The Ca²⁺ Channel CNGC19 Regulates Arabidopsis Defense Against Spodoptera Herbivory

Plant and Insect Materials

Arabidopsis (Arabidopsis thaliana) seeds (ecotype Columbia; Col-0) and mutant lines with a T-DNA insertion in the exon of AtCNGC19 (At3g17690), namely SALK_129200C and SALK_027306 (provided by TAIR; Alonso et al., 2003) were used. The absence of CNGC19 mRNA in the homozygous SALK_ 129200C and SALK_27306 were checked by RT-PCR using CNGC19 gene-specific primers. The cngc19-1 (SALK_27306) line has been characterized for its involvement in salt stress in Kugler et al. (2009). AtCNGC20 (SALK_0129133C) and AtCaM2 (SALK_114166C) were also obtained from TAIR. The cam2 line was previously characterized by Zhang et al. (2009). The bcat4 (AT3G19710) mutant line SALK_013627 was kindly provided by Stefan Binder (University of Ulm). pepr1 pepr2 double mutants were provided by Gerald Berkowitz (University of Connecticut). GCaMP3 lines were gifted by Dale Sanders (John Innes Centre). Seeds were sown in 10-cm pots and stratified for 2 d at 4°C in darkness. Afterwards, plants were moved to ventilated growth rooms with constant air flow and 60% humidity at 22°C. Plants were grown at a 10-h light/14-h dark photoperiod and a light intensity of 150 μmol m⁻² s⁻¹. Larvae of Spodoptera litura were hatched from eggs and reared on an agar-based optimal diet (Bergomaz and Boppré, 1986). The insect biomass assay was done using first-instar larvae (freshly hatched larvae grown for 3 d in light), which were preweighed before the experiment and selected to have equal weights. Three larvae were placed on a single plant and covered with perforated plastic covers. After 8 d of feeding, all larvae were removed and weighed individually. Each experiment had 10 plants and the experiments were repeated three to four times independently. For phytohormone and GS estimation, 1-d feeding was performed with fourth-instar S. litura larvae which were starved 12 h before plant feeding.

Plant Treatments

All simulated herbivory experiments were performed 6 weeks postgermination at a vegetative (prebolting) growth stage. For experiments with insect OSs, the adaxial (upper) side of the leaf was wounded with a pattern wheel (six vertical motions). Fourth-instar S. litura larvae were reared on an artificial diet of Arabidopsis leaves for 24 h before collecting OS on ice. The OS was centrifuged at 13,000 rpm for 2 min and diluted 1:1 with water. A total of 20 μL of fresh diluted OS was spread across all the holes on a single leaf (W + OS). In control plants, water was added (W+Water). coi1-1 homozygous plants were selected by CAPS marker (Xie et al., 1998). This method for simulated herbivory was also used for CNGC19 expression in the coi1-1 and pepr1 pepr2 mutant lines. For CNGC19 expression analysis in systemic leaves upon simulated herbivory, leaves were numbered, leaf number 8 was wounded with a pattern wheel, and water/OS was applied. Systemic leaves 5, 11, 13, and 16 are known to have vascular connection with leaf number 8 (Mousavi et al., 2013; Kiep et al., 2015), and were harvested after 30 min and 60 min for gene expression studies. For CNGC19 expression in Spodoptera-fed plants, fourth-instar S. litura larvae were transferred to wild-type plants and allowed to feed for the indicated time intervals. Chemically synthesized JA-Ile (Walter et al., 2007; Scholz et al., 2014) was dissolved in 100% ethanol to make 300-mM stock, which was then diluted in water to a final concentration of 500 μM. Fourteen-d–old seedlings were treated with 500-μM JA-Ile for the indicated time intervals.

