Functional Characterization of CaSpr2 in Jasmonate-Dependent Induced Defense Against Western Flower Thrips in Capsicum annuum

TL;DR

This study identifies a gene in peppers that helps defend against a harmful insect pest by regulating a key plant defense pathway.

Contribution

The study reveals CaSpr2 as a novel regulator in the jasmonic acid pathway critical for pepper resistance to western flower thrips.

Findings

Silencing CaSpr2 reduced jasmonic acid levels and increased pepper susceptibility to western flower thrips.

Thrips on CaSpr2-silenced plants showed improved fitness, including longer lifespan and higher reproduction rates.

CaSpr2 is essential for pepper resistance to thrips through its role in the jasmonic acid signaling pathway.

Abstract

Insect pests, such as the western flower thrip (WFT), poses a significant threat to global agriculture by damaging crops and reducing yield. Although the jasmonic acid (JA) signaling pathway is known to be involved in plant defense against WFTs, the key molecular components of this pathway in non-model crops like pepper remain poorly understood. This study functionally characterizes the role of suppressor of prosystemin-mediated responses2 (Spr2) in defense response against WFTs in pepper. We demonstrated that silencing CaSpr2 accumulated lower levels of JA and jasmonoyl-isoleucine (JA-Ile), and exhibited enhanced susceptibility to WFTs. Moreover, WFT individuals on CaSpr2-silenced plants exhibited enhanced fitness, including prolonged adult longevity, increased fecundity, and accelerated population growth. Our findings establish CaSpr2 as a crucial regulator within the JA-mediated signaling pathway that is essential for resistance to WFTs in pepper. This knowledge provides a valuable genetic target for breeding improved pepper cultivars with enhanced and sustainable pest resistance, potentially reducing reliance on chemical pesticides.

Insect infestation poses a significant threat to global agriculture by impairing plant growth and reducing crop yields. The western flower thrip (WFT) causes substantial damage through both direct feeding and transmission of plant viruses. Although the jasmonic acid (JA) signaling pathway is known to participate in plant defense against WFTs, the underlying molecular mechanisms in non-model crops such as peppers, remain largely elusive. This study investigates the role of suppressor of prosystemin-mediated responses2 (Spr2) within JA-mediated defense against WFTs in pepper. Through an integrated approach employing virus-induced gene silencing (VIGS), transcription analysis, phytohormone quantification, insect behavior assays and life history investigations, we demonstrated that silencing CaSpr2 significantly reduced JA and JA-Ile accumulation, and led to a strong feeding preference of WFTs for CaSpr2-silenced plants. Furthermore, the adult lifespan, survival rate, female fecundity, oviposition rate, and population parameters of WFTs were significantly improved on CaSpr2-silenced plants. Spr2 functions as an essential component within the JA signaling pathway, thereby playing a critical role in conferring resistance to WFTs in cultivated pepper. These findings provide profound insights and practical implications for breeding thrips-resistant cultivars in non-model plants, through genetic manipulation of JA signaling, offering a promising avenue for sustainable agricultural pest management.

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1. Introduction

Insect infestation is one of the most paramount factors that significantly impedes plant growth, diminishes crop productivity, and causes a myriad of other agricultural challenges [1]. Among these pests, the western flower thrip (WFT), Frankliniella occidentalis (Pergande), belonging to the family Thripidae and order Thysanoptera, emerges as one of the most detrimental phytophagous insects worldwide [2]. As a polyphagous insect pest, it damages leaves, flowers, and fruits by piercing plant tissues and ingesting cellular contents [3]. In pepper crops, thrips feeding leads to leaf curling and deformation, flower drop, a reduced fruit set rate, and abnormal fruit development [4]. The WFT also acts as a vector for plant viruses such as tomato spotted wilt virus (TSWV) and impatiens necrotic spot virus (INSV), with TSWV being a major contributor to pepper yield losses [5,6]. Furthermore, the WFT also prefers to hide in narrow crevices of plants, such as flower buds, developing fruits, or minute fissures in stems and bark. Its widespread insecticide resistance further complicates management [7]. Therefore, strategies based on plant defense responses are needed to mitigate WFT damage effectively.

