Plant materials and growth conditions
Arabidopsisthaliana (L.) Heynh. accession Columbia was used as the wild-type Arabidopsis plant (WT). For phenotypic observations and gene expression analyses, plants were grown in soil at 22 or 28 °C under long-day conditions (16 h light and 8 h dark)—18-day old Arabidopsis at 22 °C and 14-day old Arabidopsis at 28 °C. To determine the resistance of transgenic Arabidopsis against Pseudomonas syringae pv. tomato, plants were grown for 2 weeks on half-strength Murashige and Skoog solid media (pH 5.7) at 22 °C under long-day conditions. For tobacco infiltration, Nicotianabenthamiana plants were grown for 4 weeks at 22 °C under long-day conditions.
Genomic DNA and RNA isolation
Genomic DNA was isolated using the urea lysis miniprep protocol as described by Yeon et al. (2019), and total RNA was prepared using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.
cDNA synthesis and PCR
cDNA was synthesized using ReverTra Ace-α™ (Toyobo Co., Ltd, Osaka, Japan), following the manufacturer’s protocol. For cloning, genomic DNA and alternatively spliced cDNAs of AtTX14 were amplified using nPfu-forte DNA polymerase (Enzynomics Inc., Daejeon, Korea), as follows: initial denaturation at 94 °C for 3 min, 35 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min, and a final extension at 72 °C for 5 min. Semi-quantitative reverse transcription (RT) polymerase chain reaction (PCR) was performed using AccuPower® PCR PreMix (Bioneer Corp., Deajeon, Korea), while quantitative RT-PCR was performed using iQ™ SYBR® Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA). Information on all primers used is listed in Table S1 (de Oliveira et al. 2016; Zhu et al. 2014).
Protein sequence analysis and structure prediction
Based on the nucleotide sequences of AtTX14-spliced mRNAs, primary protein sequences were predicted and aligned with multiple sequence alignments using Clustal Omega and Jalview 2.11.0 (Goujon et al. 2010, Waterhouse et al. 2009). The secondary structure was predicted based on a 3D protein model of AtTX14 Full, generated using SWISS-MODEL based on the crystal structure of the RPS4 TIR domain (Protein Data Bank ID: 4C6R) (Waterhouse et al. 2018; Williams et al. 2014).
Construction of plant overexpression vector
PCR products and a pCAMBIA3300m vector were digested using BamHI and PstI (Enzynomics Inc.) and purified using the EZ-Pure™ PCR Purification Kit ver.2 (Enzynomics Inc.). The pCAMBIA3300m vector was generated from pCAMBIA3300 by adding (1) an extra CaMV 35S promoter and (2) multiple cloning sites made up of BamHI, KpnI, SacI, XbaI, and PstI recognition sequences downstream of the additional CaMV 35S promoter. AtTX14 1IR transgenic plants were generated by cloning AtTX14 genomic DNA from the start codon to the in-frame stop codon in the first intron at the multiple cloning site. In the case of AtTX14 2IR and Full transgenic plants, coding sequences amplified from cDNA were used for cloning.
Agrobacteriumtumefaciens (Agrobacterium) and plant transformation
Plant overexpression vectors were introduced into Agrobacterium strain GV3101 by the freeze-thaw method (Holsters et al. 1978). Transgenic plants were generated by the floral dipping method of WT plants using transformed Agrobacterium (Clough and Bent 1998). T1 transformants and their self-pollinated progenies were selected based on their resistance to glufosinate-ammonium (BASTA®; BASF, Ludwigshafen, Germany).
Yeast two-hybrid (Y2H) analyses
pGBKT7 and pGADT7 (Takara Bio., Inc., Shiga, Japan), containing GAL4 DNA binding domain (BD) and activation domains (AD), respectively, were used as bait and prey cloning vectors to investigate protein-protein interactions in the Y2H system. pGBKT7 and pGADT7 with the genes of interest were co-transformed into yeast AH109 strain, following the manufacturer’s protocol for Yeastmaker™ Yeast Transformation System 2 (Clontech Laboratories, Mount View, CA, USA). Transformants were grown at 30 °C for 4–5 days (d) on synthetic dropout (SD) without leucine and tryptophan (-LT) or without leucine, tryptophan, and histidine (-LTH).
Western blotting of proteins expressed in yeast
Yeast total proteins were extracted from cells grown in YPDA medium (10 g Bacto yeast extract, 20 g Bacto peptone, and 40 mg adenine hemisulfate in 1 L water) at 30 °C overnight (O/N), using Urea/SDS protein extraction buffer (40 mM Tris-HCl [pH 6.8], 5% sodium dodecyl sulfate [SDS], 30% glycerol, 2% β-mercaptoethanol, and 8 M urea). The proteins were separated on a 10% SDS-PAGE gel and transferred onto a polyvinylidene difluoride membrane (Merck Group, Darmstadt, Germany) with a transfer buffer (4.5 g Tris, 7.765 g glycine, 0.05 g SDS, 175 mL methanol in 1 L water) at 100 V for 2 hours (h). After rinsing with water, the membranes were blocked using General-Block Solution (TransLab, Daejeon, Korea) for 1 h. Membranes were washed once with PBS-T buffer (8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4, 2 mL Tween-20 in 1L water) and incubated in PBS-T with the primary antibody, HA or Myc (Clontech Laboratories), at 4 °C O/N. After washing for 1 h with PBS-T, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnologies Inc., Dallas, TX, USA) in PBS-T for 1 h. HRP activity was detected using SuperSignal® West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA) and ChemiDoc™ Touch Gel Imaging System (Bio-Rad Laboratories).
Infection of Arabidopsis with P. syringae pv. tomato
DC3000 and DC3000 (AvrRpm1), P. syringae without or with the AvrRpm1 effector protein, were infected into 2-week-old Arabidopsis, and colony-forming units (CFUs) were determined at 0 and 3 d post infiltration (dpi), as described by Ishiga et al. (2017). In brief, plants were inoculated by flooding with a bacterial suspension (4 × 106 CFU mL-1 in 0.025% v/v Silwet L-77 and 10 mM MgCl2).
Transient protein expression in tobacco by agroinfiltration and measurement of chlorophyll contents
Agroinfiltration was performed using a previously described method, with slight modifications (Kim et al. 2018; Kontra et al. 2016). In brief, seed cultures of Agrobacterium GV3101 carrying an overexpression vector for the corresponding AtTX14 AS variant and p19, an RNA silencing suppressor, were grown in LB medium containing appropriate antibiotics at 25 °C for 2 d. The main cultures were grown at 25 °C O/N after seed culture inoculation. Harvested cells were resuspended in infiltration buffer (10 mM MES [pH 5.7], 10 mM MgCl2, and 200 μM acetosyringone) and diluted to an OD600 of 1.0. After a 2-h incubation, Agrobacteria to be tested in combinations were mixed and infiltrated together onto the lower sides of 4-week-old tobacco leaves. The induction of HR was assessed 10 d after agroinfiltration. Chlorophyll content in tobacco leaf segments was measured 10 d after Agrobacterium infiltration, as described by Harris et al. (2013).