CNGC19 and BCAT4 Promoter Reporter Assays

For the promoter β-glucuronidase (GUS) fusion construct, a 2.6-kb fragment upstream of the translation start codon of CNGC19 including the first exon was amplified (∼3-kb fragment) from genomic DNA and was cloned in the pBI101.2 vector at the SalI/BamHI sites. An ∼2.2-kb genomic fragment upstream of the translation start codon of BCAT4 was amplified and cloned in the pBI101.2 vector at the SalI/BamHI sites for the BCAT4 promoter-GUS fusion construct. For GUS reporter staining, Arabidopsis leaves from ProCNGC19:GUS- and ProBCAT4:GUS-expressing transgenic plants (T₃ generation) were vacuum-infiltrated with GUS staining solution (50 mM of sodium phosphate buffer at pH 7.0, 2 mM of EDTA (w/v), 0.12% Triton, 0.4 mM of ferrocyanide, 0.4 mM of ferricyanide, and 1.0 mM of 5-bromo-4-chloro-3-indolyl-β-D-glucuronide cyclohexylammonium salt) for 5–15 min and incubated in the dark at 37°C for 1 h. Tissues were cleared of chlorophyll by treatment with 70% ethanol at 65°C for 1 h and analyzed by light microscopy. ProCNGC19:GUS and ProBCAT4:GUS expressing transgenic plants were wounded by needle or fed by Spodoptera (fourth-instar larvae). After 5 min of treatments leaf tissues were collected and incubated in GUS staining solution for 1 h. List of primer pairs used in this study are in Supplemental Table 1

Ca²⁺ Measurements

GCaMP3 reporters: Transgenic Arabidopsis (Col) expressing GCaMP3, and a cngc19-2 mutant crossed with GCaMP3 (T₂), selected to ensure equal background fluorescence, were used for Ca²⁺ measurements. Four-week–old plants under short-day conditions on soil were used for the experiments. Both wild-type and cngc19-2 GCaMP3-expressing leaf discs were also tested for their similar response to 2-M CaCl₂ with 20% ethanol after each experiment to ensure equal basal fluorescence (Supplemental Figure 5B). This ensured that the differences in background intensity of GCaMP3 do not account for varying signal intensity/spread in different plants. We only used local leaf (leaf 5) for wounding and imaging in all experiments. A stereomicroscope (Nikon) was used to visualize the fluorescence, with GFP excitation at 470 nm and emission at 500 to 550 nm. Basal fluorescence level of unwounded leaf was captured by a camera (Nikon) with 4× gain and 5-s exposure time. In leaf 5 of wild type and cngc19, leaf lamella including midrib was wounded using sharp scissors and fluorescence was captured at every 5-s interval for 5 min. Mean fluorescence of images were calculated using the software ImageJ 1.52a (U.S. National Institutes of Health). TIFF files were used for data processing and whole-leaf mean fluorescence, excluding the cut region, was calculated by selecting the leaf area using a freehand selection tool (Supplemental Figure 5). Normalized fluorescence intensity (ΔF/F) was calculated according to ΔF/F = F−F₀/F₀ (F, fluorescence after wounding; F₀, basal fluorescence intensity) and was performed as described in Toyota et al. (2018). Spodoptera feeding and visualization of signals in the same leaf is technically challenging and was not done, unlike in Toyota et al. (2018), where systemic signals upon feeding were probed.

Aequorin (Aeq) reporters: Transgenic Arabidopsis (Col-0)-expressing cytosolic apoaequorin (Knight et al., 1997) and cngc19-2 mutant seedlings transformed with pMAQ2 vector (T₂) with good discharge were used for Ca²⁺ measurements. Aeq and cngc19-pMAQ2 seedlings were grown in liquid MS medium and 12-d–old seedlings were transferred into a 96-well white plate (Thermo Fisher Scientific) containing 5-μM coelenterazine (PJK International) in the dark overnight at 21°C. Bioluminescence counts in seedlings were recorded as relative light units per s with a microplate luminometer (Luminoskan Ascent, v2.6; Thermo Fisher Scientific). After 102-s background reading, 20-nM Pep1 (GenScript) was added manually to the well and readings in relative light units per s were taken for 900-s Calibrations were performed by estimating the amount of Aeq remaining at the end of experiment by discharging all remaining Aeq with 2 M of CaCl₂ with 20% ethanol, and the counts were recorded for 360 s. The luminescence counts obtained were calibrated using the equation from Rentel and Knight (2004).