Plants have evolved both constitutive and inducible defenses to protect themselves against herbivorous insects [8]. Constitutive defenses are inherently expressed, whereas induced defenses are triggered upon herbivore attack [9]. Inducible defenses often involve the synthesis of secondary metabolites that can directly deter or harm insects [10,11]. Phytohormones serve as crucial inducing factors that orchestrate plant defense responses, predominantly encompassing jasmonic acid (JA), salicylic acid (SA), ethylene (ET), and their related derivatives [12]. Among these, the JA signaling pathway stands out as the most pivotal pathway in regulating plant defense against phytophagous insects [13,14]. It is triggered by mechanical damage from insect feeding or by the exogenous application of JA or methyl jasmonate (MeJA) [15]. Activation of JA signaling induces secondary metabolites and volatiles that reduce insect feeding efficiency, slow growth, and limit reproduction [16,17,18]. The WFT is highly susceptible to JA-related induced defenses across various plant species, including Arabidopsis thaliana [19], kidney bean (Phaseolus vulgaris) [20], Chinese cabbage (Brassica rapa) [21], Chrysanthemum [22], cotton (Aphis gossypii) [23] and tomato (Solanum lycopersicum) [24]. Nevertheless, the mechanisms underlying the defense against phytophagous insects in pepper plants remain largely elusive.

This study focuses on the suppressor of prosystemin-mediated responses2 (Spr2) gene, which encodes a chloroplast fatty acid desaturase (FAD7) involved in JA biosynthesis and systemic wound signaling [25]. FAD7 appears to modulate JA-dependent defenses against chewing insects and SA-dependent defenses against aphids via distinct effects on JA synthesis and SA signaling [26]. Arbuscular mycorrhizal fungi (AMF) colonization is significantly reduced in spr2 mutant tomato plants, aligning with other phenotypic impacts of impaired FAD7 function [27]. In tomato, spr2 mutant plants show reduced volatile organic compound levels and compromised resistance to herbivorous insects [28,29]. To investigate the role of Spr2 in pepper defense against the WFT, and considering the difficulty in obtaining pepper mutant materials, the pTRV (tobacco rattle virus)-based VIGS vectors were employed to generate Spr2-silenced pepper plants (TRV-CaSpr2). Subsequently, a comprehensive analysis was conducted, wherein the endogenous levels of JA and JA-Ile were measured in both TRV-GFP (control) and TRV-CaSpr2-infected pepper plants. Moreover, selection preference experiments were performed, and the detailed lifespan tables for WFTs were constructed to evaluate the ecological impact of Spr2 silencing. The results demonstrated that TRV-CaSpr2 plants had lower JA levels, were more attractive to WFTs and provided a more conducive environment for the survival and reproduction of the WFT population.

2. Materials and Methods

2.1. Insects and Plants

The initial population of F. occidentalis was generously donated by the Institute of Plant Protection, Hunan Academy of Agricultural Sciences, China. Subsequently, these insects were maintained on broad beans (Vicia faba) within an artificial climate incubator set at 25 °C, a relative humidity of 65 ± 5%, and a photoperiod consisting of 14 h of light followed by 10 h of darkness, with a light intensity of 4800 lx. Pepper plants (Capsicum annuum) of the edible hot pepper variety ‘Bo La’ were obtained from Hunan Xingshu Seed Company (http://www.xsseed.com, accessed on 29 March 2021). The plants were cultivated in a growth chamber under a daytime temperature of 25 °C and a nighttime temperature of 22 °C, with a light-dark cycle of 16 h and 8 h, respectively.

2.2. Sequence Comparisons and Phylogenetic Analyses

The full-length protein sequences employed for sequence comparisons and phylogenetic analyses were sourced from the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/, accessed on 15 May 2022). Pairwise sequence comparisons and calculation of sequence identities were conducted utilizing CLUSTALX (http://www.clustal.org, accessed on 20 May 2022) [30]. The average pairwise distances were determined using MEGA7.0.2 (https://www.megasoftware.net/, accessed on 21 May 2022). Phylogenetic trees were constructed via the Neighbor-Joining method, incorporating strict distance measures and employing randomized bootstrapping to evaluate the validity of branching patterns, all implemented within the MEGA7.0.2 framework. The robustness of the inferred evolutionary relationships was rigorously assessed by 1000 bootstrap replicates [31].

