Identification of a DAMP receptor and its cognate peptide ligand in sweet potato (Ipomoea batatas)

Identification of a DAMP receptor and its cognate peptide ligand in sweet potato (Ipomoea batatas) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Identification of a DAMP receptor and its cognate peptide ligand in sweet potato (Ipomoea batatas) Hsueh-Han Lu, Anja Meents, Judith Fliegmann, Ming-Jing Hwang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2263331/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Sweet potato ( Ipomoea batatas ) is an important tuber crop, but also target of numerous insect pests. Intriguingly, the abundant storage protein in tubers, sporamin, has intrinsic trypsin protease inhibitory activity. In leaves, sporamin is induced by wounding or a volatile homoterpene and enhances insect resistance. While the signaling pathway leading to sporamin synthesis is partially established, the initial event, perception of a stress-related signal is still unknown. Here, we identified a leucine-rich repeat receptor kinase ( Ib LRR-RK1) that is induced upon wounding and herbivory, and related to peptide-elicitor receptors (PEPRs) from tomato and Arabidopsis. We also identified a gene encoding a precursor protein comprising a peptide ligand ( Ib Pep1) for Ib LRR-RK1. Ib Pep1 represents a distinct signal in sweet potato, which might work in a complementary and/or parallel pathway to the previously described HypSys peptides to strengthen insect resistance. Notably, an inter-family compatibility in the Pep/PEPR system from Convolvulaceae and Solanaceae was identified. Biological sciences/Plant sciences/Plant immunity/Pattern recognition receptors in plants Biological sciences/Plant sciences/Plant stress responses/Herbivory sweet potato herbivory plant defense plant elicitor peptide receptor DMNT DAMP LRR-RLK Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Plants have evolved several mechanisms to cope with biotic and abiotic stresses. When encountering stresses, such as pathogen infection, insect feeding, and wounding, receptor kinases (RKs) or receptor-like proteins (RLPs) properly identify specific patterns derived either from the aggressors (microbe-associated molecular patterns, MAMPs and herbivore-associated molecular patterns, HAMPs) or from the perturbation of cellular integrity (danger- or damage-associated molecular patterns, DAMPs). Subsequently, these pattern recognition receptors (PRRs) trigger signal transduction pathways to activate appropriate plant immune responses, leading to pattern-triggered immunity (PTI) 1 . PTI can reduce the damages caused by the invasion of many pathogens and insects 2 . PRRs are usually composed of extracellular, transmembrane, and intracellular domains. They are classified by their extracellular domains. The extracellular leucine-rich repeat domain, a single-pass transmembrane domain, and a cytoplasmic protein kinase domain characterize leucine-rich repeat receptor kinases (LRR-RKs). LRR-RKs and LRR-RLPs are sensors for proteinaceous immunogenic ligands, such as peptides and small proteins 2 . For example, the FLS2 receptor binds a 22-amino acid epitope (flg22) conserved in bacterial flagellins 3 , EFR recognizes a conserved N-terminal fragment of bacterial elongation factor Tu 4 , and Sl Eix1 and Sl Eix2 bind Trichoderma cell wall-derived xylanase 5 . Peptide ligands play an important role in regulating the signal transduction of insect resistance and wound defense responses 6 , 7 . In Arabidopsis thaliana , eight plant elicitor peptides ( At Pep1- At Pep8) are found to participate in damage-related defense responses after recognition by a pair of LRR-RKs, the PEP receptors 1 and 2 ( At PEPR1 and 2) 8 , 9 . Each of the At Peps is derived from the carboxy terminus of their precursor protein At PROPEP1-8 10,11 , how and if the peptides are cleaved off is however mostly not known. However, a METACASPASE4 (MC4)-dependent maturation of At Pep1was recently described. High levels of [Ca 2+ ] cyt that occur only in directly damaged cells bind to MC4, which in this activated form cleaves PROPEP1 and releases At Pep1 12,13 . AtPROPEP2, AtPROPEP3 , and the receptor genes AtPEPR1/2 are strongly induced upon herbivore attack. Moreover, pepr1 pepr2 double mutant plants display a reduced resistance to Spodoptera littoralis larvae 7 , 14 , 15 . In Zea mays , the precursor of Zm Pep3, an At Pep-ortholog, can be induced by insect oral secretion and insect HAMP. The application of Zm Pep3 can induce emission of some insect herbivory-related volatile organic compounds (VOCs), biosynthesis and accumulation of phytohormones, and transcripts that are indirectly involved in defense against herbivores. Zm Pep3 also causes accumulation of proteinase inhibitor and contributes to the resistance to lepidopteran insects 16 . Systemin was the first peptide discovered in plants with signaling capacities. In Solanum lycopersicum , the injury-induced systemin can cause defense responses against insects 17 , 18 . Tomato systemin is an endogenous peptide ligand composed of 18 amino acids, which is derived from a precursor protein by phytaspase-dependent cleavage at two aspartate residues 19 . Systemin induces proteinase inhibitors and activates phospholipase A2, thereby promoting the release of jasmonic acid precursors from the cell membrane. Induction of insect-resistance defense genes by jasmonic acid signaling pathways further contributes to the resistance of herbivore attack 17 , mediated by the LRR-RK receptor Sl SYR1, which however is not necessary for wound responses 20 . Hydroxyproline-rich systemins (HypSys) are systemin-like endogenous peptide ligands in Solanaceae plants. In addition to the hydroxyproline-rich conserved sequence, the HypSys precursor protein preproHypSys has a secretion sequence at the N-terminus, which is absent from Peps and systemin precursor proteins. Similar to systemin, HypSys induces the production of jasmonates and the expression of defense genes 21 . In petunia, HypSys is described to induce the expression of the immune gene defensin1 22 . The precursors of Sl HypSys I, II, and III in tomato are synthesized and sequestered in the cell wall matrix of phloem parenchyma cells in response to systemin, wounding, and methyl jasmonate 23 . Moreover, the HypSys precursor gene IbpreproHypSys in sweet potato ( Ipomoea batatas ) can be induced by injury. The application of Ib HypSys in sweet potato induces downstream insect-resistance genes such as sporamin and ipomoelin , and improves the biosynthesis of lignin, to increase the ability to repel insects 24 , 25 . However, it is still unclear how HypSys binds to receptors and participates in defense responses. Sweet potato is the fifth largest food crop in the world and has high nutritional and economic value. Several cultivars of sweet potato have higher insect resistance than others. For example, Ipomoea batatas cv. Tainong 57, which is widely cultivated in Taiwan, has strong insect resistance and represents a suitable model crop for studying insect resistance mechanisms 26 . Sporamin, which was previously thought to be a unique storage protein in sweet potato tuberous roots, was recently described to be regulated by herbivore attack, injuries, jasmonic acid, and the homoterpene ( E )-4,8–dimethyl–1,3,7-nonatriene (DMNT) in sweet potato leaves 26 , 27 . Functional studies revealed that sporamin is a serine-type trypsin inhibitor, which acts in the insect intestine and retards insect growth and development 28 , 29 . Transgenic Nicotiana benthamiana and Brassica rapa subsp. chinensis plants overexpressing sporamin demonstrated a strong pest resistance capacity 30 , 31 , as did transgenic sweet potato plants overexpressing IbNAC1 , which is a transcription factor binding to the sporamin wounding response element ( SWRE ) region of the sporamin promoter 32 . Ib NAC1 also regulates the jasmonic acid response and ROS signaling 33 and is regulated by Ibb HLH3/4, Ib WIPK1, Ib JAZ2, and Ib EIL1 upon injury 32 . The MAPK pathway is also part of the signal transduction from wounding stress to sporamin expression 32 . However, the molecular connection between danger perception (ligands, receptors) and downstream defense responses is still elusive. In order to discover the key players upstream of the intracellular signaling cascade leading to induced resistance against herbivores we isolated candidates for both, a cell surface receptor and endogenous peptide ligands from sweet potato. Among the wound- and herbivore-induced genes in I. batatas we detected a gene encoding a leucine-rich receptor kinase related to the plant elicitor peptide (Pep) receptor (PEPR) family, Ib LRR-RK1. When heterologously expressed in N. benthamiana , this receptor candidate did not provide responsiveness to HypSys but to extracts of damaged sweet potato leaf tissue. Finally, we identified the cognate peptide ligand, Ib Pep1, characterized the specificity and sensitivity of the new receptor/ligand-pair and compared the signaling capacities of the newly identified peptide with the previously described HypSys peptides. Results Sweet potato encodes putative DAMP receptors We based our search for receptors of sweet potato which are involved in responses to wounding and herbivore attack on published sequences for DAMP-related receptors in Arabidopsis ( At PEPR1/2: AT1G73080/AT1G17750) and tomato, S. lycopersicum ( Sl SYR1/2: Soly c03g082470/Solyc03g082450.2.1; Sl PEPR1: XP_004235511). Several closely related receptor genes, designated ItLRR-RK1 to ItLRR-RK13 , were mined from the Ipomoea trifida “Sweet potato Genomic Resource database” ( http://sweetpotato.plantbiology.msu.edu/index.shtml ) (Supplementary Fig. 1). Next, two transcriptomic databases, i.e. the I. batatas cv. Tainong 57 transcriptome database 27 and I. batatas database, Ipomoea Genome Hub ( https://ipomoea-genome.org/ ) were accessed to explore putative LRR-RK genes with sequence homology to ItLRR-RKs . Five putative I. batatas receptor kinase genes ( IbLRR-RK1 to IbLRR-RK5 ) were identified. RT-PCR (Fig. 1 A) and qRT-PCR (Fig. 1 B) experiments revealed that both wounding and insect herbivory rapidly induced IbLRR-RK1 (MT210638). Upon wounding and treatment with Spodoptera larvae-derived oral secretion, the relative expression level of IbLRR-RK1 increased nearly 30-fold at 15 min, 12-fold at 30 min, and returned to normal levels at 60 min. Herbivory feeding also increased IbLRR-RK1 expression level 5.4-fold at 15 min and 1.7-fold at 30 min (Fig. 1 B). These data demonstrate that mechanical wounding and herbivory induce the receptor-like kinase Ib LRR-RK1, suggesting that this receptor might be involved in perception of a wound-related signal. The receptor candidate Ib LRR-RK1 is related to PEPRs The gene IbLRR-RK1 from I. batatas cv. Tainong 57 encodes a typical member of the PEPR family. It consists of an ectodomain composed of a signal peptide, an N-terminal cap region typically found in plant LRR-RKs, 26 repetitions of the plant-specific version of the LRR motif, and an outer juxtamembrane; this is followed by a transmembrane domain; the cytosolic part contains the inner juxtamembrane domain and a serine/threonine kinase domain (Supplementary Fig. 2). As expected, the green fluorescent protein (GFP)-tagged Ib LRR-RK1 protein, transiently expressed in either A. thaliana protoplasts or N. benthamiana leaves, localized to the plasma membrane (Fig. 1 C), like other plant LRRs such as Sl SYR1-GFP or At EFR-GFP, which were used as positive controls. Ib LRR-RK1 is most likely related to PEPRs from tomato (XP_004235511, Sl PEPR1) and Arabidopsis ( At 1g73080, At 1g17750; PEPR1 and PEPR2; Supplementary Fig. 3). Ib LRR-RK1, which is 97% identical with It LRR-RK1, shared 65% or 50% identical amino acid residues to Sl PEPR1 or At PEPR1, respectively, and 35% identity to Sl SYR1, while other putative RLK members selected from the Sweet Potato Databases never shared more than 36% identity to either of the mentioned receptors (Table 1 ). In addition, comparing the extracellular domains of different receptors also showed that Ib LRR-RK1 has a highly similar ligand-binding surface when compared with Sl PEPR1 and At PEPR1 (60 and 48% identity, respectively), and shares only 36% identical residues with tomato Sl SYR1 (Table 2 ). Thus, Ib LRR-RK1 is part of the plant elicitor peptide receptor group. Table 1 Comparison of the full-length amino acid sequence of Ib LRR-RK1 with related sequences, calculation of % identity by Vector NTI. It LRR-RLK1 Sl PEPR1 At PEPR1 At PEPR2 Sl SYR1 Sl SYR2 Ib LRR-RK1 97 65 50 49 36 35 It LRR-RK1 64 50 49 36 35 Sl PEPR1 51 48 35 35 At PEPR1 66 36 34 At PEPR2 36 35 Sl SYR1 79 At, Arabidopsis thaliana; Ib, Ipomoea batatas; It, Ipomoea trifida; Sl, Solanum lycopersicum. Table 2 Comparison of the amino acid sequence of the extracellular domain of Ib LRR-RK1 with At PEPR1, Sl PEPR1, and Sl SYR1, calculation of % identity by Vector NTI. Sl PEPR1 At PEPR1 Sl SYR1 Ib LRR-RK1 60 48 36 Sl PEPR1 49 36 At PEPR1 36 At, Arabidopsis thaliana; Ib, Ipomoea batatas; Sl, Solanum lycopersicum. Ib LRR-RK1 is a functional receptor Establishing the functionality of new receptor candidates for which the ligands are not known is challenging, and can be overcome by approaches in which chimeric versions are ectopically expressed in suitable plants 34 , 35 . To test if the kinase domain of the putative receptor from sweet potato is able to feed into the immune response pathway we generated a chimeric version with the ectodomain of tomato SYR1 a receptor with known ligand (Supplementary Fig. 4A). The chimeric receptor SYR1- Ib K as well as the original Ib LRR-RK1 and SYR1 were transiently expressed in leaves of N. benthamiana . The GFP-tagged recombinant proteins localized to the cell surface, as predicted (Fig. 1 C, Supplementary Fig. 4B). Treatment with the ligand of SYR1 resulted in the induction of an oxidative burst for the SYR1- Ib K expressing leaf pieces (Fig. 2 A, Supplementary Fig. 4C), proving the functionality of the kinase domain of Ib LRR-RK1. Several other defense-related peptides from various plants such as Sl Pep6, Sl HypSysIII, Ib HypSysIV, or At Pep1 were applied in addition to systemin (Supplementary Table 1) in bioassays with leaves expressing either the original Ib LRR-RK1 or SYR1- Ib K. Interestingly, Sl Pep6 triggered the defense pathway in the presence of Ib LRR-RK1, leading to ROS production and ethylene accumulation (Fig. 2 B, C). We than verified the recognition of Sl Pep6 by Ib LRR-RK1 in protoplasts, generated from A. thaliana Col-0 mesophyll cells. FRK1 (flg22-induced receptor-like kinase 1) is a PTI marker gene of early defense responses in Arabidopsis 36 and its promoter is widely used in combination with a luciferase reporter gene to monitor PAMP activity 37 . The co-expression of Ib LRR-RK1 with p FRK1:LUC resulted in Sl Pep6-dependent induction of the reporter (Fig. 2 D), confirming the previous experiments in N. benthamiana , while the chimeric receptor SYR1- Ib K recognized systemin, but not Sl Pep6 (Fig. 2 E). Taken together, we demonstrated that the activation of Ib LRR-RK1 can trigger plant immune responses such as ROS burst, ethylene biosynthesis, and defense gene expression and identified a ligand. Ib LRR-RK1 perceives an endogenous peptide For the molecular identification of the cognate ligand of Ib LRR-RK1 we took advantage of the fact that Sl Pep6 was functional in activating sweet potato Ib LRR-RK1. We hence used the sequence of Sl Pep6 and other Peps from the Solanaceae family as probes to search for endogenous peptides and applied scanning sequence pattern and tBlastn strategies on the sweet potato genomic resource database and the Ipomoea genome hub database (Supplementary Fig. 5). Two putative Peps were selected from the I. trifida genomic resource database. It Pep1 (LSSRPPRPGLGNSGDPQTNDTSS) consisted of 23 amino acid residues, which were present at the C-terminus of the precursor protein It PROPEP1 (itf01g30920.t1) (Supplementary Fig. 5B). The putative It Pep2 (RRGRTPPRPENLKLNLRARKHSLEDQ) with 26 residues was derived from the C-terminus of It PROPEP2 (itf07g21780.t1) (Supplementary Fig. 5C). Both candidate peptides, It Pep1 and It Pep2, were synthesized and applied to N. benthamiana leaf discs transiently expressing Ib LRR-RK1. It Pep1, but not It Pep2, activated an Ib LRR-RK1-dependent ROS burst (Fig. 3 A), and neither peptide elicited a response in the SYR1- Ib K or p19 controls (Supplementary Fig. 6A, B). Next, the cDNA of the PROPEP1 gene from I. batatas cv. TN57 was cloned by RT-PCR using oligonucleotides deduced from ItPROPEP1 . The CDS encompasses 378 bp and 125 deduced amino acid residues with a calculated molecular