Genome-wide functional analysis of hot pepper immune receptors reveals an autonomous NLR clade in seed plants

Plants possess hundreds of intracellular immune receptors encoding nucleotide-binding domain leucine-rich repeat (NLR) proteins. Full-length NLRs or a specific domain of NLRs often induce plant cell death in the absence of pathogen infection. In this study we used genome-wide transient expression analysis to identify a group of NLRs (ANLs; ancient and autonomous NLRs) carrying autoactive coiled-coil (CC^A) domains in pepper (Capsicum annuum). CC^A-mediated cell death mimics hypersensitive cell death triggered by the interaction between NLRs and pathogen effectors. Sequence alignment and mutagenesis analyses revealed that the intact α1 helix of CC^As is critical for both CC^A- and ANL-mediated cell death. Cell death induced by CC^As does not require NRG1/ADR1 or NRC type helper NLRs, suggesting ANLs may function as singleton NLRs. We also found that CC^As localize to the plasma membrane, as demonstrated for Arabidopsis singleton NLR ZAR1. Extended studies revealed that autoactive CC^As are well conserved in other Solanaceae plants as well as in rice, a monocot plant. Further phylogenetic analyses revealed that ANLs are present in all tested seed plants (spermatophytes). Our study not only uncovers the autonomous NLR clade in plants but also provides powerful resources for dissecting the underlying molecular mechanism of NLR-mediated cell death in plants.