Increasing the resilience of plant immunity to a warming climate

Plant materials

A. thaliana plants were grown in soil (2:1 Arabidopsis Mix: perlite) covered with or without standard Phifer glass mesh for 3–4 weeks at 21 °C–23 °C and 60% relative humidity under a 12 h light/12 h dark regimen (100 ± 10 µmol m−2 s−1). Accessions, mutants and transgenic lines are outlined in Supplementary Table 3. All experiments with 35S::CBP60g were performed with line no. 17, unless otherwise specified.

Seeds of rapeseed (Brassica napus) cultivar Westar, tomato (Solanum lycopersicum) cultivar Castlemart, and tobacco (Nicotiana tabacum) cultivar Xanthi were grown in Arabidopsis Mix soil supplemented with 1 g l−1 of 20-20-20 general purpose fertilizer (Peters Professional). After 2 days of imbibition, plants were grown in growth chambers (20 °C/18 °C, 16 h day/8 h night for rapeseed; 23 °C/23 °C; 12 h day/12 h night for tomato and tobacco) for 4–7 weeks.

Seeds of rice (Oryza sativa) cultivar Nippponbare were germinated on wet filter paper in petri dishes and 4- to 5-day-old seedlings were transplanted to Redi-earth soil. Seedlings were grown at 28 °C (16 h day/8 h night) for 4–5 weeks.

Generation of constructs and transgenic lines

To generate transgenic Arabidopsis harbouring 35S::uORFsTBF1-CBP60g, 35S::TGA1-4myc, or 35S::SARD1, genomic DNA (CBP60g, TGA1) or coding sequences (SARD1) were amplified and ligated into pENTR D-TOPO (Invitrogen). To clone TBF1 uORF sequence, PCR-amplified uORFsTBF139 amplicon was ligated into pENTR-AtCBP60g using HiFi DNA Assembly (New England Biolabs). The uORFsTBF1CBP60g, TGA1 or SARD1 construct was subcloned to pGWB517 through Gateway Cloning (Invitrogen). Plasmids carrying gene constructs were transformed into Agrobacterium tumefaciens GV3101, which was used for Arabidopsis transformation by floral dipping42. T1 plants were selected on half-strength Murashige and Skoog medium supplemented with hygromycin (35 mg l−1) and 1% sucrose. Homozygous T2 and T3 transgenic plants were analysed.

To generate 35S::ICS1 plants, the ICS1 cDNA was amplified from RNA extracted from infected Arabidopsis leaves and ligated into pCR Blunt TOPO (Invitrogen). Full-length cDNA with chloroplast transit sequence was confirmed and the 35S::ICS1 construct was subcloned into pCAMBIA3301 modified to remove the GUS reporter and to include a C-terminal V5-His6 tag (Invitrogen) resulting in pSM200-1. pSM200-1 was transformed into A. tumefaciens GV3101 and used to transform Arabidopsis eds16-1 mutant by floral dipping42. T1 plants were selected for glufosinolate tolerance using Finale and surviving plants were selfed and tested for presence of the insert using PCR. Homozygous T4 transgenic plants were analysed.

To generate transgenic rapeseed harbouring 35S::AtCBP60g-myc, the AtCBP60g coding sequence, amplified from Arabidopsis cDNA, or the corresponding genomic sequence was cloned into pGWB517 through Gateway reaction (Invitrogen). The binary vector was introduced into A. tumefaciens GV3101 by electroporation. B. napus cultivar Westar were transformed using Agrobacterium-mediated method43. After 7-day explant-recovery period following co-cultivation on MS medium with benzyladenine (3 mg l−1), and timentin antibiotic (300 mg l−1) to eliminate Agrobacterium, putative transformants with roots (T0) were transferred to soil. Genomic DNA was extracted from young leaves using cetyltrimethylammonium bromide method and used for PCR detection of transgene. Two primer pairs for the hygromycin phosphotransferase (HPT) and AtCBP60g genes in the transgene were used to assess transformation. About ten T0 transgenic lines were used to produce T1 transgenic plants by self-pollination. RT–qPCR was used to screen for independent T1 transgenics that robustly expressed the AtCBP60g transcript. 35S::AtCBP60g line no. 1-12 was derived from the cDNA construct, whereas 35S::AtCBP60g line no. 2-11 was derived from the genomic DNA construct.

