Volatile organic compounds emitted from the damaged hot peppers are oppositely interpreted by thrips and its predator

Volatile organic compounds emitted from the damaged hot peppers are oppositely interpreted by thrips and its predator | 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 Volatile organic compounds emitted from the damaged hot peppers are oppositely interpreted by thrips and its predator Yonggyun Kim, Mojtaba Esmaeily, Akhtar Ayoobi, Gahyeon Jin, Eeshita Mandal, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6437640/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 A plant emits diverse volatile compounds and communicates with other organisms in different trophic levels. Deciphering these compounds would be useful to understand their biorational interactions. Hot pepper, Capsicum annuum, produced different compositions of volatile organic compounds (VOCs) upon an infestation by the western flower thrips, Frankliniella occidentalis. These VOCs induced a non-preference behavior to the thrips while they attracted a natural enemy, Orius laevigatus. Among the differentially emitted VOCs, green leaf volatiles (GLVs) and terpenes were included and played crucial roles in their interactions. Z-3-methyl hexenoate, one of GLVs, induced the non-preference behavior but attracted the predator. Similarly, a terpenoid linalool attracted the predator but gave the non-preference to the thrips. Suppression of GLV or linalool biosynthesis was performed by virus-induced gene silencing of hydroperoxide lyase (HPL) or linalool synthase (LS) expression in the hot pepper and led to significant malfunction in the tri-trophic communication. The tri-trophic interactions were mediated by jasmonic acid (JA), but not salicylic acid, signal in the hot pepper. Insect resistance of the hot peppers against the thrips were positively correlated with HPL or LS expression levels, which would be useful for breeding programs for insect-resistant hot peppers. Biological sciences/Zoology/Entomology Biological sciences/Plant sciences/Plant stress responses/Herbivory Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The hot pepper, Capsicum annuum , is cultivated worldwide as an indispensable condiment for various foods because of its typical color, pungency, taste, and distinct aroma 1 . In Korea, nearly 80 different plant pathogens and insect pests have been identified as causing economic damage to hot pepper cultivation 2 . Among these, the western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), is one of the most severe insect pests. It transmits the tomato spotted wilt virus (TSWV) and also directly causes feeding damage 3 . Originating in the western part of North America, this thrips quickly spread to most temperate and subtropical regions worldwide due to its polyphagous feeding habits and relatively high tolerance to temperature extremes, facilitated by increased global trade 4 . Hot peppers infected by TSWV experience retarded growth and reduced fruit quality, resulting in significant economic losses and sometimes leading to the abandonment of harvesting 5,6 . TSWV is transmitted from plant to plant solely by F. occidentalis and eight other thrips species out of more than 7,700 thrips species 7 . Consequently, controlling the thrips is essential to prevent TSWV-related diseases in hot peppers. Several chemical insecticides including spinosad have been effective in killing thrips 8 . However, the cryptic behavior of thrips between leaves and flowers enables them to avoid exposure to sprayed insecticides 9 . This challenge has led to an increase in the frequency and dosage of insecticide spraying, which may select for thrips resistant to chemical insecticides. Consequently, this facilitates the rapid buildup of insecticide resistance in thrips. For example, frequent spraying of spinosad has selected for point-mutated individuals in the acetylcholine receptor gene, which become over 3,000-fold more tolerant to spinosad compared to the susceptible strain 10 . To effectively control this insect pest, alternative control measures should be developed. As an alternative, a biological control strategy has been designed and implemented to control F. occidentalis using seasonal inoculative releases of a predatory bug, Orius laevigatus (Fieber) (Hemiptera: Anthocoridae) 11 . However, to successfully control the thrips, the biological control method occasionally needs supplementary applications of chemical insecticides, where the insecticide-resistant O. laevigatus would be optimal to minimize toxicant damage 12,13 . Insect resistance in plants has been regarded as an alternative insect pest control strategy because it minimizes chemical spraying and the associated labor, ideally reducing hazards to humans and the environment 14 . The insect resistance includes antixenosis, which deters herbivores; antibiosis, which delivers direct toxicants to herbivores after feeding; and tolerance of the plants to herbivore damage. Indeed, this host plant resistance against thrips has successfully been applied to prevent TSWV infection in peanuts, Arachis hypogaea , exhibiting both antixenosis and antibiosis 15 . Alternatively, TSWV reduces the host plant resistance to increase the accessibility of its vector thrips to the host plants for viral transmission 16 . In addition to direct insect resistance of host plants, a tri-trophic interaction can be exploited to recruit natural enemies to attack herbivores by releasing plant odors known as volatile organic compounds (VOCs) upon herbivore damage 17 . This chemical communication led us to hypothesize that hot peppers at the primary trophic level infested by F. occidentalis at the secondary trophic level release VOCs to repel thrips as allomone and attract the natural enemy, O. laevigatus , at the tertiary trophic level as kairomone. This study analyzed the behaviors of F. occidentalis and O. laevigatus in response to thrips-infested hot peppers. Differential behaviors between the herbivore and the predator were elucidated by analyzing VOCs emitted by the host plant. The influences of salicylic acid (SA) and jasmonic acid (JA) on inducing insect behaviors and VOC production were evaluated based on plant defense signaling pathways. The functional VOCs were tested by silencing gene expressions related to VOC biosynthesis through virus-induced gene silencing (VIGS) followed by behavioral assays. Finally, the study established a correlation between the levels of VOC-biosynthetic gene expression and hot pepper resistance to F. occidentalis . Results Damaged hot pepper repels herbivore but attracts predator through JA signaling. When the hot peppers were infested by F. occidentalis , the peppers became significantly less preferred than uninfested control plants (Fig. 1A). Conversely, the damaged plants were favored by their predator, O. laevigatus . These contrasting behaviors were time-dependent (Fig. 1B). Repellency against F. occidentalis was rapidly induced, reaching maximum levels 6 h post-infestation (pi). Simultaneously, attraction towards O. laevigatus was also induced. The defense system of hot peppers utilizing JA signaling was implicated to explain the behavioral changes of the thrips and their predators, as JA plays a critical role in plant defense against herbivore infestation 18 . Its impact on tri-trophic interactions was assessed in hot peppers by examining effects from JA or SA signaling pathways (Fig. 1C). Methyl JA (Me-JA) treatment on undamaged hot peppers repelled thrips while attracting the predator. However, SA treatment did not trigger these behaviors. The Me-JA treatment prompted behavioral modifications in the thrips and its predator within a day, and the induction was dose-dependent (Fig. 1D). RNA-Seq analysis confirms that JA signaling is induced in hot peppers infested by F. occidentalis . To investigate the response of hot peppers to infestation by F. occidentalis , damaged and undamaged plants were compared in terms of their transcriptomes (Fig. 2). With the exception of one replication (‘R1’) in the ‘Damage’ treatment, more than 6.1 Gb of nucleotides were sequenced in each replication, and over 23,735 genes were predicted (Fig. 2A). RNA-Seq data from R1 of the ‘Damage’ treatment were excluded from further transcriptome analysis due to inadequate gene prediction. Using the predicted genes, their expression profiles were analyzed, revealing two distinct clusters between the damaged and undamaged hot peppers (Fig. 2B). Most (96.0%) of the genes were expressed in both treatments (Fig. 2C). Among these commonly expressed genes, 4,072 were differentially expressed (= DEG) by more than two-fold following thrips infestation (Fig. 2D). The up-regulated DEGs (= 2,584) due to thrips damage were further categorized by the number of fold changes, with 155 genes demonstrating more than 20-fold changes in their expression levels after thrips infestation (Fig. 2E). The highly expressed genes were functionally categorized into different stress responses including JA signaling and terpenoids (Fig. 2F). DEGs (= 63 genes) that exhibited over 40-fold changes are listed in Table S1. Notably, JA biosynthesis and signaling pathways are illustrated in Fig. 2G, where genes identified in the RNA-Seq analysis contributed to the synthetic and signaling components. Certain JA-associated genes were up-regulated in their expression levels following thrips infestation (Fig. 2H). PGPRs induce the antixenosis of hot peppers through JA signaling. To demonstrate the role of JA signaling in modulating insect behaviors, PGPR applications were administered to hot peppers because several PGPRs are known to enhance JA signaling in hot peppers, thereby inducing the production of VOCs to deter herbivores 19 . Of the five PGPRs tested, two (‘G-2273’ and ‘AK-0’) effectively repelled F. occidentalis from the treated hot peppers (Fig. 3A). This repellency occurred 12 h post-treatment with the PGPRs (Fig. 3B). The two PGPR treatments significantly ( P < 0.05) attracted the predator, O. laevigatus (Fig. 3C). These PGPR treatments up-regulated genes associated with JA biosynthesis such as 13-LOX , AOS , and AOC (Fig. 3D). The expression profiles of hot peppers treated with the effective PGPRs (‘AK-0’ and ‘G-2273’) differed from those treated with the ineffective PGPR (‘G-2311’) and the control (‘TSB’) according to clustering analysis. VOCs emitted by the hot peppers infested by F. occidentalis . The VOCs of the hot peppers were collected using the headspace-solid phase microextraction (HS-SPME) technique (Fig. S1A). A total of 169 VOCs were identified by GC-MS from both damaged and undamaged plants, with differential detection across the two treatments (Fig. S1B). Over 34% of the VOCs were commonly emitted across both treatments (Fig. 4A). Forty-four VOCs uniquely emitted from the damaged plants included green leaf volatiles (GLVs), terpenoids, and others (Fig. 4B). Among the GLVs, Z -3-methyl hexenoate (MeH) was unique to the VOCs of the damaged plants and was chosen to test its biological activity (Fig. 4C). MeH effectively repelled F. occidentalis and attracted O. laevigatus . The GLV biosynthetic pathway is depicted in Fig. 4D, where genes identified in the RNA-Seq analysis were discovered in the pathway components. Most of the genes associated with GLV biosynthesis were upregulated in their expression levels following thrips infestation (Fig. 4E). Notably, hydroperoxide lyase ( HPL ), which catalyzes the cleavage of C18 fatty acids to produce the six-carbon GLV skeleton, was significantly upregulated following thrips infestation or treatments with effective PGPRs (Fig. 4F). Among terpenoids, linalool was specific to VOCs of the damaged plants and used to assess its biological activity against the thrips and their predator (Fig. 5A). Linalool also repelled F. occidentalis and attracted O. laevigatus . The terpenoid biosynthetic pathway is depicted in Fig. 5B, where genes identified in the RNA-Seq analysis were located in the pathway components. Most genes associated with terpenoid biosynthesis were upregulated in their expression levels following thrips infestation (Fig. 5C). Notably, linalool synthetase ( LS ) was significantly upregulated following thrips infestation or treatments with effective PGPRs (Fig. 5D). Suppression of HPL or LS expression disrupts the tri-trophic interactions involving hot pepper- F. occidentalis - O. laevigatus . To specifically suppress HPL or LS expression in the hot peppers, the virus-induced gene silencing (VIGS) technique was employed (Fig. 6). Co-injection of two expression vectors (pTRV1 and pTRV2) successfully silenced a specific target gene, phytoene desaturase ( PDS ), causing the treated hot peppers to lose their green color in contrast to the control (Fig. 6A). When the recombinant pTRV2 containing the HPL or LS construct was administered, the VIGS treatments significantly suppressed the target genes even following thrips infestation. Conversely, the controls exhibited significant induction of the target genes following thrips infestation (Fig. 6B). Suppression of the target genes resulted in a marked reduction of MeH and linalool emitted by the hot peppers post-thrips infestation (Fig. 6C). In wild-type plants, thrips-infested hot peppers ('Damage') significantly enhanced repellency against thrips and attraction of predators (Fig. 6D) compared to undamaged plants. Under the VIGS treatment specific to HPL expression, the thrips infestation did not repel the thrips as effectively as in the control without infestation (‘Undamage in dsCON vs Damage in dsRNA’). Instead, the thrips showed a preference for the dsRNA-treated hot peppers (‘Undamage in dsCON vs Undamage in dsRNA’ or ‘Damage in dsCON vs Damage in dsRNA’) over those treated with dsCON. However, adding MeH restored the repellency by inhibiting the thrips' orientation towards the dsRNA treatment (‘Damage in dsCON vs Damage in dsRNA’). By contrast, the attraction of predators was significantly suppressed in HPL -silenced plants, whether damaged or undamaged. The HPL -silenced plants were unsuccessful in attracting predators compared