to prevent or treat diseases caused by pathogens. However, in addition to inducing toxicity in pathogens, these conventional agrochemicals pose hazards to the environment and human health [1]. Furthermore, continuous applications of conventional germicides may fail because of the emergence of resistant pathogens. Recent advances in the study of plant innate immunity, especially plant-pathogen interactions, have shown that elicitors can trigger immune responses in plants that lead to intense and persistent resistance to pathogens [2][3][4][5][6]. The application of elicitors seems to be an attractive and promising method for treating agricultural pathogens in a green and environmentally friendly manner.
Advances in research into plant innate immunity have revealed that plants employ a multilayered surveillance system to detect and respond to pathogen invasion. According to the typical zigzag model, there are two layers in plant innate immunity: namely, pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) [7]. In PTI, plant pattern recognition receptors (PRRs) on the plasma membrane recognize pathogen-/microbe-associated molecular patterns (PAMPs/MAMPs) and trigger subsequent immune responses, such as membrane depolarization, calcium influx, a spike in reactive oxygen species (ROS) levels, activation of mitogen-activated protein kinase (MAPK) cascades, induction of pathogenesis-related (PR) gene expression and callose deposition [3].

Architectures and Functions of Harpins
Harpins, the first identified cell-free elicitor causing a hypersensitive response (HR) from Gram-negative plant-pathogenic bacteria, are an important type of elicitor that is involved in the interaction between plants and pathogens. They function in many physiological responses in microbes and abiotic and biotic stresses in plants, e.g., working as virulence factors in the invasion of bacterial pathogens into a host, inducing HR and non-HR immunity in plants and promoting plant growth [16][17][18][19]. Harpins are elicitors with approximately 10-60 kDa, encoded by sequences located in the hypersensitive response and pathogenicity (hrp) gene cluster and released through the type III secretion system [16,20]. Most harpins are acidic, hydrophilic, and thermostable and are sensitive to chemically denaturing proteins because of their distinctive sequences and architectures [16,[19][20][21][22]. Harpins are enriched with glycine and serine but carry few aromatic amino acids and low levels of cysteine, which is an amino acid that contributes to disulfide bonds. The level of glycine exceeds 15% in most harpins, and these glycine residues cluster in specific protein regions [16]. Harpins usually carry many (from two to nine) α-helixes, and most do not harbor domains similar to other proteins in bacteria or plants, which makes them good elicitors in plants [16,[23][24][25].
Since the first harpin, HrpN (sometimes called HrpNea) was identified in the fire blight pathogen Erwinia amylovora, a devasting pathogen in the Rosaceae family, numerous studies have focused on the functions and signaling pathways induced by harpins in plants [14,18,20,[26][27][28][29][30]. The types of immunity primed by harpins include the common HR, through which harpin immunity was discovered, and non-HR immunity ( Table 2). Typical HR-related cell death and non-HR defense responses, such as bursts in ROS production and increased expression of pathogenesis-related genes, callose deposition and phytohormone production, have been observed in harpin-treated plants [14,[31][32][33][34].

Action Mechanisms Triggered by Harpins in Plants
Several possible explanations for harpin-induced HR are discussed herein. First, normal physiological cell membrane functions may be disrupted by harpins. Specifically, harpins bind to the plasma membrane of plant cells, which activates subsequent immune responses, and several harpins, such as HrpZ Psph and PopA, form pores in the plasma membrane that enable ion conduction [25,[35][36][37]. Second, mitochondrial function may be indirectly/directly disrupted by harpins, triggering mitochondrion-dependent programmed cell death in plants; for example, mitochondrial electron transport was inhibited by HrpN, which reduced ATP synthesis in tobacco. HrpZ1 induced the rapid release of cytochrome c from mitochondria into the cytosol, which led to the accumulation of ROS in Arabidopsis thaliana. RipX (previously named PopA) interacts with mitochondrial ATP synthase F1 subunit alpha (ATPA) and represses the transcription of atpA in Nicotiana benthamiana [29,38,39]. Third, harpins induce the expression of HR-related genes and activate MAPK signaling cascades [19,33,[40][41][42].
Several membrane-localized harpin targets in plants have been identified, and their discovery has clarified harpin mechanisms of action. For example, a small transmembrane protein in apple (Malus x domestica) HrpN-interacting protein from Malus (HIPM) and its homologue in Arabidopsis (AtHIPM) have been found to interact with the harpin HrpN, and this interaction has been shown to be critical to susceptibility to E. amylovora [28,43]. The N-terminal 198 amino acids of HrpN were required for the HrpN protein interaction with HIPM. Further physiological analyses showed that the atHIPM knockout mutant was slightly larger than the wild type. All these results indicated that AtHIPM negatively regulates HrpN functions [28]. HIPM also interacts with oxygen-evolving enhancer-like protein (MdOEE), which may affect photosynthesis and subsequent ROS production [43]. The HIPM induction of this signaling axis may explain the mechanism through which harpins affect plant growth and immunity. In addition to HIPM, plasma membrane intrinsic protein (PIP) is a plasma membrane harpin sensor. The aquaporin AtPIP1;4, which is involved in the translocation of H 2 O 2 from apoplasts to the cytoplasm to induce subsequent immune responses and membrane permeability of CO 2 and H 2 O, functions as a sensor of Hpa1 in A. thaliana. Upon Hpa1 treatment, the interaction between Hpa1 and AtPIP1;4 led to increased photosynthesis to promote plant growth, suggesting that harpins induce the AtPIP1;4 signaling pathway to promote plant growth and trigger immune responses [26,44].

