In vivo Identification of Arabidopsis SIZ1 SUMO E3 Ligase Substrates by TurboID-Mediated Proximity Labeling

To investigate how SIZ1 promotes immune activation, we employed an enzyme-catalyzed proximity-labeling approach to identify its in vivo substrates. We fused SIZ1 with TurboID, an engineered promiscuous biotin ligase, and generated stable transgenic Arabidopsis expressing pSIZ1::SIZ1g-TurboID-HA. Ten-day-old pSIZ1::SIZ1-TurboID-HA and WT plants were treated with free biotin for 5 hours to achieve effective proximity labeling, after which labeled proteins were affinity purified and identified by label-free quantitative mass spectrometry (LFQMS) with two biological replicates.