Abstract
Perception of pathogen-derived ligands by corresponding host receptors is a pivotal strategy in eukaryotic innate immunity. In plants, this is complemented by circadian anticipation of infection timing, promoting basal resistance even in the absence of pathogen threat. Here, we report that trichomes, hair-like structures on the epidermis, directly sense external mechanical forces, including raindrops, to anticipate pathogen infections in Arabidopsis thaliana. Exposure of leaf surfaces to mechanical stimuli initiates the concentric propagation of intercellular calcium waves away from trichomes to induce defence-related genes. Propagating calcium waves enable effective immunity against pathogenic microbes through the CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR 3 (CAMTA3) and mitogen-activated protein kinases. We propose an early layer of plant immunity in which trichomes function as mechanosensory cells that detect potential risks.
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Introduction
Innate immunity is an evolutionarily conserved front line of defense across the plant and animal kingdoms. In plants, pattern-recognition receptors (PRRs), such as leucine-rich repeat receptor-like kinases (LRR-RLKs) and LRR receptor proteins (LRR-RPs), specifically recognize microbe-associated molecular patterns (MAMPs) as non-self molecules, leading to the activation of pattern-triggered immunity (PTI) to limit pathogen proliferation1,2. While adapted pathogens have evolved virulence effectors that can circumvent PTI, plants also deploy disease resistance (R) genes, primarily encoding nucleotide-binding LRR proteins, which mount effector-triggered immunity (ETI)3,4,5. ETI often culminates in a hypersensitive response as well as acute and localized cell death at the site of infection accompanied by profound transcriptional changes of defense-related genes to retard pathogen growth4,5. These ligand–receptor systems are largely dependent on a transient increase in intracellular calcium concentration ([Ca2+]i), followed by the initiation of phosphorylation-dependent signaling cascades, including mitogen-activated protein kinases (MAPKs) and calcium-dependent protein kinases, that orchestrate a complex transcriptional network and the activity of immune mediators6,7.
In addition to PTI and ETI, plant immunity can be induced periodically in the absence of pathogen threat, a process controlled by the circadian clock and driven by daily oscillations in humidity as well as light–dark cycles8,9,10. Such responses enable plants to prepare for the potential increased risk of infection at the time when microbes are anticipated to be most infectious. The anticipation of potentially pathogenic microorganisms through sensing climatological changes and their specific detection thus constitute two distinct layers of the plant immune system.
Among the climatological factors that affect the outcome of plant–microbe interactions, rain is a major cause of devastating plant diseases, as fungal spores and bacteria are spread through rain-dispersed aerosols or ballistic particles splashed from neighboring infected plants. Natural raindrops contain bacteria at a concentration of 1.06 × 104 (/cm3)11, including plant pathogens such as Pseudomonas syringae12, Xanthomonas campestris, and Pantoea ananatis13. Likewise, raindrops contain fungi such as Alternaria sp., Fusarium sp., Cladosporium sp., Phoma sp., Rhizopus sp., and Botrytis cinerea14. In addition, raindrops negatively regulate stomatal closure, which facilitates pathogen entry into leaf tissues13,15,16. High humidity, which is usually associated with rain, enhances the effects of bacterial pathogen effectors, such as HopM1, and establishes an aqueous apoplast for aggressive host colonization62. The carnivorous Venus flytrap (Dionaea muscipula) captures insects by sensing mechanostimulation via sensory hairs on leaf lobes63. To monitor diverse MS applied to plants, several sensing mechanisms have been proposed that include detection of cell wall components, distortion of the plasma membrane, and displacement of the plasma membrane against the cell wall64. In all these systems, a transient increase in [Ca2+]cyt is thought to play a pivotal role in short- and long-term responses. Two successive stimulations of sensory hairs of the flytrap are required to meet the threshold of [Ca2+]cyt for rapid closure of the leaf blade63,65. As the trichome on the leaf surface is widely found in many land plants, there may be a common intercellular network of cell–cell communication that initiates calcium waves for activating immune responses.