weight of 13.25 kDa, and a pI of 4.44. The putatively bioactive 23-mer peptide, Ib Pep1, corresponds to the C-terminus of the precursor protein and is 100% identical to the one from It PROPEP1 (Supplementary Fig. 6C). As expected, the I. batatas PROPEP as well as the Pep amino acid sequences are more closely related to those from solanaceous plants than to those of Arabidopsis (Supplementary Fig. 6D, Supplementary Table 2). Ib LRR-RK1 perceives Ib Pep1 with high sensitivity and specificity Exploiting the same heterologous expression system described above we interrogated the sensitivity and the specificity of the putative ligand/receptor-pair. The dose-dependent induction of ROS by Ib Pep1 was clearly detectable in the sub-nanomolar range and the half-maximal activation of this output was estimated at 1 nM (Fig. 3 B). The tomato Pep ( Sl Pep6) was 10-times less efficient in this bioassay with Ib LRR-RK1 (Fig. 3 B). In the reciprocal approach, we cloned the Pep receptor of tomato ( Sl PEPR1, 38 ), expressed it in N. benthamiana and compared the efficiencies of the Peps for the induction of ROS. The tomato PEPR/ Sl Pep6 pair showed the same efficiency as the corresponding sweet potato pair, with an EC 50 value of 1 nM. Interestingly, Sl PEPR1 recognized also the peptide from sweet potato, albeit with a much lower sensitivity, and an estimated EC 50 value above 100 nM (Fig. 3 D). N-terminal and C-terminal truncated versions of Ib Pep1 were synthesized to investigate the specificity for the predicted sweet potato peptide on Ib LRR-RK1 (Fig. 3 C). Deleting up to three N-terminal residues did not have a major impact on the perception, the loss of arginine at position 4, however, led to a severe increase of the EC 50 value (either when deleted as in Ib Pep1 (5–23) or when changed to an alanine as in Ib Pep1 (A4)). In contrast, the C-terminus needs to be present for a sensitive perception although the last two serine residues can be replaced by alanine ( Ib Pep1 (A22A23)). Ib PROPEP1-GFP is mainly localized at the tonoplast We expressed IbPROPEP1 (OP311829) as a C-terminal fusion with GFP in N. benthamiana and A. thaliana protoplasts and observed the localization of the protein by confocal microscopy. Ib PROPEP1-GFP partially localized to the tonoplast (Fig. 4 , Supplementary Fig. 7) as reported for At PROPEP1-YFP 12 , but also aggregated into bright small globular structures, resembling bulbs 39 , inside the vacuole of Arabidopsis mesophyll protoplasts. Similarly, in N. benthamiana , Ib PROPEP1-GFP accumulated in the tonoplast and aggregated into several small globular structures which moved inside the vacuole (Supplementary Fig. 7, Supplementary Movie 1). An Ib LRR-RK1-activating DAMP is present in sweet potato leaves Preliminary data hinted that Ib PROPEP1 might be cleaved after damage to the leaf tissue and that a mature, signaling competent peptide might be liberated (data not shown), as it is known from the cognate ligands of PEPRs 6 . To simulate a corresponding scenario, we prepared first an extract from I. batatas cv. TN57 leaves and applied it on transiently Ib LRR-RK1-expressing N. benthamiana leaves. Leaf discs expressing the receptor responded to the treatment with the partially purified leaf extract with a ROS burst, which was not detectable in control leaves transformed with p19 only (Fig. 5 A). Next, we damaged sweet potato leaves by squeezing with tweezers. After 10 min, this material was harvested, in parallel to tissue from control plants. Interestingly, the elicitor activity was higher in extracts from wounded leaves in comparison to the non-damaged leaves. This activity clearly depended on the expression of Ib LRR-RK1 (Fig. 5 B). These data indicated that crude extracts of I. batatas leaves contain ligands for Ib LRR-RK1 that might accumulate upon wounding stress. Ib Pep1 and Ib HypSysIV activate complementary signaling cascades We next addressed the question whether Ib Pep1 is involved in herbivore resistance responses in sweet potato or in other processes. Therefore, whole sweet potato plants were sprayed with 25 µM Ib Pep1 and analyzed for sporamin induction. For comparison, the synthetic hydroxyproline-rich glycopeptide Ib HypSysIV, which was shown to activate sporamin expression 24 was tested at 25 µM as well. The qRT-PCR analysis after 30 min and 1 h of incubation revealed HypSys-dependent transient increases of sporamin (X60930.1) (16-fold), Ib NAC1 (GQ280387.1) (22-fold), and Ib WIPK1 (HQ434622) (68-fold) transcript levels (Fig. 6 A), confirming the ability of HypSys peptides to rapidly trigger sporamin-related signaling cascades. In contrast, Ib Pep1 transiently induced sporamin only 3.5-fold after 30 min, while Ib NAC1 and Ib WIPK1 were induced to higher and longer lasting expression levels compared to Ib HypSysIV treatment (Fig. 6 B). Moreover, when analyzing other defense-related genes we also found that Ib Pep1 and Ib HypSysIV treatments increased the expression of Ib LRR-RK1 and Ib CML-like1 (OP311828) (Fig. 6 A, B). Further, compared to water controls and Ib Pep1, the application of Ib HypSysIV resulted in a significantly increased emission of the wound-inducible volatile DMNT (Fig. 6 C) 26 . Scions incubated with the tomato-derived peptide Sl Pep6 or an inactive scrambled peptide only displayed basal DMNT levels comparable to the control treatment, confirming thereby the functionality of the peptide application method and the (species-)specificity of the Ib HypSysIV elicitor. In order to elucidate which role peptides play within the Ipomoea defense framework, local and systemic TN57 leaves were analyzed for phytohormone levels after peptide treatment. In comparison to water-treated controls, no significant differences in local and systemic jasmonic acid concentrations could be observed after Ib HypSysIV treatment (Supplementary Fig. 8A). Interestingly, Ib HypSysIV-treated leaves showed a significantly increased amount of bioactive JA-Ile, however only locally (Supplementary Fig. 8B). For the stress-related hormones SA and ABA, no significant differences to control treatments were detected, except for a local decrease in SA concentrations upon contact with Ib HypSysIV (Supplementary Fig. 8C). Treatment with Ib Pep1 did neither alter jasmonate nor SA levels although low concentrations might mask possible effects. However, exposure to Ib Pep1 resulted in decreasing amounts of ABA, mainly observed in the local leaf (Supplementary Fig. 8D). Although no tremendous changes in phytohormone levels were overall visible, we noted a clear tendency that for phytohormones regulated by Ib HypSysIV, no response would occur during exposure to Ib Pep1 and vice versa . RNAseq of I. batatas reveals differentially expressed genes (DEGs) upon Ib Pep1 and Ib HypSysIV treatment In order to better understand the similarities between Ib Pep1 and Ib HypSysIV and their particular functionalities, RNAseq experiments were conducted on single leaves treated with either peptide or water (control), respectively, for 1 h (Fig. 7 ). Overall, 24482 expressed genes were detected based on mapping onto the I. trifida reference genome from which 22702 were shared among all treatments including control samples. 241 genes were exclusively detected upon Ib HypSysIV treatment, while 485 transcripts were detected only after Ib Pep1 treatment. An additional number of 326 common transcripts was found in both peptide treatments but not in the control (Fig. 7 A). Strikingly, 179 expressed genes were mapped onto the I. trifida genome but found only in control plants, suggesting that expression of these genes is reduced upon peptide treatments. Further, spraying of Ib HypSysIV induced significant upregulation of 555 genes whereas 826 genes were significantly downregulated, compared to water-treated control leaves (data not shown). Upon Ib Pep1 incubation, an even stronger response was observed with 1749 genes up- and 2694 downregulated (data not shown). A comparison of both peptide treatments revealed that 4836 genes were significantly differentially regulated due to these different treatments, 2607 up- and 2229 downregulated, when Ib HypSysIV vs Ib Pep1 was compared (Fig. 7 B). These results support the idea that the two sweet potato peptides have distinct functions, which may be based on their ability to regulate different genes. To support this hypothesis, further confirmation with KEGG and GO pathway analyses and quantititave PCR of selected genes is necessary and will be performed. Discussion Recent evidence has shown that sweet potato exhibits DAMP-mediated activation of defenses. The volatile homoterpene, DMNT, has been demonstrated to activate resistance mechanisms in leaves leading to protection against herbivore feeding 26 , 40 . Peptide-based activation of defense reactions also has been observed in sweet potato 24 . However, the biological significance and interconnection with induced resistance against insects remained unclear. This study provides evidence for the existence of a Pep/PEPR system in sweet potato and investigates the input- and output-conditions. Briefly, we show that the system can be activated by a damage-induced endogenous elicitor, provide indirect evidence that this elicitor might be Ib Pep1, the product of Ib PROPEP1 cleavage, and that it functions in parallel and complementary to a HypSys-dependent signaling pathway. Mining the sweet potato genome databases we identified a wound- and herbivory-induced gene encoding a canonical leucine-rich repeat-containing receptor kinase, Ib LRR-RK1 (Figs. 1 A, B, and Supplementary Fig. 2). Using a chimeric receptor approach, in which we combined the cytosolic kinase domain of Ib LRR-RK1 with the extracellular recognition domain of Sl SYR1 (Supplementary Fig. 4A), we were able to generate a functional receptor after heterologous expressions in both N. benthamiana and A. thaliana (Fig. 2 , Supplementary Fig. 4C). Phylogenetic analysis suggested that Ib LRR-RK1 might be a member of the plant elicitor peptide receptors (PEPRs). Indeed, Sl Pep6 from tomato, but not At Pep1 from A. thaliana , was recognized by the native Ib LRR-RK1, and triggered the activation of typical defense responses after expression of Ib LRR-RK1-GFP in both N. benthamiana and A. thaliana (Fig. 2 ). Of note, the sweet potato peptide Ib HypSysIV, which is described to be involved in the wound response 24 was not recognized by Ib LRR-RK1. Based on the above findings, sequences of Peps and their precursor proteins (PROPEPs) from tomato and other Solanaceae plants were used to search for the related putative peptide in the sweet potato genome. We identified a 23-amino acids long peptide ligand, Ib Pep1, which is derived from the C-terminus of its precursor protein Ib PROPEP1. Ib Pep1 is capable to initiate the ROS burst in transgenic Ib LRR-RK1-expressing N. benthamiana with a tenfold-higher sensitivity in comparison to Sl Pep6 (Fig. 3 B). However, the fact that the tomato peptide was recognized by sweet potato prompted us to investigate the reciprocal scenario. Indeed, Sl PEPR1, the tomato receptor for Sl Pep6 38 recognized Ib Pep1, providing here for the first time data on interfamily (Solanaceae and Convolvulaceae) compatibility of Peps. The structure-activity characterization of the ligand of Ib LRR-RK1 using various synthetic Ib Pep1 derivatives unraveled some structural requirements for the interaction with the corresponding receptor (Fig. 3 C). As for other Peps, the C-terminus of the peptide is of utmost importance, since the C-terminaly truncated peptide ( Ib Pep1(1–20)) is at least 100-fold less efficient compared to the 23-mer Ib Pep1 (Fig. 3 C). Unlike the Peps from other plant families, however, the identity of the residues at the C-terminus seems not to be as important since the replacement of the last two residues with alanine residues only marginally decreased the affinity. Peps from sweet potato share five of the 12 highly conserved residues with the family-specific Pep-motif of the Solanaceae ( 38 in the overlapping 20-mer core region (Fig. 3 C, Supplementary Fig. 9A). Testing one of these highly conserved residues ( Ib Pep1(A4)) confirmed the importance of the arginine at that position. As illustrated in a composite consensus sequence for Peps of Solanales (the order including Solanaceae and Convolvulaceae), conserved arginine and proline residues are clustered at the N-terminus of the peptides whereas proline and asparagine residues are conserved at the C-termini (Supplementary Fig. 9B). PROPEPs have been reported to distribute to distinct subcellular localizations in Arabidopsis 11 . While At PROPEP3 is present in the cytosol, At PROPEP1 and At PROPEP6 are positioned at the tonoplast. Our findings show that Ib PROPEP1-GFP is localized at the tonoplast as well (Fig. 4 , Supplementary Fig. 7). In addition, Ib PROPEP1-GFP also appeared in vesicle-like structures attached to the tonoplast that dynamically fuse with the vacuole (Supplementary Movie 1). Whether these structures correspond to bulbs, which have been described as cytoplasmic projections into the vacuole, surrounded by a tonoplast-derived double membrane 39 , 41 , or are artefacts of dimerizing GFP with which the overexpressed PROPEP is tagged 42 remains to be investigated. However, to the best of our knowledge, this localization has never been reported for other PROPEPs. We hypothesize that the purpose of Ib PROPEP1 enrichment in bulbs could be to store sufficient amounts of the precursor and release it rapidly after cell and vacuole injury to allow cleavage into active Ib Pep1. In planta , we demonstrated the release of a specific agonist of Ib LRR-RK1. Incubation of only 10 minutes of wounded sweet potato leaves increased the amount of the elicitor in a partially purified fraction, in comparison to non-incubated leaf material (Fig. 5 ). The inherent trypsin inhibitory activity of sporamin provides strong protection against herbivory in sweet potato and other, transgenic plants species expressing sporamin 26 , 29 , 31 . Strongly induced expression of sporamin was detected in sweet potato leaves during pest attack and injury stress 30 . The 18 amino acid hydroxyprolinated peptide Ib HypSysIV, which can be extracted from sweet potato leaves, was amplifying the wounding signal and activated the expression of sporamin 24 . In the present study, we found that spraying with either peptide, Ib HypSysIV or Ib Pep1, rapidly induced the expression of wound-induced defense response genes including IbWIPK1 , IbNAC1 , sporamin , and even IbLRR-RK1 , in sweet potato leaves (Fig. 6 ). However, Ib HypSysIV treatment induced the expression of sporamin much more strongly than Ib Pep1 treatment. Previous studies have revealed that application of At Peps and the activation of At PEPR1/2 lead to increased jasmonate accumulation and induced jasmonate responses in Arabidopsis 7 . We found that the application of Ib Pep1 did not increase the amount of jasmonates, in contrast to Ib HypSysIV, which induced the accumulation of JA-Ile in sweet potato leaves slightly (Supplementary Fig. 8), suggesting that Ib HypSysIV may trigger the jasmonate pathway and associated responses in contrast to Ib Pep1. A clear discrepancy between the two peptides lies in their ability to regulate the synthesis and release of the homoterpene DMNT. This volatile danger signal is induced in sweet potato upon wounding and herbivory 26 . Only treatment with Ib HypSysIV but neither Ib Pep1 nor Sl Pep6 nor a scrambled control peptide were able to induced DMNT, indicating the specificity of this response (Fig. 6 C). Overall, our study suggests that in addition to the Ib Pep1/ Ib LRR-RK1 pair described here for the first time, there is another, as yet unidentified, DAMP receptor that specifically interacts with the Ib HypSysIV ligand in sweet potato. The latter system appears to be more active than the Pep/PEPR pair in the jasmonate pathway regulating sporamin expression. Having shown that both, Ib Pep1 and Ib HypSysIV, have a certain ability to regulate defense responses against herbivory attack and wounding, albeit with different efficacies, we have yet to define the key signaling pathway(s) regulated by Ib Pep1. Preliminary analyses of RNAseq data suggest that Ib Pep1 and Ib HypSysIV control partly distinct pathways, which will need to be further investigated in combination with real infestation and infection assays in the future. Conclusions Previous studies have shown that Peps/PEPR ligand-receptor systems are widespread in plants. Here, we identified a novel peptide ligand and its corresponding receptor from sweet potato. This adds another ligand/receptor pair to the growing list of DAMP perception systems. Understanding how the downstream gene