Ca2+ Signalling in Plant Biotic Interactions

Editorial

20 January 2023

Editorial: Ca2+ signalling in plant biotic interactions

Thomas A. DeFalco

Wolfgang Moeder

and

Keiko Yoshioka

1,748 views

3 citations

Editors

Keiko Yoshioka

University of Toronto

Wolfgang Moeder

University of Toronto

Thomas DeFalco

Western University

Impact

Review

11 January 2022

Calcium/Calmodulin-Mediated Defense Signaling: What Is Looming on the Horizon for AtSR1/CAMTA3-Mediated Signaling in Plant Immunity

Peiguo Yuan

, 1 more and

B. W. Poovaiah

AtSR1/CAMTA3 acts as a signaling switch with dependence on Ca2+-binding status of CaM during plant immune response. In non-stressful environments, there is no [Ca2+]cyt dynamic. Hence, the status of CaM is not in a saturated phase at four Ca2+ binding sites, designated as apo-CaM. Apo-CaM interacts with the IQ motifs of AtSR1. This CaM-AtSR1 complex may have an effect on the stability of AtSR1, which suppresses the transcriptional expression of defense-related genes (e.g., EDS1, ICS1, and NPR1) as an autoregulator. Thus, suppression of plant immunity maintains normal plant growth and development. Under the virulent pathogen infection, [Ca2+]cyt is transiently increased, which confers a status of mixture of Apo-CaM and Ca2+-saturated CaM. Ca2+-saturated CaM interacts with the CaMBD of AtSR1, while Apo-CaM interacts with the IQ motifs. This CaM-AtSR1 complex loses its binding activity to the cis-regulatory element “CGCG,” and de-regulates the transcriptional expression of the defense-related genes. This eventually activates the basal resistance or PTI. Under avirulent pathogen infection, [Ca2+]cyt is constitutively increased, which leads to stable binding of Ca2+ ions to CaM. The Ca2+-saturated CaM interacts only with the CaMBD of AtSR1. This CaM-AtSR1 complex regulates the ETI-triggered PCD. In the atsr1 mutant, due to the loss of negative regulation of the NPR1 transcription by AtSR1, the plants constitutively activate basal immune response, resulting in the autoimmune phenotype. Under pathogen infection in the atsr1 mutant due to the lack of fine-tuning for transcriptional regulation of defense-related genes, the immune response is disturbed in the mutants, i.e., enhanced basal resistance and failure of ETI-induced PCD to the virulent and avirulent bacterial pathogens, respectively. Refer to Yuan et al. (2021) for further details.

Calcium (Ca²⁺) signaling in plant cells is an essential and early event during plant-microbe interactions. The recognition of microbe-derived molecules activates Ca²⁺ channels or Ca²⁺ pumps that trigger a transient increase in Ca²⁺ in the cytoplasm. The Ca²⁺ binding proteins (such as CBL, CPK, CaM, and CML), known as Ca²⁺ sensors, relay the Ca²⁺ signal into down-stream signaling events, e.g., activating transcription factors in the nucleus. For example, CaM and CML decode the Ca²⁺ signals to the CaM/CML-binding protein, especially CaM-binding transcription factors (AtSRs/CAMTAs), to induce the expressions of immune-related genes. In this review, we discuss the recent breakthroughs in down-stream Ca²⁺ signaling as a dynamic process, subjected to continuous variation and gradual change. AtSR1/CAMTA3 is a CaM-mediated transcription factor that represses plant immunity in non-stressful environments. Stress-triggered Ca²⁺ spikes impact the Ca²⁺-CaM-AtSR1 complex to control plant immune response. We also discuss other regulatory mechanisms in which Ca²⁺ signaling activates CPKs and MAPKs cascades followed by regulating the function of AtSR1 by changing its stability, phosphorylation status, and subcellular localization during plant defense.

6,772 views

34 citations

Pathways in which the DEGs are significantly enriched according to the transcriptomic data. (A) Volcano plot of DEGs. (B) DEG cluster analysis. (C) Bubble chart of the top 20 metabolic pathways enriched in GO terms. (D) Bubble chart of the top 20 metabolic pathways enriched in KEGG pathways.