Methods
Plants
A. thaliana accession Columbia-0 (Col-0) was the background for all plants used in this study. WRKY33pro:EYFP-NLS (Col-0) and CBP60gpro:EYFP-NLS (Col-0) were generated as described in the subsection “Promoter-reporter imaging”66. 35Spro:GCaMP3 (Col-0)53, camta2 camta3 CAMTA3pro:CAMTA3-GFP, and camta2 camta3 CAMTA3pro:CAMTA3A855V-GFP were provided from Michael F. Thomashow (Michigan State University)45,47. gl1 [Col(gl1)] was obtained from Lehle Seeds (TX, USA) and was previously designated as gl-1 mutant. The Arabidopsis mutants fls2 (SALK_093905) and bak1-3 (SALK_034523) were obtained from the Arabidopsis Biological Resource Center (ABRC). 35Spro:GCaMP3 was introduced into the gl1 mutant background by crossing. The selection of homozygous lines was performed by genoty** using primers listed in Supplementary Data 10. Plants were grown on soil (peat moss; Super Mix A and vermiculite mixed 1:1) at 22 °C under diurnal conditions (16-h-light/8-h-dark cycles) with 50–70% relative humidity. WRKY33pro:EYFP-NLS (Col-0) and CBP60gpro:EYFP-NLS (Col-0) were sown on soil and grown in a growth room at 23 °C in constant light66. 35Spro:GCaMP3 (Col-0) and 35Spro:GCaMP3 (gl1) were grown on Murashige and Skoog (MS) plates [1× MS salts, 1% (w/v) sucrose, 0.01% (w/v) myoinositol, 0.05% (w/v) MES, and 0.5% (w/v) gellan gum, pH 5.8]53,67.
Artificial raindrop treatment
Reverse osmosis water was kept in a 500 mL beaker until the water temperature reached room temperature (22 °C). A transfusion set (NIPRO Infusion Set TI-U250P, Nipro, Osaka, Japan) was installed on a steel stand with the beaker at a height of 1.2 m (H-type Stand I3, As One, Osaka, Japan) and was adjusted to release 13 µL water droplets (Supplementary Fig. 1a). In this setting, the applied mechanical energy to the leaf surface is equivalent to one in which 5.8 µL of raindrops reach a terminal velocity of 6.96 m/s68. This size raindrop is frequently observed in nature; thus, the impact of simulated rain is comparable with that of true rain68. The adaxial side of leaves from 4-week-old plants was treated with 10 droplets for RNA-seq and 1, 4, or 10 droplets for quantitative RT-PCR (RT-qPCR). The adaxial side of leaves from 4-week-old plants was treated with 1 falling or static droplet (Supplementary Fig. 1b). Sample leaves were collected 15 min after treatment and stored at −80 °C until use.
Brush treatment
The adaxial side of leaves from 4-week-old plants was brushed once or 4 times for RNA-seq and RT-qPCR along the main veins at an angle of 30–40° (KOWA nero nylon drawing pen flat 12, Kowa, Aichi, Japan) (Supplementary Fig. 1c). Sample leaves were collected 15, 30, and 60 min after treatment for RNA-seq and 15 min after treatment for RT-qPCR and stored at −80 °C until use.
RNA-seq library construction
Total RNA was extracted from 80 to 100 mg frozen samples using Sepasol-RNA I Super G (Nacalai Tesque, Kyoto, Japan) and the TURBO DNase free kit (Thermo Fisher Scientific, IL, USA) according to the manufacturer’s protocols. Total RNA was further purified with the RNeasy RNA Isolation Kit (QIAGEN, Hilden, Germany) and assessed for quality and quantity with a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific). We used 1 µg total RNA for mRNA purification with NEBNext Oligo d(T)25 (NEBNext poly(A) mRNA Magnetic Isolation Module; New England Biolabs, MA, USA), followed by first-strand cDNA synthesis with the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs) and NEBNext Multiplex Oligo for Illumina (New England Biolabs) according to the manufacturer’s protocols. For the analysis of raindrop- and MS-induced gene expression, the amount of cDNA was determined on an Agilent 4150 TapeStation System (Agilent, CA, USA). cDNA libraries were sequenced as single-end reads for 81 nucleotides on an Illumina NextSeq 550 (Illumina, CA, USA). The reads were mapped to the Arabidopsis thaliana reference genome (TAIR10, http://www.arabidopsis.org/) on the web (BaseSpace, Illumina, https://basespace.illumina.com/). Pairwise comparisons between samples were performed with the EdgeR69 package on the web (Degust, https://degust.erc.monash.edu/).