responses to different ligands are coordinated in the genetic network is a topic that needs to be addressed in the future. Although Ib Pep1 was not able to induce the emission of DMNT, the trypsin protease inhibitor sporamin and its transcription factor Ib NAC1 were upregulated, hinting at a modular way to increase insect resistance. Peps vary widely from species to species, conserved family-specific Pep-motifs are sufficient for Pep recognition by PEPRs from different species of the same plant family 38 . In our experiment, we found that the peptide ligand Sl Pep6 of tomato belonging to Solanaceae family did interact with Ib LRR-RK1 from sweet potato belonging to Convolvulaceae family and activated downstream responses. V ice versa , the reciprocal combination was functional as well. To our knowledge, this is the first example that a peptide ligand does not follow the rule of family-specific incompatibility of Peps but suggests the conservation of a plant order-specific peptide ligand structure in Solanales. Materials And Methods Plant material and growth conditions Sweet potato scions ( Ipomoea batatas Lam.; cultivar Tainong 57) were grown in phytochambers under long day conditions (16 h light : 8 h dark) at 28°C (day) and 25°C (night) in 70% relative humidity for 3 weeks as previously described 26 . When growing for 4–5 weeks, sweet potato and Nicotiana benthamiana plants were maintained in a greenhouse with a 16 h photoperiod and a 25°C/20°C day/night program. Arabidopsis thaliana ecotype Columbia-0 (Col-0) was grown at 22°C with an 8 h photoperiod in growth chambers for 4–5 weeks. Peptides Peptides were ordered from GenScript Biotech (Leiden, Netherlands). They were dissolved prior to each experiment in BSA/NaCl (10 mg/ml, 0.1 M) solution. The list of peptides and their sequences can be found in Supplementary Table 1. RNA extraction, RT- and qRT-PCR analyses Harvested sweet potato leaves were processed and used for real-time PCR as described in 26 with the additional primer pair IbLRR-RK1, sporamin, IbWIPK1, IbNAC1, IbCML1 (Supplementary Table 3) on a Bio-Rad CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, USA). Semi-quantitative reverse transcription PCR (RT-PCR) was performed with 2X Taq DNA Polymerase (Bioman) and the primers listed in Supplementary Table 3. RNA-Seq analysis and processing RNA from single 3rd leaves treated for 1 h with Ib HypSysIV, Ib Pep1, and water (control) was extracted according to 26 using TRIzol Reagent (Invitrogen, USA). Four biological replicates per treatment were used for RNA-Seq experiments conducted by Novogene Europe (Cambridge, UK). RNA quality was monitored using NanoPhotometer® spectrophotometer (IMPLEN, CA, USA) and RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). 1 µg of RNA per sample was used as template material for further sample preparations. Sequencing libraries were generated via NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s instructions. 20 M paired end reads of 150 bp per sample were generated, sequenced on an Illumina NovaSeq 6000 instrument (San Diego, USA). Raw reads were trimmed by in-house scripts. The clean reads were mapped onto Ipomoea trifida reference genome ( http://sweetpotato.uga.edu/ ), using HISAT2 V2.0.5 with default parameter. HTSeq V0.6.1 software was used with the union mode to count read numbers mapped of genes for each sample. R package from Bioconductor, DESeq2 V1.22.2 was used to estimate gene abundance and detect differentially expressed genes (DEGs) among the sample groups. A model based on the negative binomial distribution was carried out to determinate DEGs with an adjusted p-value cutoff of 0.05. To control the False Discovery Rate (FDR) Benjamini-Hochberg adjustment was performed. Genes with a log2-fold change > = 1 and padj < 0.05 were considered as significantly DEGs. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of DEGs were implemented by the GOseq V1.34.1 R package and KOBAS V3.0 software. Wounding, insect feeding, and peptide spray treatments I. batatas and N. benthamiana plants with six to eight fully developed leaves were used in the study. For wound treatment, the third or fourth fully expanded leaves were wounded using tweezers and the wounded leaves samples were collected at different time points. For insect feeding treatment, starved Spodoptera litura larvae (2nd instar) were placed on the third or fourth fully expanded leaves and the treated leaves samples were collected at intervals. To study the local effects of peptide solutions on DMNT emission and gene expression, whole sweet potato plants with six to eight fully expanded leaves were evenly sprayed with peptide solution or double-distilled water (control) until all leaves were fully covered in liquid. After a 1 h incubation period, single plants were placed for 24 h in 2.4 L glass desiccators (VWR international) for headspace volatile collection. For RNA-Seq, qRT-PCR, and phytohormone analyses each 3rd fully expanded leaf was locally sprayed with peptide solution or ddH 2 O (control) and harvested together with the adjacent 4th leaf (systemic) after the indicated time points. VOC collection and quantification Volatiles were collected over 24 h from peptide- or water-treated sweet potato plants enclosed in 2.4 L desiccators using the closed-loop stripping technique 43 . Throughout the headspace collection, each desiccator was connected to an air circulation pump (Fürgut GmbH, Germany) containing a charcoal trap with 1.5 mg absorption material (CLSA filter, 6 cm long, 0.5 cm diameter, Gränicher & Quartero, France). After collection, volatiles were eluted and measured as described 26 with minor modifications. In this study, samples were eluted with 2 x 20 µl of dichloromethane containing 10 µg ml − 1 n -bromodecane as internal standard used for further relative quantification. Cloning of receptor and propeptide gene candidates Ib LRR-RK1 and Ib PROPEP1 genes were identified using blastn as well as tblastn on various databases using the receptor and propeptide sequences for Solanaceae plants from 38 . Used databases included Sweet Potato Genomic Resource database ( http://sweetpotato.plantbiology.msu.edu/index.shtml ), I. batatas cv. TN57 transcriptome database 27 , I. batatas database: Ipomoea Genome Hub ( https://ipomoea-genome.org/ ), and NCBI ( https://www.ncbi.nlm.nih.gov/ ). The Ib LRR-RK1 and Ib PROPEP1 coding sequences were amplified from sweet potato leaf cDNA using gene-specific primers ( Ib LRR-RLK1_FL_F, Ib LRR-RLK1_FL_R, Ib PROPEP1_FL_F, Ib PROPEP1_FL_R, as shown in Supplementary Table 2) in a PCR reaction with Q5 High-Fidelity DNA Polymerase (NEB), respectively. The coding sequence encoding the tomato Sl PEPR1 (XP_004235511) was amplified using the primers Sl PEPR1_FL_F and Sl PEPR1_FL_R (Supplementary Table 3). All full length coding sequences were cloned into the pCR8/GW/TOPO vector (Invitrogen). LR clonase (Invitrogen) was used to transfer these coding sequences from PCR8 to pMDC83 vectors 44 , generating C-terminal fusions with GFP. Generation of chimeric receptors Gene-specific level I modules for SYR1 20 and Ib LRR-RK1 (see above) were generated by proof reading PCR (Phusion High Fidelity DNA Polymerase, ThermoFisher Scientific) from existing templates using the oligonucleotide primers listed in Supplementary Table 2, subcloned, and verified by sequencing. GoldenGate cloning was used to assemble the receptor expression constructs with general level I modules (A-B p35S (G005), D-E GFP (G011), E-F nos-T (G006) and dy F-G (BB09)) into the vector backbone LIIα F 1–2 (BB10) as described 45 . Transient expression of receptor constructs and bioassays Transient expression in N. benthamiana was performed as described 34 . The oxidative burst was measured with leaf pieces floating on 100 µl water containing 20 µM L-012 (Wako) and 2 µg/ml horseradish peroxidase (Applichem), after addition of peptides, with a luminescence plate reader (Mithras LB 940, Berthold, or Infinite M200 PRO plant reader, TECAN). The amount of ethylene was measured by GC in the headspace of 4 leaf pieces floating on 500 µl water, treated for 4 h with the peptides or controls. Transient co-expression of the pFRK1:Luciferase reporter 37 with the receptor expression constructs in mesophyll protoplasts of A. thaliana Col-0 wild-type was performed as described 46 . Luminescence was recorded for up to 6 h in W5-medium containing 200 µM firefly luciferin (Synchem UG) after overnight incubation for 14 h and subsequent treatment with peptides or control solution. Subcellular localization The Ib LRR-RK1-GFP, Ib PROPEP1-GFP, and the tonoplast localization marker protein fusion γ-Tip-mCherry 47 were transiently expressed in N. benthamiana leaves and A. thaliana mesophyll protoplast as described above. Fluorescence images were taken using a TCS SP5 Confocal microscope (Leica) and analyzed by LAS AF Lite application software (Leica) or a Zeiss Axio Zoom.V16. Immunoblotting Wounded leaves of N. benthamiana transiently expressing IbPROPEP1-GFP were ground to a fine powder in liquid nitrogen. The powder was immediately supplemented with 100 µl of 2× SDS sample buffer solution (Nacalai Tesque Inc.), heated up to 95°C for 5 min at, and centrifuged for 10 min at 16,000 x g to remove cellular debris. Ten µg extracted proteins were run in 15% SDS polyacrylamide gels for IbPROPEP1-GFP and IbPep1-GFP separation, followed by semi-dry Western blotting to PVDF membranes (MILLIPORE), and incubation with anti-GFP antibodies (rabbit, 1:2000; Abcam) overnight. After a second incubation with Goat-anti-Rabbit IgG labeled with horseradish peroxidase enzyme (1:20000; Jackson), chemiluminescent substrates (Chemi-Lumi One Super; Nacalai Tesque Inc.) were supplied for detection of luminescence. Luminescent images were taken and analyzed with a KETA CLX Chemiluminescence Imaging System (Wealtec). Crude endogenous ligand extraction According to 48 , 10 g injured and non-injured sweet potato leaves were harvested, respectively. Samples were homogenized with 1% cold trifluoroacetic acid (TFA) in a blender for 2 min. After filtering the extracts through 4 layers of Miracloth to remove plant debris and centrifuging at 8,500 rpm for 20 min at 4°C, the supernatant was slowly pressed through a customized Sep-Pak C18 solid phase extraction cartridge (Waters) and eluted with 60% (v/v) methanol/0.1% (v/v) TFA. The eluate containing peptides were dried in a speed vac and re-suspended in 200 µl double-distilled H 2 O. Phytohormone extraction and quantification Local and systemic leaves collected after 1 h peptide treatment were extracted and measured as described 26 using an Agilent 1200 HPLC system (Agilent, USA) with subsequent API 5000 tandem mass spectrometer (Applied Biosystems, USA) equipped with a Turbo spray ion source employed in negative ionization mode. Statistical analysis Data generated using qRT-PCR was analyzed as described in 26 followed by a Shapiro-Wilk normality test with subsequent t-test or Mann-Whitney rank sum test based on the data distribution. Phytohormone levels were analyzed using a two-way ANOVA with initial Shapiro-Wilk-normality and equal variance test. For all analyses, phytohormone content was set as the dependent variable with treatment and leaf type as independent variables. For identification of significant differences between groups, pairwise multiple comparison procedure via the Holm-Sidak method was implemented with a significance level of p < 0.05. All statistical analyses were conducted in SigmaPlot (V 11.0). Declarations ACKNOWLEDGEMENTS We thank Y.-R. Chen (Agricultural Biotechnology Research Center of Academia Sinica, Taiwan) and Y.-L. Chen (Department of Biotechnology and Bioindustry Sciences of National Cheng Kung University, Taiwan) for peptide analysis, S.-P. Chen (Sanming Academy of Agricultural Sciences, China) for receptor cloning, C.-P. Cheng (Institute of Plant Biology of National Taiwan University, Taiwan) for suggestion on experimental design. We also thank M. Reichelt and A. Lehr for phytohormone measurements and experimental support, and E. Goschala for growing sweet potato plants. We are grateful to the staffs of Technology Commons in College of Life Science and the Instrumentation Center sponsored by Ministry of Science and Technology, National Taiwan University (Taiwan) for help with the confocal laser scanning microscopy (CLSM). We gratefully acknowledge the International Max Planck Research School supporting A.K.M. and National Science and Technology Council, Taiwan. The research was partially conducted in the context of a project supported by the German Academic Exchange Service (DAAD; PPP Project ID 57136171) and the National Science Council Taiwan National Science and Technology Council, Taiwan (110-2311-B-002-022-, 109-2311-B-002-021-, 111 -2927-I-002 -501-). We are also grateful for financial support to G.F. by the DFG-CRC1101. This publishment was supported by project 111-2311-B-002-026- from the National Science and Technology Council, Taiwan. AUTHOR CONTRIBUTIONS Dr. Kai-Wun Yeh and Dr. Axel Mithöfer conceived and planned the experiments. Dr. Judith Fliegmann and Dr. Georg Felix contributed to the design and implementation of the research, to the analysis of the results. Hsueh-Han Lu and Dr. Anja K. Meents contributed to sample preparation and carried out the experiments. Dr. Ming-Jing Hwang and Dr. Ching-Shu Suen provided bioinformatics analysis to search for novel peptide ligands in sweet potato. Hsueh-Han Lu took the lead in writing the manuscript and both Dr. Judith Fliegmann and Dr. Axel Mithöfer contributed to the final version of the manuscript with input from all authors. Dr. Kai-Wun Yeh supervised the project. All authors provided critical feedback and helped shape the research, analysis, and manuscript. All authors have read and agreed to the final version of the manuscript. COMPETING INTERESTS STATEMENT The authors declare that they have no competing interests. DATA AVAILABILITY The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. References Boller, T. & Felix, G. A Renaissance of Elicitors: Perception of Microbe-Associated Molecular Patterns and Danger Signals by Pattern-Recognition Receptors. Annual Review of Plant Biology 60 , 379–406, doi: 10.1146/annurev.arplant.57.032905.105346 (2009). Boehm, H., Albert, I., Fan, L., Reinhard, A. & Nuernberger, T. 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A salt-regulated peptide derived from the CAP superfamily protein negatively regulates salt-stress tolerance in Arabidopsis. J. Exp. Bot. 66 , 5301–5313, doi: 10.1093/jxb/erv263 (2015). Thuerig, B., Felix, G., Binder, A., Boller, T. & Tamm, L. An extract of Penicillium chrysogenum elicits early defense-related responses and induces resistance in Arabidopsis thaliana independently of known signalling pathways. Physiol Mol Plant P 67 , 180–193, doi: 10.1016/j.pmpp.2006.01.002 (2005). Additional Declarations There is NO Competing Interest. Supplementary Files Supplementaryinformation.docx Dataset 1 SupplementarymovieS1.mp4 Supplemantary Movie 1 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2263331","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":153227569,"identity":"1a9e4fdd-ea78-4596-b17b-8d52ab26442d","order_by":0,"name":"Hsueh-Han Lu","email":"","orcid":"","institution":"National Taiwan University","correspondingAuthor":false,"prefix":"","firstName":"Hsueh-Han","middleName":"","lastName":"Lu","suffix":""},{"id":153227570,"identity":"05859814-600b-4095-a987-4b4d0894132f","order_by":1,"name":"Anja Meents","email":"","orcid":"","institution":"Max Planck Institute for Chemical Ecology","correspondingAuthor":false,"prefix":"","firstName":"Anja","middleName":"","lastName":"Meents","suffix":""},{"id":153227571,"identity":"a2eb6897-302e-41a3-bed8-63fa6f1fe07c","order_by":2,"name":"Judith Fliegmann","email":"","orcid":"https://orcid.org/0000-0003-1021-8006","institution":"University of Tübingen","correspondingAuthor":false,"prefix":"","firstName":"Judith","middleName":"","lastName":"Fliegmann","suffix":""},{"id":153227572,"identity":"14185045-7e1f-44c5-804d-677ebb6cd2a8","order_by":3,"name":"Ming-Jing Hwang","email":"","orcid":"https://orcid.org/0000-0002-9657-5663","institution":"Academia Sinica","correspondingAuthor":false,"prefix":"","firstName":"Ming-Jing","middleName":"","lastName":"Hwang","suffix":""},{"id":153227573,"identity":"bf1209ef-b878-4013-98cf-169f776eceea","order_by":4,"name":"Ching-Shu Suen","email":"","orcid":"https://orcid.org/0000-0002-3096-1983","institution":"Academia Sinica","correspondingAuthor":false,"prefix":"","firstName":"Ching-Shu","middleName":"","lastName":"Suen","suffix":""},{"id":153227574,"identity":"e78a79fc-cbb9-472f-90c4-e75557fdcbd2","order_by":5,"name":"Georg Felix","email":"","orcid":"https://orcid.org/0000-0002-6746-6185","institution":"University of Tübingen","correspondingAuthor":false,"prefix":"","firstName":"Georg","middleName":"","lastName":"Felix","suffix":""},{"id":153227575,"identity":"38358072-7986-4e41-8e8d-9fb26c7fd9ca","order_by":6,"name":"Axel Mithöfer","email":"","orcid":"","institution":"Max Planck Institute for Chemical Ecology","correspondingAuthor":false,"prefix":"","firstName":"Axel","middleName":"","lastName":"Mithöfer","suffix":""},{"id":153227576,"identity":"d4383f67-ccdf-4a2c-bedd-ddd7aafd0242","order_by":7,"name":"Kai-Wun Yeh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYDACHgY2ECUH4bGRoMUYQrGBBIjUkthAtBaDM2fMHhfuuJO+Xb7HgOFD2WEGe4kEAlrO9pgbzzzzLHdnG48B44xzhxl4CGo5z7tNmrftcO6GYzwGzEAGA480kVrSDUBa/hKl5WwvWEsCWAsjMVokz5z/DvKL4YZjaQUHe86l8/Dcf4BfC9+ZtDRQiMkbHD688cGPMms59p4D+LUoAOWZGRsgqkAk4ZiUb0DSMgpGwSgYBaMAKwAAadpF97lprAAAAAAASUVORK5CYII=","orcid":"","institution":"National Taiwan University","correspondingAuthor":true,"prefix":"","firstName":"Kai-Wun","middleName":"","lastName":"Yeh","suffix":""}],"badges":[],"createdAt":"2022-11-11 13:32:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2263331/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2263331/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":29345193,"identity":"8065ff7f-5aa7-4ea5-a918-dbfd3a9a4383","added_by":"auto","created_at":"2022-11-21 17:14:41","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":172816,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReceptor kinase \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eIb\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eLRR-RK1 is induced by wounding and herbivory in sweet potato leaves.