cAMP Quantification

For cAMP quantification, leaves of 6-week–old wild-type plants and pepr1 pepr2 mutants were treated with S. litura OS for 2.5 and 5 min. Samples were ground in liquid N₂ to form fine powder and cAMP was extracted by homogenizing in 500-μL lysis buffer (50 mM of Tris-HCl at pH 7.5, 100 mM of NaCl, 0.1% Triton, 1 mM of DTT, 2 mM of EDTA, and protease inhibitor cocktail [Roche]). After 30 min of incubation on ice, samples were centrifuged at 13,000 rpm for 5 min at 4°C. Supernatant was collected and protein amount was quantified. cAMP was measured according to the user manual provided with the cAMP ELISA Kit (Cayman Chemical).

TEVC in Xenopus Oocytes

The CNGC19-YFP fragment was amplified from the pEG101-CNGC19:YFP construct and subcloned into vector pGEMHE in BamHI/EcoRI restriction sites (Liu and Luan, 2001). Oocytes were isolated by mini-laparotomy from adult female Xenopus laevis. Surgical procedures for oocyte collection were approved by the Institutional Animal Ethics Committee, which reports to the Committee for the Purpose of Control and Supervision of Experiments on Animals, Government of India. Clusters of oocytes were broken down with collagenase and individual oocytes defolliculated manually. Oocytes at stages V to VI were selected and incubated in ND-96 solution (96 mM of NaCl, 2 mM of KCl, 1 mM of MgCl₂, 1.8 of mM CaCl₂, and 5 mM of HEPES at pH 7.5, supplemented with 2.5 mM of sodium pyruvate and enriched with Horse serum) with 50-μg/mL penicillin/streptomycin at 18°C overnight before injections. Capped RNA was prepared from the linearized plasmid DNA templates using the Ampli-Cap Max T7 Message Maker kit (CellScript), following the manufacturer’s instructions. Approximately 10 ng of each cRNA was injected into each oocyte. Electrophysiological experiments were performed 48 to 96 h after injection using a TEC-10CX TEVC Amplifier (NPI Electronic) to maintain holding potentials and record membrane currents. The analog output of the amplifier was sampled at 10 kHz and filtered at 3 kHz, digitized, and stored for further analysis. The software package pCLAMP v10.2 (Molecular Devices) was used to generate voltage clamp commands, acquire membrane currents, and analyze digitized data. All experiments were performed at room temperature. All experimental data were first analyzed with Clampfit v10.2 (pCLAMP; Molecular Devices) and exported to the software Origin v8.0 (OriginLabs) for subsequent analysis and display. The bath solution was 200-mM–mannitol buffered with MES-Tris to pH 7.4. Additions made were: 300 μm of dibutyryl cAMP with a 15-min incubation period, followed by CaCl₂ (15 mM), and finally DIDS (300 μM). NaCl (10 mM) and KCl (10 mM) were added before CaCl2 on some runs to check for the selectivity of the channel. DIDS was used to inhibit Ca²⁺-activated Cl⁻ channels. Oocytes were held at −80 mV, stepped to potentials ranging from −130 mV to +40 mV, and finally brought to −60 mV. Tail currents at −60 mV were analyzed as a measure of channels open at the end of the step episode.

Confocal Microscopy to Detect Expression and Localization of CNGC19-YFP in Oocytes

Xenopus oocytes injected with either water or CNGC19-YFP and were imaged in bright field and in fluorescence confocal mode. Oocytes were imaged with a 10× Plan Apochromat objective on a LSM-780 Confocal Microscope (Zeiss) 36 to 48 h postinjection. YFP was excited using the 514-nm line of a multi-line Argon laser while the emission was recorded using a spectral detector over 523 to 675 nm. Line scans were performed on fluorescence images of both CNGC19-YFP and water-injected oocytes.

