Ptm 501

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).

Histone-enriched fractions were extracted from seedlings of Arabidopsis, rice, maize, and tobacco plants using EpiQuik Total Histone Extraction Kit (Epigentek USA, OP-0006-100). The histone-enriched fractions were used for immunoblot analysis; antibodies used in this study were anti-H3 (ab1791; Abcam), anti-H3K9ace (07-352; Millipore), anti-butyryllysine (PTM-301; PTM Biolabs), and anti-crotonyllysine (PTM-501; PTM Biolabs). Immunoblotting results were quantified using ImageJ (v1.6.0_24).

pET28 construct containing His-fused FoSir5 was expressed in BL21 E. coli. Protein expression was induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.2 mM when OD600 reached 0.6, and the culture was further grown at 16°C overnight. Cells were harvested and resuspended in lysis buffer A (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 1 mM PMSF, and Roche EDTA free protease inhibitor). Following sonication and centrifugation, the supernatant was loaded onto a nickel column pre-equilibrated with lysis buffer. The column was washed with five column volumes of wash buffer (lysis buffer with 20 mM imidazole) and the bound proteins were then eluted with elution buffer (lysis buffer with 200 mM imidazole). After purification, proteins were dialyzed at 4°C overnight. In vitro enzymatic reactions were performed as described previously (Liu et al., 2017 (link)). In brief, 50 µg native CTH (A002544, Sangon Biotech) were incubated with 0.5 µg recombinant FoSir5 protein at 30°C for 1 hr in HDCR buffer (50 mM Tris pH 7.5, 5% glycerol, 5 mM NAD⁺, 0.1 mM EDTA, 50 mM NaCl, and 0.2 mM PMSF) in the presence of 50, 100, or 200 µM crotonyl-CoA (C4282, Sigma). The assay mixture was then analyzed using Western blotting by anti-PanKcr (PTM-501, PTM Biolabs) and anti-H3 (ab1791, Abcam).