Ptm 902

For construction of the RP27:FoDLAT/FoDLAT^K148Q /FoDLAT^K148R:GFP vectors, we amplified fragments by PCR with primers FoDLAT-GFP-F/R, respectively. The fragments were then inserted into the pYF11 vector. Afterward, the constructs were transformed into Fo, ΔFoSir5, and OE-1, respectively. GFP fusion proteins in different pairs of strains were immunoprecipitated as described above. The eluted proteins were then analyzed by Western blot using anti-GFP, anti-Kcr (PTM-501, PTM Biolabs), anti-Ksu (PTM-419, PTM Biolabs), anti-Kma (PTM-902, PTM Biolabs), and anti-Kglu (PTM-1152, PTM Biolabs), followed by quantification using Quantity One (Bio-Rad).

The leaves of maize were ground in liquid nitrogen and the proteins were then purified as described (Liu et al., 2018 (link)). Briefly, the proteins (20 μg) of maize were electrophoresed on 12% gel in SDS-PAGE for 2.5 h with voltage 110 V followed by electrotransfer onto a polyvinylidene difluoride (PVDF) membrane with constant current 300 mA for 1 h. After blocking in 5% skim milk powder in TBST buffer (20 mM Tris–HCl, 150 mM NaCl, 0.05% Tween 20) for 1 h, the PVDF membrane was first incubated with anti-malonyllysine mouse mAb (1:1,000 dilution, PTM-902, PTM Biolabs) at 4°C for 12 h. Anti-mouse IgG peroxidase conjugated secondary antibody (A9044, Sigma) was used at a 1:10,000 dilution, and incubation time was 2 h at room temperature. The signals were detected using an enhanced chemiluminescence (ECL) immunoblotting detection kit (Beyotime Biotechnology).

The extracted proteins from each group of samples were separated by SDS-PAGE electrophoresis, and then transferred to a polyvinylidene difluoride membrane. The membrane was first incubated with pan anti-malonyllysine antibody (PTM-902, PTM Biolabs, Chicago, IL, USA) at 1:1000 (v/v) dilution. Goat anti-Mouse IgG peroxidase conjugated secondary antibody (31430, Thermo pierce, Rockford, IL, USA) was used at a 1:5000 (v/v) dilution. The proteins were then detected with an enhanced chemiluminescence immunoblotting detection kit (Advansta, San Jose, CA, USA).