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e The expression pattern of \u003cem\u003eIbLRR-RK1\u003c/em\u003e - \u003cem\u003eIbLRR-RK5\u003c/em\u003e receptor-like genes in response to wounding in sweet potato leaves. \u003cem\u003eIbActin1\u003c/em\u003e expression was used as internal control, and \u003cem\u003eIbNAC1\u003c/em\u003e was used as positive control of wounding by semi-quantitative RT-PCR. \u003cstrong\u003e(B) \u003c/strong\u003eSweet potato leaves were wounded and treated with \u003cem\u003eSpodoptera litura \u003c/em\u003elarvae oral secretion or exposed to feeding \u003cem\u003eS. litura\u003c/em\u003e larvae. Expression of \u003cem\u003eIb\u003c/em\u003eLRR-RK1 was analyzed by quantitative RT-PCR. Bars and error bars represent mean ± s.d. of n = 4. \u003cstrong\u003e(C) \u003c/strong\u003eCell surface localization of the GFP-tagged \u003cem\u003eIb\u003c/em\u003eLRR-RK1 in transiently transformed \u003cem\u003eArabidopsis thaliana\u003c/em\u003e protoplasts or \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaf samples, observed by confocal microscope (TCS SP5 Confocal; Leica); well-studied plasmamembrane-localized receptor proteins (SYR1 from tomato and EFR from \u003cem\u003eA. thaliana \u003c/em\u003e\u003csup\u003e\u003cem\u003e4,20\u003c/em\u003e\u003c/sup\u003e) were used as positive controls in \u003cem\u003eN. benthamiana\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2263331/v1/3e03217b8bcd9d14816b32a5.jpg"},{"id":29344937,"identity":"9ffded99-7cde-4856-aa9b-8a450245ca36","added_by":"auto","created_at":"2022-11-21 17:06:41","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":121987,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eActivation of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eIb\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eLRR-RK1 by \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSl\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ePep6 from tomato induces various immune responses.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eROS burst in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves transformed with either SYR1-\u003cem\u003eIb\u003c/em\u003eK (\u003cstrong\u003eA\u003c/strong\u003e) or \u003cem\u003eIb\u003c/em\u003eLRR-RK1 (\u003cstrong\u003eB\u003c/strong\u003e) was induced with 1 µM systemin (red circles), \u003cem\u003eSl\u003c/em\u003ePep6 (orange circles), \u003cem\u003eAt\u003c/em\u003ePep1 (black triangles), \u003cem\u003eSl\u003c/em\u003eHypSysIII (gray circles), \u003cem\u003eIb\u003c/em\u003eHypSysIV (dark gray diamonds), or the control (BSA/NaCl, open squares). Values and error bars represent mean ± s.e. of n = 4 replicates. \u003cstrong\u003e(C)\u003c/strong\u003e Ethylene production in \u003cem\u003eIb\u003c/em\u003eLRR-RK1-expressing leaf discs of \u003cem\u003eN. benthamiana\u003c/em\u003e was induced with the same selection of peptides at 1 µM, 90 ng/µl Pen extract \u003csup\u003e49\u003c/sup\u003e was used as a positive control. Values and error bars represent mean ± s.d. of n = 3 replicates. Mesophyll protoplasts from \u003cem\u003eA. thaliana\u003c/em\u003e Col-0 were co-transformed with either SYR1-\u003cem\u003eIb\u003c/em\u003eK \u003cstrong\u003e(D)\u003c/strong\u003e or \u003cem\u003eIb\u003c/em\u003eLRR-RK1 \u003cstrong\u003e(E)\u003c/strong\u003e and the reporter construct (pFRK1:luciferase), or with pFRK1:luciferase only (Supplementary Figure 4E). Induction of luminescence was monitored after treatment with either 10 nM flg22 (black squares, positive control), systemin (red circles),\u003cem\u003e Sl\u003c/em\u003ePep6 (orange circles), or \u003cem\u003eIb\u003c/em\u003eHypSysIV (dark gray diamonds) at time point 0, mock control is shown with white squares. Values and error bars represent mean ± s.d. of n = 2.\u003c/p\u003e","description":"","filename":"floatimage2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2263331/v1/492e9f3cd05f37f1457b6fe0.jpg"},{"id":29344311,"identity":"72765dc1-6392-4a35-a88b-e6c577f08422","added_by":"auto","created_at":"2022-11-21 16:58:41","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":133832,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIb\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eLRR-RK1 recognizes sweet potato \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eIb\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ePep1 with high sensitivity and specificity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eROS burst in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves transformed with p19 plus\u003cem\u003e Ib\u003c/em\u003eLRR-RK1-GFP, \u003cstrong\u003e(A)\u003c/strong\u003e in response to 1 μM of \u003cem\u003eSl\u003c/em\u003ePep6, \u003cem\u003eIt\u003c/em\u003ePep1, \u003cem\u003eIt\u003c/em\u003ePep2, 10 nM flg22 or BSA/NaCl (mock), respectively, and, \u003cstrong\u003e(B)\u003c/strong\u003e in response to the indicated concentrations of \u003cem\u003eIb\u003c/em\u003ePep1 (●) and \u003cem\u003eSl\u003c/em\u003ePep6 (■). Values and error bars in (\u003cstrong\u003eA\u003c/strong\u003e) represent mean ± s.e. of n = 4. (\u003cstrong\u003eC\u003c/strong\u003e) Sequences and specific ROS-inducing activities of various peptide derivatives of \u003cem\u003eIb\u003c/em\u003ePep1 used in this study. EC\u003csub\u003e50\u003c/sub\u003e values indicate concentrations required for induction of half-maximal ROS production in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves expressing \u003cem\u003eIb\u003c/em\u003eLRR-RK1-GFP. (\u003cstrong\u003eD\u003c/strong\u003e) Dose-response curves for \u003cem\u003eSl\u003c/em\u003ePEPR1-GFP, treated with of \u003cem\u003eIb\u003c/em\u003ePep1 (●) and \u003cem\u003eSl\u003c/em\u003ePep6 (■), filled and open symbols correspond to independent experiments. Data in (\u003cstrong\u003eB\u003c/strong\u003e) and (\u003cstrong\u003eD\u003c/strong\u003e) correspond to the integrated ROS response over 30 min. Curve fittings and calculation of EC\u003csub\u003e50\u003c/sub\u003e values were performed by nonlinear regression.\u003c/p\u003e","description":"","filename":"floatimage3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2263331/v1/4159b2734d148dbb8294478d.jpg"},{"id":29344936,"identity":"7eb545ef-631a-4098-be32-1293e5929b57","added_by":"auto","created_at":"2022-11-21 17:06:41","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":43061,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIb\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ePROPEP1 mainly localizes to the tonoplast.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIb\u003c/em\u003ePROPEP1-GFP was transiently transformed in \u003cem\u003eArabidopsis thaliana \u003c/em\u003eprotoplasts, and protoplasts were observed to monitor the subcellular localization of \u003cem\u003eIb\u003c/em\u003ePROPEP1 by confocal microscopy. Partial colocalization with the tonoplast marker γ-Tip-mCherry was observed. See Supplementary Movie 1 for the observation of moving GFP-labeled vesicles.\u003c/p\u003e","description":"","filename":"floatimage4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2263331/v1/4855457c1ec06e506b9374a4.jpg"},{"id":29344306,"identity":"21b9d642-15ad-4f40-b471-39b892a667a8","added_by":"auto","created_at":"2022-11-21 16:58:41","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":59115,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIb\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eLRR-RK1 recognizes an endogenous compound.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) A partially purified extract from \u003cem\u003eI. batatas\u003c/em\u003e leaves induces a ROS burst in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves expressing \u003cem\u003eIb\u003c/em\u003eLRR-RK1 (♦). The extract (black symbols, 1 µl) did not induce a response in leaf pieces transformed with p19 only (▲). Mock treatments are shown in gray symbols. Values and error bars represent mean ± s.e. of n = 4. \u003cstrong\u003e(B)\u003c/strong\u003e ROS burst (integrated over 30 min) in \u003cem\u003eN. benthamiana\u003c/em\u003e control leaves (p19) or leaves expressing \u003cem\u003eIb\u003c/em\u003eLRR-RK1 in response to 1 μl partially purified extract from unwounded (control) or 10 min wounded sweet potato leaves. Bars and error bars represent mean ± s.e. of n = 4.\u003c/p\u003e","description":"","filename":"floatimage5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2263331/v1/e93e9c3ae36d864ba7dbfe16.jpg"},{"id":29345194,"identity":"b79ac744-c71b-4bd4-91ab-710de9d87893","added_by":"auto","created_at":"2022-11-21 17:14:41","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":119268,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInduction of defense-related genes and volatiles in response to \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eIb\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ePep1 and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eIb\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eHypSysIV in sweet potato.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSweet potato leaves were treated with 25 μM of \u003cem\u003eIb\u003c/em\u003ePep1 (\u003cstrong\u003eA\u003c/strong\u003e) or \u003cem\u003eIb\u003c/em\u003eHypSysIV (\u003cstrong\u003eB\u003c/strong\u003e), respectively, and tested for the expression level of herbivore defense-related genes. Expression of \u003cem\u003esporamin\u003c/em\u003e, \u003cem\u003eIbNAC1\u003c/em\u003e, \u003cem\u003eIbWIPK1\u003c/em\u003e, \u003cem\u003eIbCML1\u003c/em\u003e, and \u003cem\u003eIbLRR-RK1\u003c/em\u003e were analyzed by quantitative RT-PCR. Bars and error bars represented mean ± s.e. of n = 4. Significance levels are * = p\u0026lt;0.05, ** = p\u0026lt;0.01, *** =p\u0026lt;0.001, respectively, according to one-tailed t-test. (\u003cstrong\u003eC\u003c/strong\u003e) The induced emission of (\u003cem\u003eE\u003c/em\u003e)-4,8–dimethyl–nonatriene (DMNT) in \u003cem\u003eI. batatas\u003c/em\u003e TN57 was evaluated after treatment of whole plants with 25 µM \u003cem\u003eIb\u003c/em\u003eHypSysIV (n = 10), \u003cem\u003eIb\u003c/em\u003ePep1 (n = 10), \u003cem\u003eSl\u003c/em\u003ePep6 (n = 11), or the scrambled peptide (n = 7), data are shown as fold-induction in comparison to the respective water controls. Bars represent the mean ± s.e. of DMNT emission. Significance levels are indicated by the asterisks (n.s. = not significant; * = p\u0026lt;0.05) and are based on a Shapiro-Wilk normality test followed by a Mann-Whitney rank sum test.\u003c/p\u003e","description":"","filename":"floatimage6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2263331/v1/c70ce6c8a925d4380aaf286c.jpg"},{"id":29344308,"identity":"e0f67786-f14e-43a8-a8b5-d49dfa389f0c","added_by":"auto","created_at":"2022-11-21 16:58:41","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":55864,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIb\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ePep1 and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eIb\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eHypSysIV peptides differentially alter gene expression patterns in sweet potato leaves.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e RNAseq data from \u003cem\u003eI. batatas\u003c/em\u003e leaves, treated with 25 µM \u003cem\u003eIb\u003c/em\u003eHypSysIV, \u003cem\u003eIb\u003c/em\u003ePep1, or ddH2O (control) for 1 h were mapped onto the \u003cem\u003eI. trifida\u003c/em\u003e genome. The Venn diagram shows the numbers of identified, expressed genes in each treatment (Σ 24482 genes). Overlapping circle parts represent the shared expressed genes between the treatments. (\u003cstrong\u003eB\u003c/strong\u003e) Volcano plot of statistical significance (-log10 adj. p-value \u0026lt;0.05) against differentially expressed genes (DEGs, log2-fold change \u0026gt;=1 and padj \u0026lt;0.05), by comparing both peptide treatments (25 µM each peptide, 1 h). The number of significantly upregulated genes (\u003cem\u003eIb\u003c/em\u003eHypSys vs. \u003cem\u003eIb\u003c/em\u003ePep1) is indicated in red with the downregulated ones highlighted in green. DEGs not meeting significance thresholds are depicted in blue.\u003c/p\u003e","description":"","filename":"floatimage7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2263331/v1/759b6574a1e4d20fb4e6ff78.jpg"},{"id":31219968,"identity":"6e299c1c-a86d-4fea-af61-b82969c7696f","added_by":"auto","created_at":"2023-01-06 14:01:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1282658,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2263331/v1/5d0df3db-d311-4e48-a711-fa5442d69bf4.pdf"},{"id":29344312,"identity":"89129d4d-74eb-4605-b2d4-0878230bc7cd","added_by":"auto","created_at":"2022-11-21 16:58:41","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6303485,"visible":true,"origin":"","legend":"Dataset 1","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-2263331/v1/e605b350b6edabce68a6aba4.docx"},{"id":29344940,"identity":"e7a7ca76-22b7-4665-a847-e7cdd85580ba","added_by":"auto","created_at":"2022-11-21 17:06:41","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":22283439,"visible":true,"origin":"","legend":"Supplemantary Movie 1","description":"","filename":"SupplementarymovieS1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-2263331/v1/49e1702e6a55d4785356665c.mp4"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Identification of a DAMP receptor and its cognate peptide ligand in sweet potato (Ipomoea batatas)","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlants have evolved several mechanisms to cope with biotic and abiotic stresses. When encountering stresses, such as pathogen infection, insect feeding, and wounding, receptor kinases (RKs) or receptor-like proteins (RLPs) properly identify specific patterns derived either from the aggressors (microbe-associated molecular patterns, MAMPs and herbivore-associated molecular patterns, HAMPs) or from the perturbation of cellular integrity (danger- or damage-associated molecular patterns, DAMPs). Subsequently, these pattern recognition receptors (PRRs) trigger signal transduction pathways to activate appropriate plant immune responses, leading to pattern-triggered immunity (PTI) \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. PTI can reduce the damages caused by the invasion of many pathogens and insects \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePRRs are usually composed of extracellular, transmembrane, and intracellular domains. They are classified by their extracellular domains. The extracellular leucine-rich repeat domain, a single-pass transmembrane domain, and a cytoplasmic protein kinase domain characterize leucine-rich repeat receptor kinases (LRR-RKs). LRR-RKs and LRR-RLPs are sensors for proteinaceous immunogenic ligands, such as peptides and small proteins \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. For example, the FLS2 receptor binds a 22-amino acid epitope (flg22) conserved in bacterial