Vector Construction and Expression of YFP Fusion Protein

Gateway Technology (Invitrogen) was used for the generation of pEG101 transformation constructs, which consisted of a target gene (CNGC19 ORF cDNA) bearing a C-terminal fusion to YFP under the control of an enhanced Cauliflower Mosaic Virus 35S promoter for plant transformations (Earley et al., 2006). The attB adaptor-bearing PCR primers were designed for the generation of attB PCR products for recombination with the donor vector pDONR207 via BP Clonase reactions (Invitrogen). Fully sequenced entry clones were recombined in LR Clonase (Invitrogen) reactions with the pEG101 destination vector. The binary constructs were introduced into Agrobacterium tumefaciens (GV3101) and used for transient expression of CNGC19 in N. benthamiana leaves. Overnight grown cell cultures were collected and resuspended in 1 mL of infiltration buffer (10 mM of MES-KOH at pH 5.6, 10 mM of MgCl₂, and 150 mM of acetosyringone) to optical density at 600 nm (OD₆₀₀) to 0.8. The working suspensions were prepared by mixing Agrobacterium harboring CNGC19-YFP construct, organelle markers for plasma membrane (PM-mCherry) or tonoplast (TP-mCherry; Nelson et al., 2007), and p19 silencing suppressor in (1:1:1) ratio, incubated at room temperature for 3 h and infiltrated into the abaxial surface of fully developed leaves. Plants were cultivated for 3 d under growth-room conditions (28°C temperature, 16-h light/8-h dark photoperiod with one bulb of cool white and five bulbs of wide spectrum lights with a light intensity of 150 μmol m⁻² s⁻¹) and the infiltrated leaf area was analyzed by confocal laser scanning microscopy using a TCS SP5 (Leica Microsystems) equipped with appropriate lasers (514 to 527 nm and 587 to 610 nm for YFP and mCherry, respectively). Autofluorescence of chloroplasts was detected at 650 to 700 nm. For stable line preparation, Arabidopsis flowering plants were transformed by floral dip method, as described in Clough and Bent (1998).

Protein Extraction and Immunoblot

Microsomes were prepared from CNGC19-YFP transiently expressed in N. benthamiana leaves by homogenizing in grinding buffer (50 mM of HEPES-KOH at pH 7.5, 10 mM of EDTA, 330 mM of Suc, 0.6% polyvinylpolypyrrolidone, 1 mM of DTT, 1 mM of PMSF, and 1% protease inhibitor cocktail). The extract was centrifuged at 18,000g for 10 min and the supernatant was further centrifuged at 100,000g for 1 h. The microsome pellet was then resuspended in resuspension buffer (20 mM of HEPES-KOH at pH 7.5, 1 mM of EDTA, 330 mM of Suc, 1 mM of PMSF, and 1% protease inhibitor cocktail). Crude microsome fraction was separated by SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was incubated first with rabbit anti-GFP antibody (AS15 2987; Agrisera Antibodies) and then with goat-anti rabbit IgG-HRP conjugates (Merck) followed by chemiluminescence with Clarity Western ECL substrate (Bio-Rad).

Expression Analysis by Real-Time PCR

Leaf material was ground to a fine powder in liquid N₂, and total RNA was isolated using TRIzol Reagent (Invitrogen) according to the manufacturers’ protocol. An additional DNase (Turbo DNase; Ambion) treatment was included to eliminate any contaminating DNA. RNA quantity was determined using Nanodrop (Thermo Fisher Scientific). DNA-free total RNA (1 μg) was converted into single-stranded cDNA using a mix of oligo-dT₁₈ primers from the Omniscript cDNA Synthesis Kit (QIAGEN). Gene-specific primers (placed at the exon–exon junction for specific amplification of cDNA, whenever possible) were designed using the NCBIprimer design tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast). For real-time PCR, primers producing 120- to 170-bp amplicons were used. RT-qPCR was done in optical 96-well plates on a CFX96 Real-Time PCR Detection System (Bio-Rad) using the iTaq universal SYBR green Mix (Bio-Rad). Previously optimized endogenous control RPS18 for herbivory experiments (Vadassery et al., 2012) was used for normalization of transcripts. Fold induction values of target genes were calculated with the ΔΔ^CT equation (Rao et al., 2013) and related to the mRNA level of target genes in the control leaf, which were defined as 1.0. All of the assays were run in duplicate or triplicate (biological replicates) to control for overall variability. The primer pairs used are listed in Supplemental Table 1.

