TurboID-based proximity-dependent labeling using SOBIR1 as a bait in potato leads to the identification of novel defense-related signaling partners

The plant immune system comprises a complex signaling network that is activated upon perceiving molecules derived from invading pathogens. The first line of defense at the plant cell surface is mediated by receptor-like proteins (RLPs) and receptor-like kinases (RLKs). RLPs, which lack a cytoplasmic signalling domain themselves, constitutively interact with the RLK SUPRESSOR OF BIR1-1 (SOBIR1), which is a key component initiating immune signal transduction upon pathogen perception. Therefore, elucidating the composition of the SOBIR1 protein complex will contribute to understanding the basic molecular mechanisms of plant disease resistance. Most of the studies focused on the identification of SOBIR1-interacting proteins are limited to model plants, due to technical challenges and lack of reliable genome and proteome databases in crop plants. Here, we evaluate the application of the biotin ligase TurboID (TbID)-based proximity-dependent labeling (PL) approach by transiently expressing SOBIR1 from Nicotiana benthamiana (NbSOBIR1), fused to TbID in leaves of the wild potato Solanum microdontum. We show that NbSOBIR1-YFP-TbID properly accumulates in potato and that proximal proteins are biotinylated. Quantitative proteomic analysis yielded over 130 candidate proteins to be in the proximity of the cytoplasmic kinase domain of NbSOBIR1, of which some could be linked to disease resistance by KEGG pathway and gene ontology (GO) molecular function analysis. We also studied the dynamics of the proteome in proximity of NbSOBIR1 upon perception of the INF1 elicitin of Phytophthora infestans that was co-expressed in potato with the elicitin receptor ELR, which is an RLP that constitutively interacts with SOBIR1. We found more than 80 proteins, including the NB-LRR REQUIRED FOR HR-ASSOCIATED CELL DEATH 1 (NRC1), putatively interacting with NbSOBIR1. In conclusion, we were able to successfully apply PL in potato and a future roadmap for further research on deciphering the composition of protein complexes involved in immune signaling has been established.