flagellins \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, EFR recognizes a conserved N-terminal fragment of bacterial elongation factor Tu \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, and \u003cem\u003eSl\u003c/em\u003eEix1 and \u003cem\u003eSl\u003c/em\u003eEix2 bind \u003cem\u003eTrichoderma\u003c/em\u003e cell wall-derived xylanase \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePeptide ligands play an important role in regulating the signal transduction of insect resistance and wound defense responses \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. In \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, eight plant elicitor peptides (\u003cem\u003eAt\u003c/em\u003ePep1-\u003cem\u003eAt\u003c/em\u003ePep8) are found to participate in damage-related defense responses after recognition by a pair of LRR-RKs, the PEP receptors 1 and 2 (\u003cem\u003eAt\u003c/em\u003ePEPR1 and 2) \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Each of the \u003cem\u003eAt\u003c/em\u003ePeps is derived from the carboxy terminus of their precursor protein \u003cem\u003eAt\u003c/em\u003ePROPEP1-8 \u003csup\u003e10,11\u003c/sup\u003e, how and if the peptides are cleaved off is however mostly not known. However, a METACASPASE4 (MC4)-dependent maturation of \u003cem\u003eAt\u003c/em\u003ePep1was recently described. High levels of [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003ecyt\u003c/sub\u003e that occur only in directly damaged cells bind to MC4, which in this activated form cleaves PROPEP1 and releases \u003cem\u003eAt\u003c/em\u003ePep1 \u003csup\u003e12,13\u003c/sup\u003e. \u003cem\u003eAtPROPEP2, AtPROPEP3\u003c/em\u003e, and the receptor genes \u003cem\u003eAtPEPR1/2\u003c/em\u003e are strongly induced upon herbivore attack. Moreover, \u003cem\u003epepr1 pepr2\u003c/em\u003e double mutant plants display a reduced resistance to \u003cem\u003eSpodoptera littoralis\u003c/em\u003e larvae \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. In \u003cem\u003eZea mays\u003c/em\u003e, the precursor of \u003cem\u003eZm\u003c/em\u003ePep3, an \u003cem\u003eAt\u003c/em\u003ePep-ortholog, can be induced by insect oral secretion and insect HAMP. The application of \u003cem\u003eZm\u003c/em\u003ePep3 can induce emission of some insect herbivory-related volatile organic compounds (VOCs), biosynthesis and accumulation of phytohormones, and transcripts that are indirectly involved in defense against herbivores. \u003cem\u003eZm\u003c/em\u003ePep3 also causes accumulation of proteinase inhibitor and contributes to the resistance to lepidopteran insects \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSystemin was the first peptide discovered in plants with signaling capacities. In \u003cem\u003eSolanum lycopersicum\u003c/em\u003e, the injury-induced systemin can cause defense responses against insects \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Tomato systemin is an endogenous peptide ligand composed of 18 amino acids, which is derived from a precursor protein by phytaspase-dependent cleavage at two aspartate residues \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Systemin induces proteinase inhibitors and activates phospholipase A2, thereby promoting the release of jasmonic acid precursors from the cell membrane. Induction of insect-resistance defense genes by jasmonic acid signaling pathways further contributes to the resistance of herbivore attack \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, mediated by the LRR-RK receptor \u003cem\u003eSl\u003c/em\u003eSYR1, which however is not necessary for wound responses \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHydroxyproline-rich systemins (HypSys) are systemin-like endogenous peptide ligands in \u003cem\u003eSolanaceae\u003c/em\u003e plants. In addition to the hydroxyproline-rich conserved sequence, the HypSys precursor protein preproHypSys has a secretion sequence at the N-terminus, which is absent from Peps and systemin precursor proteins. Similar to systemin, HypSys induces the production of jasmonates and the expression of defense genes \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. In petunia, HypSys is described to induce the expression of the immune gene \u003cem\u003edefensin1\u003c/em\u003e \u003csup\u003e22\u003c/sup\u003e. The precursors of \u003cem\u003eSl\u003c/em\u003eHypSys I, II, and III in tomato are synthesized and sequestered in the cell wall matrix of phloem parenchyma cells in response to systemin, wounding, and methyl jasmonate \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Moreover, the HypSys precursor gene \u003cem\u003eIbpreproHypSys\u003c/em\u003e in sweet potato (\u003cem\u003eIpomoea batatas\u003c/em\u003e) can be induced by injury. The application of \u003cem\u003eIb\u003c/em\u003eHypSys in sweet potato induces downstream insect-resistance genes such as \u003cem\u003esporamin\u003c/em\u003e and \u003cem\u003eipomoelin\u003c/em\u003e, and improves the biosynthesis of lignin, to increase the ability to repel insects \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. However, it is still unclear how HypSys binds to receptors and participates in defense responses.\u003c/p\u003e \u003cp\u003eSweet potato is the fifth largest food crop in the world and has high nutritional and economic value. Several cultivars of sweet potato have higher insect resistance than others. For example, \u003cem\u003eIpomoea batatas\u003c/em\u003e cv. Tainong 57, which is widely cultivated in Taiwan, has strong insect resistance and represents a suitable model crop for studying insect resistance mechanisms \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Sporamin, which was previously thought to be a unique storage protein in sweet potato tuberous roots, was recently described to be regulated by herbivore attack, injuries, jasmonic acid, and the homoterpene (\u003cem\u003eE\u003c/em\u003e)-4,8\u0026ndash;dimethyl\u0026ndash;1,3,7-nonatriene (DMNT) in sweet potato leaves \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Functional studies revealed that sporamin is a serine-type trypsin inhibitor, which acts in the insect intestine and retards insect growth and development \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Transgenic \u003cem\u003eNicotiana benthamiana\u003c/em\u003e and \u003cem\u003eBrassica rapa\u003c/em\u003e subsp. \u003cem\u003echinensis\u003c/em\u003e plants overexpressing sporamin demonstrated a strong pest resistance capacity \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, as did transgenic sweet potato plants overexpressing \u003cem\u003eIbNAC1\u003c/em\u003e, which is a transcription factor binding to the sporamin wounding response element (\u003cem\u003eSWRE\u003c/em\u003e) region of the \u003cem\u003esporamin\u003c/em\u003e promoter \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eIb\u003c/em\u003eNAC1 also regulates the jasmonic acid response and ROS signaling \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e and is regulated by \u003cem\u003eIbb\u003c/em\u003eHLH3/4, \u003cem\u003eIb\u003c/em\u003eWIPK1, \u003cem\u003eIb\u003c/em\u003eJAZ2, and \u003cem\u003eIb\u003c/em\u003eEIL1 upon injury \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The MAPK pathway is also part of the signal transduction from wounding stress to \u003cem\u003esporamin\u003c/em\u003e expression \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. However, the molecular connection between danger perception (ligands, receptors) and downstream defense responses is still elusive.\u003c/p\u003e \u003cp\u003eIn order to discover the key players upstream of the intracellular signaling cascade leading to induced resistance against herbivores we isolated candidates for both, a cell surface receptor and endogenous peptide ligands from sweet potato. Among the wound- and herbivore-induced genes in \u003cem\u003eI. batatas\u003c/em\u003e we detected a gene encoding a leucine-rich receptor kinase related to the plant elicitor peptide (Pep) receptor (PEPR) family, \u003cem\u003eIb\u003c/em\u003eLRR-RK1. When heterologously expressed in \u003cem\u003eN. benthamiana\u003c/em\u003e, this receptor candidate did not provide responsiveness to HypSys but to extracts of damaged sweet potato leaf tissue. Finally, we identified the cognate peptide ligand, \u003cem\u003eIb\u003c/em\u003ePep1, characterized the specificity and sensitivity of the new receptor/ligand-pair and compared the signaling capacities of the newly identified peptide with the previously described HypSys peptides.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSweet potato encodes putative DAMP receptors\u003c/h2\u003e \u003cp\u003eWe based our search for receptors of sweet potato which are involved in responses to wounding and herbivore attack on published sequences for DAMP-related receptors in Arabidopsis (\u003cem\u003eAt\u003c/em\u003ePEPR1/2: AT1G73080/AT1G17750) and tomato, \u003cem\u003eS. lycopersicum\u003c/em\u003e (\u003cem\u003eSl\u003c/em\u003eSYR1/2: \u003cem\u003eSoly\u003c/em\u003ec03g082470/Solyc03g082450.2.1; \u003cem\u003eSl\u003c/em\u003ePEPR1: XP_004235511). Several closely related receptor genes, designated \u003cem\u003eItLRR-RK1\u003c/em\u003e to \u003cem\u003eItLRR-RK13\u003c/em\u003e, were mined from the \u003cem\u003eIpomoea trifida\u003c/em\u003e \u0026ldquo;Sweet potato Genomic Resource database\u0026rdquo; (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://sweetpotato.plantbiology.msu.edu/index.shtml\u003c/span\u003e\u003cspan address=\"http://sweetpotato.plantbiology.msu.edu/index.shtml\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Supplementary Fig.\u0026nbsp;1). Next, two transcriptomic databases, i.e. the \u003cem\u003eI. batatas\u003c/em\u003e cv. Tainong 57 transcriptome database \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003eI. batatas\u003c/em\u003e database, \u003cem\u003eIpomoea\u003c/em\u003e Genome Hub (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ipomoea-genome.org/\u003c/span\u003e\u003cspan address=\"https://ipomoea-genome.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were accessed to explore putative \u003cem\u003eLRR-RK\u003c/em\u003e genes with sequence homology to \u003cem\u003eItLRR-RKs\u003c/em\u003e. Five putative \u003cem\u003eI. batatas\u003c/em\u003e receptor kinase genes (\u003cem\u003eIbLRR-RK1\u003c/em\u003e to \u003cem\u003eIbLRR-RK5\u003c/em\u003e) were identified. RT-PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and qRT-PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) experiments revealed that both wounding and insect herbivory rapidly induced \u003cem\u003eIbLRR-RK1\u003c/em\u003e (MT210638). Upon wounding and treatment with \u003cem\u003eSpodoptera\u003c/em\u003e larvae-derived oral secretion, the relative expression level of \u003cem\u003eIbLRR-RK1\u003c/em\u003e increased nearly 30-fold at 15 min, 12-fold at 30 min, and returned to normal levels at 60 min. Herbivory feeding also increased \u003cem\u003eIbLRR-RK1\u003c/em\u003e expression level 5.4-fold at 15 min and 1.7-fold at 30 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). These data demonstrate that mechanical wounding and herbivory induce the receptor-like kinase \u003cem\u003eIb\u003c/em\u003eLRR-RK1, suggesting that this receptor might be involved in perception of a wound-related signal.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe receptor candidate\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eIb\u003c/span\u003e\u003cb\u003eLRR-RK1 is related to PEPRs\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe gene \u003cem\u003eIbLRR-RK1\u003c/em\u003e from \u003cem\u003eI. batatas\u003c/em\u003e cv. Tainong 57 encodes a typical member of the PEPR family. It consists of an ectodomain composed of a signal peptide, an N-terminal cap region typically found in plant LRR-RKs, 26 repetitions of the plant-specific version of the LRR motif, and an outer juxtamembrane; this is followed by a transmembrane domain; the cytosolic part contains the inner juxtamembrane domain and a serine/threonine kinase domain (Supplementary Fig.\u0026nbsp;2). As expected, the green fluorescent protein (GFP)-tagged \u003cem\u003eIb\u003c/em\u003eLRR-RK1 protein, transiently expressed in either \u003cem\u003eA. thaliana\u003c/em\u003e protoplasts or \u003cem\u003eN. benthamiana\u003c/em\u003e leaves, localized to the plasma membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), like other plant LRRs such as \u003cem\u003eSl\u003c/em\u003eSYR1-GFP or \u003cem\u003eAt\u003c/em\u003eEFR-GFP, which were used as positive controls. \u003cem\u003eIb\u003c/em\u003eLRR-RK1 is most likely related to PEPRs from tomato (XP_004235511, \u003cem\u003eSl\u003c/em\u003ePEPR1) and Arabidopsis (\u003cem\u003eAt\u003c/em\u003e1g73080, \u003cem\u003eAt\u003c/em\u003e1g17750; PEPR1 and PEPR2; Supplementary Fig.\u0026nbsp;3). \u003cem\u003eIb\u003c/em\u003eLRR-RK1, which is 97% identical with \u003cem\u003eIt\u003c/em\u003eLRR-RK1, shared 65% or 50% identical amino acid residues to \u003cem\u003eSl\u003c/em\u003ePEPR1 or \u003cem\u003eAt\u003c/em\u003ePEPR1, respectively, and 35% identity to \u003cem\u003eSl\u003c/em\u003eSYR1, while other putative RLK members selected from the Sweet Potato Databases never shared more than 36% identity to either of the mentioned receptors (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In addition, comparing the extracellular domains of different receptors also showed that \u003cem\u003eIb\u003c/em\u003eLRR-RK1 has a highly similar ligand-binding surface when compared with \u003cem\u003eSl\u003c/em\u003ePEPR1 and \u003cem\u003eAt\u003c/em\u003ePEPR1 (60 and 48% identity, respectively), and shares only 36% identical residues with tomato \u003cem\u003eSl\u003c/em\u003eSYR1 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Thus, \u003cem\u003eIb\u003c/em\u003eLRR-RK1 is part of the plant elicitor peptide receptor group.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of the full-length amino acid sequence of \u003cem\u003eIb\u003c/em\u003eLRR-RK1 with related sequences, calculation of % identity by Vector NTI.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eIt\u003c/em\u003eLRR-RLK1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eSl\u003c/em\u003ePEPR1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eAt\u003c/em\u003ePEPR1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eAt\u003c/em\u003ePEPR2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eSl\u003c/em\u003eSYR1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eSl\u003c/em\u003eSYR2\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eIb\u003c/em\u003eLRR-RK1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eIt\u003c/em\u003eLRR-RK1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSl\u003c/em\u003ePEPR1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAt\u003c/em\u003ePEPR1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAt\u003c/em\u003ePEPR2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSl\u003c/em\u003eSYR1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003cem\u003eAt, Arabidopsis thaliana; Ib, Ipomoea batatas; It, Ipomoea trifida; Sl, Solanum lycopersicum.\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of the amino acid sequence of the extracellular domain of \u003cem\u003eIb\u003c/em\u003eLRR-RK1 with \u003cem\u003eAt\u003c/em\u003ePEPR1, \u003cem\u003eSl\u003c/em\u003ePEPR1, and \u003cem\u003eSl\u003c/em\u003eSYR1, calculation of % identity by Vector NTI.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eSl\u003c/em\u003ePEPR1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eAt\u003c/em\u003ePEPR1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eSl\u003c/em\u003eSYR1\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eIb\u003c/em\u003eLRR-RK1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSl\u003c/em\u003ePEPR1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAt\u003c/em\u003ePEPR1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003cem\u003eAt, Arabidopsis thaliana; Ib, Ipomoea batatas; Sl, Solanum lycopersicum.\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eIb\u003c/span\u003e \u003cb\u003eLRR-RK1 is a functional receptor\u003c/b\u003e \u003c/p\u003e \u003cp\u003eEstablishing the functionality of new receptor candidates for which the ligands are not known is challenging, and can be overcome by approaches in which chimeric versions are ectopically expressed in suitable plants \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. To test if the kinase domain of the putative receptor from sweet potato is able to feed into the immune response pathway we generated a chimeric version with the ectodomain of tomato SYR1 a receptor with known ligand (Supplementary Fig.