For the comparative analysis of differentially expressed genes between leaves in the gl1 mutant and Col-0, the amount of cDNA was determined by the QuantiFluor dsDNA System (Promega, WI, USA). cDNA libraries were sequenced as single-end reads for 36 nucleotides on an Illumina NextSeq 500 (Illumina). The reads were mapped to the A. thaliana reference genome (TAIR10) via Bowtie270 with the options “--all --best --strata”. Pairwise comparisons between samples were performed with the EdgeR package in the R program69. Enrichment of GO categories for biological processes was determined using BiNGO (http://www.psb.ugent.be/cbd/papers/BiNGO/Home.html) (one-sided hypergeometric test; P < 0.05)71. Pearson correlation coefficient of the expression levels between two transcriptome profiles was calculated with Excel function (PEARSON function) (r = 0–0.2: no correlation, r = 0.2–0.4: weak correlation, r = 0.4–0.7: slightly correlated, r = 0.7–1.0: correlated).
Quantification of the force density
The abaxial side of leaves from 4-week-old plants was physically attached to the measuring pan of the electronic balance QUINTIX224-1S (Sartorius Lab Instruments GmbH & Co., Göttingen, Germany) with surgical tape (3 M Company, MN, USA). The adaxial side of the leaf was treated with 1 falling droplet or brushed once (shown in the above subsections). The peak weight applied to the leaf surface was obtained as the force. The force per unit area (N/m2) is converted from the peak weight (kg) and the contact area of the brush tip (5.6 × 10−5 m2) or raindrop (9.73 × 10−6 m2).
Re-analysis of immune-related transcriptome datasets
We used the following public transcriptome datasets for comparative analysis with the RNA-seq data obtained in this study: 10-day-old Arabidopsis seedlings treated with 1 µM flg22 for 30 min (Array Express; E-NASC-76)29, 8-day-old Arabidopsis seedlings treated with 40 µM chitin for 1 h (Gene Expression Omnibus; GSE74955), leaves from 4-week-old Arabidopsis plants inoculated with Pseudomonas syringae pv. maculicola (Psm) ES4326 (24 h post inoculation) (GSE18978), 2-week-old Arabidopsis seedlings treated with 0.5 mM SA or 50 µM JA for 24 h (DNA Data Bank of Japan; DRA003119)33, 2-week-old Arabidopsis seedlings grown with MS medium and treated with water spray for 10 min or 25 min (E-MTAB-8021)23, leaves from 4-week-old Arabidopsis plants treated with bending back and forth manually for 30 min (accession is now not available)26, leaves from 4-week-old Arabidopsis plants treated with brushing for 30 min (NCBI BioProject; PRJNA473032)27, 2-week-old Arabidopsis seedlings grown with MS medium and treated with cotton swabbing for 30 min (accession is now not available)28, 12-day-old Arabidopsis GVG-NtMEK2DD seedlings treated with 2 μM DEX for 0 and 6 h (NCBI Sequence Read Archive; SRP111959)36, and 4-week-old Arabidopsis camta1 camta2 camta3 triple mutant (GSE43818)47 (Supplementary Data 11). The overlaps between differentially expressed genes in each transcriptome dataset were evaluated as Venn diagrams (http://bioinformatics.psb.ugent.be/webtools/Venn/) and Upset plot (https://asntech.shinyapps.io/intervene/).
