Chemical libraries were purchased from TargetMol (Shanghai, China) and Selleck (Houston, TX). Synthetic PACAP 1-38, PACAP 1-27, and PACAP 6-38 (≥95% purity) were obtained from GenScript (Nanjing, China) and dissolved in sterile water. The identity and purity of the synthesized PACAP peptides were confirmed by mass spectrometry (,Supplementary Figure S4).

Wild-type Arabidopsis ecotype Col-0, corresponding transgenic lines, and Nicotiana benthamiana were used in this study. Unless otherwise specified, all plants were cultivated in a controlled-environment greenhouse. Growth conditions were maintained at 20–25 °C under a 16-h light/8-h dark photoperiod.

For Arabidopsis and N. benthamiana, seeds were surface sterilized by immersion in 30% (v/v) sodium hypochlorite solution containing 0.02% (v/v) Tween-20 for 15 min with gentle agitation, followed by five rinses with sterile distilled water to remove residual sterilizing agents. The sterilized seeds were subjected to cold stratification at 4 °C for 2–3 days to break dormancy and promote synchronized germination. Seeds were then sown on 1/2 Murashige and Skoog (MS) (PhytoTechLABS, Beijing, China) basal medium (pH 5.7) supplemented with 0.5% (w/v) sucrose as a carbon source and solidified with 0.4% (w/v) Phytagel (OriLeaf, Nanjing, China). Plates were placed vertically in a controlled growth chamber under continuous white light (40 μmol·m⁻²·s⁻¹) at 22 °C for seedling cultivation. After 7–10 days, seedlings were either transferred to soil or directly used for subsequent experiments, depending on the experimental design.

For histochemical staining of transgenic lines, the predicted FRK1 promoter (~2 kb upstream of the ATG start codon) was amplified from Col-0 genomic DNA. The proFRK1::GUS construct was cloned into the pGWB3 vector and then introduced into Col-0 plants by the floral dip method. Homozygous T3 plants were selected for analysis. Seedlings were vacuum infiltrated with staining solution containing 100 mM NaH₂PO₄ (pH 7.0), 10 mM Na₂EDTA, 0.5 mM K₄[Fe(CN)₆]·3H₂O, 0.5 mM K₃;[Fe(CN)₆], 0.1% (v/v) triton X-100, and 1 mM X-Gluc (5-bromo-4-chloro-3-indolyl β-D-glucuronide, cyclohexylammonium salt). Samples were incubated at 37 °C in the dark for 14 h. Chlorophyll was removed with 95% ethanol until tissues were clear, and staining patterns were documented by photography. The histochemical assay of GUS activity was carried out as previously reported (Ye et al., 2020).

Total RNA was extracted from plant tissues using the Ultrapure RNA Kit (Cowin Biotech, China). RNA integrity was verified by agarose gel electrophoresis, and concentration and purity were measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Nanjing, China). One microgram of total RNA was treated with genomic DNA wiper and reverse-transcribed into cDNA using the HisScript II Q RT SuperMix kit (Vazyme, Nanjing, China). RT-qPCR was performed with ChamQ Universal SYBR qPCR Master Mix (Takara, Beijing, China) on a Bio-Rad CFX Connect™ Real-Time PCR Detection System. Relative expression levels were calculated using the 2^−ΔΔCt method, with EF1a as an internal control. Each assay included three biological replicates and three technical replicates.

Seven-day-old Arabidopsis seedlings grown on 1/2 MS medium under growth conditions maintained at 20–25 °C with a 16-h light/8-h dark photoperiod were transferred into 24-well plates containing sterile water one day before treatment and incubated overnight (approximately 8 h). On the following day, seedlings were treated with PACAP peptide solution for the indicated times (e.g., 15 min, 30 min, and 1 h). Frozen tissues were ground with 3-mm zirconia beads using a FastPrep 24 grinder, and total proteins were extracted in buffer containing 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 2 mM EDTA, 5 mM DTT, 1× EDTA-free protease inhibitor cocktail (Roche), and 1% Triton X-100. Equal amounts of protein (14 μL per lane) were subjected to SDS–PAGE using 4–20% gradient gels and transferred to PVDF membranes (Millipore) at 100 V for 1 h.

Phosphorylated MPK3 and MPK6 were detected using rabbit monoclonal anti-phospho-p44/42 MAPK (Erk1/2) antibody (Cell Signaling Technology, Shanghai, China; 1:2000). HRP-conjugated goat anti-rabbit IgG (Abmart, Shanghai, China; 1:12,000) was used as the secondary antibody. Blots were visualized by ECL and imaged with a Tanon 3600 system (Tanon, Beijing, China), with exposure times of 30 s–2 min. All experiments were repeated at least three times with consistent results.