Quantification of Phytohormones in Arabidopsis Leaves

Plant material was weighed (250 mg) for the wild type and cngc19-2 mutant line and frozen with liquid N and samples were kept at −80°C until used. Wild-type and cngc19-1 samples were lyophilized in another set of experiments and 10-mg fine powdered lyophilized tissue was used. For phytohormone analysis, finely ground leaf material was extracted with 1.5 mL of methanol containing 60 ng of d₆-JA (HPC Standards), 60 ng of salicylic acid-d₄ (Santa Cruz Biotechnology), 60 ng of abscisic acid-d₆ (Toronto Research Chemicals), and 12 ng of d₆-JA-Ile conjugate (HPC Standards) as internal standards. Phytohormones were quantified on an API 5000 LC-MS/MS system (Applied Biosystems) as described in Vadassery et al. (2012) and Scholz et al. (2017). Because it was observed that both the D6-labeled JA and D6-labeled JA-Ile contained 40% of the corresponding D5-labeled compounds, the sum of the peak areas of D5- and D6-compound was used for quantification.

GS Analysis

Whole plants were used for GS analysis as opposed to single leaves due to vast leaf to leaf variation (Brown et al., 2003). Samples were freeze-dried until constant weight and ground to a fine powder. Twenty-five mg of freeze-dried and pulverized material per plant was used for GS analysis. GS were extracted with 1 mL of 80% methanol solution containing 0.05-mM intact 4-hydroxybenzylglucosinolate as the internal standard. After centrifugation at 12,000 rpm for 10 min at 4°C, extracts were loaded onto DEAE Sephadex A-25 columns (flowthrough was collected for further metabolite analysis) and treated with arylsulfatase for desulfation (Sigma-Aldrich). The eluted desulfoglucosinolates were separated using high performance liquid chromatography (Agilent 1100 HPLC system; Agilent Technologies) on a reversed phase C-18 column (Nucleodur Sphinx RP; 250 × 4.6 mm, 5-µm particle size; Macherey-Nagel) with a water-acetonitrile gradient (1.5% acetonitrile for 1 min, 1.5% to 5% acetonitrile from 1 to 6 min, 5% to 7% acetonitrile from 6 to 8 min, 7% to 21% acetonitrile from 8 to 18 min, and 21% to 29% acetonitrile from 18 to 23 min, followed by a washing cycle; flow 1.0 mL min⁻¹). Detection was performed with a photodiode array detector and peaks were integrated at 229 nm. We used the response-factors aliphatic GS 2.0 and indole GS 0.5 (Burow et al., 2006) for quantification of individual GSs.

Amino Acid Analysis

Amino acids were analyzed in the flowthrough from the DEAE Sephadex A 25 columns used in GS analysis. Plant extracts were diluted with 1:10 (v/v) in water containing the ¹³C-, ¹⁵N-labeled algal amino acid mix (Isotec). Amino acids in the diluted extracts were directly analyzed by LC–MS/MS as described before in “Quantification of Phytohormones in Arabidopsis Leaves,” with the modification that an API5000 Mass Spectrometer (Applied Biosystems) was used and the chromatographic gradient was modified as follows: 0–1 min, 3% B in A; 1–2.7 min, 3–100% B in A; 2.7–3 min 100% B, 3.1–6 min 3% B in A (A: 0.05% formic acid in water; B: acetonitrile). The mobile phase flow rate was 1.1 mL/min (Crocoll et al., 2016).