2.3. TRV-Mediated Silencing Assays

For TRV-based VIGS assay [32,33,34], DNA fragments corresponding to partial sequences of CaPDS (phytoene desaturase) and CaSpr2 were independently amplified via PCR, employing specific primers (Table S1). The amplified fragments were cloned into the pTRV2-vector, thereby generating pTRV2-PDS and pTRV2-Spr2. These constructs were transformed into Agrobacterium tumefaciens strain GV3101 individually. Subsequently, Agrobacterium cultures harboring pTRV1, or one of the three plasmids (pTRV2-GFP, pTRV2-CaPDS and pTRV2-CaSpr2) were mixed at a 1:1 ratio to achieve a final OD_600_ of 1.0. This mixture was then infiltrated into the fifth to sixth leaves of three-week-old N. benthamiana plants to facilitate the proliferation of TRV, in order to achieve a higher inoculation efficiency. Four days post-infiltration, crude extracts were prepared by homogenizing the infiltrated leaf tissue in 0.01 M phosphate buffer (pH 7.0) at a 20% (w/v) ratio. The homogenate was centrifuged at 5000 rpm for 10 min at 4 °C, and the resulting supernatant was collected for mechanical inoculation onto the fifth to sixth leaves of four-week-old pepper plants. The pepper plants were cultivated in a growth chamber under a daytime temperature of 25 °C and a nighttime temperature of 22 °C, with a light-dark cycle of 16 h and 8 h, respectively.

2.4. RNA Isolation and RT-qPCR

Total RNA extraction was carried out using Trizol Reagent (Vazyme, Nanjing, China) adhering to the manufacturer’s protocol. Subsequently, cDNA was synthesized from the extracted total RNA employing a cDNA synthesis kit (TransGen, Beijing, China). For the quantitative real-time polymerase chain reaction (qPCR) analysis, SYBR Green Supermix (Vazyme, Nanjing, China) was utilized in accordance with the protocol. The reference gene CaActin served as an internal control to normalize and determine the relative expression levels of other genes. All primers employed in the qPCR experiments are detailed in Table S1. The relative gene expression was calculated by applying the geometric mean of threshold cycle (Ct) values for the reference gene CaActin, utilizing the 2^−ΔΔCt^ method. The statistical comparisons of gene expression levels were conducted using Student’s t-test (data were presented as mean ± SE of three biological replicates, with the significance threshold set at p < 0.05), and the results were visualized using GraphPad Prism 9.0.0 software (https://www.graphpad.com/, accessed on 18 October 2022).

2.5. Hormone Quantification

Leaf samples, each weighing 100 mg, were collected from pTRV-GFP and pTRV-CaSpr2 plants. These samples were then promptly flash-frozen in liquid nitrogen and subsequently stored at −80 °C. To quantify the contents of JA and JA-Ile, high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) was employed, following the previously described methodology [35]. The statistical comparisons of hormone contents between different groups were conducted using Student’s t-test (data were presented as mean ± SE of three biological replicates, with the significance threshold set at p < 0.05), and the results were visualized using GraphPad Prism 9.0.0.

2.6. Selection Preference of WFTs for Host Plants

Preference tests to evaluate WFT responses to plants were conducted using a glass Y-tube olfactometer (Fujian Minbo Glass Co., Ltd., Fuzhou, China), following the method previously described [36]. The olfactometer featured a Y-shaped tube with a central stem branching into two arms, each connected to a distinct glass odor bottle. One bottle contained TRV-GFP-infected pepper leaves, while the other held TRV-CaSpr2-infected pepper leaves. To ensure sufficient odor dispersion, the tested odors from pepper leaves were sealed in vials 5 min prior to each trial, allowing the odors to permeate the arms. For each trial, individual 2-day-old thrips, starved for 1 h, were introduced into the central tube and permitted to choose between the two arms. Each treatment involved 30 WFTs, with five replicates conducted per treatment. The statistical comparisons of selection preference experiments were conducted using chi-squared (χ^2^) test (data were presented as mean ± SE, with the significance threshold set at p < 0.05), and the results were visualized using GraphPad Prism 9.0.0.

2.7. The Life Tables of WFT Feeding on TRV-GFP and TRV-CaSpr2-Infected Pepper Plants

In alignment with the age-stage two-sex life table theory [37,38] and following the methodological framework of Li et al. [39], we carried out a systematic observational study on WFTs that were feeding on TRV-GFP or TRV-CaSpr2-infected pepper leaves. We precisely recorded the number of surviving thrips at each distinct developmental stage, closely monitored and documented their feeding conditions, and accurately tallied the egg-laying quantity of female thrips. Subsequently, leveraging the comprehensive dataset thus obtained, we constructed highly detailed life tables for the WFTs.

