E1501

HEK293T cells in 24-well plates were transfected with the SuperTOPFlash reporter plasmid, the control plasmid for β-gal and the indicated amounts of expression plasmids using Lipofectamine 2000 according to the manufacturer’s instructions. At 48 h after transfection, the luciferase assay was performed using a luciferase assay kit (E1501; Promega, Shanghai, China) according to the manufacturer’s protocol. The results are presented as the mean ± S.D. of at least three independent experiments. The luciferase activity was expressed as fold induction relative to the control.

The airway organoids (organoid size approximately 100 µm) were extracted from the Matrigel by mechanical shearing through a flamed glass Pasteur pipette, and approximately 100 organoids were infected with 1.0 × 10⁵ PFU of pseudotype SARS‐CoV‐2 in a 24‐well plate containing 500 µl airway organoid medium. The virus and organoid‐containing plates were first centrifuged at 1,100 g for 30 min and placed at 37°C with 5% CO₂ for an additional 30 min. After incubation, airway organoids were collected by centrifugation at 400 g for 5 min and re‐embedded in Matrigel with the same airway organoid medium containing pseudotype SARS‐CoV‐2. At 72 h, organoids were harvested, and the luciferase activities were measured according to the manufacturer’s instructions (E1501, Promega).

The in vitro translation assay was carried out as described previously^{4 (link),8 (link)} using the E. coli PURExpress system (New England Biolabs (NEB), E6800S). Then, 1 µl of antibiotic solution was added to 5 µl of PURExpress reaction mix. Each reaction contained 10 ng μl⁻¹ of mRNA encoding the Fluc, which was in vitro transcribed from a pIVEX-2.3MCS vector containing the Fluc gene using T7 polymerase (Thermo Fisher Scientific). The reaction mix was incubated for 30 minutes at 32 °C while shaking (600 r.p.m.). Reactions were stopped with 5 µl of kanamycin (50 mg ml⁻¹) and transferred into a 96-well microplate (Greiner Lumitrac, non-binding, white, chimney). Next, 40 µl of luciferase assay substrate solution (Promega, E1501) was added, and luminescence was measured using a plate reader (Tecan Infinite 200 PRO). Nuclease-free water was added instead of antibiotic as control. Absolute luminescence values were normalized using reactions without antibiotic. All assays were done as triplicates with individually prepared reaction mix.

MIN6-K8 mouse insulinoma cells (source: Dr. Susumu Seino, Kobe University, Japan) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat-inactivated fetal bovine serum and maintained in a humidified incubator with 95% air and 5% CO2 at 37 °C. For barr1 knockdown studies, ~1.5 × 10⁶ cells were electroporated using the Nucleofector^TM kit (VCA-1002, Lonza) and 100 nM of mouse barr1 siRNA or scrambled control siRNA (siGenome SMARTpool siRNA, a pool  of four different siRNAs; cat. # M-040976-01-0005; Horizon/Dharmacon), according to the manufacturer’s instructions. For studies involving the Pdx1-pGL3 luciferase reporter construct^{50 (link)}, cells were co-transfected with siRNA (either barr1 or control siRNA) and the Pdx1-pGL3 plasmid or empty pGL3 vector. Cells were harvested 48 h after electroporation, and luciferase activity was assessed using a Luciferase Assay System (E1501, Promega) and a multi-mode plate reader (SpectraMax M5, Molecular Devices).

The isolation and transfection of barley protoplasts was performed as described in Saur et al., 2019 [33 (link)]. In short, cDNAs of the AVR_as were co-expressed with cDNAs of Mla10, Mla22, Mla10Lrr22, and Mla22Lrr10 using the pIPKb002 vector with a strong ubiquitin promoter or with intron-containing DNA of chimeras M16666, M11166, M61111, or M66111 in a pUBI-NOS-vector (described in Shen et al., 2003 [38 (link)]) in barley cv. Golden Promise protoplasts. Protoplast solution (300 μl of 3.5 x 10⁵ cells/ml) was transfected with 4.5 μg of LUC reporter construct, 10 μg of Mla plasmid, and 6.5 μg of the respective AVR_a effector or an empty vector (EV). The protoplasts were incubated for 16 h at 21°C in a plant growth chamber and then harvested by centrifugation at 1,000 x g. Subsequently, the supernatant was removed, and protoplasts were lysed by addition of 180 μl of cell culture lysis reagent (Promega, E1531). The LUC activity of samples was measured in a luminometer (Centro, LB960) using a 96-well plate in which 50 μl of protoplast lysate were mixed with 50 μl of the LUC substrate (Promega, E1501). The relative LUC units (RLU) were calculated by setting the absolute value of the EV sample to 1.

TZM-bl cells (2 × 10⁴ cells/well seeded in a 24-well plate the previous day) were inoculated with MDM-free culture supernatant for 6 h at 37°C, washed with PBS, and maintained in DMEM-10. At 48 h postinfection, cells were lysed in cell culture lysis reagent (E153A, Promega) and analyzed for luciferase activity using a commercial kit (E1501, Promega).