\u0026nbsp;4A). The chimeric receptor SYR1-\u003cem\u003eIb\u003c/em\u003eK as well as the original \u003cem\u003eIb\u003c/em\u003eLRR-RK1 and SYR1 were transiently expressed in leaves of \u003cem\u003eN. benthamiana\u003c/em\u003e. The GFP-tagged recombinant proteins localized to the cell surface, as predicted (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, Supplementary Fig.\u0026nbsp;4B). Treatment with the ligand of SYR1 resulted in the induction of an oxidative burst for the SYR1-\u003cem\u003eIb\u003c/em\u003eK expressing leaf pieces (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, Supplementary Fig.\u0026nbsp;4C), proving the functionality of the kinase domain of \u003cem\u003eIb\u003c/em\u003eLRR-RK1. Several other defense-related peptides from various plants such as \u003cem\u003eSl\u003c/em\u003ePep6, \u003cem\u003eSl\u003c/em\u003eHypSysIII, \u003cem\u003eIb\u003c/em\u003eHypSysIV, or \u003cem\u003eAt\u003c/em\u003ePep1 were applied in addition to systemin (Supplementary Table\u0026nbsp;1) in bioassays with leaves expressing either the original \u003cem\u003eIb\u003c/em\u003eLRR-RK1 or SYR1-\u003cem\u003eIb\u003c/em\u003eK. Interestingly, \u003cem\u003eSl\u003c/em\u003ePep6 triggered the defense pathway in the presence of \u003cem\u003eIb\u003c/em\u003eLRR-RK1, leading to ROS production and ethylene accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C). We than verified the recognition of \u003cem\u003eSl\u003c/em\u003ePep6 by \u003cem\u003eIb\u003c/em\u003eLRR-RK1 in protoplasts, generated from \u003cem\u003eA. thaliana\u003c/em\u003e Col-0 mesophyll cells. \u003cem\u003eFRK1\u003c/em\u003e (flg22-induced receptor-like kinase 1) is a PTI marker gene of early defense responses in Arabidopsis \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e and its promoter is widely used in combination with a luciferase reporter gene to monitor PAMP activity \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. The co-expression of \u003cem\u003eIb\u003c/em\u003eLRR-RK1 with p\u003cem\u003eFRK1:LUC\u003c/em\u003e resulted in \u003cem\u003eSl\u003c/em\u003ePep6-dependent induction of the reporter (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), confirming the previous experiments in \u003cem\u003eN. benthamiana\u003c/em\u003e, while the chimeric receptor SYR1-\u003cem\u003eIb\u003c/em\u003eK recognized systemin, but not \u003cem\u003eSl\u003c/em\u003ePep6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Taken together, we demonstrated that the activation of \u003cem\u003eIb\u003c/em\u003eLRR-RK1 can trigger plant immune responses such as ROS burst, ethylene biosynthesis, and defense gene expression and identified a ligand.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eIb\u003c/span\u003e \u003cb\u003eLRR-RK1 perceives an endogenous peptide\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFor the molecular identification of the cognate ligand of \u003cem\u003eIb\u003c/em\u003eLRR-RK1 we took advantage of the fact that \u003cem\u003eSl\u003c/em\u003ePep6 was functional in activating sweet potato \u003cem\u003eIb\u003c/em\u003eLRR-RK1. We hence used the sequence of \u003cem\u003eSl\u003c/em\u003ePep6 and other Peps from the Solanaceae family as probes to search for endogenous peptides and applied scanning sequence pattern and tBlastn strategies on the sweet potato genomic resource database and the \u003cem\u003eIpomoea\u003c/em\u003e genome hub database (Supplementary Fig.\u0026nbsp;5). Two putative Peps were selected from the \u003cem\u003eI. trifida\u003c/em\u003e genomic resource database. \u003cem\u003eIt\u003c/em\u003ePep1 (LSSRPPRPGLGNSGDPQTNDTSS) consisted of 23 amino acid residues, which were present at the C-terminus of the precursor protein \u003cem\u003eIt\u003c/em\u003ePROPEP1 (itf01g30920.t1) (Supplementary Fig.\u0026nbsp;5B). The putative \u003cem\u003eIt\u003c/em\u003ePep2 (RRGRTPPRPENLKLNLRARKHSLEDQ) with 26 residues was derived from the C-terminus of \u003cem\u003eIt\u003c/em\u003ePROPEP2 (itf07g21780.t1) (Supplementary Fig.\u0026nbsp;5C). Both candidate peptides, \u003cem\u003eIt\u003c/em\u003ePep1 and \u003cem\u003eIt\u003c/em\u003ePep2, were synthesized and applied to \u003cem\u003eN. benthamiana\u003c/em\u003e leaf discs transiently expressing \u003cem\u003eIb\u003c/em\u003eLRR-RK1. \u003cem\u003eIt\u003c/em\u003ePep1, but not \u003cem\u003eIt\u003c/em\u003ePep2, activated an \u003cem\u003eIb\u003c/em\u003eLRR-RK1-dependent ROS burst (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), and neither peptide elicited a response in the SYR1-\u003cem\u003eIb\u003c/em\u003eK or p19 controls (Supplementary Fig.\u0026nbsp;6A, B). Next, the cDNA of the \u003cem\u003ePROPEP1\u003c/em\u003e gene from \u003cem\u003eI. batatas\u003c/em\u003e cv. TN57 was cloned by RT-PCR using oligonucleotides deduced from \u003cem\u003eItPROPEP1\u003c/em\u003e. The CDS encompasses 378 bp and 125 deduced amino acid residues with a calculated molecular weight of 13.25 kDa, and a pI of 4.44. The putatively bioactive 23-mer peptide, \u003cem\u003eIb\u003c/em\u003ePep1, corresponds to the C-terminus of the precursor protein and is 100% identical to the one from \u003cem\u003eIt\u003c/em\u003ePROPEP1 (Supplementary Fig.\u0026nbsp;6C). As expected, the \u003cem\u003eI. batatas\u003c/em\u003e PROPEP as well as the Pep amino acid sequences are more closely related to those from solanaceous plants than to those of Arabidopsis (Supplementary Fig.\u0026nbsp;6D, Supplementary Table\u0026nbsp;2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eIb\u003c/span\u003e \u003cb\u003eLRR-RK1 perceives\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eIb\u003c/span\u003e\u003cb\u003ePep1 with high sensitivity and specificity\u003c/b\u003e\u003c/p\u003e \u003cp\u003eExploiting the same heterologous expression system described above we interrogated the sensitivity and the specificity of the putative ligand/receptor-pair. The dose-dependent induction of ROS by \u003cem\u003eIb\u003c/em\u003ePep1 was clearly detectable in the sub-nanomolar range and the half-maximal activation of this output was estimated at 1 nM (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The tomato Pep (\u003cem\u003eSl\u003c/em\u003ePep6) was 10-times less efficient in this bioassay with \u003cem\u003eIb\u003c/em\u003eLRR-RK1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In the reciprocal approach, we cloned the Pep receptor of tomato (\u003cem\u003eSl\u003c/em\u003ePEPR1, \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e), expressed it in \u003cem\u003eN. benthamiana\u003c/em\u003e and compared the efficiencies of the Peps for the induction of ROS. The tomato PEPR/\u003cem\u003eSl\u003c/em\u003ePep6 pair showed the same efficiency as the corresponding sweet potato pair, with an EC\u003csub\u003e50\u003c/sub\u003e value of 1 nM. Interestingly, \u003cem\u003eSl\u003c/em\u003ePEPR1 recognized also the peptide from sweet potato, albeit with a much lower sensitivity, and an estimated EC\u003csub\u003e50\u003c/sub\u003e value above 100 nM (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eN-terminal and C-terminal truncated versions of \u003cem\u003eIb\u003c/em\u003ePep1 were synthesized to investigate the specificity for the predicted sweet potato peptide on \u003cem\u003eIb\u003c/em\u003eLRR-RK1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Deleting up to three N-terminal residues did not have a major impact on the perception, the loss of arginine at position 4, however, led to a severe increase of the EC\u003csub\u003e50\u003c/sub\u003e value (either when deleted as in \u003cem\u003eIb\u003c/em\u003ePep1 (5\u0026ndash;23) or when changed to an alanine as in \u003cem\u003eIb\u003c/em\u003ePep1 (A4)). In contrast, the C-terminus needs to be present for a sensitive perception although the last two serine residues can be replaced by alanine (\u003cem\u003eIb\u003c/em\u003ePep1 (A22A23)).\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eIb\u003c/span\u003e \u003cb\u003ePROPEP1-GFP is mainly localized at the tonoplast\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe expressed \u003cem\u003eIbPROPEP1\u003c/em\u003e (OP311829) as a C-terminal fusion with GFP in \u003cem\u003eN. benthamiana\u003c/em\u003e and \u003cem\u003eA. thaliana\u003c/em\u003e protoplasts and observed the localization of the protein by confocal microscopy. \u003cem\u003eIb\u003c/em\u003ePROPEP1-GFP partially localized to the tonoplast (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Supplementary Fig.\u0026nbsp;7) as reported for \u003cem\u003eAt\u003c/em\u003ePROPEP1-YFP \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, but also aggregated into bright small globular structures, resembling bulbs \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, inside the vacuole of Arabidopsis mesophyll protoplasts. Similarly, in \u003cem\u003eN. benthamiana\u003c/em\u003e, \u003cem\u003eIb\u003c/em\u003ePROPEP1-GFP accumulated in the tonoplast and aggregated into several small globular structures which moved inside the vacuole (Supplementary Fig.\u0026nbsp;7, Supplementary Movie 1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAn\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eIb\u003c/span\u003e\u003cb\u003eLRR-RK1-activating DAMP is present in sweet potato leaves\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePreliminary data hinted that \u003cem\u003eIb\u003c/em\u003ePROPEP1 might be cleaved after damage to the leaf tissue and that a mature, signaling competent peptide might be liberated (data not shown), as it is known from the cognate ligands of PEPRs \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. To simulate a corresponding scenario, we prepared first an extract from \u003cem\u003eI. batatas\u003c/em\u003e cv. TN57 leaves and applied it on transiently \u003cem\u003eIb\u003c/em\u003eLRR-RK1-expressing \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. Leaf discs expressing the receptor responded to the treatment with the partially purified leaf extract with a ROS burst, which was not detectable in control leaves transformed with p19 only (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Next, we damaged sweet potato leaves by squeezing with tweezers. After 10 min, this material was harvested, in parallel to tissue from control plants. Interestingly, the elicitor activity was higher in extracts from wounded leaves in comparison to the non-damaged leaves. This activity clearly depended on the expression of \u003cem\u003eIb\u003c/em\u003eLRR-RK1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). These data indicated that crude extracts of \u003cem\u003eI. batatas\u003c/em\u003e leaves contain ligands for \u003cem\u003eIb\u003c/em\u003eLRR-RK1 that might accumulate upon wounding stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eIb\u003c/span\u003e \u003cb\u003ePep1 and\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eIb\u003c/span\u003e\u003cb\u003eHypSysIV activate complementary signaling cascades\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe next addressed the question whether \u003cem\u003eIb\u003c/em\u003ePep1 is involved in herbivore resistance responses in sweet potato or in other processes. Therefore, whole sweet potato plants were sprayed with 25 \u0026micro;M \u003cem\u003eIb\u003c/em\u003ePep1 and analyzed for \u003cem\u003esporamin\u003c/em\u003e induction. For comparison, the synthetic hydroxyproline-rich glycopeptide \u003cem\u003eIb\u003c/em\u003eHypSysIV, which was shown to activate \u003cem\u003esporamin\u003c/em\u003e expression \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e was tested at 25 \u0026micro;M as well. The qRT-PCR analysis after 30 min and 1 h of incubation revealed HypSys-dependent transient increases of sporamin (X60930.1) (16-fold), \u003cem\u003eIb\u003c/em\u003eNAC1 (GQ280387.1) (22-fold), and \u003cem\u003eIb\u003c/em\u003eWIPK1 (HQ434622) (68-fold) transcript levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), confirming the ability of HypSys peptides to rapidly trigger sporamin-related signaling cascades. In contrast, \u003cem\u003eIb\u003c/em\u003ePep1 transiently induced \u003cem\u003esporamin\u003c/em\u003e only 3.5-fold after 30 min, while \u003cem\u003eIb\u003c/em\u003eNAC1 and \u003cem\u003eIb\u003c/em\u003eWIPK1 were induced to higher and longer lasting expression levels compared to \u003cem\u003eIb\u003c/em\u003eHypSysIV treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Moreover, when analyzing other defense-related genes we also found that \u003cem\u003eIb\u003c/em\u003ePep1 and \u003cem\u003eIb\u003c/em\u003eHypSysIV treatments increased the expression of \u003cem\u003eIb\u003c/em\u003eLRR-RK1 and \u003cem\u003eIb\u003c/em\u003eCML-like1 (OP311828) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). Further, compared to water controls and \u003cem\u003eIb\u003c/em\u003ePep1, the application of \u003cem\u003eIb\u003c/em\u003eHypSysIV resulted in a significantly increased emission of the wound-inducible volatile DMNT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC) \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Scions incubated with the tomato-derived peptide \u003cem\u003eSl\u003c/em\u003ePep6 or an inactive scrambled peptide only displayed basal DMNT levels comparable to the control treatment, confirming thereby the functionality of the peptide application method and the (species-)specificity of the \u003cem\u003eIb\u003c/em\u003eHypSysIV elicitor.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn order to elucidate which role peptides play within the \u003cem\u003eIpomoea\u003c/em\u003e defense framework, local and systemic TN57 leaves were analyzed for phytohormone levels after peptide treatment. In comparison to water-treated controls, no significant differences in local and systemic jasmonic acid concentrations could be observed after \u003cem\u003eIb\u003c/em\u003eHypSysIV treatment (Supplementary Fig.\u0026nbsp;8A). Interestingly, \u003cem\u003eIb\u003c/em\u003eHypSysIV-treated leaves showed a significantly increased amount of bioactive JA-Ile, however only locally (Supplementary Fig.\u0026nbsp;8B). For the stress-related hormones SA and ABA, no significant differences to control treatments were detected, except for a local decrease in SA concentrations upon contact with \u003cem\u003eIb\u003c/em\u003eHypSysIV (Supplementary Fig.\u0026nbsp;8C). Treatment with \u003cem\u003eIb\u003c/em\u003ePep1 did neither alter jasmonate nor SA levels although low concentrations might mask possible effects. However, exposure to \u003cem\u003eIb\u003c/em\u003ePep1 resulted in decreasing amounts of ABA, mainly observed in the local leaf (Supplementary Fig.\u0026nbsp;8D). Although no tremendous changes in phytohormone levels were overall visible, we noted a clear tendency that for phytohormones regulated by \u003cem\u003eIb\u003c/em\u003eHypSysIV, no response would occur during exposure to \u003cem\u003eIb\u003c/em\u003ePep1 and \u003cem\u003evice versa\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRNAseq of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eI. batatas\u003c/span\u003e \u003cb\u003ereveals differentially expressed genes (DEGs) upon\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eIb\u003c/span\u003e\u003cb\u003ePep1 and\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eIb\u003c/span\u003e\u003cb\u003eHypSysIV treatment\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn order to better understand the similarities between \u003cem\u003eIb\u003c/em\u003ePep1 and \u003cem\u003eIb\u003c/em\u003eHypSysIV and their particular functionalities, RNAseq experiments were conducted on single leaves treated with either peptide or water (control), respectively, for 1 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Overall, 24482 expressed genes were detected based on mapping onto the \u003cem\u003eI. trifida\u003c/em\u003e reference genome from which 22702 were shared among all treatments including control samples. 