Bingxiao Wang, Zhaoyang Zhou, Jian-Min Zhou, and Jiejie Li

PNAS; June 14, 2024; 121 (25) e2312415121; https://doi.org/10.1073/pnas.2312415121

Significance

Our study demonstrates that myosin XIK acts as a key regulator in the spatial confinement of the FLS2/BIK1 complex within nanodomains on the plasma membrane. This myosin-dependent nanodomain targeting ensures the stable formation of immune receptor complexes, which is essential for the rapid and robust activation of immune responses upon pathogen perception. By elucidating the intricate interplay between myosin XIK, nanodomain organization, and immune receptor complexes, our research provides valuable insights into the regulatory mechanisms underlying surface immune complex assembly. This understanding has broader implications for deciphering immune signaling dynamics and opens broad avenues for studying nanodomain-based protein complex organization in diverse biological processes.

Abstract

Plants rely on immune receptor complexes at the cell surface to perceive microbial molecules and transduce these signals into the cell to regulate immunity. Various immune receptors and associated proteins are often dynamically distributed in specific nanodomains on the plasma membrane (PM). However, the exact molecular mechanism and functional relevance of this nanodomain targeting in plant immunity regulation remain largely unknown. By utilizing high spatiotemporal resolution imaging and single-particle tracking analysis, we show that myosin XIK interacts with remorin to recruit and stabilize PM-associated kinase BOTRYTIS-INDUCED KINASE 1 (BIK1) within immune receptor FLAGELLIN SENSING 2 (FLS2)-containing nanodomains. This recruitment facilitates FLS2/BIK1 complex formation, leading to the full activation of BIK1-dependent defense responses upon ligand perception. Collectively, our findings provide compelling evidence that myosin XI functions as a molecular scaffold to enable a spatially confined complex assembly within nanodomains. This ensures the presence of a sufficient quantity of preformed immune receptor complex for efficient signaling transduction from the cell surface.

See https://www.pnas.org/doi/10.1073/pnas.2312415121

Figure 1: Myosin XIK interacts with FLS2/BIK1 complex. (A) Myosin XIK can coimmunoprecipitate with FLS2 and BIK1, but not with BRI1. The pXIK::XIK-YFP/xi3ko protoplasts were transformed with FLS2-FLAG, BIK1-FLAG, or BRI1-FLAG. Co-IP was carriedout with an anti-FLAG agarose, and the proteins were analyzed using western blots with anti-GFP and anti-FLAG antibodies. Asterisks denote nonspecific bands from BRI1 degradation. (B and C) The interaction between XIK and endogenous FLS2 (B) and BIK1 (C). Co-IP was carried out in pXIK::XIK-YFP/xi3ko seedlings with an anti-GFP agarose, and the proteins were analyzed using western blots with anti-GFP, anti-FLS2, and anti-BIK1 antibodies. Seven-day-old pXIK::XIK-YFP/xi3ko seedlings were treated with mock or 1 µM flg22 for 30 min prior to protein extraction. (D and E) In vitro protein pull-down assay demonstrates the direct interaction of XIK-GTD with FLS2 (D) and BIK1 (E). Purified, recombinant His-tagged FLS2-KD and BIK1 cosedimented with purified XIK-GTD fused to a GST tag (GST-XIK-GTD) but not with purified GST alone. (F and G) FRET-FLIM analysis confirms the interaction of XIK with FLS2 and BIK1, but not with a PM marker LTi6b. (F) Intensity and lifetime maps of XIK-YFP when expressed on its own or coexpressed with indicated proteins. (Scale bar, 5 µm.) Scale varies from the shortest lifetime of 2 ns to the longest lifetime of 3 ns. (G) Quantification of the average fluorescence lifetime of XIK-YFP when expressed alone or coexpressed with indicated proteins. pXIK::XIK-YFP/xi3ko protoplasts were transformed with indicated constructs. Experiments were repeated three times with similar results. Sample sizes in (G) are 6, 8, 7, and 7 cells in each respective group from left to right. Values are means ± SD. Different letters indicate significant differences at P < 0.05, as determined by two-way ANOVA with Tukey’s multiple comparisons test.