PCR primers are listed in Supplementary Table 4 and sequences were confirmed by Sanger sequencing.

Agrobacterium-mediated transient expression in rapeseed and tobacco

For transient expression in rapeseed, Agrobacterium GV3101 harbouring 35S::mRFP-4myc or 35S::AtCBP60g-4myc was grown in Luria-Bertani (LB) medium, resuspended in infiltration buffer (10 mM MES (pH 5.7), 10 mM MgCl2 and 500 µM acetosyringone) at OD600 = 0.1, and infiltrated to the first and second true leaves of rapeseed plants using a needleless syringe. For transient expression in tobacco (N. tabacum), Agrobacterium GV3101 harbouring 35S::eGFP-GBPL3 or 35S::mRFP-MED15-flag was grown in LB medium, resuspended in the same infiltration buffer at OD600 = 0.1, and infiltrated to fully expanded leaves of tobacco plants using a needleless syringe. Agroinfiltrated rapeseed or tobacco plants were incubated for 2–3 days at 21–23 °C before experiments.

Temperature conditions

Based on previous studies15,44,45,46, Arabidopsis plants were acclimated at 23 °C (ambient) or 28 °C (elevated) for 24 h before chemical treatment and/or 48 h before pathogen infiltration, unless otherwise specified. Four- to five-week-old rapeseed plants were incubated at ambient (23 °C) or elevated temperatures (28 °C) for 48 h before pathogen infiltration or chemical treatments. Four- to five-week-old tomato plants were incubated at ambient (23 °C) or elevated temperatures (28 °C–32 °C) for 48 h before chemical treatments. Five-week-old rice plants were incubated at ambient (28 °C) or elevated temperatures (35 °C) before chemical treatments. Four- to seven-week-old tobacco plants were incubated at ambient (23 °C) or elevated temperatures (28 °C) for 48 h before chemical treatments. All plants were grown with a 12 h day/12 h night cycle, except for rice and rapeseed plants, which were grown with a 16 h day/8 h night cycle.

Growth and developmental phenotyping

For growth biomass measurements, aboveground parts of 4- or 6-week-old pre-flowering plants were weighed, and representative plants were photographed. For flowering time measurements, the first instance of floral appearance for each individual plant was recorded.

BTH and flg22 treatments

Arabidopsis plants were infiltrated or sprayed with mock (0.1% DMSO), benzo(1,2,3)thiadiazole-7-carbothioic acid-S-methyl ester (BTH; Chem Service, 100 µM, 0.1% DMSO) or flg22 peptide (EZBiolab, 200 nM in 0.1% DMSO). For tomato or rapeseed, 50 µM (rapeseed) or 100 µM (tomato) of BTH solution (0.02% Silwet L-77 and 0.1% DMSO) or solvent control was sprayed. Plants were further incubated for 24 h. For rice, 200 µM of BTH solution (0.1% Silwet L-77 and 0.1% DMSO) or solvent control was sprayed. Rice plants were further incubated for 24 h and their 4th leaves were used for analyses.

Basal disease-resistance assay

Plants were infiltrated with 0.5 to 1.5 × 106 CFU ml−1 (OD600 = 0.0005; for Arabidopsis) or 0.5 to 1.5 × 105 CFU ml−1 (OD600 = 0.00005; for rapeseed) of Pst DC3000, 0.5 to 1.5 × 108 CFU ml−1 of Pst DC3000 ΔhrcC (OD600 = 0.05; for Arabidopsis) or 0.5 to 1.5 × 106 CFU ml−1 of P. syringae (Ps) pv. tabaci 11528 (for tobacco) as described previously15. Plants were returned to growth chambers at the appropriate temperature and 60% relative humidity. Bacterial levels were measured as previously described15,47.

ETI assay

Plants were dipped in 0.5 to 1.5 × 108 CFU ml−1 of Pst DC3000(avrPphB)48 and Pst DC3000(avrRps4)49 (OD600 = 0.05) as described previously24,47. Plants were left at room temperature for 1 h with a cover dome to maintain high humidity and then returned to the growth chamber without covering at either 23 °C or 28 °C (60% relative humidity). Bacterial growth was measured as described in the previous section.