to the control without infestation (‘Undamage in dsCON vs Damage in dsRNA’). Instead, predators showed a preference for the control plant over the HPL -silenced plants (‘Undamage in dsCON vs Undamage in dsRNA’ or ‘Damage in dsCON vs Damage in dsRNA’) compared to dsRNA-treated hot peppers. However, the introduction of MeH restored the attractiveness by inhibiting the orientation of the predators towards the control plants (‘Damage in dsCON vs Damage in dsRNA’). The VIGS treatments targeting LS expression in the hot peppers also resulted in similar behavioral changes in both thrips and predators, as observed in the VIGS treatments targeting HPL expression. Suppression of LS expression reduced both thrips repellency and attractiveness towards O. laevigatus in the hot peppers infested by F. occidentalis . Linalool treatment restored the normal responses of thrips and their predators to the LS -silenced plants. Differential expression levels of HPL and LS influence the antixenosis of various hot pepper varieties. Ten distinct hot pepper varieties were evaluated for their repellency against F. occidentalis (Fig. 7A). In a choice test using a tunnel, two varieties (‘KM’ and ‘TD’) were found to be less preferred by the thrips. Additionally, two varieties (‘KM’ and ‘MS’) were more preferred by O. laevigatus when infested by the thrips (Fig. 7B). VOCs were collected from these 10 varieties and categorized into 499 different compounds, including GLVs, terpenoids, and others (Table S2). These VOCs were analyzed across the 10 hot pepper varieties, revealing two clusters with the three varieties (‘TD’, ‘MS’, and ‘KM’) not being grouped (Fig. 7C). Notably, MeH was only detected in the three resistant varieties (‘TD’, ‘MS’, and ‘KM’), and linalool was exclusively found in two resistant varieties (‘TD’ and ‘KM’) (Fig. 7D). All ten hot pepper varieties up-regulated the gene expression levels of HPL and LS following thrips infestation, although the induction levels varied among the varieties (Fig. 7E). The expression levels of HPL across the 10 hot pepper varieties strongly correlated with their repellent activities against F. occidentalis (Fig. 7F). The expression levels of LS also correlated with their repellent activities against F. occidentalis . Discussion This study analyzed a tri-trophic interaction among hot peppers, F. occidentalis , and O. laevigatus through VOCs produced by hot peppers in response to thrips infestation. To elucidate the role of VOCs in biological activity, the behaviors of the herbivore and its predator toward the damaged plants were investigated. VOC production was validated by comparing the transcriptomes of undamaged and damaged host plants, monitoring gene expressions related to the biosynthetic pathways of the candidate VOCs. Ultimately, the functional VOCs were analyzed for the expression levels of the biosynthetic genes across different hot pepper varieties, highlighting how susceptibility to thrips varies. This approach demonstrated the pivotal roles of GLV and terpenes in the chemical communication within the tri-trophic interaction. Thrips infestation prompted hot peppers to employ antixenosis, enhancing non-preference behavior over the control plants, as a defense against herbivore attack. Intriguingly, the damaged plants attracted the predator, O. laevigatus . Augmentative biological control programs have employed the predator bug, O. laevigatus , particularly in greenhouses cultivating vegetable crops including hot peppers, to combat thrips pests 20 . Natural enemies are generally affected both directly and indirectly in tri-trophic interactions with host plants 21 . Direct interactions between natural enemies and host plants involve physical characteristics, such as leaf trichomes, waxy leaves, and leaf thickness 22-24 , and biochemical properties including exudates, nectars, and odors 25 . Indirect effects involve the herbivores' biological traits, such as developmental time, size, weight, fecundity, survival, longevity, establishment, and digestibility 21,26,27 . These biological characteristics of herbivores are influenced by the host plants, illustrating functional tri-trophic interactions. The damaged hot peppers' modulation of behaviors that repel F. occidentalis and attract O. laevigatus suggested a chemical communication across tri-trophic levels: host plant (hot pepper), herbivore ( F. occidentalis ), and predator ( O. laevigatus ). The targeted communication calling specific natural enemies by the infested host plants has been demonstrated in various plants, including corn and cabbage, depending on the herbivores present 28,29 . In cabbages, the damaged host plants released different VOCs depending on the herbivores' species and infestation density 30 . The study reported an up-regulation of VOCs in damaged cabbages, including GLVs (( Z )-3-hexenol, n-heptanal, and ( Z )-3-hexenyl acetate), terpenes (α-pinene, sabinene, myrcene, limonene, camphor, and ( E )-4,8-dimethyl-1,3,7-nonatriene), and another hydrocarbon (α-copaene). Our current study identified the VOCs, including GLVs and terpenes, released from hot peppers infested by F. occidentalis upon capturing the volatile compounds using HS-SPME. The antixenosis induced by the functional VOCs is likely mediated by the JA signaling pathway in the hot pepper infested by F. occidentalis . In addition to the MeJA effect on the induction of antixenosis in hot peppers, DEG analysis between control and infested hot peppers showed that genes associated with JA biosynthesis and signaling components were significantly upregulated in response to thrips infestation, along with the increased expression of GLV and terpene biosynthetic genes. Notably, the expression levels of 13-LOX, which oxygenates α-linolenic acid in a regio- and stereo-selective manner to produce 13( S )-hydroperoxylinolenic acid in JA synthesis, increased more than 200-fold after thrips infestation. Indeed, disruption of 13-LOX expression suppressed the JA pathway in peppers, leading to enhanced susceptibility to F. occidentalis 31 . Moreover, two PGPRs (‘AK-0’ and ‘G-2273’) among five bacterial isolates effectively induced antixenosis in hot peppers against F. occidentalis and up-regulated the expression of 13-LOX and other JA-synthetic genes. It is well-known that PGPRs assist plants in defending against various pathogens and herbivores. For example, in cotton insect resistance, gossypol is considered a primary antibiosis factor against insect pests, and its production is stimulated by treatment with a PGPR classified into Bacillus sp., enhancing JA biosynthesis 32 . Insect resistance can be induced by suppressing a plant factor negatively associated with insect resistance. The increased JA synthesis by PGPRs suppressed a specific microRNA, mir814 , which antagonizes the insect resistance of Arabidopsis thaliana following treatment with Bacillus amyloliquefasciens FZB42, leading to induced systemic resistance 33 . In our current study, the selected PGPRs activate JA synthesis, potentially causing antixenosis by producing VOCs that repel F. occidentalis and attract O. laevigatus . In our study, Z -3-methyl hexenoate (MeH), a GLV produced by infested hot peppers, was particularly effective in repelling thrips and attracting predators. GLVs are widely produced by green plants and their production can be induced by abiotic stimuli 34 , herbivores 35 , or pathogens 36 via the HPL branch of the oxylipin pathway, often within seconds of stress exposure. While GLV emission is typically short-lived, it can persist for days during herbivory or repeated wounding 37 . Furthermore, GLVs serve as semiochemicals that guide insects to locate food or prey 38 . MeH is synthesized from hexenol through JA signaling in a medicinal herb 39 , and this precursor is derived from C18 unsaturated fatty acids such as linoleic acid and linolenic acid, following catalytic cleavage by hydroperoxide lyase (HPL). Our VIGS analysis, which aimed to suppress HPL expression, corroborated the biosynthetic pathway and physiological role of MeH. Additionally, MeH was detected solely in hot pepper varieties that are resistant to F. occidentalis . The expression levels of HPL were positively correlated with the degree of non-preference exhibited by F. occidentalis towards the hot peppers. This functional GLV may also prime hot pepper plants against further attacks from herbivores or pathogens. GLVs induce defense priming in plants, enabling them to respond faster or more robustly to insect herbivores and pathogens. For instance, a tomato ( Solanum lycopersicum ) infested by the whitefly, Bemisia tabaci , releases Z -3-hexenol through JA signaling, which increases the expression of defense genes and flavonoid levels 40 . This indicates that the production and release of other GLVs, as well as MeH, may be stimulated by JA signaling, contributing to antixenosis. Linalool, a terpene produced by hot peppers infested by F. occidentalis , is catalyzed by LS, a terpene synthase. The suppression of gene expression by gene-specific VIGS treatment prevented the upregulation of linalool titers in the hot peppers following thrips damage. Indirectly, linalool may also prime the host plant to produce ROS, as demonstrated in Arabidopsis infested by P. xylostella , causing oxidative damage to the herbivore 41 . The expression of terpene synthase genes is regulated by the MYC2 transcription factor under the JA immune signaling pathway in hot peppers 42 . This suggests that JA signaling initiates the synthesis of various terpenes, including linalool. Various hot pepper varieties differ in their susceptibility to F. occidentalis infestation as well as in their ability to produce GLVs and terpenes. Even without damage, hot peppers emit a range of VOCs. For instance, the Habanero pepper, known for its intense pungency and fruity aroma, releases 66 volatile compounds, including multiple short-chain hydrocarbons such as 6-methyl-( E )-4-heptenyl 3-methylbutanoate, 6-methyl-( E )-4-heptenyl 2-methylpropanoate, 6-methyl-( E )-4-heptenyl 2-methylbutanoate, 2-methylbutanoate, and 6-methyl-( E )-4-heptenol 43 . However, it is primarily the various GLVs and terpenes that characterize the VOCs of damaged hot peppers. Our current data indicate that the JA signaling pathway, which is triggered by thrips infestation, upregulates biosynthetic gene expressions such as HPL and LS , and these are positively correlated with antixenosis resistance to F. occidentalis . In other words, hot pepper varieties expressing high levels of HPL and LS genes tend to be resistant to F. occidentalis . This positive correlation provides crucial information for the breeding of resistant hot pepper varieties against F. occidentalis , a significant insect pest that transmits TSWV to high-value crops 3 . Besides VOCs, established tri-trophic interactions include the relationship among hot peppers, F. occidentalis , and O. laevigatus , which is mediated by the aggregation pheromone released by F. occidentalis . This pheromone facilitates chemical communication among the thrips, but functions as a kairomone for both adult and nymph stages of O. laevigatus , particularly in its active components mixture of ( R )-lavandulyl acetate and neryl ( S )-2-methylbutanoate 44 . Conversely, tri-trophic interactions may be compromised by viral infections such as TSWV that reduce predator access to the plants, thus promoting the virus's horizontal transmission. F. occidentalis larvae thrive better on TSWV-infected host plants than on non-infected hosts and evade predation by predatory mites 45 . This complexity in tri-trophic interactions have led to the identification of chemical signals originating from the primary trophic level, which are interpreted differently by the herbivore and its predator. Additionally, this study highlights resistant genetic markers like HPL and LS that deter thrips from hot peppers, beneficial for future breeding programs. Methods Insect rearing. Adults of F. occidentalis were collected from a hot pepper field in Andong, Korea, and reared on sprouted bean seed kernels under specific laboratory conditions: a constant temperature of 25 ± 1°C, a photoperiod of 16:8 h (L:D), and a relative humidity of 60 ± 5%. Under these conditions, the thrips underwent two instars (L1 and L2), prepupa, and pupa before maturing into adults. The predator, O. laevigatus , colony, donated by Oal, Inc. (Seoul, Korea), was cultivated under similar laboratory conditions. These predators were maintained on hot pepper plants with larvae of F. occidentalis providedas food. Plant growth-promoting rhizobacteria (PGPR). Five different PGPR bacteria of Paenibacillus polymyxa GYUN-2273 (NCBI accession number: OR883773), Bacillus subtilis GYUN-2311 (KACC accession number: 92471), B. velezensis AK-0 (KACC accession number: 92099), B. tequilensis G-300 (KACC accession number: 81153), and Brevibacillus halotolerans B-4359 (NCBI GenBank number: CP139435) were isolated from the rhizosphere of agricultural regions in Andong or donated by the Nakdonggang National Institute of Biological Resources, Sangju, Korea. These bacteria were inoculated into 100 mL of tryptic soy broth (TSB: Difco, Sparks, MD, USA) and cultured at 28°C with an agitation of 180 rpm. After 48 h culture, 10 mL of the broth was evenly applied to the soil in a pot (10 cm diameter and 10 cm height) containing young hot pepper plants at the six-leaf stage. At various times, the effects of the PGPR on the insect resistance of the hot peppers were evaluated. Cultivating different hot pepper varieties. All hot pepper varieties, including C. annuum , seedlings, were cultivated in a nursery under conditions of 25 ± 1°C, a 12:12 h (L:D) photoperiod, and approximately 60% RH. Biological activities were evaluated using the Gguari variety (Hanlim, Seoul, Korea) as a susceptible reference. The other test hot pepper varieties included PR Daekan (Jenong S&T, Jeju, Korea), Dabokhangajeong (Sakata Korea, Seoul, Korea), PR 911 (Hana Seed, Anseong, Korea), Kaltanmiso (Seedland, Cheongju, Korea), Meotjinsanai (Syngenta Korea, Seoul, Korea), Subicho (Yeongyang, Korea), Meaunkaltan (Pepper & Breeding Institute, Gimje, Korea), Callazzang (Nongwoobio, Suwon, Korea), Titan Daebak (Farmhannong, Seoul, Korea), and Kaltanyeonseung (Nongwoobio, Suwon, Korea). The assays were conducted using 6-week-old plants at the six-leaf stage to ensure uniformity and developmental consistency. Chemicals. Methyl hexenoate (99%), linalool (97%), salicylic acid (99%), methyl jasmonate (95%), and cyclopentanone (99%) were purchased from Sigma Aldrich Korea (Seoul, Korea). These compounds were subsequently dissolved in dimethyl sulfoxide (DMSO) to prepare the test solutions. DNA Taq polymerase, RT Premix, and Power SYBR Green PCR Master Mix were sourced from GeneALL Biotechnology Company (Seoul, Korea), Intron Biotechnology (Seoul, Korea), and Life Technologies (Carlsbad, CA, USA), respectively. Furthermore, the restriction enzymes (BamHI and XbaI) were sourced from Takara (Kyoto, Japan). Insect damage treatment to hot peppers with F. occidentalis. The young hot pepper plants at the six-leaf stage were infested with 50 L2 larvae of F. occidentalis per plant for 8 h under controlled culturing conditions. Afterwards, the insects were removed, and the plants were immediately used for further experiments. Behavioral bioassay using Y-tube olfactometer. To assess the behavioral responses to chemicals, a Y-tube olfactometer was utilized as described by Khan et al 46 . In each trial, 20 adults of F. occidentalis or O. laevigatus were placed in the Y-tube for observation. Positive responses included insects trapped on a sticky card (2 cm × 2 cm) or observed feeding on pepper plants. Those that did not cross the fork were recorded as no response (‘NR’). All trials were conducted in darkness at 25 ± 1°C and 65% RH, each lasting 6 h and replicated three times. Behavioral bioassay using a tunnel assay. The tunnel assay was employed to evaluate the behavioral choice between two hot pepper varieties for F. occidentalis and O. laevigatus 47 . All assessments were performed in the dark at 25 ± 1°C and 65% RH, and each lasted 6 h. In each test, 50 adults of F. occidentalis or O. laevigatus were introduced into the tunnel's midpoint through an entrance hole. Positive responses were those where insects were trapped on a sticky card (24 cm × 14 cm) or seen feeding on the plants. After each session, the tunnel was cleaned thoroughly, with at least one hour between trials. Each treatment was replicated three times. RNA extraction, cDNA synthesis, RT-PCR, and RT-qPCR. Plant leaves were collected and flash-frozen in liquid nitrogen before storage at -80°C to prevent cross-contamination and RNA degradation. Each sample involved grinding 0.1 g of plant tissues thoroughly with a cold (4 o C) mortar and pestle, which had been preheated at 180°C for 20 min to inactivate RNases. RNA was extracted using Trizol reagent according to the manufacturer's instructions. After extraction, RNA was resuspended in nuclease-free water, and its concentration was measured using a spectrophotometer (NanoDrop, Thermo Scientific, Wilmington, DE, USA). RNA was then used for cDNA synthesis with an RT Premix containing oligo-dT primer, following the manufacturer’s instructions. RT-PCR involved DNA Taq polymerase and proceeded under these conditions: initial denaturation at 94°C for 5 min, 35 cycles of denaturation at 94°C for 1 min, annealing at various temperatures (Table S3) for 1 min, extension at 72°C for 1 min, and a final extension at 72°C for 10 min using gene-specific primers. Each 20 µL RT-PCR reaction contained the cDNA template, dNTPs (each at 2 mM), 10 pmol of each forward and reverse primer, and 2 units/µL of Taq polymerase. Gene expression levels were assessed with a real-time PCR machine (Step One Plus Real-Time PCR System, Applied Biosystems, Singapore) using Power SYBR Green PCR Master Mix as per Bustin et al 48 . The expression of β-actin served as a reference to normalize target gene expression levels under various treatments. Melting curve analysis confirmed the specificity of each PCR product. Quantitative analysis used the comparative CT (2 -∆∆CT ) method 49 , with each experiment replicated three times with independent cohorts or sample preparations. RNA-Seq analysis of hot peppers after thrips damage. Hot peppers infested by F. occidentalis were used to extract RNA as previously described. Each treatment was replicated three times. The concentration and purity of the RNA samples were assessed using a 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) and the samples were sent to Macrogen (Seoul, Korea) for RNA library construction and next-generation sequencing using the Illumina NovaSeq 6000 system (Illumina, San Diego, CA, USA). The sequenced FASTQ data were uploaded to the NCBI database under the project number PRJNA104655. Data analysis was conducted using the bioinformatics software CLC Genomic Workbench (version 22.0.1, Qiagen, Hilden, Germany). The raw sequenced data were trimmed to remove low-quality sequences and mapped to the C. annuum genome. RPKM (reads per kilobase per million mapped reads) values were used to calculate relative mRNA expression levels. Gene IDs were retrieved from the gene symbols with LOC numbers in the mapped reads, using the general feature format (GFF) file of the C. annuum genome. Differentially expressed genes (DEGs) were identified based on a minimum two-fold change and a p -value of less than 0.05 by comparing RPKM values. Gene ontology (GO) analysis was conducted using Blast2GO 6.0 (BioBam, Valencia, Spain) to classify biological, cellular, and molecular functional categories of selected DEGs. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed to determine the functional categories of genes as defined in the KEGG database, using the KEGG mapper (https://www.genome.jp/kegg/mapper/search.html) and the BioDBnet (Biological Database Network, https://biodbnetabcc.ncifcrf.gov/db/db2dbRes.php) ID conversion tool. Transcript analysis of the damaged hot peppers in the JA pathway. Genes involved in the JA biosynthetic pathway were selected based on the model proposed by Wang et al. 50 and our current transcriptome data of hot peppers to compare expression levels across treatments. The analyzed genes include phospholipase A 1 ( PLA1 ), 13-lipoxygenase ( 13-LOX ), allene oxide synthase ( AOS ), allene oxide cyclase ( AOC ), JA-amino acid synthetase ( JASSY ), L-3-ketoacyl CoA thiolase ( KAT ), multifunctional protein ( MCX ), acyl-CoA oxidase ( ACX ), oxophytodienoic acid reductase 3 ( OPR3 ), coronatine insensitive 1 ( COI1 ), jasmonate zim domain ( JAZ ), and MYC2 . Genes involved in the terpene biosynthetic pathway were identified using the pathway outlined by Mani et al. 51 from our current transcriptome of the hot peppers: acetyl-CoA acetyltransferase ( AACT , XP_016580453.1), hydroxymethylglutaryl-CoA synthase ( HMGS , XP_016551730.1), 3-hydroxy-3-methylglutaryl-coenzyme A reductase ( HMGR , XP_016557834.2), mevalonate kinase ( MK , XP_016538569.2), phosphomevalonate kinase ( PMK , XP_016574993.2), diphosphomevalonate decarboxylase ( MPD , XP_016551358.2), farnesyl pyrophosphate synthase ( FPS , NP_001311799.1), sesquiterpene synthase ( STPS , XP_016566183.2), (-)-germacrene D synthase ( GDS , XP_016564626.1), 1-deoxy-D-xylulose-5-phosphate synthase ( DXS , XP_047256568.1), 1-deoxy-D-xylulose-5-phosphate reductase ( DXR , XP_016563443.1), 2-methyl-D-erythritol 4-phosphate cytidylyltransferase ( CMS , XP_016538918.1), 2-methyl-D-erythritol 2,4-cyclodiphosphate synthase ( MCS , XP_016566381.1), 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase ( HDS , XP_016547325.1), 4-hydroxy-3-methylbut-2-en-1-yl diphosphate reductase ( HDR , XP_016539712.1), geranylgeranyl diphosphate synthase ( GPPS , XP_016556802.1), and linalool synthase ( LS , XP_016539311.2). Genes associated with the GLV biosynthetic pathway were selected via the method proposed by Scala et al. 38 from our current transcriptome of the hot peppers: lipoxygenase ( LOX , NP_001311748.1), hydroperoxide lyase ( HPL , NP_001311810.1), alcohol dehydrogenase ( ADH , XP_016568293.2), aldo-keto reductase ( AKR , XP_016543252.1), aldehyde reductase ( ADR , XP_016578140.2), and alcohol acetyltransferase ( AAT , XP_016541551.2). These genes were used to compare gene expression levels in the terpene pathway between damaged and undamaged hot pepper plants. Virus-induced gene silencing (VIGS). Virus-induced gene silencing was performed following the methods of Zhang & Liu 52 with slight modifications.Open reading frames (ORFs) of HPL (GenBank accession number: U51674.1) and LS (GenBank accession number: XM_047394954.1) genes were cloned into the pCR2.1-TOPO vector using the TOPO TA Cloning Kit (Invitrogen) with ORF primers (Table S3). The confirmed clones were cultured in LB medium containing ampicillin (100 µg/mL), and their plasmids were extracted using a spin column (Plasmid SV, GeneAll). Extracted pCR2.1-HPL and pCR2.1- LS plasmids were sequenced at Macrogen. Upon confirmation, the recombinant vectors were digested with BamHI and XbaI, and the resulting inserts were ligated to construct the recombinant pTRV2- HPL and pTRV2- LS expression vectors. These vectors were transformed into Escherichia coli (DH5α) competent cells and cultured in LB containing kanamycin (100 mg/mL). Finally, 10 μL of the validated recombinant plasmid of pTRV2- HPL and pTRV2- LS were transferred into Agrobacterium tumefaciens strain GV3101 via electroporation and cultured on LB with kanamycin (100 µg/mL) and rifampicin (50 µg/mL) at 28°C for 72 h. Subsequent clones were resuspended in infiltration buffer (10 mM MES, 10 mM MgCl 2 , and 200 μM acetosyringone) to achieve a bacterial concentration between 1.3 and 1.5 at OD 600 . For injection, equal volumes of bacterial suspension containing pTRV1 and either pTRV2- HPL or pTRV2- LS were mixed and injected into the abaxial side of cotyledons using a needless syringe. The inoculated seedlings were then cultured under standard plant culturing conditions. Analysis of VOCs using headspace-solid phase microextraction (HS-SPME). The described test leaves were harvested and pulverized using a mortar and pestle under liquid nitrogen. Two grams of the pulverized leaf samples were placed into a 20 mL headspace vial containing an internal standard (cyclohexanone, 8,000 ppm), which was subsequently stored at -80°C until analysis. The samples were equilibrated at 40°C for 10 min in a thermostatic autosampler tray, followed by a 40-min exposure to the divinylbenzene /carboxyl/polydimethylsiloxane (DVB/CAR/PDMS) fiber (50/30 μm, Supelco, Bellefonte, PA, USA). The fiber was then thermally desorbed in the GC injector port at 250°C for 10 min (spitless). The GC–MS system (Agilent 8890A/5977B MSD Series, Agilent Technologies, Santa Clara, CA, USA) was operated in the electron impact (EI, ionization energy: 70 eV) mode with a scan range of m/z 35–550. VOCs were separated on a DB-WAX capillary column (30 m × 0.25 mm i.d., 0.25 μm film thickness; Agilent). The temperature program for the GC oven started at 35°C for 2 min, increased from 35–45°C at a rate of 2°C/min, then from 45–130°C at 5°C/min, and finally held at 225°C at a rate of 10°C/min for 5 min. Helium served as the carrier gas at a constant flow rate of 1.5 mL/min. The transfer line temperature was maintained at 250°C. VOC identification was conducted by matching mass spectra against the standard National Institute of Standards and Technology (NIST library version 20). Quantification of methyl hexanoate and linalool relied on calibration curves of pure standards. Each experiment was replicated three times with individual sample preparation. Classification of VOCs emitted from hot peppers and pattern analysis. The VOCs identified via GC-MS were systematically classified into distinct groups such as GLVs and terpenes, according to their chemical structures. GLVs, exemplified by compounds like hexanal and its derivatives, exhibit straight-chain and branched-chain hydrocarbons with a basic C6 skeleton 53 . In contrast, terpenes consisted of isoprene unit compounds, which included monoterpenes (C10) and sesquiterpenes (C15) 54 in this study. Statistical analysis. All tests involved three independent biological replicates. Means were compared using the least significant difference (LSD) test in a one-way analysis of variance (ANOVA) conducted with PROC GLM of the SAS program 55 . Significant differences were established at a Type I error of 0.05. 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06:50:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6437640/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6437640/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82000293,"identity":"395abcb3-ae88-4c75-8286-20eaf3cd97ab","added_by":"auto","created_at":"2025-05-05 19:25:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":273844,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModifications in host preference behaviors of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eF. occidentalis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e (‘Fo’) and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eO. laevigatus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e (‘Ol’) towards hot peppers following thrips damage.\u003c/strong\u003e\u003cem\u003e \u003c/em\u003e(A) Choice test comparison between damaged and undamaged hot peppersusing a tunnel assay. (B) Contrasting responses of prey (Fo) and predator (Ol) to damaged hot peppers over time, analyzed using the tunnel assay. (C) Effects of methyl jasmonic acid (‘Me-JA’, 10 mM) and salicylic acid (‘SA’, 10 mM) on these behaviors, evaluated with a Y-tube choice test. (D) Impact of Me-JA treatment on hot peppers regarding these behaviors, exploring different times and concentrations using a Y-tube test. Each Y-tube testtrial involved 20 adults of either \u003cem\u003eF. occidentalis\u003c/em\u003e or \u003cem\u003eO.\u003c/em\u003e \u003cem\u003elaevigatus\u003c/em\u003e. Each trial in the tunnel assay used 50 adults of \u003cem\u003eF. occidentalis\u003c/em\u003e or \u003cem\u003eO.