Plant Response to Harpins Promotes Biotic and Abiotic Resistance and Growth
Treatment with harpins contributes to pathogen resistance in plants (Table 2), which is an attribute with practical usefulness in agriculture. For example, the application of Messenger (a commercial product that contains Harpin (Ea) as the main active component), harpin (Axiom, Rx Green Technologies) and HpaXpm reduced the severity of diseases caused by Phytophthora infestans and Botrytis cinerea, Pythium aphanidermatum, and Tobacco mosaic virus (TMV) in tomato, hemp seedlings, and tobacco, respectively [14,19,45,46]. Transgenic plants expressing harpins, e.g., transgenic soybean expressing hrpZpsta from P. syringae pv. tabaci and hrf2 from Xanthomonas oryzae pv. oryzicola, tobacco expressing ripX and hpa1, and transgenic cotton expressing hpa1 Xoo from X. oryzae pv. oryzae have shown increased immune responses [29][30][31]47,48].
In addition to increases in immune responses and pathogen resistance, harpins induce other physiological activities, such as growth and development, tolerance to drought and high salinity (Table 2) [31,[49][50][51][52][53][54]. Specifically, treatment with HpaXpm promoted increased root length and fresh weight in A. thaliana, and the expression of SSB Xoc and Hpa1 in N. benthamiana promoted increased root length and plant growth [19,31,49]. This harpin-promoted growth may have been related to an increase in photosynthesis due to increased chlorophyll levels, CO 2 conduction rates, and greater changes in the levels of phytohormones, e.g., ethylene (ET), gibberellin (GA), and salicylic acid (SA) [14,18,26,27,31,53]. Notably, exogenous PopW (a harpin from Ralstonia solanacearum ZJ3721) treatment led to greater drought resistance in Medicago sativa L. and tomato, and this effect was related to multiple mechanisms, such as the action of phytohormones, e.g., abscisic acid (ABA), GA, SA, and drought-related gene expression [15,52].
A particular partial harpin sequence has been shown to be sufficient for harpin function, although fragments are different in different harpins. Specifically, three heptads in the N-terminal coiled-coil domain of Hpa1 from X. oryzae pv. Oryzae, a 23-amino acid fragment of HpaG from Xanthomonas axonopodis pv. glycines, a 24-amino acid peptide in the HrpZ protein of P. syringae, were sufficient to initiate HR in plants [31,55,56]. Further studies showed that this particular segment of harpins induced a more robust response than that induced by harpins [22]. All these results suggest that certain harpin sequences are essential for the plant response, which may indicate that these elicitors can be leveraged to induce plant resistance to pathogens.
Different harpins show antagonistic effects. For example, HrpW(ea) from E. amylovora administered at subnanomolar concentrations decreased the defense responses that had been triggered by another harpin from this bacterium, HrpN(ea). This result may be attributable to the opposing anion channel changes triggered by HrpW(ea) and HrpN(ea) [57].
Studies have mainly focused on the architectures of harpins and the functions and action mechanisms involved in microbes and host plants for 30 years. Practical application of harpins has been successful; i.e., Messenger was registered in the United States and allowed to use on all crops in 2000; now, it has been used in many countries. More effective harpins remain to be used efficiently in practice and play more roles in agriculture and gardening.