Calcium influx was measured using the Col-Q (Columbia aequorin) Arabidopsis line as previously reported (Choi et al., 2014). A 100 μL mixture containing CaCl₂ (1 M, 100×), MES (200 mM, 100×), and coelenterazine (CTZ, 1 mM, 100×) was prepared and added to each well of a 96-well plate. Seven-day-old seedlings grown on 1/2 MS medium were transferred into the plate and incubated overnight (approximately 8 h) to allow aequorin reconstitution.

On the following day, 50 μL of treatment solutions were added to each well. PACAP and PACAP 6–38 were dissolved in sterile water and applied at final concentrations of 20, 50, and 100 μM. Luminescence signals were recorded for 30 min using a High Sensitivity Plate Luminescence Detector (BLT Lux-P110, Guangzhou, China), and peak luminescence values were used for quantification. Each data point was measured using 8 seedlings (technical replicates), and the experiment was independently repeated three times, yielding consistent results.

Imaging was performed using a Tanon 3600 imaging system (Tanon, Beijing, China) to quantify calcium influx, with 10–15 seedlings per well. The experiment was repeated three times.

P. syringae pv. tomato DC3000 was cultured on King’s B (KB) agar medium (12.5 g/L King’s B base, 15 g/L agar, 10 mL/L glycerol) at 28 °C for 48 h. Single colonies were inoculated into 4 mL of KB liquid medium and grown overnight at 28 °C with shaking at 200 rpm until the OD₆₀₀ reached 0.6–0.8. The bacterial cells were harvested by centrifugation and resuspended in 10 mM MgCl₂ to an OD₆₀₀ of 0.002 for inoculation.

To examine whether peptide pretreatment enhances plant resistance, fully expanded leaves of 4-week-old Arabidopsis plants were infiltrated with PACAP or its antagonist PACAP 6–38 one day before bacterial inoculation using a 1 mL needleless syringe. Control plants were treated with sterile water (mock). After 24 h, leaves were inoculated with P. syringae pv. tomato DC3000 suspension as described above.

After infiltration, excess bacterial solution was removed with sterile filter paper, and plants were maintained under high-humidity conditions in a growth chamber. At 2 or 3 days post-infection (dpi), three leaf discs (0.5–0.6 cm diameter) per leaf were collected, surface-sterilized with 75% ethanol for 30 s, rinsed twice with sterile distilled water, and homogenized in 10 mM MgCl₂. Serial dilutions of the homogenates were plated on KB agar containing rifampicin (50 μg/mL). Plates were incubated at 28 °C for 48 h before counting colony-forming units (CFUs). For each biological replicate, at least nine individual plants were analyzed, and the experiment was repeated three times with consistent results.

For the R. solanacearum soaking infection assay, two-week-old Nicotiana tabacum cv. Yabuli seedlings grown on 1/2 MS agar medium plates were gently removed and recovered in sterile ddH₂O overnight prior to inoculation. Roots were slightly wounded using a sterile blade to facilitate bacterial entry. Seedlings were then infiltrated with PACAP, PACAP 6–38, or ddH₂O as a mock control. Twelve hours after peptide or mock treatment, a suspension of R. solanacearum at a concentration of 1 × 10⁶ CFU mL⁻¹ was infiltrated into the same tissues.

Samples were collected at 1 day post-inoculation for bacterial quantification. Tissues were weighed, homogenized in sterile water, and serial dilutions were plated onto solid CPG medium. Colony-forming units (CFUs) were counted after incubation at 28 °C for 2 days and expressed as CFU per gram of fresh tissue.

Leaf discs (0.125 cm²) were excised from the 7th to 9th true leaves of 4-week-old Arabidopsis plants and placed in 96-well plates containing 100 µL sterile water. Samples were incubated overnight under low light conditions. The water was then replaced with 100 µL of reaction solution containing 20 µM L-012 (TargetMol Chemicals Inc., Shanghai, China), 10 µg/mL horseradish peroxidase (Beyotime Biotechnology, Shanghai, China), and either flg22 or the test chemicals. Chemiluminescence was recorded immediately for 40–90 min using a High Sensitivity Plate Luminescence Detector (BLT Lux-P110). The experiment was repeated three times with consistent results.