Gene expression analyses

RNA extraction and quantitative PCR analyses were performed as described previously15. Twenty to sixty milligrams of fresh leaf tissues were flash-frozen in liquid nitrogen and ground using a TissueLyser (Qiagen). Plant RNA was extracted using a Qiagen Plant RNeasy Mini Kit following the manufacturer’s protocol, including on-column DNase I digestion. cDNA was synthesized by adding 100–300 ng of RNA to a solution of oligo-dT primers, dNTPs and M-MLV reverse transcriptase (Invitrogen). Approximately 1.5 ng of cDNA was mixed with the appropriate primers (Supplementary Table 4) and SYBR master mix (Applied Biosystems). Quantitative PCR (qPCR) was run on a 7500 Fast Real-Time PCR system or QuantStudio 3 Real-Time PCR system (Applied Biosystems), with 2–4 biological replicates (and 3 technical replicates for each biological replicate) per experimental treatment. StepOnePlus (Applied Biosystems) was used for data acquisition and analysis. Gene expression values were calculated as described previously15 with the following internal controls: PP2AA3 (Arabidopsis), SlARD2 (tomato), OsUBC (rice), NtAct (tobacco) and BnaGDI1 (rapeseed). RT–qPCR primer sequences are listed in Supplementary Table 4.

Transcriptome analyses

For RNA-seq in Fig. 1, Arabidopsis Col-0 plants were inoculated with mock (0.25 mM MgCl2) or Pst DC3000 suspension, and then incubated at 23 °C or 30 °C for 24 h. For RNA-seq in Fig. 3, Arabidopsis Col-0 and 35S::CBP60g were inoculated with Pst DC3000 suspension, and then incubated at 23 °C or 28 °C for 24 h. Total RNA was extracted as described above. RNA samples for each treatment were checked for quality and cDNA libraries were prepared, as described previously15. All 12 libraries per experiment were pooled in equimolar amounts for multiplexed sequencing. Pools were quantified using the Kapa Biosystems Illumina Library Quantification qPCR kit, and loaded on one lane (Fig. 1) or two lanes (Fig. 3) of Illumina HiSeq 4000 Rapid Run flow cells. RNA-seq and analyses were performed as described previously15. For Fig. 1, results were filtered for Pst DC3000-induced or -repressed genes using a pathogen/mock fold change > 2. Temperature-downregulated, neutral and upregulated target genes were analysed for Gene Ontology (GO) enrichment using the Database for Annotation, Visualization and Integrated Discovery50 (DAVID; https://david.ncifcrf.gov/). For Fig. 3, results were further filtered for genes with RPKM values above 1 and 23 °C/28 °C RPKM ratios with at least twofold change. Filtered genes were grouped into four clusters. Cluster 1 had genes more downregulated at 28 °C in Col-0 (that is, Col/35S::CBP60g ratios of 23 °C/28 °C RPKM values > 2). Cluster 2 had genes more upregulated at 28 °C in Col-0 (that is, Col/35S::CBP60g ratios of 23 °C/28 °C RPKM values < 0.5). Cluster 3 had genes similarly downregulated, whereas cluster 4 had genes similarly upregulated in Col-0 and 35S::CBP60g, respectively (that is, Col/35S::CBP60g ratios of 23 °C/28 °C RPKM values between 2 and 0.5). GO enrichment analyses were also conducted using DAVID50.