\u003c/em\u003e \u003cem\u003elaevigatus\u003c/em\u003e. Every experiment was replicated three times. An asterisk (*) denotes a significant difference compared to the control at Type I error = 0.05 (t-test), while 'ns' indicates no significant difference. 'NR' stands for no response in the choice tests. Different letters above standard error bars signify significant differences among means at Type I error = 0.05 (LSD test).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6437640/v1/7b6c591d8a4fb9e7c7908012.png"},{"id":82000608,"identity":"b609c1d5-fcf5-4371-8ad1-146a714a4661","added_by":"auto","created_at":"2025-05-05 19:33:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":537349,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUp-regulation of jasmonic acid ('JA') signaling in hot peppers following infestation by \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eF. occidentalis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e (A) RNA-Seq summary for hot peppers that were either damaged or undamaged. Each plant, at the six-leaf stage, was exposed to 50 L2 larvae for 8 h. Three replicates ('R1-R3') were performed for each of two conditions (‘Undamaged’ and ‘Damaged’). (B) A heatmap analysis showing the differential transcriptomes between the two conditions across replicates. (C) Comparison of 22,801 gene contigs distributed between the two conditions in hot peppers. (D) Identification of differentially expressed genes (DEGs) in hot peppers in response to thrips infestation. DEGs were selected based on RPKM fold changes ≥ 2 among common contigs. (E) Distribution of up-regulated DEGs (= 2,584) classified by fold-changes. (F) Functional categorization of highly upregulated DEGs (= 155), focusing on plant defense signaling pathways using the KEGG mapper (https://www.genome.jp/kegg/mapper/search.html) and bioDBnet (Biological Database Network, https://biodbnetabcc.ncifcrf.gov/db/db2dbRes.php) ID conversion tool. (G) Genes involved in JA biosynthesis and signaling pathway\u003csup\u003e50\u003c/sup\u003e after thrips damage, with colored genes indicating the up-regulated DEGs in this study’s RNA-Seq. (H) A heatmap illustrating the enhanced JA signal components in hot peppers following thrips infestation. Asterisks (*) denote a significant difference at a Type I error rate of 0.05 (t-test) between the two conditions. 'ns' indicates no statistical difference. The right-hand bar shows expression levels, indicated by varying color intensities.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6437640/v1/7c987173ea6ddc2df598f3f6.png"},{"id":82000297,"identity":"fc6a05be-758b-4111-b1d3-9b23a6cc9164","added_by":"auto","created_at":"2025-05-05 19:25:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":205053,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlteration in host preference behaviors of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eF. occidentalis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e (‘Fo’) and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eO. laevigatus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e to hot peppers in response to treatments with certain plant growth-promoting rhizobacteria (PGPR) mediated by jasmonic acid (JA) signaling. \u003c/strong\u003e(A) Choice test involving thrips' preference for hot peppers, comparing the bacterial culture medium (‘TSB’) and five PGPR treatments. Each PGPR was applied at the six-leaf stage of young hot peppers using 10 mL of 10\u003csup\u003e8\u003c/sup\u003e CFU/mL. The behavior assay was conducted 8 h later using a tunnel assay. (B) Time-dependency of the repellent behavior of the thrips exposed to two effective PGPRs: G-2273 and AK-0. (C) Choice test of \u003cem\u003eO. laevigatus\u003c/em\u003e regarding hot peppers, comparing TSB and the two effective PGPR treatments. Each PGPR was applied to young hot peppers (six leaf stage) at a volume of 10 mL of 10\u003csup\u003e8\u003c/sup\u003e CFU/mL. The behavior assay was conducted 8 h later using a tunnel assay. In each tunnel assay task, 50 adults of \u003cem\u003eF. occidentalis\u003c/em\u003e or \u003cem\u003eO.\u003c/em\u003e \u003cem\u003elaevigatus\u003c/em\u003e were used. All experiments were replicated three times. (D) Heatmaps displaying the expression analyses of three JA signal component genes in response to the three PGPR treatments. Internal \u003cem\u003eβ-actin\u003c/em\u003e gene expression levels were used as the normalization standard for the JA signal gene expression levels. Each treatment was replicated three times. Analysis of gene expression patterns by a phylogenetic tree was performed using the ClustVis online tool (https://biit.cs.us.ee/clustvis/). An asterisk (*) indicates a significant difference compared to the control at Type I error = 0.05 (t-test), while 'ns' denotes no significant difference. 'NR' indicates no response in the choice tests. Different letters above standard error bars denote significant differences among means at Type I error = 0.05 (LSD test).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6437640/v1/9069509934762c3117beddd5.png"},{"id":82000291,"identity":"0edfd5bb-4018-444f-be7e-658ceff9330c","added_by":"auto","created_at":"2025-05-05 19:25:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":330343,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEmission of green leaf volatiles (GLVs) fromhot peppers infested by \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eF. occidentalis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand how they influence the behavior of thrips and the predator, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eO. laevigatus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, towards the plants.\u003c/strong\u003e (A) Volatile organic compounds (VOCs = 168) were collected from both damaged and undamaged hot peppers by \u003cem\u003eF. occidentalis \u003c/em\u003eusing HS-SPME. (B) Categorization of the 44 VOCs specific to damaged hot peppers into three distinct chemical groups. (C) A Y-tube choice test involved thrips and predators choosing between hot peppers treated with the solvent (‘Hexane’) and methyl hexenoate ('MeH'). For each Y-tube test, 20 adult specimens of \u003cem\u003eF. occidentalis\u003c/em\u003e or \u003cem\u003eO.\u003c/em\u003e \u003cem\u003elaevigatus\u003c/em\u003e were utilized. All experiments were replicated three times. (D) The expressed genes categorized into the GLV biosynthesis pathway post-thrips damage, where the red-colored genes denote the up-regulated contigs in the RNA-Seq of this study. GLVs and their derivatives are indicated in blue. (E) Heatmap showing expression analyses of six genes involved in GLV biosynthesis in response to thrip infestation. qPCR was conducted8 h post-infestation. (F) Up-regulation of gene expression levels of hydroperoxide lyase (\u003cem\u003eHPL\u003c/em\u003e) followingthrips infestation or effective PGPR treatments, analyzed 48 h after the application. Internal \u003cem\u003eβ-actin\u003c/em\u003e gene expression levels were used for normalization. Each treatment was repeated three times. An asterisk(*) indicates a significant difference compared to the control at a Type I error of 0.05 (t-test), while 'ns' signifies no significant difference. 'NR' representsno response in the choice tests. Distinct letters above standard error bars denote significant differences among means at a Type I error of 0.05 (LSD test).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6437640/v1/e3d97af6712b425d827697b6.png"},{"id":82001043,"identity":"989d8671-86e7-47cf-a216-eeb767fd2a35","added_by":"auto","created_at":"2025-05-05 19:41:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":423730,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEmission of a terpene, linalool, from hot peppers infested by \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eF. occidentalis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, influences the behaviors of the thrips and the predator, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eO. laevigatus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, towards the plants.\u003c/strong\u003e (A) Y-tube choice test of the thrips and the predator on hot peppers, comparing the solvent (‘Hexane’) and linalool treatments. Each run of the Y-tube test utilized 20 adults of \u003cem\u003eF. occidentalis\u003c/em\u003e or \u003cem\u003eO.\u003c/em\u003e \u003cem\u003elaevigatus\u003c/em\u003e. All experiments were replicated three times. (B) Genes involved in the terpene biosynthesis pathway were categorized following thrips damage, illustrating two distinct pathways: a mevalonate (‘MVA’) pathway in the cytoplasm and the methyl erythritol phosphate (‘MEP’) pathway\u0026nbsp;in the plastids\u003csup\u003e51\u003c/sup\u003e. The red-colored genes indicate the up-regulated contigs in the RNA-Seq of this study. (C) Heatmap showing the expression analyses of MEP and MVA component genes in response to thrips infestation. qPCR was conducted at 8 h post-infestation. (D) Up-regulation of linalool synthase (\u003cem\u003eLS\u003c/em\u003e) gene expression following thrips infestation or effective PGPR treatments. In the PGPR treatments, gene expression was analyzed 48 h after application. Internal \u003cem\u003eβ-actin\u003c/em\u003e gene expression levels served as normalization controls. Each treatment was replicated three times. An asterisk (*) indicates a significant difference compared to the control at a Type I error rate of 0.05 (t-test), while 'ns' indicates no significant difference. 'NR' denotes no response in the choice tests. Different letters above standard error bars signify significant differences among means at a Type I error of 0.05 (LSD test).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6437640/v1/70f7b3f40041ad81211d3cc0.png"},{"id":82000300,"identity":"e755f3ea-186c-476b-a966-21d10606af3f","added_by":"auto","created_at":"2025-05-05 19:25:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":435825,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional assay of hydroperoxide lyase (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eHPL\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e) and linalool synthase (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLS\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e) gene expression in the resistance of hot peppers to \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eF. occidentalis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e (A) Suppression of \u003cem\u003eHPL\u003c/em\u003e or \u003cem\u003eLS\u003c/em\u003e expression was achieved using the virus-induced gene silencing (VIGS) technique by infecting with dual vectors of pTRV1 and pTRV2. For treatment, dsRNA constructs specific to \u003cem\u003eHPL\u003c/em\u003eor \u003cem\u003eLS\u003c/em\u003e were cloned into the pTRV2, termed dsHPL or dsLS, respectively. Tocontrol RNAi efficiency, pTRV2 recombinant with dsRNA specific to the \u003cem\u003ephytoene desaturase\u003c/em\u003e gene (‘dsPDS’) was injected into hot peppers. After 28 days, decoloration of leaves was observed compared with those injected with the control vector (‘dsCON’). (B) The effect of VIGS on \u003cem\u003eHPL\u003c/em\u003e and \u003cem\u003eLS\u003c/em\u003e expression levels, with or without thrips infestation, was examined. At 28 days post-VIGS treatment, the hot peppers were exposed to thrips. After 8 h, gene expression analysis was conducted with three replications per treatment, with internal \u003cem\u003eβ-actin\u003c/em\u003e gene levels used to normalize expression. (C) Changes in methyl hexanoate (‘MeH’) or linalool levels post-VIGS treatment were assessed. Following 28 days of VIGS treatment and subsequent thripsexposure after 8 h, HS-SPME was utilized to collect volatile organic compounds, with three replications per treatment. (D) A choice test assessing preference of thrips and the predator, \u003cem\u003eO. laevigatus\u003c/em\u003e, between wild type (‘WT’) and VIGS-treated hot peppers was conducted using a tunnel assay. During each assay, 50 adults of \u003cem\u003eF. occidentalis\u003c/em\u003e or \u003cem\u003eO.\u003c/em\u003e \u003cem\u003elaevigatus\u003c/em\u003e were used. Each choice test was repeated three times with independently prepared samples. An asterisk (*) signifies a significant difference compared to the control at a Type I error rate of 0.05 (t-test), 'ns' denotes no significant difference, and 'NR' indicates no response in the choice tests. Different letters above the standard error bars depict significant differences among means at a Type I error rate of 0.05 (LSD test).\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6437640/v1/91cf7d2d75086d5cb818ba37.png"},{"id":82000611,"identity":"af053e6a-6036-4823-ac1b-169d2c8aade6","added_by":"auto","created_at":"2025-05-05 19:33:15","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":441705,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of methyl-hexenoate (‘MeH’) and linalool production on insect resistance in hot peppers against thrips.\u003c/strong\u003e (A) Screening of ten hot pepper varieties in a tunnel choice test involving \u003cem\u003eF. occidentalis\u003c/em\u003e ('Fo'), with the susceptible Gguary variety as a reference. Varieties tested include '911' for PR 911, ‘DG’ for Dabokhangajeong, ‘DK’ for PR Daekan, ‘KJ’ for Callazzang, ‘KM’ for Kaltanmiso, ‘KY’ for Kaltanyeonseung, ‘MK’ for Meaunkaltan, ‘MS’ for Meotjinsanai, ‘SB’ for Subicho, and ‘TD’ for Titan Daebak. (B) A similar screening involving the predator, \u003cem\u003eO. laevigatus\u003c/em\u003e(‘Ol’), was conducted to assess preference among ten hot pepper varieties using a tunnel assay. In each tunnel assay run, 50 adults of \u003cem\u003eF. occidentalis\u003c/em\u003e or \u003cem\u003eO.