Mbp beads

EMSAs were conducted using a LightShift™ EMSA Optimization & Control Kit (Thermo Prod#20148X) and a Chemiluminescent Nucleic Acid Detection Module (Thermo 89,880) in accordance with the manufacturer’s protocol. The recombinant maltose-binding protein (MBP)-ZjSEP3 protein and MBP protein were purified from E. coli BL21 using MBP beads (New England BioLabs). The DNA fragments and mutated sequences of the LHY promoter were synthesized and labeled with biotin at the 5′ DNA terminus for serving as biotin probes. Biotin-unlabeled fragments of the same sequences or mutated sequences were used as cold probes. MBP alone was used as the negative control.

PsoRPM3 protein IP experiments and LC-MS analysis was conducted according to the method reported by Xiao et al. [71 ]. Briefly, the PsoRPM3 carrying the MBP tag protein was constructed into the prokaryotic expression vector pMal-c2x, and the fusion protein MBP-PsoRPM3 was induced to express overnight at 37 °C and 180 rpm. After sonication, the supernatant was collected and the fusion protein was collected using MBP beads (New England Biolabs, Ipswich, MA, USA). The total protein derived from the root inoculated with 10,000 pre-J2 of M. incognita after 3 days, and the total protein of roots that had not been inoculated with M. incognita were used as controls. Incubate in a shaker at 4 °C for 2 h. After centrifugation and elution, the protein was sent to Beijing Qinglian Biotech Co., Ltd. for the LC/MS tests. The acquired mass spectrometric data were analyzed using ProteomeDiscoverer2.1 and then anatomized for searches of the NCBI (https://www.ncbi.nlm.nih.gov, accessed on 12 September 2019) database and M. incognita (https://www6.inrae.fr/meloidogyne_incognita/, accessed on 15 September 2019) database.

The ChIP assay was adapted from Zhao et al.^{45 (link)}. Total DNA of three-week-old seedlings of Nipponbare rice was extracted. The total DNA was sheared into 100–500 bp fragments using an ultrasonic crusher. E.coli expressed MBP-OsNAC42 and MBP-OsNAC42M proteins were purified. MBP-OsNAC42, MBP-OsNAC42M and sheared rice DNA fragments were co-incubated with amylase Resin (NEB, E8021S, MBP beads) for 8 h. The incubation buffer included: 140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 1% methanol. After 8 hours co-incubation, 125 mM glycine was further added into the beads mixture and incubated for 10 min. MBP beads were washed three times using incubation buffer. For each of 400 ml volume of the samples, add 16 μL 5 M NaCl and was incubated for another 8 h to breakdown associated DNA with MBP-OsNAC42 and MBP-OsNAC42M proteins. Subsequently, DNA fragments were extracted using the phenol–chloroform method for further ChIP-qPCR analysis with a Bio-Rad CFX96 real-time PCR detection system. PCR were performed in triplicate for each sample, and the expression levels were normalized to the input sample and only MBP control treatment for enrichment detection. The enrichment folds were calculated against the amount of bound DNA of rice ubiquitin promoter. Relevant primer sequences were given in Supplementary Data 2.