The life history and population parameters of the WFTs fed on TRV-GFP or TRV-CaSpr2-infected pepper plants were calculated and analyzed employing the two-sex life table analysis method, in conjunction with the paired bootstrap test in TWOSEX-MSChart software v.2020 [39,40], and the results were visualized using SigmaPlot v.12.0 (https://systat-sigmaplot.com/, accessed on 10 January 2023).

3. Results

3.1. Identification and Silencing of CaSpr2 in Pepper

To investigate the function of CaSpr2, the amino acid sequences of Spr2 from various solanaceous crops (pepper, tomato, potato, tobacco, etc.) were downloaded from the GenBank database, and further phylogenetic and homology analyses were conducted. The Spr2 proteins derived from three pepper species were clustered within the same sub-clade, distinctly separated from those of tobacco, tomato, and potato (Figure 1A). These Spr2 proteins have been previously implicated in JA biosynthesis.

Given the substantial challenges associated with acquiring transgenic pepper materials, we opted to employ a TRV-based VIGS to silence CaSpr2 in pepper plants. Meanwhile, TRV-GFP served as the negative control and TRV-CaPDS was utilized as the positive control due to its characteristic photobleaching phenotype [41]. Following this, the full-length coding sequences (CDS) of CaSpr2 and CaPDS were amplified (Figure S1A,B). Leveraging the VIGS online tool (https://vigs.solgenomics.net/, accessed on 6 July 2022), specific sequences of CaSpr2 and CaPDS were elected and amplified (Figure S1C,D), which were then individually cloned into the pTRV2-vector. Initially, N. benthamiana plants were infected with the TRV construct, and then the infected tissue was used to inoculate pepper plants. Compared with the control TRV-GFP-infected plants, a marked bleaching phenotype was evident on the CaPDS-silenced pepper leaves, and CaSpr2-silenced pepper leaves exhibited a mild chlorosis (Figure 1B). The transcript levels of CaPDS and CaSpr2 were quantified by reverse-transcription quantitative polymerase chain reaction (RT-qPCR) analysis. The results revealed that, relative to the GFP control, the expression level of CaPDS was reduced by approximately 80%, and the expression level of CaSpr2 was diminished by roughly 70% (Figure 1C). These findings underscore the efficacy of the TRV-based VIGS system in effectively suppressing gene expression in pepper plants.

3.2. Silencing of CaSpr2 Exerts a Negative Impact on JA Accumulation and Attracts the Feeding of WFTs

To delve into the impact of CaSpr2 silencing on the accumulation of JA and its bioactive conjugate JA-Ile [42], we evaluated the phytohormone levels in leaves of pepper plants infected with either TRV-GFP or TRV-CaSpr2. The results revealed that silencing of CaSpr2 led to a reduction in the levels of JA and JA-Ile (Figure 2A,B). Subsequently, we conducted a further investigation into the effect of CaSpr2 silencing on the accumulation of JA and JA-Ile in pepper plants in response to thrips infestation. Statistically significant differences were observed at both 12 h and 24 h post-infestation for JA and JA-Ile levels (Figure 2C,D). These findings indicated that CaSpr2 silencing compromised the induction of JA and JA-Ile in pepper leaves subjected to thrips feeding. Collectively, they also underscore that CaSpr2 is pivotal for the biosynthesis of JA and JA-Ile in response to herbivore attack.

To investigate the behavioral response of WFTs to CaSpr2-silenced pepper plants, we evaluated the orientation preference patterns of thrips exposed solely to plant odors (odors from either TRV-GFP or TRV-CaSpr2-infected pepper leaves) under conditions devoid of any additional visual, gustatory, or tactile cues. The results revealed that 74.2% of WFTs exhibited a pronounced preference for TRV-CaSpr2-infected pepper leaves, demonstrating a statistically significant difference compared to those exposed to TRV-GFP-infected pepper leaves (Figure 2E). These results underscore the WFTs were attracted by the plant odors of CaSpr2-silenced pepper plants.