FLG22-INDUCED RECEPTOR-LIKE KINASE 1 (FRK1) is an early marker gene of PTI in Arabidopsis (Chinchilla et al., 2007; Thor et al., 2020). In this study, a FRK1 promoter-based high-throughput screening system was employed using transgenic pFRK1-GUS seedlings, in which the FRK1 promoter is fused to the β-glucuronidase (GUS) reporter gene (Figure 1A). A large-scale screening was conducted using pFRK1-GUS Arabidopsis reporter seedlings, which were exposed to approximately 10,000 compounds from various commercial libraries. Five-day-old seedlings were grown under uniform conditions in 96-well plates and treated with individual compounds, and GUS staining was evaluated 24 hours after treatment. Primary hits were selected based on visibly intensified blue coloration compared to the negative control (Figure 1A).

Since GUS staining served only as a preliminary indicator of immune activation, a subset of these candidates underwent secondary validation through immune response assays, including MAPK phosphorylation, defense gene expression, and ROS production, to eliminate false positives and confirm genuine immune activation (Ma et al., 2025). Following this validation, it was discovered that the neuropeptide PACAP and its receptor antagonist fragment PACAP 6–38 function as plant immune elicitors. Notably, both PACAP 1–27 and PACAP 6–38 are truncated derivatives of PACAP 1-38 (Figure 1B). All three PACAP peptides activated the expression of pFRK1-GUS in a concentration-dependent manner (Figure 1C; ,Supplementary Figure S1). Subsequent experiments employed 20 μM PACAP based on these dose-response observations, as lower concentrations produced minimal or inconsistent immune responses. Together, these results indicate that PACAP may play a role in plant defense responses by activating early immune gene expression.

Calcium serves as a crucial second messenger in all eukaryotic cells and plays a vital role in plant immune signaling (Jiang and Ding, 2023). Under normal physiological conditions, the concentration of free calcium in the apoplast is significantly higher than in the cytosol (Demidchik et al., 2018). Upon pathogen infection or elicitor recognition, a rapid calcium influx is triggered, which initiates downstream immune signaling (Zipfel and Oldroyd, 2017; Yuan et al., 2017). To investigate the effects of PACAP on the induction of free cytosolic calcium ([Ca²⁺]_cyt), an aequorin-based Ca²⁺ reporter system combined with a luminescence microplate reader was used. The quantitative analysis showed that PACAP 1–38 rapidly induced [Ca²⁺]_cyt in Arabidopsis in a dose-dependent manner. Notably, both PACAP 1–38 and PACAP 6–38 induced significant calcium signaling, while PACAP 1–27 exhibited relatively weak activity compared to the other two variants (Figure 2A). Additionally, the in vivo bioluminescence imaging results further confirmed the increase in [Ca²⁺]_cyt after PACAP 1–38 and PACAP 6–38 treatments (Figure 2B).

To explore whether PACAP and its truncated form, PACAP 6-38, could activate other hallmark events in plant immunity, the activation of MAPK was examined by immunoblot analysis. The results demonstrated that treatment with either PACAP 1–38 or PACAP 6–38 effectively induced MAPK phosphorylation in Arabidopsis seedlings (Figure 2C). And this induction was detectable as early as 15 minutes after treatments, which mirrored PAMP-induced MAPK phosphorylation (Figure 2C). RT-qPCR analysis indicated that PACAP-induced FRK1 expression, which was consistent with the pFRK1-GUS results (Figure 2D). Furthermore, the expression of WRKY29, another immune marker gene, was induced by PACAP1–38 in a time-dependent manner (Figure 2D). Interestingly, although PACAP triggered Ca²⁺ influx, MAPK phosphorylation, and defense gene expression, none of the three variants (PACAP 1–38, 1–27, 6–38) induced a detectable ROS burst at 20–100 μM over two hours. Control assays with flg22 confirmed the assay’s sensitivity, indicating that PACAP activates immune responses without a canonical ROS burst (,Supplementary Figure S2).

Next, it was examined whether PACAP-triggered early immune responses are sufficient to confer effective pathogen resistance in plants. Following inoculation with pathogenic strain P. syringae pv. tomato DC3000, the results indicated that both PACAP 1–38 and PACAP 6–38 enhanced plant resistance to this pathogen (Figure 3A). Consistently, PACAP 1-38, PACAP 1–27 and PACAP 6–38 also enhanced plant resistance to R. solanacearum (Figure 3B). Additionally, in vitro growth assays were conducted to evaluate the antimicrobial activity of PACAP, which revealed that PACAP did not directly inhibit the growth of the tested pathogens (,Supplementary Figures S3A, B). In summary, through the chemical screening and functional validation, it was identified that PACAP is an effective plant immunity inducer that activates several hallmark responses associated with early immune signaling.