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 and leucine-rich repeat (NLR) proteins. Autoactive NLRs, some cases a specific domain of NLR, induce plant cell death in the absence of pathogen infection. In this study, we identified a group of NLRs (ANLs; ancient and autonomous NLRs) carrying autoactive coiled-coil (CCA ) domains in pepper (Capsicum annuum L.) by genome-wide transient expression analysis. The CCA -mediated cell death mimics hypersensitive cell death triggered by interaction between NLR and pathogen effectors. Sequence alignment and mutagenesis analyses revealed that the intact α1 helix of CCA is critical for both CCA - and ANL-mediated cell death. The cell death induced by CCA does not require NRG1/ADR1 or NRC type helper NLRs, suggesting ANLs may function as singleton NLRs. We also found that CCA localize in the plasma membrane as Arabidopsis singleton NLR ZAR1. Extended studies revealed that autoactive CCA s are well conserved in other Solanaceae plants as well as a monocot plant, rice. Further phylogenetic analyses revealed that ANLs are present in all tested seed plants (spermatophytes). Our studies not only uncover the autonomous NLR clade in plants but also provide powerful resources for dissecting the underlying molecular mechanism of NLR-mediated cell death in plant.