GoKawiilMolecular cloning
Coding sequences for the near full-length NPR1 (amino acids 40–564; AT1G64280), NPR3 (amino acids 33–561; AT5G45110), NPR4 (amino acids 30–552; AT4G19660), MED15A-FL (amino acids 1–1,335; AT1G15780), MED15A-KIX (amino acids 17–98; AT1G15780), MED15A-KIX long (amino acids 1–98), MED15E-KIX (amino acids 1–75; AT2G10440), MED15D-FL (amino acids 1–189; AT1G15790), MED15D-KIX (amino acids 1–93), NIMIN1-FL (amino acids 1–142; AT1G02450), NIMIN1-NTD (amino acids 1–108), NIMIN1-αH long (amino acids 31–72), NIMIN1-αH short (amino acids 36–68), NIMIN1-CTD (amino acids 109–142) and TGA3 (amino acids 87–384; AT1G22070) were amplified from the A. thaliana cDNA library with primers pairs that contain linker sequences at the 5′ ends for T4 DNA polymerase (NEB) treatment (forward: 5′-AAAACCTCTACTTCCAATCG-3′, reverse: 5′-CCACACTCATCCTCCGGTTA-3′) for ligation-independent cloning. To generate the Avi-tagged MED15A-KIX, MED15A-KIX (amino acids 17–98) was synthesized with an Avi tag followed by a GSGSGSGS linker at the 5′ by Integrated DNA Technologies and fused after the TEV cleavage site. The loop-deletion NPR1-SBD (amino acids 415–564 with amino acids 460–483 replaced with a GSGSG linker) and the mammalianized MBP (mMBP)51 were synthesized by Azenta/Genewiz. The mMBP–NPR1-SBD fusion construct, where mMBP was used as a crystallization chaperone, was generated by fusing the loop-deletion NPR1-SBD to the last α-helix of mMBP through overlap PCR with a linker sequence encoding five alanine residues (A5) between the two52. The NPR1 and MED15A-KIX mutant inserts were made either through overlap PCR or using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs). For His–MBP-tagged constructs, the inserts were cloned into the pAL vector for Escherichia coli expression system or into HTB vector for insect cell. For His–Venus, the pACE vector that was modified for expressing in both E. coli and the insect cell systems was used. For His–Msb (E. coli acidic protein msyB)53, a pET vector was used. T4 DNA polymerase-treated inserts were incubated with T4 DNA polymerase-treated vectors at a 2:1 molar ratio at room temperature for 5 min before transformation into DH5α-competent or Stbl3-competent cells. For all constructs, the inserts were fused with a 6×His tag followed by a MBP, Venus or Msb protein and a TEV cleavage site at the N terminus. Plasmids were miniprepped with Qiagen miniprep kit following the manufacturer’s instructions and confirmed via Sanger sequencing or whole-plasmid sequencing at Genewiz (Azenta). Positive plasmids were either transformed into BL21-competent cells for expression in E. coli or into DH10Bac cells for bacmids. For insect cell expression, DH10Bac cells were cultured in LB overnight at 37 °C before being resuspended, lysed and then neutralized with 250 µl of P1, 250 µl of P2 and 350 µl of N3 buffers from the Qiagen miniprep kit, respectively. The mixture was incubated on ice for 10 min before being centrifuged at top speed for 10 min. The supernatant was mixed with equal volume of 2-propanol and incubated on ice for 30 min before centrifuging for 15 min at room temperature. The resulting pellet was washed with 500 µl of 70% ice-cold ethanol twice, air dried and eventually dissolved in 40 µl of ddH 2 O. Recombinant virus was made using Sf9 monolayer cells (Thermo Fisher Scientific, Gibco), and proteins were expressed in Hi5 monolayer cells (Thermo Fisher Scientific, HighFive) in the baculovirus expression vector system (Invitrogen).
Protein expression and purification
E. coli cells expressing the target proteins were cultured at 37 °C to optical density at 600 nm (OD 600 ) of 0.8–1 and induced with 0.2–0.5 mM IPTG at 16 °C overnight. Cells were harvested by centrifugation at 3,000g, resuspended and lysed in lysis buffering (200 mM NaCl, 20 mM Tris HCl pH 8, 0.5 mM TCEP and 20 mM imidazole pH 8) and 1 mM PMSF by sonicating with 30% amplitude, 0.5 s ON–0.5 s OFF for 5 min. The sonicated crude was then centrifuged at 39,375g for 50 min at 4 °C. The supernatant or lysate was loaded on a column that contained 2 ml of nickel or amylose resin and was pre-equilibrated with lysis buffer. After being washed with lysis buffer, protein on the resin was eluted with lysis buffer containing either 200 mM imidazole (for nickel resin) or 10 mM maltose (for amylose resin). The eluted protein was mixed with 4× Laemmli buffer (277.8 mM Tris-HCl, pH 6.8, 44.4% (v/v) glycerol, 4.4% SDS and 0.02% bromophenol blue) and confirmed on an SDS–PAGE gel and by TEV cleavage. Purification procedures for proteins expressed in insect cell were the same as above with a few modifications. Insect cells were pelleted by centrifugation at 2,000g for 15 min, resuspended and lysed in the same lysis buffer supplemented with 1 mM PMSF and protease inhibitors including leupeptin, aprotinin and pepstatin. The elution was then concentrated, centrifuged at top speed at 4 °C for 10 min before size exclusion using Superdex 200 increase 10/30 gl (Cytiva) in sizing buffer (150 mM NaCl, 20 mM HEPES pH 7.5 and 0.5 mM TCEP). Unless otherwise stated, NPR1 (amino acids 40–564) and MED15A-KIX (amino acids 17–98) were used in this study.