Original Research

21 September 2022

Flagellin C decreases the expression of the Gossypium hirsutum cation/proton exchanger 3 gene to promote calcium ion, hydrogen peroxide, and nitric oxide and synergistically regulate the resistance of cotton to Verticillium wilt

Heng Zhou

, 4 more and

Canming Tang

2,319 views

4 citations

Defense priming has no significant effect on stimulus-induced calcium concentration changes in systemic tissue. (A–C) Flg22-induced calcium changes were recorded in systemic leaf disks 2 days after pre-treatment via infiltration of local leaves with 10 mM MgCl2 (mock) or 200 nM flg22 (primed). As triggering stimulus in the systemic leaf, 200 nM flg22 was applied, leading to the samples mock – triggered (T) or primed – triggered (PT), respectively. For detailed experimental setup, see Figure 4. As a control, inactive flg22 from A. tumefaciens was used as triggering stimulus with local mock pre-treatment. Data are averages of R-GECO1 fluorescence over time normalized to R-GECO1 fluorescence intensity at time point of flg22 application (t0). Error bars denote mean ± SD for n replicates (RGmT nT = 7, nPT = 7, ninactive flg22 = 3, (B), cpk5xRGmT nT = 6, nPT = 6 and (C), CPK5#7xRGmT nT = 9, nPT = 9). Parameters of flg22 induced calcium changes of measurements shown in panels (A–C) are analyzed in panels (D–F). In panel (D) dot plots represent the amplitude of the maximal signal change, while in panel (E) the correlating time points of maximum signal after flg22 treatment are shown. Dots represent the individual measurements. Shown are means ± SD of 6 – 9 biological replicates per line for each treatment (T vs. PT). (F) Apparent resting FR–GECO1/FmT in systemic leaf disks of local mock (10 mM MgCl2, control = C) or 200 nM flg22 (primed = P) treated plants. Shown are means ± SD (n ≥ 6). Dots represent the individual measurements. Two-way ANOVA and Bonferroni post test (p ≤ 0.05) reveals no significant differences between the pre-treatments but significant differences between genotypes indicated by different letters.

Original Research

14 December 2021

Plant Immune Memory in Systemic Tissue Does Not Involve Changes in Rapid Calcium Signaling

Bernadette Eichstädt

, 7 more and

Tina Romeis

Upon pathogen recognition, a transient rise in cytoplasmic calcium levels is one of the earliest events in plants and a prerequisite for defense initiation and signal propagation from a local site to systemic plant tissues. However, it is unclear if calcium signaling differs in the context of priming: Do plants exposed to a first pathogen stimulus and have consequently established systemic acquired resistance (SAR) display altered calcium responses to a second pathogen stimulus? Several calcium indicator systems including aequorin, YC3.6 or R-GECO1 have been used to document local calcium responses to the bacterial flg22 peptide but systemic calcium imaging within a single plant remains a technical challenge. Here, we report on an experimental approach to monitor flg22-induced calcium responses in systemic leaves of primed plants. The calcium-dependent protein kinase CPK5 is a key calcium sensor and regulator of the NADPH oxidase RBOHD and plays a role in the systemic calcium-ROS signal propagation. We therefore compared flg22-induced cytoplasmic calcium changes in Arabidopsis wild-type, cpk5 mutant and CPK5-overexpressing plants (exhibiting constitutive priming) by introgressing the calcium indicator R-GECO1-mTurquoise that allows internal normalization through mTurquoise fluorescence. Aequorin-based analyses were included for comparison. Based on the R-GECO1-mTurquoise data, CPK5-OE appears to reinforce an “oscillatory-like” Ca²⁺ signature in flg22-treated local tissues. However, no change was observed in the flg22-induced calcium response in the systemic tissues of plants that had been pre-challenged by a priming stimulus – neither in wild-type nor in cpk5 or CPK5-OE-lines. These data indicate that the mechanistic manifestation of a plant immune memory in distal plant parts required for enhanced pathogen resistance does not include changes in rapid calcium signaling upstream of CPK5 but rather relies on downstream defense responses.