241 genes were exclusively detected upon \u003cem\u003eIb\u003c/em\u003eHypSysIV treatment, while 485 transcripts were detected only after \u003cem\u003eIb\u003c/em\u003ePep1 treatment. An additional number of 326 common transcripts was found in both peptide treatments but not in the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Strikingly, 179 expressed genes were mapped onto the \u003cem\u003eI. trifida\u003c/em\u003e genome but found only in control plants, suggesting that expression of these genes is reduced upon peptide treatments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurther, spraying of \u003cem\u003eIb\u003c/em\u003eHypSysIV induced significant upregulation of 555 genes whereas 826 genes were significantly downregulated, compared to water-treated control leaves (data not shown). Upon \u003cem\u003eIb\u003c/em\u003ePep1 incubation, an even stronger response was observed with 1749 genes up- and 2694 downregulated (data not shown). A comparison of both peptide treatments revealed that 4836 genes were significantly differentially regulated due to these different treatments, 2607 up- and 2229 downregulated, when \u003cem\u003eIb\u003c/em\u003eHypSysIV \u003cem\u003evs Ib\u003c/em\u003ePep1 was compared (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). These results support the idea that the two sweet potato peptides have distinct functions, which may be based on their ability to regulate different genes. To support this hypothesis, further confirmation with KEGG and GO pathway analyses and quantititave PCR of selected genes is necessary and will be performed.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eRecent evidence has shown that sweet potato exhibits DAMP-mediated activation of defenses. The volatile homoterpene, DMNT, has been demonstrated to activate resistance mechanisms in leaves leading to protection against herbivore feeding \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Peptide-based activation of defense reactions also has been observed in sweet potato \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. However, the biological significance and interconnection with induced resistance against insects remained unclear. This study provides evidence for the existence of a Pep/PEPR system in sweet potato and investigates the input- and output-conditions. Briefly, we show that the system can be activated by a damage-induced endogenous elicitor, provide indirect evidence that this elicitor might be \u003cem\u003eIb\u003c/em\u003ePep1, the product of \u003cem\u003eIb\u003c/em\u003ePROPEP1 cleavage, and that it functions in parallel and complementary to a HypSys-dependent signaling pathway.\u003c/p\u003e \u003cp\u003eMining the sweet potato genome databases we identified a wound- and herbivory-induced gene encoding a canonical leucine-rich repeat-containing receptor kinase, \u003cem\u003eIb\u003c/em\u003eLRR-RK1 (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B, and Supplementary Fig.\u0026nbsp;2). Using a chimeric receptor approach, in which we combined the cytosolic kinase domain of \u003cem\u003eIb\u003c/em\u003eLRR-RK1 with the extracellular recognition domain of \u003cem\u003eSl\u003c/em\u003eSYR1 (Supplementary Fig.\u0026nbsp;4A), we were able to generate a functional receptor after heterologous expressions in both \u003cem\u003eN. benthamiana\u003c/em\u003e and \u003cem\u003eA. thaliana\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Supplementary Fig.\u0026nbsp;4C). Phylogenetic analysis suggested that \u003cem\u003eIb\u003c/em\u003eLRR-RK1 might be a member of the plant elicitor peptide receptors (PEPRs). Indeed, \u003cem\u003eSl\u003c/em\u003ePep6 from tomato, but not \u003cem\u003eAt\u003c/em\u003ePep1 from \u003cem\u003eA. thaliana\u003c/em\u003e, was recognized by the native \u003cem\u003eIb\u003c/em\u003eLRR-RK1, and triggered the activation of typical defense responses after expression of \u003cem\u003eIb\u003c/em\u003eLRR-RK1-GFP in both \u003cem\u003eN. benthamiana\u003c/em\u003e and \u003cem\u003eA. thaliana\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Of note, the sweet potato peptide \u003cem\u003eIb\u003c/em\u003eHypSysIV, which is described to be involved in the wound response \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e was not recognized by \u003cem\u003eIb\u003c/em\u003eLRR-RK1.\u003c/p\u003e \u003cp\u003eBased on the above findings, sequences of Peps and their precursor proteins (PROPEPs) from tomato and other Solanaceae plants were used to search for the related putative peptide in the sweet potato genome. We identified a 23-amino acids long peptide ligand, \u003cem\u003eIb\u003c/em\u003ePep1, which is derived from the C-terminus of its precursor protein \u003cem\u003eIb\u003c/em\u003ePROPEP1. \u003cem\u003eIb\u003c/em\u003ePep1 is capable to initiate the ROS burst in transgenic \u003cem\u003eIb\u003c/em\u003eLRR-RK1-expressing \u003cem\u003eN. benthamiana\u003c/em\u003e with a tenfold-higher sensitivity in comparison to \u003cem\u003eSl\u003c/em\u003ePep6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). However, the fact that the tomato peptide was recognized by sweet potato prompted us to investigate the reciprocal scenario. Indeed, \u003cem\u003eSl\u003c/em\u003ePEPR1, the tomato receptor for \u003cem\u003eSl\u003c/em\u003ePep6 \u003csup\u003e38\u003c/sup\u003e recognized \u003cem\u003eIb\u003c/em\u003ePep1, providing here for the first time data on interfamily (Solanaceae and Convolvulaceae) compatibility of Peps.\u003c/p\u003e \u003cp\u003eThe structure-activity characterization of the ligand of \u003cem\u003eIb\u003c/em\u003eLRR-RK1 using various synthetic \u003cem\u003eIb\u003c/em\u003ePep1 derivatives unraveled some structural requirements for the interaction with the corresponding receptor (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). As for other Peps, the C-terminus of the peptide is of utmost importance, since the C-terminaly truncated peptide (\u003cem\u003eIb\u003c/em\u003ePep1(1\u0026ndash;20)) is at least 100-fold less efficient compared to the 23-mer \u003cem\u003eIb\u003c/em\u003ePep1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Unlike the Peps from other plant families, however, the identity of the residues at the C-terminus seems not to be as important since the replacement of the last two residues with alanine residues only marginally decreased the affinity. Peps from sweet potato share five of the 12 highly conserved residues with the family-specific Pep-motif of the Solanaceae (\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e in the overlapping 20-mer core region (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, Supplementary Fig.\u0026nbsp;9A). Testing one of these highly conserved residues (\u003cem\u003eIb\u003c/em\u003ePep1(A4)) confirmed the importance of the arginine at that position. As illustrated in a composite consensus sequence for Peps of Solanales (the order including Solanaceae and Convolvulaceae), conserved arginine and proline residues are clustered at the N-terminus of the peptides whereas proline and asparagine residues are conserved at the C-termini (Supplementary Fig.\u0026nbsp;9B).\u003c/p\u003e \u003cp\u003ePROPEPs have been reported to distribute to distinct subcellular localizations in Arabidopsis \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. While \u003cem\u003eAt\u003c/em\u003ePROPEP3 is present in the cytosol, \u003cem\u003eAt\u003c/em\u003ePROPEP1 and \u003cem\u003eAt\u003c/em\u003ePROPEP6 are positioned at the tonoplast. Our findings show that \u003cem\u003eIb\u003c/em\u003ePROPEP1-GFP is localized at the tonoplast as well (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Supplementary Fig.\u0026nbsp;7). In addition, \u003cem\u003eIb\u003c/em\u003ePROPEP1-GFP also appeared in vesicle-like structures attached to the tonoplast that dynamically fuse with the vacuole (Supplementary Movie 1). Whether these structures correspond to bulbs, which have been described as cytoplasmic projections into the vacuole, surrounded by a tonoplast-derived double membrane \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, or are artefacts of dimerizing GFP with which the overexpressed PROPEP is tagged \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e remains to be investigated. However, to the best of our knowledge, this localization has never been reported for other PROPEPs. We hypothesize that the purpose of \u003cem\u003eIb\u003c/em\u003ePROPEP1 enrichment in bulbs could be to store sufficient amounts of the precursor and release it rapidly after cell and vacuole injury to allow cleavage into active \u003cem\u003eIb\u003c/em\u003ePep1. \u003cem\u003eIn planta\u003c/em\u003e, we demonstrated the release of a specific agonist of \u003cem\u003eIb\u003c/em\u003eLRR-RK1. Incubation of only 10 minutes of wounded sweet potato leaves increased the amount of the elicitor in a partially purified fraction, in comparison to non-incubated leaf material (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe inherent trypsin inhibitory activity of sporamin provides strong protection against herbivory in sweet potato and other, transgenic plants species expressing sporamin \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Strongly induced expression of \u003cem\u003esporamin\u003c/em\u003e was detected in sweet potato leaves during pest attack and injury stress \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. The 18 amino acid hydroxyprolinated peptide \u003cem\u003eIb\u003c/em\u003eHypSysIV, which can be extracted from sweet potato leaves, was amplifying the wounding signal and activated the expression of \u003cem\u003esporamin\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. In the present study, we found that spraying with either peptide, \u003cem\u003eIb\u003c/em\u003eHypSysIV or \u003cem\u003eIb\u003c/em\u003ePep1, rapidly induced the expression of wound-induced defense response genes including \u003cem\u003eIbWIPK1\u003c/em\u003e, \u003cem\u003eIbNAC1\u003c/em\u003e, \u003cem\u003esporamin\u003c/em\u003e, and even \u003cem\u003eIbLRR-RK1\u003c/em\u003e, in sweet potato leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). However, \u003cem\u003eIb\u003c/em\u003eHypSysIV treatment induced the expression of \u003cem\u003esporamin\u003c/em\u003e much more strongly than \u003cem\u003eIb\u003c/em\u003ePep1 treatment. Previous studies have revealed that application of \u003cem\u003eAt\u003c/em\u003ePeps and the activation of \u003cem\u003eAt\u003c/em\u003ePEPR1/2 lead to increased jasmonate accumulation and induced jasmonate responses in Arabidopsis \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. We found that the application of \u003cem\u003eIb\u003c/em\u003ePep1 did not increase the amount of jasmonates, in contrast to \u003cem\u003eIb\u003c/em\u003eHypSysIV, which induced the accumulation of JA-Ile in sweet potato leaves slightly (Supplementary Fig.\u0026nbsp;8), suggesting that \u003cem\u003eIb\u003c/em\u003eHypSysIV may trigger the jasmonate pathway and associated responses in contrast to \u003cem\u003eIb\u003c/em\u003ePep1. A clear discrepancy between the two peptides lies in their ability to regulate the synthesis and release of the homoterpene DMNT. This volatile danger signal is induced in sweet potato upon wounding and herbivory \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Only treatment with \u003cem\u003eIb\u003c/em\u003eHypSysIV but neither \u003cem\u003eIb\u003c/em\u003ePep1 nor \u003cem\u003eSl\u003c/em\u003ePep6 nor a scrambled control peptide were able to induced DMNT, indicating the specificity of this response (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Overall, our study suggests that in addition to the \u003cem\u003eIb\u003c/em\u003ePep1/\u003cem\u003eIb\u003c/em\u003eLRR-RK1 pair described here for the first time, there is another, as yet unidentified, DAMP receptor that specifically interacts with the \u003cem\u003eIb\u003c/em\u003eHypSysIV ligand in sweet potato. The latter system appears to be more active than the Pep/PEPR pair in the jasmonate pathway regulating sporamin expression.\u003c/p\u003e \u003cp\u003eHaving shown that both, \u003cem\u003eIb\u003c/em\u003ePep1 and \u003cem\u003eIb\u003c/em\u003eHypSysIV, have a certain ability to regulate defense responses against herbivory attack and wounding, albeit with different efficacies, we have yet to define the key signaling pathway(s) regulated by \u003cem\u003eIb\u003c/em\u003ePep1. Preliminary analyses of RNAseq data suggest that \u003cem\u003eIb\u003c/em\u003ePep1 and \u003cem\u003eIb\u003c/em\u003eHypSysIV control partly distinct pathways, which will need to be further investigated in combination with real infestation and infection assays in the future.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003ePrevious studies have shown that Peps/PEPR ligand-receptor systems are widespread in plants. Here, we identified a novel peptide ligand and its corresponding receptor from sweet potato. This adds another ligand/receptor pair to the growing list of DAMP perception systems. Understanding how the downstream gene responses to different ligands are coordinated in the genetic network is a topic that needs to be addressed in the future. Although \u003cem\u003eIb\u003c/em\u003ePep1 was not able to induce the emission of DMNT, the trypsin protease inhibitor sporamin and its transcription factor \u003cem\u003eIb\u003c/em\u003eNAC1 were upregulated, hinting at a modular way to increase insect resistance. Peps vary widely from species to species, conserved family-specific Pep-motifs are sufficient for Pep recognition by PEPRs from different species of the same plant family \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. In our experiment, we found that the peptide ligand \u003cem\u003eSl\u003c/em\u003ePep6 of tomato belonging to Solanaceae family did interact with \u003cem\u003eIb\u003c/em\u003eLRR-RK1 from sweet potato belonging to Convolvulaceae family and activated downstream responses. V\u003cem\u003eice versa\u003c/em\u003e, the reciprocal combination was functional as well. To our knowledge, this is the first example that a peptide ligand does not follow the rule of family-specific incompatibility of Peps but suggests the conservation of a plant order-specific peptide ligand structure in Solanales.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003ePlant material and growth conditions\u003c/h2\u003e \u003cp\u003eSweet potato scions (\u003cem\u003eIpomoea batatas\u003c/em\u003e Lam.; cultivar Tainong 57) were grown in phytochambers under long day conditions (16 h light : 8 h dark) at 28\u0026deg;C (day) and 25\u0026deg;C (night) in 70% relative humidity for 3 weeks as previously described \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. When growing for 4\u0026ndash;5 weeks, sweet potato and \u003cem\u003eNicotiana benthamiana\u003c/em\u003e plants were maintained in a greenhouse with a 16 h photoperiod and a 25\u0026deg;C/20\u0026deg;C day/night program. \u003cem\u003eArabidopsis thaliana\u003c/em\u003e ecotype Columbia-0 (Col-0) was grown at 22\u0026deg;C with an 8 h photoperiod in growth chambers for 4\u0026ndash;5 weeks.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePeptides\u003c/h2\u003e \u003cp\u003ePeptides were ordered from GenScript Biotech (Leiden, Netherlands). They were dissolved prior to each experiment in BSA/NaCl (10 mg/ml, 0.1 M) solution. The list of peptides and their sequences can be found in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction, RT- and qRT-PCR analyses\u003c/h2\u003e \u003cp\u003eHarvested sweet potato leaves were processed and used for real-time PCR as described in \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e with the additional primer \u003cem\u003epair IbLRR-RK1, sporamin, IbWIPK1, IbNAC1, IbCML1\u003c/em\u003e (Supplementary Table\u0026nbsp;3) on a Bio-Rad CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, USA). Semi-quantitative reverse transcription PCR (RT-PCR) was performed with 2X Taq DNA Polymerase (Bioman) and the primers listed in Supplementary Table\u0026nbsp;3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eRNA-Seq analysis and processing\u003c/h2\u003e \u003cp\u003eRNA from single 3rd leaves treated for 1 h with \u003cem\u003eIb\u003c/em\u003eHypSysIV, \u003cem\u003eIb\u003c/em\u003ePep1, and water (control) was extracted according to \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e using TRIzol Reagent (Invitrogen, USA). Four biological replicates per treatment were used for RNA-Seq experiments conducted by Novogene Europe (Cambridge, UK). RNA quality was monitored using NanoPhotometer\u0026reg; spectrophotometer (IMPLEN, CA, USA) and RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). 1 \u0026micro;g of RNA per sample was used as template material for further sample preparations. Sequencing libraries were generated \u003cem\u003evia\u003c/em\u003e NEBNext\u0026reg; UltraTM RNA Library Prep Kit for Illumina\u0026reg; (NEB, USA) following manufacturer\u0026rsquo;s instructions. 20 M paired end reads of 150 bp per sample were generated, sequenced on an Illumina NovaSeq 6000 instrument (San Diego, USA). Raw reads were trimmed by in-house scripts. The clean reads were mapped onto \u003cem\u003eIpomoea trifida\u003c/em\u003e reference genome (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://sweetpotato.uga.edu/\u003c/span\u003e\u003cspan address=\"http://sweetpotato.uga.