Hormone profiling

Plant hormones were extracted and quantified using a previously described protocol15, with minor modifications. Methanolic extraction was performed with abscisic acid (ABA)-d6, SA-d4 or SA-13C6 as an internal control. Filtered extracts were analysed using an Acquity Ultra Performance Liquid Chromatography system coupled to a Quattro Premier XE MS/MS (Waters) or a 1260 infinity High Performance Liquid Chromatography system coupled to a 6460 Triple Quadrupole mass spectrometer (Agilent). Column temperature was set at 40 °C with a 0.4 ml min−1 flow rate and a gradient of mobile phases water + 0.1% formic acid (A) and methanol (B) was used as follows: 0–0.5 min 2% B; 0.5–3 min 70% B; 3.5–4.5 100% B; 4.51–6 min 2% B; followed by additional 1 min for equilibration. Eluted analytes were introduced into Agilent jet stream electro spray ionization ion source and analysed in negative ion mode with delta EMV (–) of 200. The following parameters were used for the mass spectrometer source: gas temperature, 300 °C; gas flow, 5  min−1; nebulizer, 45 psi; sheath gas temperature, 250 °C; sheath gas flow, 11 l min−1; capillary voltage, 3,500 V; nozzle voltage, 500 V. The following parameters were used for data acquisition in multiple reaction monitoring (MRM) mode: dwell time, 50 ms; cell accelerator voltage, 4 V; fragmentor voltage, 90 V and collision energy, 16 V for SA and SA-d4; fragmentor voltage, 130 V and collision energy, 9 V for ABA-d6. The following MRM transitions were monitored: SA (m/z 137→93), SA-d4 (m/z 141→97) and ABA-d6 (m/z 269.1→159.1). Peak selection and integration of acquired MRM data files was done using QuanLynx v4.1 software (Waters) or Quantitative Analysis (for QQQ) program in MassHunter software (Agilent). Analyte levels were calculated as previously indicated15.

Nuclear–cytoplasmic fractionation

Approximately 0.1–0.2 g of ground plant tissues (pre-frozen, stored at −80 °C for less than 1 week) were dissolved in nuclei isolation buffer (20 mM Tris-Cl pH 7.5, 25% glycerol, 20 mM KCl, 2.5 mM MgCl2, 2 mM EDTA, 250 mM sucrose, 1× protease inhibitor cocktail (Roche)) on ice (NPR1–YFP protein analysis) or at 23 °C or 28 °C (GBPL3 protein analysis). After removing debris by filtering with two layers of Miracloth (Millipore), collected extracts were centrifuged at 1,000g for 10 min at cold room or at 23 °C or 28 °C using a temperature-controlled centrifuge. Supernatants were collected as the cytosolic fraction and pellets were suspended in nuclei washing buffer (nuclei isolation buffer supplemented with 0.1 % Triton X-100) (Sigma-Aldrich) by gentle tapping and centrifuged at 1,000g for 10 min at 4 °C. After washing twice, pellets were resuspended in nuclei isolation buffer and collected as nuclear fractions, which were further used for analysis.

Chromatin immunoprecipitation

ChIP was performed as previously reported51, with some modifications. Collected fresh leaf tissues were fixed (1% formaldehyde in 1× phosphate buffered saline (PBS)) by vacuum infiltration and incubated for 10–15 min to crosslink at room temperature. After quenching the remaining fixation solution with 125 mM glycine solution for 5 min, plant tissues were flash-frozen in liquid nitrogen and ground by mortar and pestle. Six-hundred milligrams of ground powder were dissolved in 2 ml of nuclei isolation buffer and crude extracts were filtered with two layers of Miracloth (Millipore). To collect nuclei, the filtrate was centrifuged at 10,000g at 4 °C for 5 min and the pellet was suspended in 75 µl of nuclei lysis buffer (50 mM Tris pH 8.0, 10 mM EDTA pH 8.0, 1% SDS). After 30 min incubation on ice, 625 µl of ChIP dilution buffer (16.7 mM Tris pH 8.0, 167 mM NaCl, 1.2 mM EDTA, 1.1% Triton X-100, 0.01% SDS) were added and the samples were sonicated for 1 min in the cold room using Sonic Dismembrator (Thermo Fisher) or 5–6 min using Bioruptor (Diagenode). After adding 200 µl of ChIP dilution buffer and 100 µl of 10% Triton X-100, samples were spun at full speed for 5 min to remove debris. For pre-clearing, samples were incubated with 25 µl of magnetic protein A or G beads (Thermo Fisher) for 2 h in the cold room. Twenty microlitres of samples were removed as 2% input samples. To capture the DNA–protein complex, antibodies (Supplementary Table 5) were used for immunoprecipitation and samples were incubated (with rotation) overnight in the cold room using a tube rotator. After washing, DNA samples were recovered using elution buffer and incubated overnight at 65 °C to remove crosslinking. DNA samples were collected and purified using a QIAquick PCR Purification Kit (Qiagen). ChIP–qPCR was performed as described in ‘Gene expression analyses’. ChIP–qPCR primer sequences are listed in Supplementary Table 4.