4,908 views

13 citations

The defense not death 1 (dnd1) mutant has an impaired [Ca2+]cyt response to eATP. Cotyledons were excised from 14-day-old Col-0 and dnd1; cytosolic aequorin was reconstituted with coelenterazine (in 10 mM CaCl2, 0.1 mM KCl, 2 mM Tris/MES, pH 5.8) as described by Mohammad-Sidik et al. (2021). Luminescence was recorded from individual samples bathed in 10 mM CaCl2, 0.1 mM KCl, 2 mM Tris/MES, pH 5.8 as described by Mohammad-Sidik et al. (2021). As with flg22, ATP causes pronounced acidification of solutions, and so in this study, pH of eATP test solutions was maintained by buffers to avoid acidic pH artefactual responses which can impair interpretation (Westphal et al., 2019). [Ca2+]cyt values were calculated as described by Matthus et al. (2019b). (A) Mean ± SEM time course of [Ca2+]cyt response of Col-0 and dnd1 to control solution (10 mM CaCl2, 0.1 mM KCl, 2 mM Tris/MES, pH 5.8) added at 35s (inverted triangle). (B) Touch-induced [Ca2+]cyt increase from pre-stimulus baseline and estimated total [Ca2+]cyt mobilized [area under the curve (AUC), after baseline subtraction]. (C) Mean ± SEM time course of [Ca2+]cyt response of Col-0 and dnd1 to 0.1 mM eATP added at 35s (inverted triangle). (D) Touch-induced [Ca2+]cyt increase from pre-stimulus baseline, peak eATP-induced [Ca2+]cyt increase from baseline, and estimated AUC. (E) As (C) but with 1 mM eATP. (F) Mean ± SEM time course of the eATP response shown in (E). (G) As (D) but for 1 mM eATP. Results are from 3 independent trials with 9 samples in total per genotype in control trials and 19–21 samples in total per genotype in eATP trials. Each dot in box plots represents an individual recording. The middle line and the triangle in the box plot are the median and mean, respectively. Student’s t-test was used for analyzing statistical difference (***p < 0.001; *p < 0.05; n.s, not significant).

Perspective

02 December 2021

Damage Signaling by Extracellular Nucleotides: A Role for Cyclic Nucleotides in Elevating Cytosolic Free Calcium?

Jian Sun

, 3 more and

Julia M. Davies

3,448 views

9 citations

Review

25 November 2021

TypiCal but DeliCate Ca++re: Dissecting the Essence of Calcium Signaling Network as a Robust Response Coordinator of Versatile Abiotic and Biotic Stimuli in Plants

Neelesh Patra

, 4 more and

Joydeep Banerjee

Plant growth, development, and ultimately crop productivity are largely impacted by the interaction of plants with different abiotic and biotic factors throughout their life cycle. Perception of different abiotic stresses, such as salt, cold, drought, heat, and heavy metals, and interaction with beneficial and harmful biotic agents by plants lead to transient, sustained, or oscillatory changes of [calcium ion, Ca²⁺]_cyt within the cell. Significant progress has been made in the decoding of Ca²⁺ signatures into downstream responses to modulate differential developmental and physiological responses in the whole plant. Ca²⁺ sensor proteins, mainly calmodulins (CaMs), calmodulin-like proteins (CMLs), and others, such as Ca²⁺-dependent protein kinases (CDPKs), calcineurin B-like proteins (CBLs), and calmodulin-binding transcription activators (CAMTAs) have played critical roles in coupling the specific stress stimulus with an appropriate response. This review summarizes the current understanding of the Ca²⁺ influx and efflux system in plant cells and various Ca²⁺ binding protein-mediated signal transduction pathways that are delicately orchestrated to mitigate abiotic and biotic stresses. The probable interactions of different components of Ca²⁺ sensor relays and Ca²⁺ sensor responders in response to various external stimuli have been described diagrammatically focusing on established pathways and latest developments. Present comprehensive insight into key components of the Ca²⁺ signaling toolkit in plants can provide an innovative framework for biotechnological manipulations toward crop improvability in near future.