P-010 - Biochemical features of a modulator NLR regulating immune receptor networks in Nicotiana benthamiana.

NLRs (Nucleotide-binding leucine-rich repeat proteins) recognize pathogen effectors and induce hypersensitive cell death response. In Solanaceae plants, sensor NLRs and helper NLRs, known as NRC (NLR required for cell death) subfamily members, function coordinately and form NLR networks. Activated NRCs form homo-hexameric resistosomes and execute downstream immune signaling on the plasma membrane. However, the regulatory mechanisms of the NRC networks remain poorly understood. A previous report revealed that gene silencing of NRCX, a member of the NRC family, impairs Nicotiana benthamiana growth. Genetical analyses revealed that NRCX suppresses the cell death response triggered by its paralogs, NRC2 and NRC3. Here, we conducted blue-native PAGE (BN-PAGE) and membrane fractionation analyses using NRCX and activated NRC2. These biochemical experiments showed that NRCX does not suppress the NRC2 resistosome formation and its enrichment in the membrane fraction. Interestingly, the BN-PAGE experiment indicated that NRCX constitutively forms higher-order complex. Furthermore, mutations into the N-terminal conserved motif (MADA motif) of NRCX compromised its cell death suppression activity and protein accumulation in the membrane fraction. These findings suggest that the higher-order complex of NRCX modulates excessive immune response on the plasma membrane and the MADA motif plays a crucial role in the NRCX-mediated immune regulation.