The NLR helper protein NRC3 but not NRC1 is required for Pto-mediated cell death inNicotiana benthamiana

ABSTRACT Intracellular immune receptors of the nucleotide-binding leucine-rich repeat (NB-LRR or NLR) proteins often function in pairs, with “helper” proteins required for the activity of “sensors” that mediate pathogen recognition. The NLR helper NRC1 ( N B-LRR protein r equired for HR-associated c ell death 1 ) has been described as a signalling hub required for the cell death mediated by both cell surface and intracellular immune receptors in the model plant Nicotiana benthamiana . However, this work predates the availability of the N. benthamiana genome and whether NRC1 is indeed required for the reported phenotypes has not been confirmed. Here, we investigated the NRC family of solanaceous plants using a combination of genome annotation, phylogenetics, gene silencing and genetic complementation experiments. We discovered that a paralog of NRC1 , we termed NRC3 , is required for the hypersensitive cell death triggered by the disease resistance protein Pto but not Rx and Mi-1.2. NRC3 may also contribute to the hypersensitive cell death triggered by the receptor-like protein Cf-4. Our results highlight the importance of applying genetic complementation to validate gene function in RNA silencing experiments.