ROS-mediated plasmodesmal regulation requires a network of an Arabidopsis receptor-like kinase, calmodulin-like proteins, and callose synthases

Abstract

Plasmodesmata (PD) play a critical role in symplasmic communication, coordinating plant activities related to growth & development, and environmental stress responses. Most developmental and environmental stress signals induce reactive oxygen species (ROS)-mediated signaling in the apoplast that causes PD closure by callose deposition. Although the apoplastic ROS signals are primarily perceived at the plasma membrane (PM) by receptor-like kinases (RLKs), such components involved in PD regulation are not yet known. Here, we show that an Arabidopsis NOVEL CYS-RICH RECEPTOR KINASE (NCRK), a PD-localized protein, is required for plasmodesmal callose deposition in response to ROS stress. We identified the involvement of NCRK in callose accumulation at PD channels in either basal level or ROS-dependent manner. Loss-of-function mutant (ncrk) of NCRK induces impaired callose accumulation at the PD under the ROS stress resembling a phenotype of the PD-regulating GLUCAN SYNTHASE-LIKE 4 (gsl4) knock-out plant. The overexpression of transgenic NCRK can complement the callose and the PD permeability phenotypes of ncrk mutants but not kinase-inactive NCRK variants or Cys-mutant NCRK, in which Cys residues were mutated in Cys-rich repeat ectodomain. Interestingly, NCRK mediates plasmodesmal permeability in mechanical injury-mediated signaling pathways regulated by GSL4. Furthermore, we show that NCRK interacts with calmodulin-like protein 41 (CML41) and GSL4 in response to ROS stress. Altogether, our data indicate that NCRK functions as an upstream regulator of PD callose accumulation in response to ROS-mediated stress signaling pathways.

Keywords: ROS perception; abiotic and biotic stress; callose; plasmodesmata; receptor-like kinase (RLK).

Figure 1

NCRK is a plasmodesmata-localized RLK that regulates basal PD permeability. (A) Phylogenetic tree of NCRK across species. (B) Conserved cysteine-rich repeat domain homolog sequence across species. (C) OsNCRK (upper panels) and NCRK (middle and bottom panels) co-localize with PD markers during transient expression, and NCRK (bottom panels) co-localizes with callose in the transgenic plant. (D) PD callose staining and its quantification in the leaf epidermal cells of wild-type Col-0 and ncrk-1 (n≥12). Scale bar: 20 μm. (E) DANS assays on Col-0 and ncrk-1. The diameter of the fluorescent circle shows the level of cell-to-cell permeability of symplasmic dye (n≥12). Scale bar: 200 μm. (F) Plant images and quantification of rosette leaf diameter, Col-0 (n=31), ncrk-1 (n=31), gsl4 (n=34). Scale bar: 1 cm. The data are summarized in box plots in which the line within the box marks the median, the box signifies the upper and lower quartiles. The whiskers go down to the smallest value and up to the largest. Data were analyzed by a Student’s t-test. ns, not significant. ^** P <0.01, ^*** P < 0.001.

Figure 2

NCRK is required for H₂O₂-induced callose deposition. (A) Images of basal and ROS-dependent callose deposition in Col-0 and ncrk-1, gsl4 and gsl8+/- mutant treated with H₂O₂ and their quantification. Scale bar: 20 μm. (n≥12). (B) Images of basal and ROS-dependent PD permeability of Col-0 and ncrk-1 mutant treated with H₂O₂ and their quantification. Scale bar: 200 μm, (n≥12). (C, D) Diagram of NCRK extracellular domain and its mutants. The Cys residues are marked as magenta, the Ala residues are marked as red. The disulfide bonds of the Cys-rich motif are shown. Quantification of basal and ROS-dependent callose deposition of ncrk-1 lines complemented with NCRK and NCRK mutants for Cys residues overexpressed using native NCRK promoter summarized in panel (E, F), respectively. Scale bar: 20 μm. (n≥12). Data were analyzed by Student’s t-test. ns, not significant. ^*** P < 0.001.

Figure 3

The extracellular domain of NCRK is required for basal and ROS-dependent callose deposition. (A) Diagram of NCRK and HPCA1/NCRK fusion protein and 3D protein structure of NCRK protein and HPCA1/NCRK fusion protein. SP, signal peptide; TMD, transmembrane domain; CRM-RD, Cys-rich motif-receptor domain; KD, kinase domain; HPD, hydrogen peroxide domain; LRR-RD, Leu-rich repeat-receptor domain. (B) Localization of NCRK and HPCA1-NCRK fusion protein in N. benthamiana. (C) Images of PD callose deposition of Col-0, ncrk-1, and HPCA1-NCRK complementation line treated with H₂O₂ and quantification of callose levels depicted in (D) (n≥12). Scale bar: 20 μm. Data were analyzed by Student’s t-test. ns, not significant. ^*** P < 0.001.

Figure 4

NCRK interacts with CML and GSL members. (A) Interaction of NCRK with its partners in vivo by BiFC assay. Scale bar: 20 μm. (B) Interaction of NCRK with its partners by Co-IP. (C, D) ROS-induced NCRK interaction investigated using BiFC assay and visualized by confocal microscopy. (E) Callose deposition of Col-0, ncrk-1, gsl4 single mutant, and ncrk gsl4 double mutant. Data were analyzed by Student’s t-test. ns, not significant. ^*** P < 0.001.

Figure 5

NCRK kinase activity is required for ROS-induced callose accumulation. (A) The kinase activity of NCRK^CD wild-type, NCRK(K238E)^CD and NCRK(D339L)^CD mutant proteins was analyzed by autoradiography. (B) NCRK phosphorylates ROP4 and CML41. (C) Partial complementation of the ncrk-1 phenotype by expressing kinase-inactive variants NCRK. (D) Fluorescence intensity quantification of callose deposition of Col-0 and ncrk-1 mutant following paraquat (MV) treatment. (E) Fluorescence intensity quantification of callose deposition in Col-0 and ncrk-1 mutant following wounding treatment. (F) Callose deposition of Col-0 and ncrk-1 mutant following flg22 treatment and (G) their intensity quantification. Scale bar: 200 μm. (n≥12). (H) Evaluation of ncrk-1 mutant plant susceptibility to Pst DC3000 cor^-; quantification of bacterial growth in Col-0 and ncrk-1 upon 0 and 3 d post‐inoculation of Pst DC3000 cor^- suspension. Data were analyzed by Student’s t-test. ns, not significant. *P < 0.05, ^** P < 0.01, ^*** P < 0.001.