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), using HISAT2 V2.0.5 with default parameter. HTSeq V0.6.1 software was used with the union mode to count read numbers mapped of genes for each sample.\u003c/p\u003e \u003cp\u003eR package from Bioconductor, DESeq2 V1.22.2 was used to estimate gene abundance and detect differentially expressed genes (DEGs) among the sample groups. A model based on the negative binomial distribution was carried out to determinate DEGs with an adjusted p-value cutoff of 0.05. To control the False Discovery Rate (FDR) Benjamini-Hochberg adjustment was performed. Genes with a log2-fold change\u0026thinsp;\u0026gt;\u0026thinsp;=\u0026thinsp;1 and padj\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered as significantly DEGs. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of DEGs were implemented by the GOseq V1.34.1 R package and KOBAS V3.0 software.\u003c/p\u003e \u003cp\u003e \u003cb\u003eWounding, insect feeding, and peptide spray treatments\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eI. batatas\u003c/em\u003e and \u003cem\u003eN. benthamiana\u003c/em\u003e plants with six to eight fully developed leaves were used in the study. For wound treatment, the third or fourth fully expanded leaves were wounded using tweezers and the wounded leaves samples were collected at different time points. For insect feeding treatment, starved \u003cem\u003eSpodoptera litura\u003c/em\u003e larvae (2nd instar) were placed on the third or fourth fully expanded leaves and the treated leaves samples were collected at intervals.\u003c/p\u003e \u003cp\u003eTo study the local effects of peptide solutions on DMNT emission and gene expression, whole sweet potato plants with six to eight fully expanded leaves were evenly sprayed with peptide solution or double-distilled water (control) until all leaves were fully covered in liquid. After a 1 h incubation period, single plants were placed for 24 h in 2.4 L glass desiccators (VWR international) for headspace volatile collection. For RNA-Seq, qRT-PCR, and phytohormone analyses each 3rd fully expanded leaf was locally sprayed with peptide solution or ddH\u003csub\u003e2\u003c/sub\u003eO (control) and harvested together with the adjacent 4th leaf (systemic) after the indicated time points.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eVOC collection and quantification\u003c/h2\u003e \u003cp\u003eVolatiles were collected over 24 h from peptide- or water-treated sweet potato plants enclosed in 2.4 L desiccators using the closed-loop stripping technique \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Throughout the headspace collection, each desiccator was connected to an air circulation pump (F\u0026uuml;rgut GmbH, Germany) containing a charcoal trap with 1.5 mg absorption material (CLSA filter, 6 cm long, 0.5 cm diameter, Gr\u0026auml;nicher \u0026amp; Quartero, France). After collection, volatiles were eluted and measured as described \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e with minor modifications. In this study, samples were eluted with 2 x 20 \u0026micro;l of dichloromethane containing 10 \u0026micro;g ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e \u003cem\u003en\u003c/em\u003e-bromodecane as internal standard used for further relative quantification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCloning of receptor and propeptide gene candidates\u003c/h2\u003e \u003cp\u003e \u003cem\u003eIb\u003c/em\u003eLRR-RK1 and \u003cem\u003eIb\u003c/em\u003ePROPEP1 genes were identified using blastn as well as tblastn on various databases using the receptor and propeptide sequences for Solanaceae plants from \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Used databases included Sweet Potato Genomic Resource database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://sweetpotato.plantbiology.msu.edu/index.shtml\u003c/span\u003e\u003cspan address=\"http://sweetpotato.plantbiology.msu.edu/index.shtml\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), \u003cem\u003eI. batatas\u003c/em\u003e cv. TN57 transcriptome database \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eI. batatas\u003c/em\u003e database: Ipomoea Genome Hub (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ipomoea-genome.org/\u003c/span\u003e\u003cspan address=\"https://ipomoea-genome.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and NCBI (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The \u003cem\u003eIb\u003c/em\u003eLRR-RK1 and \u003cem\u003eIb\u003c/em\u003ePROPEP1 coding sequences were amplified from sweet potato leaf cDNA using gene-specific primers (\u003cem\u003eIb\u003c/em\u003eLRR-RLK1_FL_F, \u003cem\u003eIb\u003c/em\u003eLRR-RLK1_FL_R, \u003cem\u003eIb\u003c/em\u003ePROPEP1_FL_F, \u003cem\u003eIb\u003c/em\u003ePROPEP1_FL_R, as shown in Supplementary Table\u0026nbsp;2) in a PCR reaction with Q5 High-Fidelity DNA Polymerase (NEB), respectively. The coding sequence encoding the tomato \u003cem\u003eSl\u003c/em\u003ePEPR1 (XP_004235511) was amplified using the primers \u003cem\u003eSl\u003c/em\u003ePEPR1_FL_F and \u003cem\u003eSl\u003c/em\u003ePEPR1_FL_R (Supplementary Table\u0026nbsp;3). All full length coding sequences were cloned into the pCR8/GW/TOPO vector (Invitrogen). LR clonase (Invitrogen) was used to transfer these coding sequences from PCR8 to pMDC83 vectors \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, generating C-terminal fusions with GFP.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eGeneration of chimeric receptors\u003c/h2\u003e \u003cp\u003eGene-specific level I modules for SYR1 \u003csup\u003e20\u003c/sup\u003e and \u003cem\u003eIb\u003c/em\u003eLRR-RK1 (see above) were generated by proof reading PCR (Phusion High Fidelity DNA Polymerase, ThermoFisher Scientific) from existing templates using the oligonucleotide primers listed in Supplementary Table\u0026nbsp;2, subcloned, and verified by sequencing. GoldenGate cloning was used to assemble the receptor expression constructs with general level I modules (A-B p35S (G005), D-E GFP (G011), E-F nos-T (G006) and dy F-G (BB09)) into the vector backbone LIIα F 1\u0026ndash;2 (BB10) as described \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eTransient expression of receptor constructs and bioassays\u003c/h2\u003e \u003cp\u003eTransient expression in \u003cem\u003eN. benthamiana\u003c/em\u003e was performed as described \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The oxidative burst was measured with leaf pieces floating on 100 \u0026micro;l water containing 20 \u0026micro;M L-012 (Wako) and 2 \u0026micro;g/ml horseradish peroxidase (Applichem), after addition of peptides, with a luminescence plate reader (Mithras LB 940, Berthold, or Infinite M200 PRO plant reader, TECAN). The amount of ethylene was measured by GC in the headspace of 4 leaf pieces floating on 500 \u0026micro;l water, treated for 4 h with the peptides or controls. Transient co-expression of the pFRK1:Luciferase reporter \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e with the receptor expression constructs in mesophyll protoplasts of \u003cem\u003eA. thaliana\u003c/em\u003e Col-0 wild-type was performed as described \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Luminescence was recorded for up to 6 h in W5-medium containing 200 \u0026micro;M firefly luciferin (Synchem UG) after overnight incubation for 14 h and subsequent treatment with peptides or control solution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSubcellular localization\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eIb\u003c/em\u003eLRR-RK1-GFP, \u003cem\u003eIb\u003c/em\u003ePROPEP1-GFP, and the tonoplast localization marker protein fusion γ-Tip-mCherry \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e were transiently expressed in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves and \u003cem\u003eA. thaliana\u003c/em\u003e mesophyll protoplast as described above. Fluorescence images were taken using a TCS SP5 Confocal microscope (Leica) and analyzed by LAS AF Lite application software (Leica) or a Zeiss Axio Zoom.V16.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eImmunoblotting\u003c/h2\u003e \u003cp\u003eWounded leaves of \u003cem\u003eN. benthamiana\u003c/em\u003e transiently expressing IbPROPEP1-GFP were ground to a fine powder in liquid nitrogen. The powder was immediately supplemented with 100 \u0026micro;l of 2\u0026times; SDS sample buffer solution (Nacalai Tesque Inc.), heated up to 95\u0026deg;C for 5 min at, and centrifuged for 10 min at 16,000 x g to remove cellular debris. Ten \u0026micro;g extracted proteins were run in 15% SDS polyacrylamide gels for IbPROPEP1-GFP and IbPep1-GFP separation, followed by semi-dry Western blotting to PVDF membranes (MILLIPORE), and incubation with anti-GFP antibodies (rabbit, 1:2000; Abcam) overnight. After a second incubation with Goat-anti-Rabbit IgG labeled with horseradish peroxidase enzyme (1:20000; Jackson), chemiluminescent substrates (Chemi-Lumi One Super; Nacalai Tesque Inc.) were supplied for detection of luminescence. Luminescent images were taken and analyzed with a KETA CLX Chemiluminescence Imaging System (Wealtec).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCrude endogenous ligand extraction\u003c/h2\u003e \u003cp\u003eAccording to \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, 10 g injured and non-injured sweet potato leaves were harvested, respectively. Samples were homogenized with 1% cold trifluoroacetic acid (TFA) in a blender for 2 min. After filtering the extracts through 4 layers of Miracloth to remove plant debris and centrifuging at 8,500 rpm for 20 min at 4\u0026deg;C, the supernatant was slowly pressed through a customized Sep-Pak C18 solid phase extraction cartridge (Waters) and eluted with 60% (v/v) methanol/0.1% (v/v) TFA. The eluate containing peptides were dried in a speed vac and re-suspended in 200 \u0026micro;l double-distilled H\u003csub\u003e2\u003c/sub\u003eO.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003ePhytohormone extraction and quantification\u003c/h2\u003e \u003cp\u003eLocal and systemic leaves collected after 1 h peptide treatment were extracted and measured as described \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e using an Agilent 1200 HPLC system (Agilent, USA) with subsequent API 5000 tandem mass spectrometer (Applied Biosystems, USA) equipped with a Turbo spray ion source employed in negative ionization mode.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData generated using qRT-PCR was analyzed as described in \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e followed by a Shapiro-Wilk normality test with subsequent t-test or Mann-Whitney rank sum test based on the data distribution. Phytohormone levels were analyzed using a two-way ANOVA with initial Shapiro-Wilk-normality and equal variance test. For all analyses, phytohormone content was set as the dependent variable with treatment and leaf type as independent variables. For identification of significant differences between groups, pairwise multiple comparison procedure via the Holm-Sidak method was implemented with a significance level of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All statistical analyses were conducted in SigmaPlot (V 11.0).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Y.-R. Chen (Agricultural Biotechnology Research Center of Academia Sinica, Taiwan) and Y.-L. Chen (Department of Biotechnology and Bioindustry Sciences of National Cheng Kung University, Taiwan) for peptide analysis, S.-P. Chen (Sanming Academy of Agricultural Sciences, China) for receptor cloning, C.-P. Cheng (Institute of Plant Biology of National Taiwan University, Taiwan) for suggestion on experimental design. We also thank M. Reichelt and A. Lehr for phytohormone measurements and experimental support, and E. Goschala for growing sweet potato plants. We are grateful to the staffs of Technology Commons in College of Life Science and the Instrumentation Center sponsored by Ministry of Science and Technology, National Taiwan University (Taiwan) for help with the confocal laser scanning microscopy (CLSM). We gratefully acknowledge the International Max Planck Research School supporting A.K.M. and National Science and Technology Council, Taiwan. The research was partially conducted in the context of a project supported by the German Academic Exchange Service (DAAD; PPP Project ID 57136171) and the National Science Council Taiwan National Science and Technology Council, Taiwan (110-2311-B-002-022-, 109-2311-B-002-021-, 111 -2927-I-002 -501-). We are also grateful for financial support to G.F. by the DFG-CRC1101. This publishment was supported by project 111-2311-B-002-026- from the National Science and Technology Council, Taiwan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDr. Kai-Wun Yeh and Dr. Axel Mith\u0026ouml;fer conceived and planned the experiments. Dr. Judith Fliegmann and Dr. Georg Felix contributed to the design and implementation of the research, to the analysis of the results. Hsueh-Han Lu and Dr. Anja K. Meents contributed to sample preparation and carried out the experiments. Dr. Ming-Jing Hwang and Dr. Ching-Shu Suen provided bioinformatics analysis to search for novel peptide ligands in sweet potato. Hsueh-Han Lu took the lead in writing the manuscript and both Dr. Judith Fliegmann and Dr. Axel Mith\u0026ouml;fer contributed to the final version of the manuscript with input from all authors. Dr. Kai-Wun Yeh supervised the project. All authors provided critical feedback and helped shape the research, analysis, and manuscript. All authors have read and agreed to the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBoller, T. \u0026amp; Felix, G. A Renaissance of Elicitors: Perception of Microbe-Associated Molecular Patterns and Danger Signals by Pattern-Recognition Receptors. 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Physiol Mol Plant P \u003cb\u003e67\u003c/b\u003e, 180\u0026ndash;193, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.pmpp.2006.01.002\u003c/span\u003e\u003cspan address=\"10.1016/j.pmpp.2006.01.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2005).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"sweet potato, herbivory, plant defense, plant elicitor peptide receptor, DMNT, DAMP, LRR-RLK","lastPublishedDoi":"10.21203/rs.3.rs-2263331/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2263331/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSweet potato (\u003cem\u003eIpomoea batatas\u003c/em\u003e) is an important tuber crop, but also target of numerous insect pests. Intriguingly, the abundant storage protein in tubers, sporamin, has intrinsic trypsin protease inhibitory activity. In leaves, sporamin is induced by wounding or a volatile homoterpene and enhances insect resistance. While the signaling pathway leading to sporamin synthesis is partially established, the initial event, perception of a stress-related signal is still unknown. Here, we identified a leucine-rich repeat receptor kinase (\u003cem\u003eIb\u003c/em\u003eLRR-RK1) that is induced upon wounding and herbivory, and related to peptide-elicitor receptors (PEPRs) from tomato and Arabidopsis. We also identified a gene encoding a precursor protein comprising a peptide ligand (\u003cem\u003eIb\u003c/em\u003ePep1) for \u003cem\u003eIb\u003c/em\u003eLRR-RK1. \u003cem\u003eIb\u003c/em\u003ePep1 represents a distinct signal in sweet potato, which might work in a complementary and/or parallel pathway to the previously described HypSys peptides to strengthen insect resistance. Notably, an inter-family compatibility in the Pep/PEPR system from Convolvulaceae and Solanaceae was identified.\u003c/p\u003e","manuscriptTitle":"Identification of a DAMP receptor and its cognate peptide ligand in sweet potato (Ipomoea batatas)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-11-21 16:58:36","doi":"10.21203/rs.3.rs-2263331/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e6bb55ed-513a-4356-9c63-d183c8a5e7ae","owner":[],"postedDate":"November 21st, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":17072953,"name":"Biological sciences/Plant sciences/Plant immunity/Pattern recognition receptors in plants"},{"id":17072954,"name":"Biological sciences/Plant sciences/Plant stress responses/Herbivory"}],"tags":[],"updatedAt":"2023-01-06T14:01:00+00:00","versionOfRecord":[],"versionCreatedAt":"2022-11-21 16:58:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2263331","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2263331","identity":"rs-2263331","version":["v1"]},"buildId":"J0_U0BvcaRcwD8yVFaRlm","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}