Immunoblot

Ground plant tissues (0.2 g per 1 ml LDS buffer (Genscript)) or fractionated protein samples (1:1 v/v) were mixed with 2× LDS buffer in the presence or absence of 2-mercaptoethanol (Sigma-Aldrich) and boiled at 70 °C for 5 min. After removing debris by centrifugation, protein samples were resolved using SDS–PAGE (SurePAGE, Genscript) and transferred to PDVF membrane (Millipore) using a wet transfer system (Bio-Rad; transfer buffer from Thermo Scientific) for further analysis. Transferred blot was incubated in PBS-T (1× PBS, 0.05 % Tween-20) supplemented with 5% non-fat dried milk for 1h and relevant proteins were detected using specific antibodies. Chemiluminescence from blots was generated after adding Supersignal West dura or West femto substrate (Thermo Scientific) and detected by a ChemiDoc MP imaging system (Bio-Rad) or iBright CL 1500 (Thermo Scientific). Relative protein quantification was performed using iBright CL 1500 (Thermo Scientific) and FIJI/ImageJ software (win64 1.52i version). Experimental conditions for antibodies are in Supplementary Table 5.

Confocal laser scanning microscopy and image analysis of Arabidopsis and tobacco cells

Images were acquired with the Zeiss confocal laser scanning microscopy 880 system and Zen black software (Carl Zeiss). Pre-treated leaves of 4- to 5-week-old plants (35S::eGFP-GBPL3) were imaged with an inverted Zeiss 880 single point scanning confocal attached to a fully motorized Zeiss Axio Observer microscope base, with Marzhauser linearly encoded stage and a 63× NA 1.4 oil plan apochromatic oil immersion objective lens. Images were acquired by frame (line) scanning unidirectionally at 0.24 microseconds using the galvanometer-based imaging mode, with a voxel size of 0.22 µm × 0.22 µm × 1 µm and an area size of 224.92 µm × 224.92 µm × 1 µm µm in Zeiss Zen Black Acquisition software and saved as CZI files. eGFP and chlorophyll was excited at 488 nm excitation laser from argon laser source and detected at 490–526 or 653–683 nm, respectively. Equal acquisition conditions (for example, excitation laser source intensity, range of acquired emission light range and exposure condition) were used for every image in each experiment. To maintain appropriate temperature during experiments, a portable temperature chamber and temperature-controlled specimen chamber of confocal microscope were used. To analyse images, FIJI/ImageJ software (Windows 64 1.52i version) was used.

Prediction of intrinsically disordered region of A. thaliana MED15

The A. thaliana MED15 protein (encoded by At1g15780) disordered region was calculated with the Predictor of Natural Disordered Regions online tool (http://www.pondr.com/). The MED15 amino acid sequence was obtained from The Arabidopsis Information Resource (TAIR; https://www.arabidopsis.org/).

Experimental design and statistical analysis of dataset

Sample size and statistical analyses are described in the relevant figure legends. Sample size was determined based on previous publications with similar experiments to allow for sufficient statistical analyses. There were no statistical methods used to predetermine sample sizes. Three to four plants (biological replicates) per genotype per treatment were analysed per individual experiment. Plants of different genotypes were grown side by side in environmentally controlled growth chambers (light, temperature, humidity) to control other covariates and to minimize unexpected environmental variations. Leaf samples of similar ages were collected and assessed randomly for each genotype. Researchers were not blinded to allocation during experiments and outcome assessment. This is in part because different plant genotypes, temperatures and treatments investigated exhibit quite distinct and obvious phenotypes visually; thus, blinding was not possible in these cases. Routine practices included more than one author observing/assessing phenotypes, whenever possible. Three or more independent experiments were performed for all assays, unless specified otherwise. The following statistical analyses were employed: (1) Student’s t-test with Bonferroni test for significance was used for pairwise comparisons; (2) one-way analysis of variance (ANOVA) with Bartlett’s test for significance was used for multi-sample experiments with one variable; and (3) two-way ANOVA followed by Tukey’s honest significant difference test was used for multi-variable analyses. Statistical tests are described in the figure legends. Bar graphs and dot plots were generated with GraphPad Prism 9 and show the mean ± s.d. or mean ± s.e.m. and individual data points.

Graphic design

Figs. 1a2f and 4f and Extended Data Fig. 7a were created in part using BioRender.com.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this paper.

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