3.3. Silencing of CaSpr2 Is Beneficial for the Developmental Durations of WFTs

The duration of the immature stages of WFTs fed on TRV-GFP and TRV-CaSpr2-infected pepper leaves were stated in Table 1. With the exception of a marginal disparity in the developmental time during the second instar larval stage, there was virtually no difference in the other developmental time between the two treatment groups (Table 1). Similarly, no significant variations were detected for the survival rates from egg to adult stage (preadult survival rate). Notably, the longevity of both female and male thrips that fed on CaSpr2-silenced leaves was significantly prolonged compared to those that fed on control leaves (Table 1). Regarding the total preoviposition period (TPOP), no statistically significant difference was observed between two treatment groups. Furthermore, the mean fecundity of females exposed to TRV-CaSpr2-infected pepper leaves reached 79.58 eggs, a value that was markedly elevated compared to the mean fecundity (48.64 eggs) of females subjected to TRV-GFP-infected pepper leaves (Table 1). These findings collectively suggest that the dietary intake of CaSpr2-silenced pepper leaves by WFTs primarily exerts an influence on their longevity and fecundity.

3.4. The Survival Rates of WFTs Reared on CaSpr2-Silenced Leaves Significantly Increased

The age-stage-specific survival rate (s_xj_) of WFTs reared on CaSpr2-silenced and control leaves delineated the probability of a newly oviposited egg surviving to age x and stage j (Figure 3A,B). The survival curves for consecutive life stages overlapped substantially, reflecting the natural variation in developmental rates among individual thrips. Male thrips fed on CaSpr2-silenced leaves exhibited greater longevity, surviving up to 40 days compared to 34 days for males on control leaves (Figure S2A). Adult survival rates were also significantly higher on CaSpr2-silenced leaves than on control leaves. The peak survival rate for females on TRV-CaSpr2 leaves was 0.44, whereas it reached only 0.37 on TRV-GFP leaves (Figure S2B).

The age-specific survival rate (l_x_), age-stage-specific fecundity (f_x_), age-specific fecundity (m_x_), and age-specific net maternity (l_x_m_x_) of WFTs fed on CaSpr2-silenced leaves and control leaves were presented in Figure 3C,D. The l_x_ curve revealed a decline in survival probability as the age of the thrips increased. In the control group, survivorship decreased rapidly beginning on 14th day, with all WFTs succumbing by the 42nd day. Also, for the CaSpr2-silenced group, survivorship declined rapidly from the 20th day onwards, and complete mortality was observed by the 44th day. The age-stage-specific fecundity curve (f_x_) indicated that female adults in both groups commenced reproduction at 13 days of age. The peak of mean fecundity was recorded at 18.5 days, with 4.46 eggs laid for WFTs fed on control leaves and 4.83 eggs for those on CaSpr2-silenced leaves (Figure 3C,D). Notably, the net maternity of WFTs was higher when they were fed on CaSpr2-silenced leaves compared to the control leaves. The peak value of l_x_m_x_ for the TRV-CaSpr2 group was 2.13 at 18.5 days, while the peak for the TRV-GFP group was 1.26 at the same age (Figure S2C).

3.5. The Life Expectancy and Reproductive Values of WFTs Fed on TRV-CaSpr2-Infected Pepper Leaves Are Prolonged

The life expectancy (e_xj_) represents the probability that an individual of age x and stage j will survive to age and stage. The mean longevity of WFTs, defined as the life expectancy at age zero (e01), was 21.92 days on TRV-GFP-infected plants, and 26.62 days on TRV-CaSpr2-infected plants (Figure 3E,F). The age-stage reproductive value (v_xj_) quantifies the contribution of an individual at age x and stage j to the future population. The v_xj_ demonstrated an initial increase followed by a decrease with advancing age of WFTs (Figure 3G,H). Furthermore, the peak v_xj_ values for female adults were recorded at 18 days, with values of 33.44/d on TRV-CaSpr2-infected leaves and 28.45/d on TRV-GFP-infected leaves (Figure S2D).

3.6. WFTs Demonstrates Better Population Parameters When Feeding on CaSpr2-Silenced Leaves

The impacts of CaSpr2-silenced leaves and control leaves on the population parameters of WFTs are detailed in Table 2. When compared to WFTs fed on control leaves, those consuming CaSpr2-silenced leaves displayed a marked and statistically significant increase in their population parameters. Specifically, the intrinsic rate of increase (r = 0.174 d^−1^), the finite rate of increase (λ = 1.190 d^−1^), and net reproductive rate (R0 = 35.105) were all substantially higher for WFTs on CaSpr2-silenced leaves than for those on control leaves (r = 0.143 d^−1^, λ = 1.154 d^−1^, R0 = 19.661, respectively). There was no statistically significant difference in the mean generation time (T) between WFTs reared on CaSpr2-silenced leaves (T = 20.393d) and those on control leaves (T = 20.701 d). These findings suggest that WFTs demonstrate enhanced fecundity or accelerated developmental rates when feeding on CaSpr2-silenced leaves.

