Phanta flash master mix dye plus

Four pairs of primers were designed to amplify the whole length of VRN-A1. To sequence VRN-A1, the whole gene was divided into two segments prior to amplification. PCRs were performed using 2× Phanta Flash Master Mix Dye Plus (Vazyme) with the following procedure: 98 °C for 30 s, followed by 35 cycles of annealing (98 °C for 10 s, 60–65 °C for 5 s and 72 °C for 1 min 30 s), and 72 °C for 1 min. Then, the PCR product was cloned using the pEASY^®-Blunt zero-cloning vector (Transgen) at 37 °C, 1:7 for 30 min. After transformation into receptive cells, the universal primer M13 was used for detection, and the positive clones were cultured overnight. All primers used are described in Table S3.

Total RNA was extracted from the E09-inoculated leaf tissue of the resistant line TA3575 using RNA isolater Total RNA Extraction Reagent (Vazyme, Nanjing, China). Reverse transcription was performed using HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, Nanjing, China) following the manufacturer’s instructions. The full-length CDS of the AelMLKL gene was amplified using 2 × Phanta Flash MasterMix (Dye Plus) (Vazyme, Nanjing, China) by PCR with the gene-specific primer AelMLKL_CDS (Supplementary Data 6). The PCR product was cloned into the One step ZTOPO-Blunt/TA vector (ZOMANBIO, Beijing, China). The inserted fragment in the ZTOPO-Blunt/TA-AelMLKL_CDS construct was verified by Sanger sequencing at Sangon Biotech, Shanghai, China.

We constructed overexpression vectors for DNAAF5, PFKL and USP39 genes using pCDNA3.1-puro-Flag, pCDNA3.1-G418-myc and pCDNA3.1-Hygro-HA plasmids, respectively. The extracted RNA was reverse transcribed to cDNA using the HiScript III 1^st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme Biotech Co., Ltd), after which the corresponding fragment was amplified using 2 × Phanta Flash Master Mix (Dye Plus) (Vazyme Biotech Co., Ltd) and a ClonExpress II One The Step Cloning Kit (Vazyme Biotech Co., Ltd) to ligate the fragment to the corresponding vector. Complete plasmids were used for subsequent experiments after sequences had been verified to be correct.
The LentiCRISPRv2 plasmid was digested using Esp3I (R0734, NEB), after which the annealed primer fragment was ligated into it using T4 ligase (M0202, NEB).
The PLKO.1-Hygro vector was used to silence the corresponding gene. After double-digestion with AgeI-HF (R3552, NEB) and EcoRI-HF (R3101, NEB), T4 ligase (M0202, NEB) was used to link the annealed primer fragments into the vector.
Depending on the progress and needs of the experiment, the Lipo8000 (C0533, Beyotime) transfection reagent was used to transfer different plasmids into various cells after which corresponding antibiotics were added to screen and obtain equivalent stable cell lines.