\u003c/em\u003e \u003cem\u003elaevigatus\u003c/em\u003e were used. The experiments were replicated three times. Different letters above the standard error bars indicate significant differences among means at a Type I error rate of 0.05 (LSD test). (C) A comparison of the volatile organic compounds (VOCs) emitted by the ten hot pepper varieties was performed. VOC emission patterns were analyzed using a phylogenetic tree via the ClustVis online tool (https://biit.cs.us.ee/clustvis/). VOCs were categorized into four groups salicylic acid derivatives (‘SA’), green leaf volatiles (‘GLV’), terpenes (‘Terpene’), and unclassified (‘UC’). (D) A comparison of individual compounds, specifically GLVs and terpenes, released from the ten hot pepper varieties was conducted. Twenty-seven GLVs are identified, including methyl-hexenoate (‘MeH’), 3-ethyl-2-methyl-(\u003cem\u003eZ\u003c/em\u003e)-1,3-hexadiene (‘GLV2’), 1-hexanol (‘GLV3’), 2-ethyl-1-hexanol (‘GLV4’), 3,5,5-trimethyl-1-hexene (‘GLV5’), 3-methyl-2-hexanol (‘GLV6’), (\u003cem\u003eE\u003c/em\u003e)-2-hexen-4-yn-1-ol (‘GLV7’), 2-hexenal (‘GLV8’), (\u003cem\u003eE\u003c/em\u003e)-2-hexenal (‘GLV9’), 3,5,5-trimethyl-2-hexene (‘GLV10’), 2-hexenoic acid methyl ester (‘GLV11’), (\u003cem\u003eE\u003c/em\u003e)-2-hexenoic acid methyl ester (‘GLV12’), (\u003cem\u003eE\u003c/em\u003e)-3-hexen-1-ol (‘GLV13’), (\u003cem\u003eZ\u003c/em\u003e)-3-hexen-1-ol (‘GLV14’), (\u003cem\u003eE\u003c/em\u003e)-3-hexen-1-ol acetate (‘GLV15’), (\u003cem\u003eZ\u003c/em\u003e)-3-hexen-1-ol acetate (‘GLV16’), 3-hexenal (‘GLV17’), 3-hexenoic acid methyl ester (‘GLV18’), (\u003cem\u003eE\u003c/em\u003e)-3-hexenoic acid methyl ester (‘GLV19’), 3-hydroxy-4-methoxybenzaldehyde (‘GLV20’), 4-methyl-2-hexanol (‘GLV21’), hexanal (‘GLV22’), 1-chloro-hexane (‘GLV23’), hexanoic acid (‘GLV24’), 4-hexen-1-yl ester hexanoic acid (‘GLV25’), and hexanoic acid methyl ester (‘GLV26’). Eight terpenes are categorized, including linalool, eucalyptol (‘Terpene 2’), geraniol (‘Terpene 3’), geranyl formate (‘Terpene 4’), geranyl vinyl ether (‘Terpene 5’), linalyl acetate (‘Terpene 6’), trans-farnesol (‘Terpene 7’), and (\u003cem\u003eE\u003c/em\u003e)-geranylgeraniol (‘Terpene 8’). (E) Induction of \u003cem\u003eHPL\u003c/em\u003e and \u003cem\u003eLS\u003c/em\u003eexpression followed thrips infestation in ten hot pepper varieties. An asterisk (*) denotes a significant difference in the damaged treatment compared to the undamaged control at a Type I error rate of 0.05 (t-test). (F) A correlation study was conducted to evaluate the relationships between \u003cem\u003eHPL\u003c/em\u003e or \u003cem\u003eLS\u003c/em\u003eexpression levels and the repellency against \u003cem\u003eF. occidentalis\u003c/em\u003ein hot pepper varieties.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-6437640/v1/8c506177125c7357f603c2d1.png"},{"id":87302144,"identity":"a5e027b8-4dda-4f9c-99b2-52bb826dc72e","added_by":"auto","created_at":"2025-07-22 13:34:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4306499,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6437640/v1/b27acd2b-4e4f-4c45-8698-2f3872e91b03.pdf"},{"id":81999890,"identity":"161fd69d-8bfd-4fb3-9df7-f98483241b77","added_by":"auto","created_at":"2025-05-05 19:17:14","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":261749,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6437640/v1/1569c1b4651fe1aad3653136.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Volatile organic compounds emitted from the damaged hot peppers are oppositely interpreted by thrips and its predator","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe hot pepper, \u003cem\u003eCapsicum annuum\u003c/em\u003e, is cultivated worldwide as an indispensable condiment for various\u0026nbsp;foods because of\u0026nbsp;its\u0026nbsp;typical color, pungency, taste,\u0026nbsp;and distinct aroma\u003csup\u003e1\u003c/sup\u003e.\u0026nbsp;In Korea, nearly\u0026nbsp;80 different plant pathogens and insect pests have been identified\u0026nbsp;as causing\u0026nbsp;economic damage to hot\u0026nbsp;pepper cultivation\u003csup\u003e2\u003c/sup\u003e. Among these, the western flower thrips, \u003cem\u003eFrankliniella occidentalis\u003c/em\u003e (Pergande) (Thysanoptera: Thripidae), is one of the most\u0026nbsp;severe\u0026nbsp;insect pests. It transmits the\u0026nbsp;tomato spotted wilt virus (TSWV)\u0026nbsp;and also directly causes feeding damage\u003csup\u003e3\u003c/sup\u003e\u003csub\u003e.\u003c/sub\u003e Originating in the western part of North America, this thrips\u0026nbsp;quickly spread to most temperate and subtropical regions\u0026nbsp;worldwide due to its polyphagous feeding habits\u0026nbsp;and relatively high tolerance to temperature extremes, facilitated by increased global trade\u003csup\u003e4\u003c/sup\u003e.\u0026nbsp;Hot\u0026nbsp;peppers infected by TSWV\u0026nbsp;experience\u0026nbsp;retarded growth and\u0026nbsp;reduced\u0026nbsp;fruit quality,\u0026nbsp;resulting\u0026nbsp;in\u0026nbsp;significant\u0026nbsp;economic losses\u0026nbsp;and sometimes\u0026nbsp;leading to\u0026nbsp;the\u0026nbsp;abandonment of harvesting\u003csup\u003e5,6\u003c/sup\u003e. TSWV is transmitted from plant to\u0026nbsp;plant solely\u0026nbsp;by \u003cem\u003eF. occidentalis\u003c/em\u003e and eight other thrips species\u0026nbsp;out of more than\u0026nbsp;7,700 thrips species\u003csup\u003e7\u003c/sup\u003e.\u0026nbsp;Consequently, controlling\u0026nbsp;the thrips\u0026nbsp;is essential\u0026nbsp;to prevent TSWV-related diseases in\u0026nbsp;hot\u0026nbsp;peppers.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSeveral chemical insecticides including spinosad have been effective in killing thrips\u003csup\u003e8\u003c/sup\u003e. However, the cryptic behavior of thrips between leaves and flowers enables them to avoid exposure to sprayed insecticides\u003csup\u003e9\u003c/sup\u003e. This challenge has led to an increase in the frequency and dosage of insecticide spraying, which may select for thrips resistant to chemical insecticides. Consequently, this facilitates the rapid buildup of insecticide resistance in thrips. For example, frequent spraying of spinosad has selected for point-mutated individuals in the acetylcholine receptor gene, which become over 3,000-fold more tolerant to spinosad compared to the susceptible strain\u003csup\u003e10\u003c/sup\u003e. To effectively control this insect pest, alternative control measures should be developed. As an alternative, a biological control strategy has been designed and implemented to control \u003cem\u003eF. occidentalis\u003c/em\u003e using seasonal inoculative releases of a predatory bug,\u0026nbsp;\u003cem\u003eOrius laevigatus\u003c/em\u003e (Fieber) (Hemiptera: Anthocoridae)\u003csup\u003e11\u003c/sup\u003e. However, to successfully control the thrips, the biological control method occasionally needs supplementary applications of chemical insecticides, where the insecticide-resistant \u003cem\u003eO. laevigatus\u003c/em\u003e would be optimal to minimize toxicant damage\u003csup\u003e12,13\u003c/sup\u003e. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInsect resistance in plants has been regarded as an alternative insect pest control strategy because it minimizes chemical spraying and the associated labor, ideally reducing hazards to humans and the environment\u003csup\u003e14\u003c/sup\u003e. The insect resistance includes antixenosis, which deters herbivores; antibiosis, which delivers direct toxicants to herbivores after feeding; and tolerance of the plants to herbivore damage. Indeed, this host plant resistance against thrips has successfully been applied to prevent TSWV infection in peanuts, \u003cem\u003eArachis hypogaea\u003c/em\u003e, exhibiting both antixenosis and antibiosis\u003csup\u003e15\u003c/sup\u003e. Alternatively, TSWV reduces the host plant resistance to increase the accessibility of its vector thrips to the host plants for viral transmission\u003csup\u003e16\u003c/sup\u003e. In addition to direct insect resistance of host plants, a tri-trophic interaction can be exploited to recruit natural enemies to attack herbivores by releasing plant odors known as volatile organic compounds (VOCs) upon herbivore damage\u003csup\u003e17\u003c/sup\u003e. This chemical communication led us to hypothesize that hot peppers at the primary trophic level infested by \u003cem\u003eF. occidentalis\u003c/em\u003e at the secondary trophic level release VOCs to repel thrips as allomone and attract the natural enemy, \u003cem\u003eO. laevigatus\u003c/em\u003e, at the tertiary trophic level as kairomone.\u003c/p\u003e\n\u003cp\u003eThis study analyzed the behaviors of \u003cem\u003eF. occidentalis\u003c/em\u003e and \u003cem\u003eO. laevigatus\u003c/em\u003e in response to thrips-infested hot peppers. Differential behaviors between the herbivore and the predator were elucidated by analyzing VOCs emitted by the host plant. The influences of salicylic acid (SA) and jasmonic acid (JA) on inducing insect behaviors and VOC production were evaluated based on plant defense signaling pathways. The functional VOCs were tested by silencing gene expressions related to VOC biosynthesis through virus-induced gene silencing (VIGS) followed by behavioral assays. Finally, the study established a correlation between the levels of VOC-biosynthetic gene expression and hot pepper resistance to \u003cem\u003eF. occidentalis\u003c/em\u003e. \u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eDamaged hot pepper repels herbivore but attracts predator through JA signaling.\u0026nbsp;\u003c/strong\u003eWhen the hot peppers were infested by \u003cem\u003eF. occidentalis\u003c/em\u003e, the peppers became significantly less preferred than uninfested control plants (Fig. 1A). Conversely, the damaged plants were favored by their predator, \u003cem\u003eO. laevigatus\u003c/em\u003e. These contrasting behaviors were time-dependent (Fig. 1B). Repellency against \u003cem\u003eF. occidentalis\u003c/em\u003e was rapidly induced, reaching maximum levels 6 h post-infestation (pi). Simultaneously, attraction towards \u003cem\u003eO. laevigatus\u003c/em\u003e was also induced.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe defense system of hot peppers utilizing JA signaling was implicated to explain the behavioral changes of the thrips and their predators, as JA plays a critical role in plant defense against herbivore infestation\u003csup\u003e18\u003c/sup\u003e. Its impact on tri-trophic interactions was assessed in hot peppers by examining effects from JA or SA signaling pathways (Fig. 1C). Methyl JA (Me-JA) treatment on undamaged hot peppers repelled thrips while attracting the predator. However, SA treatment did not trigger these behaviors. The Me-JA treatment prompted behavioral modifications in the thrips and its predator within a day, and the induction was dose-dependent (Fig. 1D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA-Seq analysis confirms that JA signaling is induced in hot peppers infested by\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eF. occidentalis\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eTo investigate the response of hot peppers to infestation by \u003cem\u003eF. occidentalis\u003c/em\u003e, damaged and undamaged plants were compared in terms of their transcriptomes (Fig. 2). With the exception of one replication (‘R1’) in the ‘Damage’ treatment, more than 6.1 Gb of nucleotides were sequenced in each replication, and over 23,735 genes were predicted (Fig. 2A). RNA-Seq data from R1 of the ‘Damage’ treatment were excluded from further transcriptome analysis due to inadequate gene prediction. Using the predicted genes, their expression profiles were analyzed, revealing two distinct clusters between the damaged and undamaged hot peppers (Fig. 2B). Most (96.0%) of the genes were expressed in both treatments (Fig. 2C). Among these commonly expressed genes, 4,072 were differentially expressed (= DEG) by more than two-fold following thrips infestation (Fig. 2D). The up-regulated DEGs (= 2,584) due to thrips damage were further categorized by the number of fold changes, with 155 genes demonstrating more than 20-fold changes in their expression levels after thrips infestation (Fig. 2E). The highly expressed genes were functionally categorized into different stress responses including JA signaling and terpenoids (Fig. 2F). DEGs (= 63 genes) that exhibited over 40-fold changes are listed in Table S1. Notably, JA biosynthesis and signaling pathways are illustrated in Fig. 2G, where genes identified in the RNA-Seq analysis contributed to the synthetic and signaling components. Certain JA-associated genes were up-regulated in their expression levels following thrips infestation (Fig. 2H). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePGPRs induce the antixenosis of hot peppers through JA signaling.\u0026nbsp;\u003c/strong\u003eTo demonstrate the role of JA signaling in modulating insect behaviors, PGPR applications were administered to hot peppers because several PGPRs are known to enhance JA signaling in hot peppers, thereby inducing the production of VOCs to deter herbivores\u003csup\u003e19\u003c/sup\u003e. Of the five PGPRs tested, two (‘G-2273’ and ‘AK-0’) effectively repelled \u003cem\u003eF. occidentalis\u003c/em\u003e from the treated hot peppers (Fig. 3A). This repellency occurred 12 h post-treatment with the PGPRs (Fig. 3B). The two PGPR treatments significantly (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) attracted the predator, \u003cem\u003eO. laevigatus\u003c/em\u003e (Fig. 3C). These PGPR treatments up-regulated genes associated with JA biosynthesis such as \u003cem\u003e13-LOX\u003c/em\u003e, \u003cem\u003eAOS\u003c/em\u003e, and \u003cem\u003eAOC\u003c/em\u003e (Fig. 3D). The expression profiles of hot peppers treated with the effective PGPRs (‘AK-0’ and ‘G-2273’) differed from those treated with the ineffective PGPR (‘G-2311’) and the control (‘TSB’) according to clustering analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVOCs emitted by the hot peppers infested by\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eF. occidentalis\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eThe VOCs of the hot peppers were collected using the headspace-solid phase microextraction (HS-SPME) technique (Fig. S1A). A total of 169 VOCs were identified by GC-MS from both damaged and undamaged plants, with differential detection across the two treatments (Fig. S1B). Over 34% of the VOCs were commonly emitted across both treatments (Fig. 4A). Forty-four VOCs uniquely emitted from the damaged plants included green leaf volatiles (GLVs), terpenoids, and others (Fig. 4B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmong the GLVs, \u003cem\u003eZ\u003c/em\u003e-3-methyl hexenoate (MeH) was unique to the VOCs of the damaged plants and was chosen to test its biological activity (Fig. 4C). MeH effectively repelled \u003cem\u003eF. occidentalis\u003c/em\u003e and attracted \u003cem\u003eO. laevigatus\u003c/em\u003e. The GLV biosynthetic pathway is depicted in Fig. 4D, where genes identified in the RNA-Seq analysis were discovered in the pathway components. Most of the genes associated with GLV biosynthesis were upregulated in their expression levels following thrips infestation (Fig. 4E). Notably, hydroperoxide lyase (\u003cem\u003eHPL\u003c/em\u003e), which catalyzes the cleavage of C18 fatty acids to produce the six-carbon GLV skeleton, was significantly upregulated following thrips infestation or treatments with effective PGPRs (Fig. 4F).