RT-qPCR
Total RNA was extracted from 30–40 mg leaf tissues with Sepasol-RNA I Super G and the TURBO DNase free kit (Thermo Fisher Scientific) according to the manufacturer’s protocols, followed by reverse transcription with the PrimeScript RT reagent kit (Takara Bio, Shiga, Japan) using oligo dT primers. RT-qPCR was performed on the first-strand cDNAs diluted 20-fold in water using KAPA SYBR FAST qPCR Master Mix (2×) kit (Roche, Basel, Switzerland) and gene-specific primers in a LightCycler 96 (Roche). Primer sequences are listed in Supplementary Data 10.
Quantification of plant hormones
The adaxial side of leaves from 4-week-old plants was treated with 10 raindrops (raindrop), brushed once (MS), or cut (wounding). Sample leaves (0.07–0.1 g) were collected 5 min or 15 min after treatment and stored at −80 °C until use. Frozen samples were ground to a fine powder, mixed with 4 mL extraction solution [80% (v/v) acetonitrile, 1% (v/v) acetic acid], and stored for 1 h at 4 °C to extract plant hormones. After centrifugation at 3000 g for 10 min, the supernatants were evaporated in a vacuum centrifugal evaporator EC-57CS (Sakuma, Tokyo, Japan) and dissolved in 1% (v/v) acetic acid. Samples were loaded onto a reverse-phase solid-phase extraction cartridge (Oasis HLB 1 cc; Waters Corporation, MA, USA). The cartridge was washed with 1 mL 1% acetic acid, and hormones were eluted with 2 mL extraction solution. The eluent was evaporated to obtain samples in 1 mL 1% acetic acid and subjected to cation exchange chromatography on an Oasis MCX 1 cc extraction cartridge (Waters Corporation). The acidic fraction was eluted with 1 mL extraction solution. The acidic eluent was analyzed for SA, and the remaining fraction was evaporated, dissolved in 1% acetic acid, and loaded onto an Oasis WAX 1-cc extraction cartridge (Waters Corporation). The cartridge was washed with 1% acetic acid and the remaining hormones were eluted with extraction solution. The elute was analyzed for JA, JA-Ile, ABA, IAA, and GA4. The contents of these hormones were quantified using liquid chromatography–electrospray tandem mass spectrometry (LC–ESI–MS/MS) (triple quadrupole mass spectrometer with 1260 high-performance LC, G6410B; Agilent Technologies Inc., CA, USA) equipped with a ZORBAX Eclipse XDB-C18 column (Agilent Technologies Inc.)72.
ChIP assay
Approximately 0.7 g of 2-week-old camta2 camta3 CAMTA3pro:CAMTA3A855V-GFP seedlings was fixed in 25 mL 1% formaldehyde under vacuum for three cycles of 2 min ON/2 min OFF using an aspirator (SIBATA, Tokyo, Japan). Subsequently, 1.5 mL of 2 M glycine was added to quench the cross-linking reaction under vacuum for 2 min. The samples were then washed with 50 mL double-distilled water and stored at −80 °C until use. Frozen samples were ground to a fine powder with a mortar and pestle in liquid nitrogen and dissolved in 2.5 mL nuclei extraction buffer (10 mM Tris-HCl pH 8.0, 0.25 M sucrose, 10 mM MgCl2, 40 mM β-mercaptoethanol, protease inhibitor cocktail)33,73. Samples were filtered through two layers of Miracloth (Calbiochem, CA, USA) and centrifuged at 17,700g at 4 °C for 5 min. The pellets were resuspended in 75 μL nuclei lysis buffer [50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% (w/v) SDS]. After incubation first at room temperature for 20 min and then on ice for 10 min, the samples were mixed with 225 μL ChIP dilution buffer without Triton [16.7 mM Tris-HCl pH 8.0, 167 mM NaCl, 1.2 mM EDTA, 0.01% (w/v) SDS]. Chromatin samples were sonicated for 35 cycles of 30 sec ON/30 s OFF using a Bioruptor UCW-201 (Cosmo Bio, Tokyo, Japan) to produce DNA fragments, followed by the addition of 375 μL ChIP dilution buffer without