NLPs
Necrosis-and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) constitute another group of proteinaceous elicitors widely secreted by bacteria, fungi, and oomycetes. NLPs contain characteristic NPP1 domains (PF05630) and are involved in multiple processes such as virulence, conidiospore production, formation of appressoria, abiotic stresses in microbes and triggering immune responses in plants [11,13,58,74,75]. On the basis of their impacts on host plants, NLPs are classified into two forms: cytotoxic to eudicot and monocot plants and noncytotoxic [59,65,76]. Cytotoxic NLPs are expressed when a pathogen converts from biotrophy into necrotrophy and function as pore-forming toxins (PFTs), and these NLPs bind to the terminal monomeric hexose moieties of glycosyl inositol phosphoryl ceramide (GIPC) to cause cytolysis [65,[76][77][78]. Noncytotoxic NLPs induce an immune response without cytolysis. Since the first NLP protein, Nep1, was characterized in culture filtrates of Fusarium oxysporum f. sp. erythroxyli in 1995, many advances have been made to reveal the function and action mechanism of NLPs [79].

Taxonomy of NLPs
NLPs have been classified into types I, II, and III, on the basis of their amino acid sequences. Type I NLPs are the most abundant NLPs and can be found in bacteria, fungi, and especially oomycetes. Type II NLPs are found in bacteria, fungi and several oomycetes. Type I and type II NLPs have been characterized based on the number of cysteine residues they carry because cysteine is essential for their activity [76,80]. Type I NLPs usually carry 2 cysteine residues, which form one disulfide bridge, while type II NLPs generally harbor a second disulfide bridge, which is not required for necrosis induction [76]. Type II NLPs harbor a calcium-binding motif, which is essential for their cytotoxicity [81]. Type III NLPs, which exist exclusively in ascomycete fungi, are less conserved, with most NLPs harboring six cysteine residues [76,81]. Different species encode considerably different numbers of NLPs (including both cytotoxic and noncytotoxic types), ranging from only one or two in most bacteria to dozens in oomycetes, suggesting horizontal gene transfer in the evolution of NLPs [76].
Most NLPs are small proteins (which are approximately 25 kDa). They are singledomain (NPP1 domain, PF05630) proteins with signal peptides (SPs) on the N-terminus, and the NPP1 domain carries a conserved -HRH-W-fragment, which is important in the coordination of divalent cations and contributes to typical NLP features and physiological functions [76,82,83]. The protein is localized to the extracellular space through the classical secretion route and is further processed [76].

Involvement of NLPs in Immune Responses in Plants
As mentioned above, NLPs are involved in multiple processes in both microbes and plants. In this subsection, we focus on their effects on host plants (Table 2). Treatments with cytotoxic NLPs induce rapid cytolysis and result in plant cell necrosis [65,82]. In addition to NLPs functioning as PFTs, noncytotoxic NLPs and conserved fragments function as PAMPs/MAMPs to prime innate immunity in plants. Exogenous NLP or NLP fragment treatment or ectopic expression of NLP or conserved fragments (nlp20/nlp24) triggers plant immune responses, such as the expression of immune-responsive genes, activation of MAPK signaling cascades, bursts in ROS production, induction of SA, and enhanced plant immunity against pathogens in addition to necrosis and ethylene-induction [13,63,65,74,[83][84][85].