\u003c/p\u003e\n\u003cp\u003eAmong terpenoids, linalool was specific to VOCs of the damaged plants and used to assess its biological activity against the thrips and their predator (Fig. 5A). Linalool also repelled \u003cem\u003eF. occidentalis\u003c/em\u003e and attracted \u003cem\u003eO. laevigatus\u003c/em\u003e. The terpenoid biosynthetic pathway is depicted in Fig. 5B, where genes identified in the RNA-Seq analysis were located in the pathway components. Most genes associated with terpenoid biosynthesis were upregulated in their expression levels following thrips infestation (Fig. 5C). Notably, linalool synthetase (\u003cem\u003eLS\u003c/em\u003e) was significantly upregulated following thrips infestation or treatments with effective PGPRs (Fig. 5D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSuppression of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eHPL\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;or\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eLS\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;expression disrupts the tri-trophic interactions involving hot pepper-\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eF. occidentalis\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eO. laevigatus\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eTo specifically suppress \u003cem\u003eHPL\u003c/em\u003e or \u003cem\u003eLS\u003c/em\u003e expression in the hot peppers, the virus-induced gene silencing (VIGS) technique was employed (Fig. 6). Co-injection of two expression vectors (pTRV1 and pTRV2) successfully silenced a specific target gene, phytoene desaturase (\u003cem\u003ePDS\u003c/em\u003e), causing the treated hot peppers to lose their green color in contrast to the control (Fig. 6A). When the recombinant pTRV2 containing the \u003cem\u003eHPL\u003c/em\u003e or \u003cem\u003eLS\u003c/em\u003e construct was administered, the VIGS treatments significantly suppressed the target genes even following thrips infestation. Conversely, the controls exhibited significant induction of the target genes following thrips infestation (Fig. 6B). Suppression of the target genes resulted in a marked reduction of MeH and linalool emitted by the hot peppers post-thrips infestation (Fig. 6C).\u003c/p\u003e\n\u003cp\u003eIn wild-type plants, thrips-infested hot peppers ('Damage') significantly enhanced repellency against thrips and attraction of predators (Fig. 6D) compared to undamaged plants. Under the VIGS treatment specific to \u003cem\u003eHPL\u003c/em\u003e expression, the thrips infestation did not repel the thrips as effectively as in the control without infestation (‘Undamage in dsCON vs Damage in dsRNA’). Instead, the thrips showed a preference for the dsRNA-treated hot peppers (‘Undamage in dsCON vs Undamage in dsRNA’ or ‘Damage in dsCON vs Damage in dsRNA’) over those treated with dsCON. However, adding MeH restored the repellency by inhibiting the thrips' orientation towards the dsRNA treatment (‘Damage in dsCON vs Damage in dsRNA’). By contrast, the attraction of predators was significantly suppressed in \u003cem\u003eHPL\u003c/em\u003e-silenced plants, whether damaged or undamaged. The \u003cem\u003eHPL\u003c/em\u003e-silenced plants were unsuccessful in attracting predators compared to the control without infestation (‘Undamage in dsCON vs Damage in dsRNA’). Instead, predators showed a preference for the control plant over the \u003cem\u003eHPL\u003c/em\u003e-silenced plants (‘Undamage in dsCON vs Undamage in dsRNA’ or ‘Damage in dsCON vs Damage in dsRNA’) compared to dsRNA-treated hot peppers. However, the introduction of MeH restored the attractiveness by inhibiting the orientation of the predators towards the control plants (‘Damage in dsCON vs Damage in dsRNA’). The VIGS treatments targeting \u003cem\u003eLS\u003c/em\u003e expression in the hot peppers also resulted in similar behavioral changes in both thrips and predators, as observed in the VIGS treatments targeting \u003cem\u003eHPL\u0026nbsp;\u003c/em\u003eexpression. Suppression of \u003cem\u003eLS\u003c/em\u003e expression reduced both thrips repellency and attractiveness towards \u003cem\u003eO. laevigatus\u003c/em\u003e in the hot peppers infested by \u003cem\u003eF. occidentalis\u003c/em\u003e. Linalool treatment restored the normal responses of thrips and their predators to the \u003cem\u003eLS\u003c/em\u003e-silenced plants. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDifferential expression levels of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eHPL\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eLS\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;influence the antixenosis of various hot pepper varieties.\u0026nbsp;\u003c/strong\u003eTen distinct hot pepper varieties were evaluated for their repellency against \u003cem\u003eF. occidentalis\u003c/em\u003e (Fig. 7A). In a choice test using a tunnel, two varieties (‘KM’ and ‘TD’) were found to be less preferred by the thrips. Additionally, two varieties (‘KM’ and ‘MS’) were more preferred by \u003cem\u003eO. laevigatus\u003c/em\u003e when infested by the thrips (Fig. 7B). VOCs were collected from these 10 varieties and categorized into 499 different compounds, including GLVs, terpenoids, and others (Table S2). These VOCs were analyzed across the 10 hot pepper varieties, revealing two clusters with the three varieties (‘TD’, ‘MS’, and ‘KM’) not being grouped (Fig. 7C). Notably, MeH was only detected in the three resistant varieties (‘TD’, ‘MS’, and ‘KM’), and linalool was exclusively found in two resistant varieties (‘TD’ and ‘KM’) (Fig. 7D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll ten hot pepper varieties up-regulated the gene expression levels of \u003cem\u003eHPL\u003c/em\u003e and \u003cem\u003eLS\u003c/em\u003e following thrips infestation, although the induction levels varied among the varieties (Fig. 7E). The expression levels of \u003cem\u003eHPL\u003c/em\u003e across the 10 hot pepper varieties strongly correlated with their repellent activities against \u003cem\u003eF. occidentalis\u003c/em\u003e (Fig. 7F). The expression levels of \u003cem\u003eLS\u003c/em\u003e also correlated with their repellent activities against \u003cem\u003eF. occidentalis\u003c/em\u003e. \u0026nbsp; \u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study analyzed a tri-trophic interaction among hot peppers, \u003cem\u003eF. occidentalis\u003c/em\u003e, and \u003cem\u003eO. laevigatus\u003c/em\u003e through VOCs produced by hot peppers in response to thrips infestation. To elucidate the role of VOCs in biological activity, the behaviors of the herbivore and its predator toward the damaged plants were investigated. VOC production was validated by comparing the transcriptomes of undamaged and damaged host plants, monitoring gene expressions related to the biosynthetic pathways of the candidate VOCs. Ultimately, the functional VOCs were analyzed for the expression levels of the biosynthetic genes across different hot pepper varieties, highlighting how susceptibility to thrips varies. This approach demonstrated the pivotal roles of GLV and terpenes in the chemical communication within the tri-trophic interaction.\u003c/p\u003e\n\u003cp\u003eThrips infestation prompted hot peppers to employ antixenosis, enhancing non-preference behavior over the control plants, as a defense against herbivore attack. Intriguingly, the damaged plants attracted the predator, \u003cem\u003eO. laevigatus\u003c/em\u003e. Augmentative biological control programs have employed the predator bug, \u003cem\u003eO. laevigatus\u003c/em\u003e, particularly in greenhouses cultivating vegetable crops including hot peppers, to combat thrips pests\u003csup\u003e20\u003c/sup\u003e. Natural enemies are generally affected both directly and indirectly in tri-trophic interactions with host plants\u003csup\u003e21\u003c/sup\u003e. Direct interactions between natural enemies and host plants involve physical characteristics, such as leaf trichomes, waxy leaves, and leaf thickness\u003csup\u003e22-24\u003c/sup\u003e, and biochemical properties including exudates, nectars, and odors\u003csup\u003e25\u003c/sup\u003e. Indirect effects involve the herbivores\u0026apos; biological traits, such as developmental time, size, weight, fecundity, survival, longevity, establishment, and digestibility\u003csup\u003e21,26,27\u003c/sup\u003e. These biological characteristics of herbivores are influenced by the host plants, illustrating functional tri-trophic interactions. The damaged hot peppers\u0026apos; modulation of behaviors that repel \u003cem\u003eF. occidentalis\u003c/em\u003e and attract \u003cem\u003eO. laevigatus\u003c/em\u003e suggested a chemical communication across tri-trophic levels: host plant (hot pepper), herbivore (\u003cem\u003eF. occidentalis\u003c/em\u003e), and predator (\u003cem\u003eO. laevigatus\u003c/em\u003e). The targeted communication calling specific natural enemies by the infested host plants has been demonstrated in various plants, including corn and cabbage, depending on the herbivores present \u003csup\u003e28,29\u003c/sup\u003e. In cabbages, the damaged host plants released different VOCs depending on the herbivores\u0026apos; species and infestation density\u003csup\u003e30\u003c/sup\u003e. The study reported an up-regulation of VOCs in damaged cabbages, including GLVs ((\u003cem\u003eZ\u003c/em\u003e)-3-hexenol, n-heptanal, and (\u003cem\u003eZ\u003c/em\u003e)-3-hexenyl acetate), terpenes (\u0026alpha;-pinene, sabinene, myrcene, limonene, camphor, and (\u003cem\u003eE\u003c/em\u003e)-4,8-dimethyl-1,3,7-nonatriene), and another hydrocarbon (\u0026alpha;-copaene). Our current study identified the VOCs, including GLVs and terpenes, released from hot peppers infested by \u003cem\u003eF. occidentalis\u003c/em\u003e upon capturing the volatile compounds using HS-SPME.\u003c/p\u003e\n\u003cp\u003eThe antixenosis induced by the functional VOCs is likely mediated by the JA signaling pathway in the hot pepper infested by \u003cem\u003eF. occidentalis\u003c/em\u003e. In addition to the MeJA effect on the induction of antixenosis in hot peppers, DEG analysis between control and infested hot peppers showed that genes associated with JA biosynthesis and signaling components were significantly upregulated in response to thrips infestation, along with the increased expression of GLV and terpene biosynthetic genes. Notably, the expression levels of 13-LOX, which oxygenates \u0026alpha;-linolenic acid in a regio- and stereo-selective manner to produce 13(\u003cem\u003eS\u003c/em\u003e)-hydroperoxylinolenic acid in JA synthesis, increased more than 200-fold after thrips infestation. Indeed, disruption of \u003cem\u003e13-LOX\u003c/em\u003e expression suppressed the JA pathway in peppers, leading to enhanced susceptibility to \u003cem\u003eF.\u0026nbsp;\u003c/em\u003eoccidentalis\u003csup\u003e31\u003c/sup\u003e. Moreover, two PGPRs (\u0026lsquo;AK-0\u0026rsquo; and \u0026lsquo;G-2273\u0026rsquo;) among five bacterial isolates effectively induced antixenosis in hot peppers against \u003cem\u003eF. occidentalis\u003c/em\u003e and up-regulated the expression of \u003cem\u003e13-LOX\u003c/em\u003e and other JA-synthetic genes. It is well-known that PGPRs assist plants in defending against various pathogens and herbivores. For example, in cotton insect resistance, gossypol is considered a primary antibiosis factor against insect pests, and its production is stimulated by treatment with a PGPR classified into \u003cem\u003eBacillus\u003c/em\u003e sp., enhancing JA biosynthesis\u003csup\u003e32\u003c/sup\u003e. Insect resistance can be induced by suppressing a plant factor negatively associated with insect resistance. The increased JA synthesis by PGPRs suppressed a specific microRNA, \u003cem\u003emir814\u003c/em\u003e, which antagonizes the insect resistance of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e following treatment with \u003cem\u003eBacillus amyloliquefasciens\u003c/em\u003e FZB42, leading to induced systemic resistance\u003csup\u003e33\u003c/sup\u003e. In our current study, the selected PGPRs activate JA synthesis, potentially causing antixenosis by producing VOCs that repel \u003cem\u003eF. occidentalis\u003c/em\u003e and attract \u003cem\u003eO. laevigatus\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eIn our study, \u003cem\u003eZ\u003c/em\u003e-3-methyl hexenoate (MeH), a GLV produced by infested hot peppers, was particularly effective in repelling thrips and attracting predators. GLVs are widely produced by green plants and their production can be induced by abiotic stimuli\u003csup\u003e34\u003c/sup\u003e, herbivores\u003csup\u003e35\u003c/sup\u003e, or pathogens\u003csup\u003e36\u003c/sup\u003e via the HPL branch of the oxylipin pathway, often within seconds of stress exposure. While GLV emission is typically short-lived, it can persist for days during herbivory or repeated wounding\u003csup\u003e37\u003c/sup\u003e. Furthermore, GLVs serve as semiochemicals that guide insects to locate food or prey\u003csup\u003e38\u003c/sup\u003e. MeH is synthesized from hexenol through JA signaling in a medicinal herb\u003csup\u003e39\u003c/sup\u003e, and this precursor is derived from C18 unsaturated fatty acids such as linoleic acid and linolenic acid, following catalytic cleavage by hydroperoxide lyase (HPL). Our VIGS analysis, which aimed to suppress \u003cem\u003eHPL\u003c/em\u003e expression, corroborated the biosynthetic pathway and physiological role of MeH. Additionally, MeH was detected solely in hot pepper varieties that are resistant to \u003cem\u003eF. occidentalis\u003c/em\u003e. The expression levels of \u003cem\u003eHPL\u003c/em\u003e were positively correlated with the degree of non-preference exhibited by \u003cem\u003eF. occidentalis\u003c/em\u003e towards the hot peppers. This functional GLV may also prime hot pepper plants against further attacks from herbivores or pathogens. GLVs induce defense priming in plants, enabling them to respond faster or more robustly to insect herbivores and pathogens. For instance, a tomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e) infested by the whitefly, \u003cem\u003eBemisia tabaci\u003c/em\u003e, releases \u003cem\u003eZ\u003c/em\u003e-3-hexenol through JA signaling, which increases the expression of defense genes and flavonoid levels\u003csup\u003e40\u003c/sup\u003e. This indicates that the production and release of other GLVs, as well as MeH, may be stimulated by JA signaling, contributing to antixenosis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLinalool, a terpene produced by hot peppers infested by \u003cem\u003eF. occidentalis\u003c/em\u003e, is catalyzed by LS, a terpene synthase. The suppression of gene expression by gene-specific VIGS treatment prevented the upregulation of linalool titers in the hot peppers following thrips damage. Indirectly, linalool may also prime the host plant to produce ROS, as demonstrated in \u003cem\u003eArabidopsis\u003c/em\u003e infested by \u003cem\u003eP. xylostella\u003c/em\u003e, causing oxidative damage to the herbivore\u003csup\u003e41\u003c/sup\u003e.