Triton, 200 μL ChIP dilution buffer with Triton [16.7 mM Tris-HCl pH 8.0, 167 mM NaCl, 1.2 mM EDTA, 0.01% (w/v) SDS, 1.1% (w/v) Triton X-100], and 35 μL 20% (w/v) Triton X-100. After centrifugation at 17,700 g at 4 °C for 5 min, 900 μL solubilized sample was split into two 2.0 mL PROKEEP low-protein-binding tubes (Watson Bio Lab USA, CA, USA) and incubated with 0.75 μL anti-GFP antibody (Cat#ab290; Abcam, Cambridge, UK) (1:600 dilution) or Rabbit IgG-Isotype Control (Input) (Cat#ab37415; Abcam) (1:600 dilution) for 4.5 h with gentle rocking, and an 18 μL aliquot was used as the input control. Then, samples from camta2 camta3 CAMTA3pro:CAMTA3A855V-GFP were mixed with 50 µL of a slurry of Protein A agarose beads (Upstate, Darmstadt, Germany) and incubated at 4 °C for 1 h with gentle rocking. Beads were washed twice with 1 mL low-salt wash buffer [20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 0.1% (w/v) SDS, 1% (w/v) Triton X-100], twice with 1 mL high-salt wash buffer [20 mM Tris-HCl pH 8.0, 500 mM NaCl, 2 mM EDTA, 0.1% (w/v) SDS, 1% (w/v) Triton X-100], twice with 1 mL LiCl wash buffer [10 mM Tris-HCl pH 8.0, 0.25 M LiCl, 1 mM EDTA, 1% (w/v) sodium deoxycholate, 1% (w/v) Nonidet P-40], and twice with 1 mL TE buffer [10 mM Tris-HCl pH 8.0, 1 mM EDTA]. After washing, beads were resuspended in 100 μL elution buffer [1% (w/v) SDS, 0.1 M NaHCO3] and incubated at 65 °C for 30 min. For the input controls, 41.1 μL TE buffer, 8.7 μL 10% (w/v) SDS, and 21 μL elution buffer were added to 18 μL of each solubilized sample. Both supernatant and input samples were mixed with 4 μL of 5 M NaCl and incubated at 65 °C overnight to reverse the cross-linking, followed by digestion with 1 μL Proteinase K (20 mg/ml) (Invitrogen, CA, USA) at 37 °C for 1 h. ChIP samples were mixed with 500 μL Buffer NTB and purified using the PCR clean-up gel extraction kit following the manufacturer’s instructions (MACHEREY-NAGEL, Düren, Germany).
ChIP-seq library construction
ChIP-seq libraries for the input and two biological replicates were constructed from 2 ng purified DNA samples with the NEB Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) according to the manufacturer’s instructions. The amount of DNA was determined on an Agilent 4150 TapeStation System (Agilent). All ChIP-seq libraries were sequenced as 81-nucleotide single-end reads using an Illumina NextSeq 550 system.
Analysis of ChIP-seq
Reads were mapped to the Arabidopsis thaliana reference genome (TAIR10, http://www.arabidopsis.org/) using Bowtie2 with default parameters70. The Sequence Alignment/Map (SAM) file generated by Bowtie2 was converted to a Binary Alignment/Map (BAM) format file by SAMtools74. To visualize mapped reads, Tiled Data Files file were generated from each BAM file using the igvtools package in the IGV48. ChIP-seq peaks were called by comparing the IP with the Input using MACS2 with the “-p 0.05 -g 1.19e8” option (binomial distribution; P < 0.05)75. The peaks were annotated using the nearest gene using the Bioconductor and the ChIPpeakAnno packages in the R program, from which we identified 2011 genes detected in both biological replicates. Enrichment of GO categories of the set of 314 genes overlap** between raindrop- and MS-induced genes for biological processes was determined using BiNGO (http://www.psb.ugent.be/cbd/papers/BiNGO/Home.html)71. Sequences of the peaks were extracted from the Arabidopsis thaliana genome as FASTA files with BEDtools76. To identify the candidates of CAMTA3-binding motifs, the FASTA files were subjected to MEME-ChIP with the default parameters (-meme-minw 6-meme-maxw 10)77, and a density plot of the distribution of the motifs were generated.
