A calmodulin-like protein regulates plasmodesmal closure during bacterial immune responses

Abstract

Plants sense microbial signatures via activation of pattern recognition receptors (PPRs), which trigger a range of cellular defences. One response is the closure of plasmodesmata, which reduces symplastic connectivity and the capacity for direct molecular exchange between host cells. Plasmodesmal flux is regulated by a variety of environmental cues but the downstream signalling pathways are poorly defined, especially the way in which calcium regulates plasmodesmal closure. Here, we identify that closure of plasmodesmata in response to bacterial flagellin, but not fungal chitin, is mediated by a plasmodesmal-localized Ca²⁺ -binding protein Calmodulin-like 41 (CML41). CML41 is transcriptionally upregulated by flg22 and facilitates rapid callose deposition at plasmodesmata following flg22 treatment. CML41 acts independently of other defence responses triggered by flg22 perception and reduces bacterial infection. We propose that CML41 enables Ca²⁺ -signalling specificity during bacterial pathogen attack and is required for a complete defence response against Pseudomonas syringae.

Keywords: At3g50770; biotic stress; cell-to-cell communication; electrophoresis mobility shift; maltose-binding protein; pathogen-associated molecular pattern (PAMP).

Figure 1

CML41 is induced by flg22 in leaves and binds Ca²⁺. (a) GUS histochemical staining of 24‐d‐old proCML41::GUS plants treated with either H₂O or flg22 infiltration for 4 h, as well as nontreatment control as indicated. Both flg22 and H₂O injection at the wound/infiltration site is indicated by arrowheads, there was a localized increase in GUS activity induced by both flg22 and H₂O injection at the wound site. (b) Quantitative RT‐PCR analysis of CML41 in the leaves of 5–6‐wk‐old wildtype Arabidopsis Col‐0 plants grown in short‐day conditions (with 9 h : 15 h, light : dark) pre‐infiltrated with either H₂O (green) or 1 μM flg22 (magenta) for 12 h. Gene transcript level was relative to GAPDH‐A (At3g26650). Data represent the mean ± standard error of the mean (SEM), n = 3 biological replicates. Primer pairs used for (b) listed in Supporting Information Table S1. Statistical difference was determined by Student's t‐test, asterisks indicate statistical significance, P‐value as indicated. (c) Gel shift Ca²⁺ binding assay, purified recombinant MBP‐CML41 protein was separated on 8% SDS‐PAGE gel in the presence of 1 mM CaCl₂ or 10 mM EGTA, the mobility of proteins was determined by comparison with the Precision Plus Protein™ Standards as indicated.

Figure 2

CML41 localizes to plasmodesmata. (a–c) Confocal image of CML41 tagged with GFP in the leaves of 5–6‐wk‐old 35S::CML41‐GFP Arabidopsis plant; bars, 20 μm. (d–f) Co‐localization of CML41‐GFP (d) with callose stained by aniline blue (e) in the leaves of 5–6‐wk‐old 35S::CML41‐GFP Arabidopsis leaf; bars, 10 μm. (g–i) Co‐localization of CML41‐GFP (g) with PDLP1‐mCherry (h), transiently expressed in Nicotiana benthamiana; bars, 10 μm.

Figure 3

CML41 negatively modulates plasmodesmatal permeability and positively regulates callose production and plant defence. (a) Plasmodesmata permeability of wildtype (WT) Arabidopsis and CML41 transgenic lines (CML41‐amiRNA‐1, ‐4 and CML41‐OEX‐2) in response to 100 nM flg22. Plants were bombarded with constructs capable of producing GFP. Diffusion of GFP to surrounding cells provided a measure of molecular flux through plasmodesmata. Plants were infiltrated with flg22 2 h after bombardment. In each box‐plot, the white line indicates the median value, the shaded area represents the lower and upper quartiles, and the error bars indicate the minimum and maximum values, n = 187 cells for WT (control), 137 for WT (flg22), 85 for CML41‐amiRNA‐1 (control), 103 for CML41‐amiRNA‐1 (flg22), 145 for CML41‐amiRNA‐4 (control), 163 for CML41‐amiRNA‐1 (flg22), 666 for CML41‐OEX‐2 (control) and 198 for CML41‐OEX‐2 (flg22). Statistical difference from the WT control was determined by Student's t‐test, asterisks indicate statistical significance, P‐values as indicated. (b) Confocal images of aniline blue stained plasmodesmal callose in the leaves of 5–6‐wk‐old Arabidopsis WT, CML41‐OEX‐2 and CML41‐amiRNA‐1 lines upon H₂O, flg22 and EGTA treatment for 30 min; bars, 20 μm. (c, d) Quantification of PD callose fluorescence intensity in the leaves of 5–6‐wk‐old Arabidopsis WT, CML41‐OEX‐2 and CML41‐amiRNA‐1 lines following flg22 (c) and EGTA treatment (d). Box plots in (c) and (d) are as mentioned earlier, n = 24 (c) and 27 (d). Statistical difference from the WT control was determined by Student's t‐test, asterisks indicate statistical significance, P‐values as indicated. (e) Macroscopy images and (f) quantification of callose deposition upon flg22 or H₂O treatments in WT Col‐0, fls2,CML41‐amiRNA‐1, ‐4 and CML41‐OEX‐2, ‐12 lines upon 1 μM flg22 for 24 h, as indicated; bars, 200 μm in (e). Data represent the mean ± SEM, n = 18 leaves. Statistical difference as determined by one‐way analysis of variance (ANOVA), asterisks indicate statistical significance, P‐values as indicated. (g) Reactive oxygen species (ROS) production stimulated by 1 μM flg22 was monitored in Arabidopsis WT, fls2,CML41‐amiRNA‐1, ‐4 and CML41‐OEX‐2, ‐12 leaf discs recorded at every minute using a luminol assay in a microplate reader, fls2 was used as a control. Data are given as relative luminescence units and represent in mean ± SEM, from three independent trials with six technical replicates per biological replicate, n = 6. (h) Evaluation of CML41 transgenic plant susceptability to Pst DC3000 cor ⁻; quantification of bacterial growth in fls2,CML41‐amiRNA‐1, ‐4,CML41‐OEX‐2, ‐12 lines and Arabidopsis WT plants upon 0 and 3 d post‐inoculation of Pst DC3000 cor ⁻ suspension. The bacterial colony number was counted in colony‐forming unit (CFU) per cm². Data represent mean ± SEM, n = 6. Statistical difference as determined by multiple Student's t‐test, asterisks indicate statistical significance from WTcontrol or flg22 treated, P‐values as indicated. The experiments were repeated three times with similar results. See Supporting Information Fig. S7 for the equivalent assays using Pst DC3000.