Dynamic accumulation of a helper NLR at the plant-pathogen interface underpins pathogen recognition

Abstract

Plants employ sensor-helper pairs of NLR immune receptors to recognize pathogen effectors and activate immune responses. Yet the subcellular localization of NLRs pre- and post-activation during pathogen infection remains poorly known. Here we show that NRC4, from the NRC solanaceous helper NLR family, undergoes dynamic changes in subcellular localization by shuttling to and from the plant-pathogen haustorium interface established during infection by the Irish potato famine pathogen Phytophthora infestans. Specifically, prior to activation, NRC4 accumulates at the extra-haustorial membrane (EHM), presumably to mediate response to perihaustorial effectors, that are recognized by NRC4-dependent sensor NLRs. However not all NLRs accumulate at the EHM, as the closely related helper NRC2, and the distantly related ZAR1, did not accumulate at the EHM. NRC4 required an intact N-terminal coiled coil domain to accumulate at the EHM, whereas the functionally conserved MADA motif implicated in cell death activation and membrane insertion was dispensable for this process. Strikingly, a constitutively autoactive NRC4 mutant did not accumulate at the EHM and showed punctate distribution that mainly associated with the plasma membrane, suggesting that post-activation, NRC4 probably undergoes a conformation switch to form clusters that do not preferentially associate with the EHM. When NRC4 is activated by a sensor NLR during infection however, NRC4 formed puncta mainly at the EHM and to a lesser extent at the plasma membrane. We conclude that following activation at the EHM, NRC4 may spread to other cellular membranes from its primary site of activation to trigger immune responses. Significance statementPlant NLR immune receptors function as intracellular sensors of pathogen virulence factors known as effectors. In resting state, NLRs localize to subcellular sites where the effectors they sense operate. However, the extent to which NLRs alter their subcellular distribution during infection remains elusive. We describe dynamic changes in spatiotemporal localization of an NLR protein in infected plant cells. Specifically, the NLR protein accumulates at the newly synthesized plant-pathogen interface membrane, where the corresponding effectors are deployed. Following immune recognition, the activated receptor re-organizes to form punctate structures that target the cell periphery. We propose that NLRs are not necessarily stationary immune receptors, but instead may spread to other cellular membranes from the primary site of activation to boost immune responses.