\u003cem\u003e\u0026nbsp;\u003c/em\u003eThe expression of terpene synthase genes is regulated by the MYC2 transcription factor under the JA immune signaling pathway in hot peppers\u003csup\u003e42\u003c/sup\u003e. This suggests that JA signaling initiates the synthesis of various terpenes, including linalool.\u003c/p\u003e\n\u003cp\u003eVarious hot pepper varieties differ in their susceptibility to \u003cem\u003eF. occidentalis\u003c/em\u003e infestation as well as in their ability to produce GLVs and terpenes. Even without damage, hot peppers emit a range of VOCs. For instance, the Habanero pepper, known for its intense pungency and fruity aroma, releases 66 volatile compounds, including multiple short-chain hydrocarbons such as 6-methyl-(\u003cem\u003eE\u003c/em\u003e)-4-heptenyl 3-methylbutanoate, 6-methyl-(\u003cem\u003eE\u003c/em\u003e)-4-heptenyl 2-methylpropanoate, 6-methyl-(\u003cem\u003eE\u003c/em\u003e)-4-heptenyl 2-methylbutanoate, 2-methylbutanoate, and 6-methyl-(\u003cem\u003eE\u003c/em\u003e)-4-heptenol\u003csup\u003e43\u003c/sup\u003e. However, it is primarily the various GLVs and terpenes that characterize the VOCs of damaged hot peppers. Our current data indicate that the JA signaling pathway, which is triggered by thrips infestation, upregulates biosynthetic gene expressions such as \u003cem\u003eHPL\u003c/em\u003e and \u003cem\u003eLS\u003c/em\u003e, and these are positively correlated with antixenosis resistance to \u003cem\u003eF. occidentalis\u003c/em\u003e. In other words, hot pepper varieties expressing high levels of \u003cem\u003eHPL\u003c/em\u003e and \u003cem\u003eLS\u003c/em\u003e genes tend to be resistant to \u003cem\u003eF. occidentalis\u003c/em\u003e. This positive correlation provides crucial information for the breeding of resistant hot pepper varieties against \u003cem\u003eF. occidentalis\u003c/em\u003e, a significant insect pest that transmits TSWV to high-value crops\u003csup\u003e3\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eBesides VOCs, established tri-trophic interactions include the relationship among hot peppers, \u003cem\u003eF. occidentalis\u003c/em\u003e, and \u003cem\u003eO. laevigatus\u003c/em\u003e, which is mediated by the aggregation pheromone released by \u003cem\u003eF. occidentalis\u003c/em\u003e. This pheromone facilitates chemical communication among the thrips, but functions as a kairomone for both adult and nymph stages of \u003cem\u003eO. laevigatus\u003c/em\u003e, particularly in its active components mixture of (\u003cem\u003eR\u003c/em\u003e)-lavandulyl acetate and neryl (\u003cem\u003eS\u003c/em\u003e)-2-methylbutanoate\u003csup\u003e44\u003c/sup\u003e. Conversely, tri-trophic interactions may be compromised by viral infections such as TSWV that reduce predator access to the plants, thus promoting the virus\u0026apos;s horizontal transmission. \u003cem\u003eF. occidentalis\u003c/em\u003e larvae thrive better on TSWV-infected host plants than on non-infected hosts and evade predation by predatory mites\u003csup\u003e45\u003c/sup\u003e. This complexity in tri-trophic interactions have led to the identification of chemical signals originating from the primary trophic level, which are interpreted differently by the herbivore and its predator. Additionally, this study highlights resistant genetic markers like \u003cem\u003eHPL\u003c/em\u003e and \u003cem\u003eLS\u003c/em\u003e that deter thrips from hot peppers, beneficial for future breeding programs.\u0026nbsp;\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eInsect rearing.\u0026nbsp;\u003c/strong\u003eAdults of \u003cem\u003eF.\u003c/em\u003e \u003cem\u003eoccidentalis\u003c/em\u003e were collected from a hot pepper field in Andong, Korea, and reared on sprouted bean seed kernels under specific laboratory conditions: a constant temperature of 25 ± 1°C, a photoperiod of 16:8 h (L:D), and a relative humidity of 60 ± 5%. Under these conditions, the thrips underwent two instars (L1 and L2), prepupa, and pupa before maturing into adults. The predator,\u0026nbsp;\u003cem\u003eO. laevigatus\u003c/em\u003e, colony, donated by Oal, Inc. (Seoul, Korea), was cultivated under similar laboratory conditions. These predators were maintained on hot pepper plants with \u003cem\u003elarvae\u003c/em\u003e of \u003cem\u003eF. occidentalis\u0026nbsp;\u003c/em\u003eprovidedas food.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlant growth-promoting rhizobacteria (PGPR).\u0026nbsp;\u003c/strong\u003eFive different PGPR bacteria of \u003cem\u003ePaenibacillus polymyxa\u003c/em\u003e GYUN-2273 (NCBI accession number: OR883773), \u003cem\u003eBacillus subtilis\u003c/em\u003e GYUN-2311 (KACC accession number: 92471), \u003cem\u003eB. velezensis\u003c/em\u003e AK-0 (KACC accession number: 92099), \u003cem\u003eB. tequilensis\u003c/em\u003e G-300 (KACC accession number: 81153), and \u003cem\u003eBrevibacillus halotolerans\u003c/em\u003e B-4359 (NCBI GenBank number: CP139435) were isolated from the rhizosphere of agricultural regions in Andong or donated by the Nakdonggang National Institute of Biological Resources, Sangju, Korea. These bacteria were inoculated into 100 mL of tryptic soy broth (TSB: Difco, Sparks, MD, USA) and cultured at 28°C with an agitation of 180 rpm. After 48 h culture, 10 mL of the broth was evenly applied to the soil in a pot (10 cm diameter and 10 cm height) containing young hot pepper plants at the six-leaf stage. At various times, the effects of the PGPR on the insect resistance of the hot peppers were evaluated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCultivating different hot pepper varieties.\u0026nbsp;\u003c/strong\u003eAll hot pepper varieties, including \u003cem\u003eC. annuum\u003c/em\u003e, seedlings, were cultivated in a nursery under conditions of 25 ± 1°C, a 12:12 h (L:D) photoperiod, and approximately 60% RH. Biological activities were evaluated using the Gguari variety (Hanlim, Seoul, Korea) as a susceptible reference. The other test hot pepper varieties included PR Daekan (Jenong S\u0026amp;T, Jeju, Korea), Dabokhangajeong (Sakata Korea, Seoul, Korea), PR 911 (Hana Seed, Anseong, Korea), Kaltanmiso (Seedland, Cheongju, Korea), Meotjinsanai (Syngenta Korea, Seoul, Korea), Subicho (Yeongyang, Korea), Meaunkaltan (Pepper \u0026amp; Breeding Institute, Gimje, Korea), Callazzang (Nongwoobio, Suwon, Korea), Titan Daebak (Farmhannong, Seoul, Korea), and Kaltanyeonseung (Nongwoobio, Suwon, Korea). The assays were conducted using 6-week-old plants at the six-leaf stage to ensure uniformity and developmental consistency.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChemicals.\u0026nbsp;\u003c/strong\u003eMethyl hexenoate (99%), linalool (97%), salicylic acid (99%), methyl jasmonate (95%), and cyclopentanone (99%) were purchased from Sigma Aldrich Korea (Seoul, Korea). These compounds were subsequently dissolved in dimethyl sulfoxide (DMSO) to prepare the test solutions. DNA Taq polymerase, RT Premix, and Power SYBR Green PCR Master Mix were sourced from GeneALL Biotechnology Company (Seoul, Korea), Intron Biotechnology (Seoul, Korea), and Life Technologies (Carlsbad, CA, USA), respectively. Furthermore, the restriction enzymes (BamHI and XbaI) were sourced from Takara (Kyoto, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInsect damage treatment to hot peppers with \u003cem\u003eF. occidentalis.\u0026nbsp;\u003c/em\u003e\u003c/strong\u003eThe young hot pepper plants at the six-leaf stage were infested with 50 L2 larvae of \u003cem\u003eF. occidentalis\u003c/em\u003e per plant for 8 h under controlled culturing conditions. Afterwards, the insects were removed, and the plants were immediately used for further experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBehavioral bioassay using Y-tube olfactometer.\u0026nbsp;\u003c/strong\u003eTo assess the behavioral responses to chemicals, a Y-tube olfactometer was utilized as described by Khan et al\u003csup\u003e46\u003c/sup\u003e. In each trial, 20 adults of \u003cem\u003eF. occidentalis\u003c/em\u003e or \u003cem\u003eO. laevigatus\u003c/em\u003e were placed in the Y-tube for observation. Positive responses included insects trapped on a sticky card (2 cm × 2 cm) or observed feeding on pepper plants. Those that did not cross the fork were recorded as no response (‘NR’). All trials were conducted in darkness at 25 ± 1°C and 65% RH, each lasting 6 h and replicated three times.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBehavioral bioassay using a tunnel assay.\u0026nbsp;\u003c/strong\u003eThe tunnel assay was employed to evaluate the behavioral choice between two hot pepper varieties for \u003cem\u003eF. occidentalis\u003c/em\u003e and \u003cem\u003eO. laevigatus\u003c/em\u003e\u003csup\u003e47\u003c/sup\u003e. All assessments were performed in the dark at 25 ± 1°C and 65% RH, and each lasted 6 h. In each test, 50 adults of \u003cem\u003eF. occidentalis\u0026nbsp;\u003c/em\u003eor \u003cem\u003eO. laevigatus\u003c/em\u003e were introduced into the tunnel's midpoint through an entrance hole. Positive responses were those where insects were trapped on a sticky card (24 cm × 14 cm) or seen feeding on the plants. After each session, the tunnel was cleaned thoroughly, with at least one hour between trials. Each treatment was replicated three times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA extraction, cDNA synthesis, RT-PCR, and RT-qPCR.\u0026nbsp;\u003c/strong\u003ePlant leaves were collected and flash-frozen in liquid nitrogen before storage at -80°C to prevent cross-contamination and RNA degradation. Each sample involved grinding 0.1 g of plant tissues thoroughly with a cold (4\u003csup\u003eo\u003c/sup\u003eC) mortar and pestle, which had been preheated at 180°C for 20 min to inactivate RNases. RNA was extracted using Trizol reagent according to the manufacturer's instructions. After extraction, RNA was resuspended in nuclease-free water, and its concentration was measured using a spectrophotometer (NanoDrop, Thermo Scientific, Wilmington, DE, USA). RNA was then used for cDNA synthesis with an RT Premix containing oligo-dT primer, following the manufacturer’s instructions. RT-PCR involved DNA Taq polymerase and proceeded under these conditions: initial denaturation at 94°C for 5 min, 35 cycles of denaturation at 94°C for 1 min, annealing at various temperatures (Table S3) for 1 min, extension at 72°C for 1 min, and a final extension at 72°C for 10 min using gene-specific primers. Each 20 µL RT-PCR reaction contained the cDNA template, dNTPs (each at 2 mM), 10 pmol of each forward and reverse primer, and 2 units/µL of Taq polymerase. Gene expression levels were assessed with a real-time PCR machine (Step One Plus Real-Time PCR System, Applied Biosystems, Singapore) using Power SYBR Green PCR Master Mix as per Bustin et al\u003csup\u003e48\u003c/sup\u003e. The expression of \u003cem\u003eβ-actin\u003c/em\u003e served as a reference to normalize target gene expression levels under various treatments. Melting curve analysis confirmed the specificity of each PCR product. Quantitative analysis used the comparative CT (2\u003csup\u003e-∆∆CT\u003c/sup\u003e) method\u003csup\u003e49\u003c/sup\u003e, with each experiment replicated three times with independent cohorts or sample preparations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA-Seq analysis of hot peppers after thrips damage.\u0026nbsp;\u003c/strong\u003eHot peppers infested by \u003cem\u003eF. occidentalis\u003c/em\u003e were used to extract RNA as previously described. Each treatment was replicated three times. The concentration and purity of the RNA samples were assessed using a 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) and the samples were sent to Macrogen (Seoul, Korea) for RNA library construction and next-generation sequencing using the Illumina NovaSeq 6000 system (Illumina, San Diego, CA, USA).