Immunoblot analysis for detection of MPK3 and MPK6 phosphorylation
The adaxial side of leaves from 4-week-old plants was brushed four times or treated with four raindrops, and samples (0.1–0.15 g) were snap-frozen in liquid nitrogen. Total proteins were extracted in protein extraction buffer [50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM DTT, 2.5 mM NaF, 1.5 mM Na3VO4, 0.5% (w/v) Nonidet P-40, 50 mM β-glycerophosphate, and proteinase inhibitor cocktail] and centrifuged once at 6000 g, 4 °C, for 20 min and twice at 17,000 g, 4 °C for 10 min. The supernatant was mixed with SDS sample buffer [50 mM Tris-HCl pH 6.8, 2% (w/v) SDS, 5% (w/v) glycerol, 0.02% (w/v) bromophenol blue, and 200 mM DTT] and heated at 70 °C for 20 min. The protein samples were subjected to SDS-PAGE electrophoresis and transferred onto a nitrocellulose membrane (GE Healthcare, IL, USA). The membrane was incubated with an anti-phospho-p44/42 MAPK polyclonal antibody (Cat#9101; Cell Signaling Technology, MA, USA) (1:1000 dilution) and goat anti-rabbit IgG(H + L)-HRP secondary antibody (Cat#170-6515; BIO-RAD, CA, USA) (1:2000 dilution). The bands for MPK3/6 were visualized using chemiluminescence solution mixed 5:1 with ImmunoStar Zeta (FUJIFILM Wako Chemicals, Osaka, Japan) and SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific). The Rubisco bands were stained with Ponceau S (Merck Sharp & Dohme Corp., NJ, USA) as a loading control. The phosphorylation levels of MPK3 and MPK6 were quantified with the blot analysis plug-in in ImageJ (https://imagej.nih.gov/ij/).
Treatment with the calcium ionophore A23187
Twelve-day-old Col-0 seedlings were treated with 50 µM calcium ionophore A23187 (Sigma-Aldrich Co., MO, USA) for 15, 30, and 60 min. Samples were processed for the phosphorylation of MPK3 and MPK6 as described in the “Immunoblot analysis for detection of MPK3 and MPK6 phosphorylation” section. The leaf tissue was stored at −80 °C until use.
Promoter–reporter imaging
The 3.0-kbp promoters for WRKY33 and CBP60g, both of which covered the previously analyzed respective regulatory sequences, were amplified from Col-0 genomic DNA by PCR and cloned into the pENTR/D-TOPO vector (Invitrogen). The promoter regions were recombined using Gateway technology into the binary vector pBGYN. The resulting pBGYN-pWRKY33-EYFP-NLS and pBGYN-pCBP60g-YFP-NLS vectors were introduced into A. tumefaciens GV3101 (pMP90) and then into Arabidopsis Col-0 plants using the floral dip method. A representative homozygous line was selected for each construct for further detailed analyses.
Promoter-reporter imaging was performed using an MA205FA automated stereomicroscope (Leica Microsystems, Wetzlar, Germany) and DFC365FX CCD camera (Leica Microsystems) in 12-bit mode. Chlorophyll autofluorescence and YFP fluorescence were detected through Texas Red (TXR) (excitation 560/40 nm, extinction 610 nm) and YFP (excitation 510/20 nm, extinction 560/40 nm) filters (Leica Microsystems). To image fluorescence emanating from the WRKY33pro:EYFP-NLS (Col-0) and CBP60gpro:EYFP-NLS (Col-0) plants66, the leaves of 3-week-old Arabidopsis plants were brushed 10 times at an interval of 15 min for 2 h or left untreated.
