Infinite f200 luminometer

Both allelic forms (QQ and qq) of the 1010 bp open chromatin fragment were synthesized and cloned into the pGL3-basic and pGL3-promoter luciferase reporter vectors (Promega Corporation). The sequence and orientation of the inserts were confirmed by sequencing. For cell culture, DF1 (a chicken fibroblast cell line) cells were cultured in 24-well plates with DMEM (Gibico, Carlsbad, CA, USA) supplemented with 10% FBS (Gibico, Carlsbad, CA, USA) in a 37 °C incubator with 5% CO₂. Using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s recommendations, we transfected per well cell in a 24 well plate (~80%–90% confluency) with a mixture comprising 720 ng of the pGL3 firefly luciferase reporter construct, 90 ng of the pRL-TK Renilla luciferase construct (Promega Corporation) and 3 µl Lipofectamine 2000. The luciferase assay was performed 48 h after transfection using the Dual Luciferase Reporter Assay system (Promega Corporation) and an Infinite F200 Luminometer (Tecan, Switzerland). Ratios of firefly luminescence/Renilla luminescence were calculated. For each test construct, one expression value was obtained as the average of three technical replicates in each plate (Supplementary Data 11).

The pBI121-3×FLAG vector inserted into 3 × FLAG tag to fuse with the pBI121 vector [43 (link)] was used for this assay. The full-length cDNA sequence of AopN was cloned into the pBI121-eGFP vector. The Flg22-elicited ROS assay was performed with the transient expression of EV (empty vector) or AopP fused FLAG using A. tumefaciens GV3101 at an optical density at 600 nm (OD₆₀₀) of 0.5. The 4–6-week-old N. benthamiana leaves were used in this assay. Ten leaf disks (4 mm in diameter) were collected from each inoculated site 24 h after inoculation and floated in 100 μL of sterile distilled water in a 96-well plate. Next, the water was replaced with 100 μL of solution containing 100 nM flg22, 20 μg/mL horseradish peroxidase, and 100 μM luminol. Luminescence was recorded immediately using a Tecan Infinite F200 luminometer (Tecan, Mannedorf, Switzerland) for 40 min. The experiment was independently repeated three times. All primers used in the PCR are listed in Table S2.

The full-length cDNA sequence of aopV was cloned and fused with pBI121-eGFP vector [46 (link)]. All primers used for PCR are listed in Table S2. Agrobacterium tumefaciens GV3101 was used to transiently express EV (pBI121-eGFP empty vector) and AopV at an optical density (OD₆₀₀) of 0.5. A Flg22-induced ROS assay was conducted with N. benthamiana leaves 36 h after inoculation. Twelve leaf discs (4 mm in diameter) were collected from each inoculation area and floated on 100 μL sterilized distilled water in a 96-well plate. The water was then removed, and 100 μL of a solution containing 100 nM flg22, 20 μg/mL horseradish peroxidase, and 100 μM luminol was added to each well. The luminescence was recorded immediately for 1 h using Tecan Infinite F200 luminometer (Tecan, Männedorf, Switzerland) [47 (link)].

The full-length cDNA sequence of aopP was cloned and fused with the pBI121-3 × FLAG vector. Flg22-elicited ROS assays were performed with the transient expression of EV (empty vector, pBI121-3 × FLAG) or AopP using A. tumefaciens GV3101 at an optical density at 600 nm (OD₆₀₀) of 0.5. Seven-week-old leaves of N. benthamiana were used in this assay. After 36 h, 12 leaf disks (4 mm in diameter) were collected from each inoculated region and were allowed to float in 100 μL of sterile distilled water in a 96-well plate. Next, the water was removed, and a 100 μL solution containing 100 nM flg22, 20 μg/mL horseradish peroxidase, and 100 μM luminol were added to each well. Luminescence was recorded immediately using a Tecan Infinite F200 luminometer (Tecan, Männedorf, Switzerland) for 60 min. Diaminobenzidine (DAB) staining was performed to evaluate differences between watermelon cotyledons inoculated with the WT strain and aopP mutant at 1 × 10⁸ CFU/mL using a syringe. After 24 h, the leaves were treated as described previously (Priller et al., 2016 (link); Sang and Macho, 2017 (link)).

Two fragments containing PDSS2(-103C-G) were generated with PCR and cloned into pGL3 Basic vector (Promega), a longer 643 bp fragment (70,486,018–70,486,660 bp) and a shorter 405 bp fragment (70,486,256–70,486,660 bp) in the “forward” (toward PDSS2) orientation. A longer 752 bp fragment (70,486,339–70,487,090 bp) and a shorter 399 bp fragment (70,486,444–70,486,842 bp) in the “reverse” (toward SOBP) orientation were also amplified and cloned into pGL3 Promoter vector (Promega). NheI and XhoI sites were selected to construct the vector. DF1 cells plated on 24 wells were transfected at 70–80% confluency with 720 ng of the pGL3 reporter plasmid and 80 ng of pRL-TK Renilla luciferase construct by 2 µl Lipofectamine 2000 (Invitrogen) for each well. The luciferase activity was measured 23–24 h after transfection using the Dual-Glo Luciferase Assay System (Promega) and an Infinite F200 Luminometer (Tecan, Switzerland). Ratios of firefly luminescence/Renilla luminescence were calculated, and normalized to control samples (Basic vector). For each test construct, one expression value was the average of three technical replicates in each plate and three separate operations were carried out to represent the final value. The pGL3 Basic vector, pGL3 Promoter vector and pGL3 Control vector were used as control.