Y2H

The CNGC19 C-terminal was fused to the GAL4 DNA binding domain in pGBKT7-BD vector (Clontech Laboratories). CaM1, CaM2, CaM3, CaM4, CaM6, CaM7, and CML 42 were cloned into the pGADT7-AD vector (Clontech Laboratories) containing the GAL4 activation domain. All constructs were made using the restriction-based cloning. The pGBKT7-BD and PGADT7-AD constructs were cotransformed into the Y2H Gold strain of yeast (Saccharomyces cerevisiae) using a Matchmaker Gold Yeast Two-Hybrid System Kit (Clontech Laboratories), and selected for transformed cells on SD-Leu/-Trp (DDO) plates lacking Leu and Trp (−LT) and were confirmed by colony PCR. pGBKT7-T antigen was cotransformed with pGADT7-p53 or pGADT7-Lam as a positive and negative control, respectively. Y2H Gold strain cotransformed with the empty vectors, pGBKT7-BD and pGADT7-AD, was also used as a negative control. Transformed cells were grown in liquid double-dropout media (−LT) for 2 d, and adjusted to an OD₆₀₀ of 1, 0.1, 0.01, and 10 μL. Each dilution was spotted on selection plates: DDO (−LT) and TDO (−LTH) + 2.5 mM 3-AT. For additional verification of the interaction, X α-gal, a substrate for α-galactosidase (MEL1 reporter), was added to the TDO plates. Plates were grown at 30°C for 3 d.

Split Ubiquitin Assay

For protein–protein interaction studies between CNGC19 and PEPR1/PEPR2/BAK1, pDONR207 clones were generated by BP Clonase reaction. For split ubiquitin assay, CNGC19 was subcloned into pMETYC vector (Grefen et al., 2009) using LR Clonase to generate the CNGC19:Cub construct, while PEPR1/PEPR2/BAK1 were cloned similarly into pXN22 vector to generate Nub constructs. CNGC19 and other constructs including empty vectors were transformed in mating yeast strains THY.AP4 and THY.AP5, respectively, and plated on appropriate dropout-media–containing plates. For protein–protein interaction studies, THY.AP4 harboring CNGC19:Cub and THY.AP5 with other constructs was allowed for mating in Yeast extract-Peptone-Dextrose medium and then plated on synthetic complete (SC) medium (lacking −Ade, −His, −Trp, and −Leu) and SC+AH containing plates to check the interaction. Empty Nub vector and CNGC19 fused with NubWt served as the negative and positive controls for the experiment.

BiFC Assay

The full-length cDNA fragments (without stop codon) of PEPRs (PEPR1 and PEPR2), BAK1, CaMs (CaM1, CaM2, CaM3, CaM4, CaM6, and CaM7), and CML42 were amplified using the attB adaptor-bearing PCR primers listed in Supplemental Table 1 and cloned into the Gateway entry vector pDONR 207 by BP Clonase reaction (Invitrogen). For BiFC experiments, CNGC19 was cloned into pSITE-nEYFP-N1 (CD3-1650; Martin et al., 2009) using LR Clonase to generate the CNGC19:nEYFP construct, while CaMs and CML42, respectively, were cloned similarly into pSITE-cEYFP-C1 (CD3-1649) to generate cEYFP constructs. For CNGC19 and PEPRs interaction studies, PEPR1, PEPR2, BAK1, and CaM2, respectively, were cloned into pSITE-cEYFP-N1 (CD3-1651) to generate cEYFP constructs. All BIFC constructs and empty vectors were transformed in Agrobacterium strain GV3101. For N. benthamiana transient expression, overnight grown cell cultures were harvested and resuspended in the above-mentioned infiltration buffer to optical density (OD₆₀₀) 0.8. The working suspensions were prepared by mixing appropriate clones containing the BiFC constructs and the gene-silencing inhibitor p19 at 1:1:1 ratio and incubated for 3 h at room temperature. The A. tumefaciens suspensions were then coinfiltrated into the abaxial surface of N. benthamiana leaves. Infiltrated leaf areas were examined using a SP5 Confocal Microscope (Leica) at 3 d postinoculation. For fluorescence quantification, 6-mm leaf discs were cut out of the infiltrated areas and placed in a black 96-well plate. Fluorescence intensities of leaf discs were measured in a FLUOstar Omega Plate Reader (BMG LABTECH) using a 485- ± 12-nm excitation and a 520-nm emission filter.