Promoter analysis
The statistical analysis for overrepresented transcriptional regulatory elements across transcriptome datasets described above was calculated using a prediction program41. The P values were calculated using Statistical Motif Analysis in Promoter or Upstream Sequences (https://www.arabidopsis.org/tools/bulk/motiffinder/index.jsp). Figures of promoter motif sequences are generated with WebLogo (https://weblogo.berkeley.edu/logo.cgi).
Real-time [Ca2+]cyt imaging
We used 4-week-old and 3-week-old plants expressing the GFP-based cytosolic Ca2+ concentration ([Ca2+]cyt) indicator GCaMP352,53. To image the fluorescence from the GCaMP3 reporter (in Col-0 and gl1) in whole leaves, the adaxial sides of leaves from 4-week-old plants were brushed. To monitor the calcium waves propagating from trichomes, a single trichome from a 2-week-old seedling was flicked with a silver chloride wire. Samples were imaged with a motorized fluorescence stereomicroscope (SMZ-25; Nikon, Tokyo, Japan) equipped with a 1× objective lens (NA = 0.156, P2-SHR PLAN APO; Nikon) and an sCMOS camera (ORCA-Flash 4.0 V2; Hamamatsu Photonics, Shizuoka, Japan)53.
To detect the accumulation levels of GCaMP3 protein in 35Spro:GCaMP3/Col-0 and 35Spro:GCaMP3/gl1, leaves from 4-week-old plants (0.1–0.15 g) were snap-frozen in liquid nitrogen. Total proteins were extracted in protein extraction buffer [50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM DTT, 0.5% (w/v) Nonidet P-40, and proteinase inhibitor cocktail] and centrifuged once at 6000 g, 4 °C, for 20 min and twice at 17,000g, 4 °C for 10 min. The supernatant was mixed with SDS sample buffer [50 mM Tris-HCl pH 6.8, 2% (w/v) SDS, 5% (w/v) glycerol, 0.02% (w/v) bromophenol blue, and 200 mM DTT] and heated at 70 °C for 20 min. The protein samples were subjected to SDS-PAGE electrophoresis and transferred onto a nitrocellulose membrane (GE Healthcare). The membrane was incubated with an anti-GFP antibody (Cat#ab290; Abcam) (1:4000 dilution) and a goat anti-rabbit IgG(H + L)-HRP secondary antibody (Cat#170-6515; BIO-RAD) (1:2000 dilution). GCaMP3 proteins were visualized using chemiluminescence solution mixed 5:1 with ImmunoStar Zeta (FUJIFILM Wako Chemicals) and SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific). Rubisco proteins were stained with Ponceau S (Merck Sharp & Dohme Corp.) as the loading control. The protein levels of GCaMP3 were quantified with the blot analysis plug-in in ImageJ.
Propidium iodide staining
A stock solution of 10 mM propidium iodide (PI) was prepared with phosphate-buffered saline pH7.5. Rosette leaves of 4-week-old Col-0 plants were cut into 5 mm squares, floated in a glass petri dish with 20 µM PI solution, and incubated for 1 h at room temperature. Stained tissues were observed under the all-in-one fluorescence microscope (BZ-X800; Keyence Corporation, Osaka, Japan) equipped with a 20× objective lens (CFI S Plan Fluor LWD ADM 20XC, Nikon) and TRITC dichroic mirror (excitation 545/25 nm, extinction 605/70 nm) (KEYENCE).
Bacterial infection
MS was applied to the adaxial leaf surface of 4-week-old plants by brushing 4 times at an interval of 15 min for 3 h. Sample leaves were then inoculated by infiltration, using a plastic syringe (Terumo Tuberculin Syringe 1 mL; TERUMO), with Psm ES4326 (OD600 = 0.001) resuspended in 10 mM MgCl2. Bacterial growth was measured 2 days after inoculation as described previously78.
