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Dynamic localization of a helper NLR at the plant–pathogen interface underpins pathogen recognition
Friday, 2021/08/27 | 07:53:58
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Cian Duggan, Eleonora Moratto, Zachary Savage, Eranthika Hamilton, Hiroaki Adachi, Chih-Hang Wu, Alexandre Y. Leary, Yasin Tumtas, Stephen M. Rothery, Abbas Maqbool, Seda Nohut, Toby Ross Martin, Sophien Kamoun, and Tolga Osman Bozkurt PNAS August 24, 2021 118 (34) e2104997118 SignificancePlant NLRs function as intracellular immune sensors of pathogen virulence factors known as effectors. In the 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 reorganizes to form punctate structures that target the cell periphery. We propose that NLRs are not necessarily stationary receptors but instead may spread to other cellular membranes from the primary site of activation to boost immune responses. AbstractPlants employ sensor–helper pairs of NLR immune receptors to recognize pathogen effectors and activate immune responses. Yet, the subcellular localization of NLRs pre- and postactivation during pathogen infection remains poorly understood. 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 extrahaustorial 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 postactivation, NRC4 may undergo 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 forms 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.
See: https://www.pnas.org/content/118/34/e2104997118
Figure 4: NRC4L9E is nonfunctional for HR and P. infestans resistance but still focally accumulates at the EHM. (A) NRC4-GFP, but not NRC4L9E mutant or EV-GFP, genetically complements CRISPR/Cas mutation of Rpi-blb2nrc4a/b plants by triggering HR when coexpressed with AVRblb2 and resistance when expressed alone and infected 1 dpai. The HR assay was repeated with the same results in 34 plants over two independent experiments. Images were taken at 3 dpai. Scattered points in scatter violin plot represent the area (mm2) occupied by infection spots measured on white light images, where each construct had n > 17 plants/leaves. Three independent experimental replicates were conducted as indicated by color of dots. Ultraviolet (representative images for three constructs) and white light imaging was taken at 8 dpi. Wilcoxon unpaired tests gave P values of 5.8 × 10−8 for NRC4L9E-GFP to NRC4-GFP, 0.28 for NRC4L9E-GFP to GUS-GFP, and 7.6 × 10−8 for NRC4-GFP to GUS-GFP. (B and C) Single-plane confocal micrographs showing NRC4L9E-GFP focally accumulates at EHM with RFP-Rem1.3 but not the EV-GFP control, which remains cytoplasmic. (Scale bars, 10 μm.). |
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