The autoactivity of tomato helper NLR immune proteins of the NRC clade is unaltered in prf mutants of Nicotiana benthamiana

Nucleotide-binding domain and leucine-rich repeat immune receptors (NLRs) can function in networks of sensors and helpers to induce hypersensitive cell death and immunity against pathogens. The tomato sensor NLR Prf guards the Pto kinase from AvrPto and AvrPtoB effector perturbation and activates the downstream helpers NRC2 and NRC3. Prf is conserved across the Solanaceae and its ortholog in the model species Nicotiana benthamiana is also required for detection of AvrPto/AvrPtoB function on Pto. A recent study reported that cell death induction after transient expression of an autoactive mutant of tomato NRC3 is abolished upon RNAi silencing of Prf in N. benthamiana. Here we generated loss-of-function prf mutants in N. benthamiana and demonstrate that autoactive mutants of eight canonical tomato NRCs (NRC0, NRC1, NRC2, NRC3, NRC4a, NRC4b, NRC6, and NRC7) still induce hypersensitive cell death when expressed transiently in the prf mutant background. Autoactive tomato NRCs also triggered cell death when expressed in lettuce (Lactuca sativa), an Asteraceae plant that does not have a Prf ortholog. These results confirm a unidirectional dependency of sensors and helpers in the NRC network and underscore the value of the N. benthamiana and lettuce model systems for studying functional relationships between paired and networked NLRs.