The nucleotide-binding leucine-rich repeat protein (NLR) required for cell death (NRC) family represents a group of helper NLRs that are required by sensor NLRs to execute hypersensitive cell death during pathogen infection. NRCs contain an N-terminal coiled-coil (CC) domain essential for their function, yet our knowledge of how this domain contributes to NRC function remains limited. Using site-directed mutagenesis and transient expression in Nicotiana benthamiana, we screened conserved hydrophobic residues among NRCs and identified seven required for NRC4-mediated cell death, revealing a hydrophobic feature within the CC domain that contributes to NRC-mediated immunity. Structural analysis revealed that four of these residues form a hydrophobic core in the CC domain. This hydrophobic core is important for NRC4 subcellular localization, oligomerization, and phospholipid association, but not for NRC4 focal accumulation at the extrahaustorial membrane during Phytophthora infestans infection. Sequence analysis and functional assays revealed that this core is highly conserved in NRCs and some singleton NLRs but has degenerated in NRC-dependent sensor NLRs. Our study identifies a hydrophobic feature in the CC domain of NRCs and reveals its contribution to NLR-mediated immunity.
Wang et al. (Sun,) studied this question.