In plant immunity research, Brian J. Staskawicz are key components of plant resistance to pathogen invasion. These proteins recognize effectors secreted by pathogens, induce the oligomerization of NLRs to form resistosomes, and initiate immune responses by regulating calcium ion levels in the cytoplasm. Among them, a class of critical NLRs is called "helper" NLRs (hNLRs), which work together with "sensor" NLRs (sNLRs) to regulate plant immunity.
On September 5, 2024, Brian Staskawicz's team from the University of California, Berkeley published a research paper titled "Activation of the helper NRC4 immune receptor forms a hexameric resistosome" in Cell, analyzing the physiological process of NRC4 as an important hNLR involved in plant immune activation.
Figure 1. Mechanism of action of NRC4 resistosome. (Liu, et al., 2025)
In this study, the researchers used a plant protein expression system to express and purify high-quality hNLR complexes, namely NRC4 resistosome. Subsequently, the researchers used cryo-electron microscopy to resolve the high-resolution structure of the resistosome. Surprisingly, unlike the tetrameric or pentamer structures formed by previously reported NLRs, the NRC4 in the activated state formed a hexameric oligomeric morphology. Further biochemical analysis showed that the activated NRC4 triggers an immune response by mediating calcium ions across the cell membrane into the cytoplasm.
The researchers also expressed and purified other NRC family resistosomes from Solanaceae plants, including NRC0, NRC2 and NRC3. The study found that these NRCs in the activated state all adopted a hexameric oligomeric form similar to NRC4 and had similar functions.
During the structural analysis, the researchers also found that NRC4 presented a double-layer dumbbell-like dodecamer form. The NLRC4 inflammasome of mouse has also been reported to form a similar double-layer structure, in which the important N-terminal sequence that initiates the immune response is wrapped, thereby inhibiting its role in the immune response, indicating that higher-order structures may play a regulatory role in the immune system.
To further understand the working mechanism of NRC family resistosomes, the researchers explored the calcium pumping mode of NRC family proteins through a series of electrophysiological experiments. The results showed that the calcium pumping mode caused by NRC and previously reported hNLR NRG1 activation was similar. When nonspecific cation channel blockers or intracellular calcium release blockers were used, the initiation of NRC4 resistosome-mediated calcium pumping was only slightly delayed, while the treatment of calcium blocker lanthanum chloride significantly reduced the calcium pumping. In addition, the additional increase of extracellular calcium ions enhanced the calcium pumping mediated by NRC4 resistosome, indicating that calcium ions entering cells through the cell membrane is the main source of calcium ion signals.
To further study the calcium ion pumping mechanism induced by NRC resistance proteins, researchers conducted relevant experiments in animal cell expression systems. However, the results showed that NRC family proteins in animal cells could not effectively pump calcium ions. Therefore, NRC resistosomes may require certain plant-specific factors to exert their functions, which on the other hand shows that NRC family proteins have different characteristics from previously reported resistosomes.
This study clarifies the unique activation mechanism of hNLR-sNLR immune receptor pairs in plants and reveals a signal transduction process that is completely different from that in mammals. In addition, the different oligomerization states of NRC4 resistosomes have greatly expanded our understanding of the regulatory mechanism of NLR proteins. These findings not only deepen our understanding of the structure and function of NRC resistance proteins, but also reveal the important role of calcium ion signals in NRC-mediated immune responses. Therefore, these findings provide an important theoretical basis for the development of new plant disease resistance strategies.
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