Action Mechanisms of NLPs in Plant Immune Responses
The cytolytic mechanism triggered by cytotoxic NLPs has been described. As mentioned above, cytotoxic NLPs target and bind to the terminal hexose residues of glycosylinositol phosphorylceramide (GIPC) sphingolipids, which vary in total amount and concentration among different plants, which are located at the outer leaflet of the envelope membrane in plants [59,65]. The binding of GIPC-NLP is driven by electrostatic interactions, which can be strengthened in the presence of sterols and results in NLP conformational changes [65,77,86]. After binding, NLPs form functional oligomers and then form shallow transient pores that leak small molecules [77,87]. Tryptophan155 at the bottom of loop L3, an important amino acid involved in the NLP-GIPC interaction in model NLP Pya , was identified by mutation and crystallization [65,82,86,88]. Moreover, differences between cytotoxic and noncytotoxic NLPs have been identified by mutation and crystallization analysis and have been attributed to hydrophobic amino acid residues in the Lc1, L2 and L3 loops of NLPs [86].
In addition to sphingolipids as NLP receptors, protein receptors of NLPs in plants have been identified. Specifically, in A. thaliana, a leucine-rich repeat (LRR) receptor named RLP23 has been reported. RLP23 associates with the LRR-receptor kinase (LRR-RK) SOBIR1 in the absence of NLPs. Upon ligand binding, another LRR-RK, BAK1, binds to form a tripartite complex and induces intracellular innate immune responses. In this complex, the LRR domain is essential to the function of RLP23 (Figure 1) [89]. Unlike its positive roles in immune responses triggered by flg22, the convergent and central immune hub BIK1 plays a negative role in the immune responses induced by NLPs in A. thaliana [90]. The immune responses induced by NLPs were milder and slower than those induced by flg22 [90]. Furthermore, an LRR-only protein, NTCD4, has been reported to be involved in NLP oligomerization, which is an important step in pore formation caused by NLPs in A. thaliana [91]. Although involvements, action mechanisms and differences between cytotoxic and noncytotoxic NLPs in plant immunity have been elucidated, there is a lack of practical applications in agriculture and gardening.
Life 2023, 13, x FOR PEER REVIEW 8 of 16 Figure 1. The BAK1-RLP23-SOBIR1 complex recognizes NLP peptides and mediates immune responses. RLP23 and SOBIR1 complex together and are isolated from BAK1 without NLP peptide perception. NLP peptides from pathogens are perceived by RLP23 and form tripartite receptor complexes of BAK1-RLP23-SOBIR1, which initiate downstream immune responses, including ROS burst, MAPK cascade activation, PR gene transcription and ethylene production. NTCD4, an LRRonly protein, promotes NLP oligomerization that induces pore formation on the plasma membrane, which is cytotoxic and triggers cell death. Figure 1. The BAK1-RLP23-SOBIR1 complex recognizes NLP peptides and mediates immune responses. RLP23 and SOBIR1 complex together and are isolated from BAK1 without NLP peptide perception. NLP peptides from pathogens are perceived by RLP23 and form tripartite receptor complexes of BAK1-RLP23-SOBIR1, which initiate downstream immune responses, including ROS burst, MAPK cascade activation, PR gene transcription and ethylene production. NTCD4, an LRRonly protein, promotes NLP oligomerization that induces pore formation on the plasma membrane, which is cytotoxic and triggers cell death.

Elicitins
Elicitins are small elicitors secreted by oomycetes (particularly Phytophthora and Pythium) that trigger necrosis and immune responses in a variety of plants, especially in tobacco and certain Brassicaceae species, which are the main sources of vegetables [92,93]. Since the first elicitins, cryptogein and capsicein (both are approximately 10 kDa), were identified in Phytophthora cryptogea and Phytophthora capsici, respectively, dozens of elicitins, e.g., parasiticein and INF1, have been identified [72,92]. Similar to harpins, elicitin structures are highly conserved with no similarity to plant proteins, which makes them good elicitors [94].

Taxonomy of Elicitins
According to a phylogenetic analysis, elicitins were divided into four elicitin (ELI) and 13 elicitin-like (ELL) clades, in which ELI proteins shared a highly conserved 98amino-acid domain (SMART 01187, PFAM 00964) with at least 66% sequence identity and contain six cysteine residues that form three disulfide bridges, while ELL proteins showed more diversity in terms of the length of the elicitin domain and cysteine spacing patterns; although similar to ELI proteins, they carry six cysteine residues [93,95]. ELI and ELL proteins carry of a signal peptide at the N-terminus, an elicitin domain and variable C-terminal domains, which tend to be enriched with threonine, serine, and proline residues, except for the ELI-1 clade proteins [93,94]. Even though elicitins share high sequence similarity, they differ in net charge, which is the basis for classifying them into acidic α-elicitins and basic β-elicitins; among these proteins, β-elicitins show 100-fold more necrosis-inducing effects than α-elicitins. The difference results from the difference in amino acid residues at position 13, with α-elicitins harboring a valine residue and β-elicitins harboring a lysine residue. The residue at position 13 not only determines the isoelectric point (pI) and induces necrosis but also participates in ligand/receptor binding [96][97][98][99]. The numbers of ELI and ELL genes vary among species, with each gene showing differential expression patterns and HR-inducing activities [100,101].