\u003c/p\u003e\n\u003cp\u003eThe sequenced FASTQ data were uploaded to the NCBI database under the project number PRJNA104655. Data analysis was conducted using the bioinformatics software CLC Genomic Workbench (version 22.0.1, Qiagen, Hilden, Germany). The raw sequenced data were trimmed to remove low-quality sequences and mapped to the \u003cem\u003eC. annuum\u003c/em\u003e genome. RPKM (reads per kilobase per million mapped reads) values were used to calculate relative mRNA expression levels. Gene IDs were retrieved from the gene symbols with LOC numbers in the mapped reads, using the general feature format (GFF) file of the \u003cem\u003eC. annuum\u003c/em\u003e genome.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDifferentially expressed genes (DEGs) were identified based on a minimum two-fold change and a \u003cem\u003ep\u003c/em\u003e-value of less than 0.05 by comparing RPKM values. Gene ontology (GO) analysis was conducted using Blast2GO 6.0 (BioBam, Valencia, Spain) to classify biological, cellular, and molecular functional categories of selected DEGs. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed to determine the functional categories of genes as defined in the KEGG database, using the KEGG mapper (https://www.genome.jp/kegg/mapper/search.html) and the BioDBnet (Biological Database Network, https://biodbnetabcc.ncifcrf.gov/db/db2dbRes.php) ID conversion tool.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscript analysis of the damaged hot peppers in the JA pathway.\u0026nbsp;\u003c/strong\u003eGenes involved in the JA biosynthetic pathway were selected based on the model proposed by Wang et al.\u003csup\u003e50\u003c/sup\u003e and our current transcriptome data of hot peppers to compare expression levels across treatments. The analyzed genes include \u003cem\u003ephospholipase A\u003csub\u003e1\u003c/sub\u003e\u003c/em\u003e (\u003cem\u003ePLA1\u003c/em\u003e), \u003cem\u003e13-lipoxygenase\u003c/em\u003e (\u003cem\u003e13-LOX\u003c/em\u003e), \u003cem\u003eallene oxide synthase\u003c/em\u003e (\u003cem\u003eAOS\u003c/em\u003e), \u003cem\u003eallene oxide cyclase\u003c/em\u003e (\u003cem\u003eAOC\u003c/em\u003e), \u003cem\u003eJA-amino acid synthetase\u003c/em\u003e (\u003cem\u003eJASSY\u003c/em\u003e), \u003cem\u003eL-3-ketoacyl CoA thiolase\u003c/em\u003e (\u003cem\u003eKAT\u003c/em\u003e), \u003cem\u003emultifunctional protein\u003c/em\u003e (\u003cem\u003eMCX\u003c/em\u003e), \u003cem\u003eacyl-CoA oxidase\u003c/em\u003e (\u003cem\u003eACX\u003c/em\u003e), \u003cem\u003eoxophytodienoic acid reductase 3\u003c/em\u003e (\u003cem\u003eOPR3\u003c/em\u003e), \u003cem\u003ecoronatine insensitive 1\u003c/em\u003e (\u003cem\u003eCOI1\u003c/em\u003e), \u003cem\u003ejasmonate zim domain\u003c/em\u003e (\u003cem\u003eJAZ\u003c/em\u003e), and \u003cem\u003eMYC2\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGenes involved in the terpene biosynthetic pathway were identified using the pathway outlined by Mani et al.\u003csup\u003e51\u003c/sup\u003e from our current transcriptome of the hot peppers: \u003cem\u003eacetyl-CoA acetyltransferase\u003c/em\u003e (\u003cem\u003eAACT\u003c/em\u003e, XP_016580453.1), \u003cem\u003ehydroxymethylglutaryl-CoA synthase\u003c/em\u003e (\u003cem\u003eHMGS\u003c/em\u003e, XP_016551730.1), \u003cem\u003e3-hydroxy-3-methylglutaryl-coenzyme A reductase\u003c/em\u003e (\u003cem\u003eHMGR\u003c/em\u003e, XP_016557834.2), \u003cem\u003emevalonate kinase\u003c/em\u003e (\u003cem\u003eMK\u003c/em\u003e, XP_016538569.2), \u003cem\u003ephosphomevalonate kinase\u0026nbsp;\u003c/em\u003e(\u003cem\u003ePMK\u003c/em\u003e, XP_016574993.2), \u003cem\u003ediphosphomevalonate decarboxylase\u003c/em\u003e (\u003cem\u003eMPD\u003c/em\u003e, XP_016551358.2), \u003cem\u003efarnesyl pyrophosphate synthase\u003c/em\u003e (\u003cem\u003eFPS\u003c/em\u003e, NP_001311799.1), \u003cem\u003esesquiterpene synthase\u003c/em\u003e (\u003cem\u003eSTPS\u003c/em\u003e, XP_016566183.2), \u003cem\u003e(-)-germacrene D synthase\u003c/em\u003e (\u003cem\u003eGDS\u003c/em\u003e, XP_016564626.1), \u003cem\u003e1-deoxy-D-xylulose-5-phosphate synthase\u003c/em\u003e (\u003cem\u003eDXS\u003c/em\u003e, XP_047256568.1), \u003cem\u003e1-deoxy-D-xylulose-5-phosphate reductase\u003c/em\u003e (\u003cem\u003eDXR\u003c/em\u003e, XP_016563443.1), \u003cem\u003e2-methyl-D-erythritol 4-phosphate cytidylyltransferase\u003c/em\u003e (\u003cem\u003eCMS\u003c/em\u003e, XP_016538918.1), \u003cem\u003e2-methyl-D-erythritol 2,4-cyclodiphosphate synthase\u003c/em\u003e (\u003cem\u003eMCS\u003c/em\u003e, XP_016566381.1), \u003cem\u003e4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase\u003c/em\u003e (\u003cem\u003eHDS\u003c/em\u003e, XP_016547325.1), \u003cem\u003e4-hydroxy-3-methylbut-2-en-1-yl diphosphate reductase\u003c/em\u003e (\u003cem\u003eHDR\u003c/em\u003e, XP_016539712.1), \u003cem\u003egeranylgeranyl diphosphate synthase\u003c/em\u003e (\u003cem\u003eGPPS\u003c/em\u003e, XP_016556802.1), and \u003cem\u003elinalool synthase\u003c/em\u003e (\u003cem\u003eLS\u003c/em\u003e, XP_016539311.2).\u003c/p\u003e\n\u003cp\u003eGenes associated with the GLV biosynthetic pathway were selected via the method proposed by Scala et al.\u003csup\u003e38\u003c/sup\u003e from our current transcriptome of the hot peppers: \u003cem\u003elipoxygenase\u003c/em\u003e (\u003cem\u003eLOX\u003c/em\u003e, NP_001311748.1), \u003cem\u003ehydroperoxide lyase\u003c/em\u003e (\u003cem\u003eHPL\u003c/em\u003e, NP_001311810.1), \u003cem\u003ealcohol dehydrogenase\u003c/em\u003e (\u003cem\u003eADH\u003c/em\u003e, XP_016568293.2), \u003cem\u003ealdo-keto reductase\u003c/em\u003e (\u003cem\u003eAKR\u003c/em\u003e, XP_016543252.1), \u003cem\u003ealdehyde reductase\u003c/em\u003e (\u003cem\u003eADR\u003c/em\u003e, XP_016578140.2), and \u003cem\u003ealcohol acetyltransferase\u003c/em\u003e (\u003cem\u003eAAT\u003c/em\u003e, XP_016541551.2). These genes were used to compare gene expression levels in the terpene pathway between damaged and undamaged hot pepper plants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVirus-induced gene silencing (VIGS).\u0026nbsp;\u003c/strong\u003eVirus-induced gene silencing was performed following the methods of Zhang \u0026amp; Liu\u003csup\u003e52\u003c/sup\u003e with slight modifications.Open reading frames (ORFs) of \u003cem\u003eHPL\u003c/em\u003e (GenBank accession number: U51674.1) and\u003cem\u003e\u0026nbsp;LS\u003c/em\u003e (GenBank accession number: XM_047394954.1) genes were cloned into the pCR2.1-TOPO vector using the TOPO TA Cloning Kit (Invitrogen) with ORF primers (Table S3). The confirmed clones were cultured in LB medium containing ampicillin (100 µg/mL), and their plasmids were extracted using a spin column (Plasmid SV, GeneAll). Extracted pCR2.1-HPL and pCR2.1-\u003cem\u003eLS\u0026nbsp;\u003c/em\u003eplasmids were sequenced at Macrogen. Upon confirmation, the recombinant vectors were digested with BamHI and XbaI, and the resulting inserts were ligated to construct the recombinant pTRV2-\u003cem\u003eHPL\u003c/em\u003e and pTRV2-\u003cem\u003eLS\u003c/em\u003e expression vectors. These vectors were transformed into \u003cem\u003eEscherichia coli\u003c/em\u003e (DH5α) competent cells and cultured in LB containing kanamycin (100 mg/mL). Finally, 10 μL of the validated recombinant plasmid of pTRV2-\u003cem\u003eHPL\u003c/em\u003e and pTRV2-\u003cem\u003eLS\u003c/em\u003e were transferred into \u003cem\u003eAgrobacterium\u003c/em\u003e\u003cem\u003e\u0026nbsp;tumefaciens\u003c/em\u003e strain GV3101 via electroporation and cultured on LB with kanamycin (100 µg/mL) and rifampicin (50 µg/mL) at 28°C for 72 h. Subsequent clones were resuspended in infiltration buffer (10 mM MES, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, and 200 μM acetosyringone) to achieve a bacterial concentration between 1.3 and 1.5 at OD\u003csub\u003e600\u003c/sub\u003e. For injection, equal volumes of bacterial suspension containing pTRV1 and either pTRV2-\u003cem\u003eHPL\u003c/em\u003e or pTRV2-\u003cem\u003eLS\u003c/em\u003e were mixed and injected into the abaxial side of cotyledons using a needless syringe. The inoculated seedlings were then cultured under standard plant culturing conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of VOCs using headspace-solid phase microextraction (HS-SPME).\u0026nbsp;\u003c/strong\u003eThe described test leaves were harvested and pulverized using a mortar and pestle under liquid nitrogen. Two grams of the pulverized leaf samples were placed into a 20 mL headspace vial containing an internal standard (cyclohexanone, 8,000 ppm), which was subsequently stored at -80°C until analysis. The samples were equilibrated at 40°C for 10 min in a thermostatic autosampler tray, followed by a 40-min exposure to the divinylbenzene /carboxyl/polydimethylsiloxane (DVB/CAR/PDMS) fiber (50/30 μm, Supelco, Bellefonte, PA, USA). The fiber was then thermally desorbed in the GC injector port at 250°C for 10 min (spitless). The GC–MS system (Agilent 8890A/5977B MSD Series, Agilent Technologies, Santa Clara, CA, USA) was operated in the electron impact (EI, ionization energy: 70 eV) mode with a scan range of m/z 35–550. VOCs were separated on a DB-WAX capillary column (30 m × 0.25 mm i.d., 0.25 μm film thickness; Agilent). The temperature program for the GC oven started at 35°C for 2 min, increased from 35–45°C at a rate of 2°C/min, then from 45–130°C at 5°C/min, and finally held at 225°C at a rate of 10°C/min for 5 min. Helium served as the carrier gas at a constant flow rate of 1.5 mL/min. The transfer line temperature was maintained at 250°C. VOC identification was conducted by matching mass spectra against the standard National Institute of Standards and Technology (NIST library version 20). Quantification of methyl hexanoate and linalool relied on calibration curves of pure standards. Each experiment was replicated three times with individual sample preparation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClassification of VOCs emitted from hot peppers and pattern analysis.\u0026nbsp;\u003c/strong\u003eThe VOCs identified via GC-MS were systematically classified into distinct groups such as GLVs and terpenes, according to their chemical structures. GLVs, exemplified by compounds like hexanal and its derivatives, exhibit straight-chain and branched-chain hydrocarbons with a basic C6 skeleton\u003csup\u003e53\u003c/sup\u003e. In contrast, terpenes consisted of isoprene unit compounds, which included monoterpenes (C10) and sesquiterpenes (C15)\u003csup\u003e54\u0026nbsp;\u003c/sup\u003ein this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis.\u0026nbsp;\u003c/strong\u003eAll tests involved three independent biological replicates. Means were compared using the least significant difference (LSD) test in a one-way analysis of variance (ANOVA) conducted with PROC GLM of the SAS program\u003csup\u003e55\u003c/sup\u003e. Significant differences were established at a Type I error of 0.05. Pattern analysis involved generating a phylogenetic tree using the ClustVis online tool (https://biit.cs.us.ee/clustvis/), and the corresponding heatmap was visualized using Prism 10.0.1 (GraphPad Software, Boston, MA, USA). \u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work was supported by a grant (No. 2022R1A2B5B03001792) from the National Research Foundation (NRF), funded by the Ministry of Science, ICT and Future Planning, Republic of Korea. This study was also supported by a research grant from Andong National University.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGovindarajan, V.S.: Capsicum production, technology, chemistry and quality. Part I. History, botany, cultivation and primary processing. Crit. Rev. Food Sci. 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SAS/STAT user\u0026rsquo;s guide, Release 6.03, Ed Cary, NC: (1989)\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"","lastPublishedDoi":"10.21203/rs.3.rs-6437640/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6437640/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"A plant emits diverse volatile compounds and communicates with other organisms in different trophic levels. Deciphering these compounds would be useful to understand their biorational interactions. Hot pepper, Capsicum annuum, produced different compositions of volatile organic compounds (VOCs) upon an infestation by the western flower thrips, Frankliniella occidentalis. These VOCs induced a non-preference behavior to the thrips while they attracted a natural enemy, Orius laevigatus. Among the differentially emitted VOCs, green leaf volatiles (GLVs) and terpenes were included and played crucial roles in their interactions. Z-3-methyl hexenoate, one of GLVs, induced the non-preference behavior but attracted the predator. Similarly, a terpenoid linalool attracted the predator but gave the non-preference to the thrips. Suppression of GLV or linalool biosynthesis was performed by virus-induced gene silencing of hydroperoxide lyase (HPL) or linalool synthase (LS) expression in the hot pepper and led to significant malfunction in the tri-trophic communication. The tri-trophic interactions were mediated by jasmonic acid (JA), but not salicylic acid, signal in the hot pepper. Insect resistance of the hot peppers against the thrips were positively correlated with HPL or LS expression levels, which would be useful for breeding programs for insect-resistant hot peppers.","manuscriptTitle":"Volatile organic compounds emitted from the damaged hot peppers are oppositely interpreted by thrips and its predator","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-05 19:17:10","doi":"10.21203/rs.3.rs-6437640/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":"9a38ad64-13a6-4d22-ae36-8182c0629f5b","owner":[],"postedDate":"May 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":47874226,"name":"Biological sciences/Zoology/Entomology"},{"id":47874227,"name":"Biological sciences/Plant sciences/Plant stress responses/Herbivory"}],"tags":[],"updatedAt":"2025-07-22T13:26:43+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-05 19:17:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6437640","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6437640","identity":"rs-6437640","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}