Microarray

Total RNA was isolated from 24 h S. litura larvae fed on Col-0 wild type and cngc19-2 leaf tissues using an RNeasy Plant Mini Kit (QIAGEN) from three independent biological replicate pools. RNA samples were checked and quantified by agarose gel, nano drop (Thermo Fisher Scientific), and Agilent 2100 Bioanalyzer (Agilent Technologies). Bioanalyzer quality-test–passed RNA samples (RIN > 7) were used for microarray experiment. GeneChips were scanned using a GeneChip Scanner 3000 7G (Affymetrix) and GeneChip Command Console Software (AGCC v3.2.4; Affymetrix), and six CEL files were generated. All six CEL files were normalized in freely available Expression Console software v.1.3.1 (Affymetrix) using the Robust Multi-array Average algorithm, and probe-set summarization files (CHP files) were generated for genes. The normalized CHP files were processed using the freely available secondary analysis tool, Transcriptome Analysis Console software v.3.1.0.5 (Affymetrix), to generate the .csv files containing differentially expressed genes using the combined criteria of >1.5-fold change with ANOVA P value of <0.05. GO enrichment for various differentially expressed transcripts was performed using the BiNGO plug-in (v3.0.3) on the Cytoscape platform (v3.2.1; http://www.cytoscape.org/). Arabidopsis GO information for biological process and molecular function was used for GO enrichment analysis. We considered a P value cutoff of ≤0.05 as significant and applied the hypergeometric test to identify enriched GO terms in BiNGO. The software MapMan (v3.5.1; https://mapman.gabipd.org/) was used for functional annotation of differentially expressed transcripts. Heat maps were generated by the software MeV_4_9_0 (https://sourceforge.net/projects/mev-tm4/files/mev-tm4/MeV%204.9.0/).

Statistical Analysis

Statistical differences between different groups were detected by one-way ANOVA and a posthoc Student-Newman-Keuls-(SNK) test or Student’s t test in the software SigmaStat 2.03 (Systat Software). Different letters indicate significant difference between treatments.

Accession Numbers

The TAIR accession numbers of major genes mentioned in this study are as follows: CNGC19 (At3g17690), CNGC20 (At3g17700), BCAT4 (AT3g19710), PEPR1 (At1g73080), PEPR2 (At1g17750), and CaM2 (AT2g41110).

Supplemental Data

• Supplemental Figure 1. Arabidopsis CNGC expression analysis upon simulated herbivory.

• Supplemental Figure 2. Verification of cngc19 T-DNA insertion lines.

• Supplemental Figure 3. CNGC20 has no functional role in S. litura herbivory.

• Supplemental Figure 4. CNGC19 expression coincides with cAMP production in Arabidopsis.

• Supplemental Figure 5. Measuring wound-induced cytosolic Ca²⁺ levels in local leaves of the wild type and cngc19-2 mutant.

• Supplemental Figure 6. CNGC19 is expressed in the Xenopus oocyte.

• Supplemental Figure 7. Subcellular localization of CNGC19 at the plasma membrane.

• Supplemental Figure 8. cAMP levels were unaltered in pepr1 pepr2 mutants upon simulated herbivory.

• Supplemental Figure 9. CNGC19 does not interact with PEPRs and BAK1 in planta.

• Supplemental Figure 10. Defense-related phytohormone levels upon herbivory in cngc19 mutants.

• Supplemental Figure 11. Functional categorization of differentially expressed genes in microarray.

• Supplemental Figure 12. Analysis of CNGC19-CaMs interaction strength on quadruple dropout medium and BiFC.

• Supplemental Figure 13. Expression profile of CaMs upon simulated herbivory.

• Supplemental Table 1. List of primer pairs used in this study.

• Supplemental Table 2. GS content in rosette leaves of cngc19 mutant lines.

• Supplemental Table 3. 2-way ANOVA on GS content of cngc19 mutant lines upon herbivory.

• Supplemental Data Set. List of genes up- and downregulated in the microarray of cngc19 +S. litura/wild type + S. litura with functional annotation by the software MapMan.

• Supplemental Movie S1. Mechanical wounding in wild-type plant.

• Supplemental Movie S2. Mechanical wounding in cngc19 mutant plant.

• Supplemental Movie S3. Mechanical wounding in wild-type plant.

• Supplemental Movie S4. Mechanical wounding in cngc19 mutant plant.