Fungal infection
Alternaria brassicicola strain Ryo-1 was cultured on 3.9% (w/v) potato dextrose agar plates (PDA; Becton, Dickinson and Company, NJ, USA) for 4–20 days at 28 °C in the dark. After incubation of the agar plates for 3–7 days under ultraviolet C light, a conidial suspension of A. brassicicola was obtained by mixing with RO water79. The adaxial side of leaves from 4-week-old plants was treated with 10 droplets or MS by brushing 4 times at an interval of 15 min for 3 h, followed by spotting with 5 µL conidia suspension (2 × 105 per mL) of A. brassicicola on the adaxial side of leaves. Inoculated plants were placed at 22 °C under diurnal conditions (16-h-light/8-h-dark cycles) with 100% relative humidity. The lesion size of fungal infection was measured with ImageJ 3 days after inoculation.
Statistics and reproducibility
GraphPad Prism 9 (GraphPad Software, CA, USA) was used for all statistical analyses. One-sided or two-sided Tukey’s multiple comparison test, one-sided Šídák’s multiple comparison test, or two-way analysis of variance (two-way ANOVA) were used for multiple comparisons. In all graphs, asterisks indicate statistical significance tested by one-sided or two-sided Tukey’s multiple comparison test, one-sided Šídák’s multiple comparison test, or two-way ANOVA (multiple groups).
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
Date availability
The authors declare that all data supporting the findings of this study are available within this article and its Supplementary Information files. RNA-seq and ChIP-seq data have been deposited in the DDBJ Sequence Read Archive at the DNA Data Bank (http://www.ddbj.nig.ac.jp/) with the accession numbers DRA011970, DRA009248, and DRA011123. Source data are provided with this paper.
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Acknowledgements
We thank M.F. Thomashow for the seeds of CAMTA3 variants, S. Ishiguro for the seeds of cer1-1 and cer3-1, Y. Takano for Alternaria brassicicola Ryo-1, N. Nakayama, T. Kawasaki, T. Matsushita, and G. Goshima for discussion of the work. This work was supported by JSPS KAKENHI Grant Numbers JP23120520, JP25120718, and JP18K19334 (to Y.T.), JP15H05955 (to Y.T. and T.K.), and JP19H05363, JP21H00366 (to M.N.) and by Cooperative Research Grant #1707 of the Plant Transgenic Design Initiative (PTraD) by Gene Research Center, Tsukuba-Plant Innovation Research Center, the University of Tsukuba (to Y.T.).
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M.M., M.N., S.H.S., and Y.T. designed the research. M.M. and T.I. established the artificial rain device. M.M. optimized the protocols for artificial raindrop and brush treatment. M.M. and M.N. constructed the Illumina sequencing libraries for RNA-seq and ChIP-seq. T.S. performed RNA-seq and analysis. M.N. performed the ChIP-seq and analysis of CAMTA3. T.M. and I.C.M. performed the quantification of phytohormones. M.M., Y.H., and T.K. performed the detection of MPK3 and MPK6 phosphorylation. M.M., T.M., and Y.Y.Y. performed the promoter analysis. Y.A. and M.T. generated 35Spro:GCaMP3 (gl1) plants, and M.N., M.T., and Y.A. visualized real-time [Ca2+]cyt. M.I. and S.B. generated the transgenic lines WRKY33pro:EYFP-NLS (Col-0) and CBP60gpro:EYFP-NLS (Col-0). M.M., M.I., and S.B. performed promoter-reporter imaging. M.N. and M.M. performed the rest of the experiments. M.M., M.N., M.J.S., S.H.S., and Y.T. wrote the paper with input from all authors.
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Matsumura, M., Nomoto, M., Itaya, T. et al. Mechanosensory trichome cells evoke a mechanical stimuli–induced immune response in Arabidopsis thaliana. Nat Commun 13, 1216 (2022). https://doi.org/10.1038/s41467-022-28813-8
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DOI: https://doi.org/10.1038/s41467-022-28813-8
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