In vitro pulldown assays
To detect the interaction between MED15s and NPRs, His–MBP–MED15s and His–Venus–NPRs were expressed in E. coli separately as described above. As His–Venus-tagged NPRs were associated with multiple proteolytic products, to control the amount of the input proteins for the pulldown experiments, 4 µg of each protein was run on an SDS–PAGE gel to calculate the percentage of the intact protein in the sample. The bands of the intact proteins were quantified with the Image Lab (v5.1) software (Bio-Rad). The concentration of the intact proteins was calculated accordingly. A total of 20 µg of His–MBP–MED15 was mixed with His–Venus–NPR at a 1:2 molar ratio with or without 200 µM SA and incubated on ice for 20 min. The mixture was then applied to 20 µl amylose resin (NEB) that was pre-equilibrated with sizing buffer, gently mixed and incubated on ice for 1 h. The supernatant was removed after centrifugation at 600g for 1 min. The resin was washed three times with 200 µl of sizing buffer. To elute, the resin was resuspended with 40 µl of 1× sample buffer and boiled at 100 °C for 10 min. After centrifugation at top speed for 1 min, the supernatant was collected and loaded on an SDS–PAGE gel that was run in 1× Rapid Running Buffer Solution (Nacalai Tesque) at 210 V for 25 min. For gel source data, see Supplementary Fig. 1.
For the pulldown experiment involving full-length NPR1 and various NIMIN1 constructs, His–MBP–NPR1 was used to pull down His–Venus–NIMIN1s. In the experiment assessing the interaction between NIMIN1 αH and various NPR1 constructs, the His–MBP-tagged NIMIN1-αH long (amino acids 31–72) was co-expressed with His–Msb-tagged NPR1-FL, NPR1-BTB-ANK or NPR1-SBD and purified with amylose resin. In the experiment in which the interaction between NPR1-SBD and MED15A-KIX or NIMIN1-αH long (amino acids 31–72) was examined, His–mMBP-tagged NPR1-SBD was used to pull down His–Venus-tagged MED15A-KIX or His–Venus-tagged NIMIN1-αH long. To pull down the MED15A-FL, His–MBP–NPR1 and His–Venus–MED15A were expressed together in the insect cell. The co-expression samples were assessed side by side with the two separately expressed proteins. For the SA-treated samples, 200 µM SA was included in buffers throughout the pulldown assays. For in-gel Venus florescence detection, protein samples were incubated in 1× sample buffer at room temperature for 5 min, and unboiled samples were loaded to the SDS–PAGE gel; before Coomassie blue staining, Venus fluorescence was detected with DyLight 488 Blot, and protein ladder was imaged with DyLight 680 Blot using the Image Lab Touch software (v3.0.1.14) built in the ChemiDoc MP imaging system (Bio-Rad).
Biolayer interferometry
The binding between NPR1 and MED15A-KIX with or without SA was detected using Octet Red 96 (ForteBio, Pall Life Sciences). The Octet streptavidin biosensors (Sartorius, for kinetics) were equilibrated in buffer for 60 s, pre-activated with 100 nM biotinylated Venus nanobody for 20 s and quenched with 200 nM biocytin for 60 s. His–Venus-tagged MED15A-KIX (200 nM) was then immobilized on the Venus nanobody-coated biosensors for 22 s. After being rinsed in buffer for 60 s, the MED15A-KIX-loaded biosensors were then dipped into a serial dilution of His–MBP–NPR1 purified from insect cell or His–mMBP–NPR1-SBD with or without SA for association and back to buffer for dissociation. All reactions were carried out at 30 °C in a Greiner black 96-well microplate containing 200 μl of sizing buffer containing 0.02% Tween-20 and 0.1% ovalbumin. The buffer-only well served as the instrument internal control, and the MED15A-KIX-absent well was the reference. Data collection was done using the Octet BLI Discovery software (v13.0.3.26). The dissociation constant between MED15A-KIX and NPR1 was determined from the steady-state equilibrium responses using the Octet BLI Analysis (v12.2).
AlphaLISA binding assays
... continue reading