4. Discussion

The regulatory mechanisms of JA in non-model plants like pepper (C. annuum) remain incompletely understood due to limited genetic and genomic resources. Recent advances, including VIGS and pepper genomic data, now allow functional dissection of hormone-regulated defense networks. These methodological breakthroughs provide a valuable opportunity to bridge fundamental plant molecular biology with applied insect pest management research.

The Spr gene family was identified in tomato as suppressors of 35S::prosystemin-mediated phenotypes, regulating systemic induction of defense genes triggered by herbivory or wounding [43,44]. Extensive studies on spr mutants reveal that these genes not only modulate JA accumulation but also influence herbivore behavior, performance, and population dynamics. For example, the spr1 mutant exhibits defects in prosystemin perception and the transmission of systemic wound signals [45], while the spr2 mutants show impaired wound-induced and constitutive JA synthesis [28]. Interestingly, it exhibits increased susceptibility to infestation by tobacco hornworm and silverleaf whitefly [29], but negatively impacts the settling behavior, survival rate, and fecundity of the potato aphid [26]. Additionally, the spr6 mutant displays deficiencies in wound- and prosystemin-induced defense gene expression, and its responsiveness to exogenous JA shows a dosage dependency [46]. Notably, spr2 mutants display contrasting effects on different insect guilds, influencing phytophagous insects in distinct ways, highlighting the ecological complexity of JA-mediated defenses and their role in shaping plant–insect interactions. These findings from tomato mutants lay a foundation for investigating the effects of Spr homologs on insect resistance in solanaceous crops and provide a reference for validating the function of Spr genes in pepper.

Building upon these findings, our study extends the functional characterization of Spr homologs to pepper, a non-model but globally important crop that suffers severe losses from WFTs. By integrating VIGS, phytohormone quantification, insect behavioral assays, and life-history analyses, we demonstrate that CaSpr2 functions as a positive regulator of JA and JA-Ile accumulation and restricts WFT growth, survival, and reproduction. The role of CaSpr2 in regulating JA accumulation also raises questions regarding potential growth–defense trade-offs. JA signaling antagonizes growth-related pathways, and excessive or constitutive JA activation can impair photosynthesis, biomass accumulation, and reproductive output [47]. Thus, while loss of CaSpr2 compromises JA-mediated defense and increases WFT susceptibility, our data do not suggest that constitutive CaSpr2 overexpression would enhance resistance without fitness costs. Instead, CaSpr2 appears to function as a regulatory node that contributes to balancing defense activation with plant physiological performance.

This study confirms the essential role of CaSpr2 in JA biosynthesis and defense against western flower thrips (WFTs) in pepper, offering direct evidence for JA-mediated regulation of insect resistance in a non-model plant. From an applied perspective, CaSpr2 should be considered a strategic target for the fine-tuned modulation of JA signaling rather than a single-gene solution. For instance, tissue-specific, inducible, or stress-responsive regulation of CaSpr2 or its downstream components could enhance resistance during herbivory while mitigating potential growth penalties under non-stress conditions. Beyond direct genetic manipulation, these findings suggest alternative routes for translating CaSpr2-associated mechanisms into pest management. CaSpr2 and related JA biosynthetic elements may serve as molecular markers in breeding programs to select pepper genotypes with optimized basal defense. Moreover, targeting specific downstream outputs of CaSpr2-mediated JA signaling, such as herbivore-induced volatiles or defensive metabolites, could provide a more selective means of deterring WFTs without broadly activating JA responses. This approach would be particularly valuable within an integrated pest management framework.

Current thrips management in pepper cultivation depends predominantly on chemical insecticides, a practice that has resulted in widespread pest resistance, environmental contamination, and the disruption of beneficial arthropod communities. Consequently, augmenting endogenous JA-mediated resistance via precisely regulated genetic or molecular breeding offers an environmentally compatible supplement to, rather than a substitute for, conventional control measures. Together, these results establish CaSpr2 as a crucial regulatory element in pepper defense against WFTs and furnish a conceptual and experimental framework for future endeavors to harness hormone-mediated resistance within sustainable pest management strategies.