Involvement and Action Mechanisms of Elicitins in Plant Immune Responses
Elicitins triggered HR and immune responses, e.g., the burst of ROS, electrolyte leakage, activation of MAPK cascades, induction of PR genes and phytohormone in tobacco, tomato, potato, pigeon pea, citrus, grapevine, pepper, oak and some Brassicaceae cultivars but not in many other herbaceous and woody plants (Table 2) [73,92]. The responses to elicitins in different plants were highly variable.
Upon LRR-receptor-like protein (RLP) elicitin response (ELR) recognition of the conserved elicitin domain, where leucine41 in the ω-loop region plays an important role, the constitutive ELR-SOBIR1 complex associates with the immune coreceptor BAK1/SERK3 in an elicitin-inducible manner; this interaction triggers immune responses, e.g., membrane depolarization, ion flux, an early-phase ROS burst, activation of MAPK signaling cascades, induction of pathogenesis-related genes, and an increase in phytoalexin and phytohormone levels and the necrosis rate [66,67,69,70,99,[102][103][104]. As the first PRR to be identified, ELR recognizes a broad range of elicitins via their conserved elicitin domain [70]. In addition to ELR, an LRR-RLP REli from N. benthamiana has been identified recently as the receptor of PoEli8 from Pythium oligandrum [73].
Distinctive biphasic ROS accumulation has been observed in plants in which the immune response has been triggered by elicitins [92,93,105]. Moreover, early-phase ROS production was induced quickly after elicitin treatment via the existing NADPH oxidases RBOHA and RBOHB, while second-phase or late-phase ROS production occurred hours later and was induced by the de novo expression of the rbohb gene, which was triggered by the activation of the MAPK-WRKY7/8/9/11 signaling cascade, causing HR [93,[106][107][108][109]. The involvement of nitric oxide (NO), which is partially mediated by the S-nitrosation of RBOHD1 and phosphorylation of MAPK kinases, has been reported in elicitin-triggered immune activation in plants [96,106,[110][111][112]. The subsequent activation of PR genes, induction of cell death and increased SA, JA and ET levels have been detected [67,96].
Similar to harpins and NLPs, treatment with elicitins contributes to pathogen resistance in plants (Table 2), which would be practically useful in agriculture and gardening. For instance, the infiltration of PoEli8 improves resistance against P. capsici in tomato and pepper, and soaking in β-CRY improves resistance against P. neolycopersici in tomato [67,73].

Other Elicitors
In addition to the above-mentioned classes of elicitors, many other proteinaceous elicitors have been studied, e.g., RNA replicases from Cucumber mosaic viru and coat protein from turnip crinkle virus, CBEL from Phytophthora parasitica var. nicotianae; PeaT1 and Hrip1 from Alternaria tenuissima; MoHrip1, MoHrip2 and MoGluB from Magnaporthe oryzae; and BcGS1 and PebC1 from Botrytis cinerea [113][114][115][116][117][118][119][120][121][122][123][124]. A 2a polymerase of Cucumber mosaic virus induced HR responses in cowpea, and the induction was independent of its replicase activity [122]. The coat protein of turnip crinkle virus elicited HR responses in Arabidopsis [123]. When administered at a 150 nM concentration, CBEL, a 34 kDa glycoprotein elicitor located in the P. parasitica cell wall, displayed cellulose-binding and elicitor-like defense activity in plants as well as lectin-like activity, eliciting necrosis and defense-related gene expression in tobacco [121]. Further work showed that two intact cellulose-binding domains were sufficient and indispensable for the induction of a defense mode in the dicot plants tobacco and A. thaliana [125]. Despite showing low sequence homology, MoHrip1 and MoHrip2 were necessary for the virulence of fungi and induced defense responses via the SA and GA pathways, increased drought tolerance via the ABA pathway and increased plant growth [12,115,117,[126][127][128][129]. PebC1 has been shown to be involved in increased plant growth, tolerance to drought, and disease and insect pest resistance [119,[130][131][132].

Prospects and Challenges
In this mini-review, we summarized the taxonomy, structures and features of proteinaceous elicitors, described their involvement in plant immunity and responses to abiotic stresses, and discussed their mechanisms of action. Advances in the study of elicitors from pathogens that trigger plant immunity are helpful not only to increase the knowledge of plant-microbe interactions but also for practical use in agriculture and gardening. Although efforts have been made to identify the involvement and mechanisms of proteinaceous elicitors in plant immunity, the mechanisms remain to be discovered. Moreover, a wider range of elicitors remains to be identified, especially elicitors that trigger intense and persistent immune and abiotic stress responses in plants. The interactions of different elicitors also remain largely uncharacterized, even though several have been assessed and reported on certain interactions. What is the molecular basis by which elicitors cause different responses in hosts and nonhosts? What are the evolutionary dynamics of elicitors? Do elicitors cause an immune response in a wider range of plants than has been discovered to date? How can proteinaceous elicitors be made more effective and durable for commercial applications? Ultimately, a better understanding of proteinaceous elicitors in the plant response to stresses will help to establish healthier and more environmentally friendly agricultural practices worldwide.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: All data generated or analysed during this study are included in this article.