Calcium Signalling in Plant Biotic Interactions.

Calcium (Ca2+) is a universal second messenger involved in various cellular processes, leading to plant development and to biotic and abiotic stress responses. Intracellular variation in free Ca2+ concentration is among the earliest events following the plant perception of environmental change. These Ca2+ variations differ in their spatio-temporal properties according to the nature, strength and duration of the stimulus. However, their conversion into biological responses requires Ca2+ sensors for decoding and relaying. The occurrence in plants of calmodulin (CaM) but also of other sets of plant-specific Ca2+ sensors such as calmodulin-like proteins (CMLs), Ca2+-dependent protein kinases (CDPKs) and calcineurin B-like proteins (CBLs) indicate that plants possess specific tools and machineries to convert Ca2+ signals into appropriate responses. Here, we focus on recent progress made in monitoring the generation of Ca2+ signals at the whole plant or cell level and their long distance propagation during biotic interactions. The contribution of CaM/CMLs and CDPKs in plant immune responses mounted against bacteria, fungi, viruses and insects are also presented.

CML8, an Arabidopsis Calmodulin-Like Protein, Plays a Role in Pseudomonas syringae Plant Immunity.

Calcium is a universal second messenger involved in various cellular processes including plant development and stress responses. Its conversion into biological responses requires the presence of calcium sensor relays such as calmodulin (CaM) and calmodulin-like (CML) proteins. While the role of CaM is well described, the functions CML proteins remain largely uncharacterized. Here, we show that Arabidopsis CML8 expression is strongly and transiently induced by Pseudomonas syringae, and reverse genetic approaches indicated that the overexpression of CML8 confers on plants a better resistance to pathogenic bacteria compared with wild-type, knock-down and knock-out lines, indicating that CML8 participates as a positive regulator in plant immunity. However, this difference disappeared when inoculations were performed using bacteria unable to inject effectors into a plant host cell or deficient for some effectors known to target the salicylic acid (SA) signaling pathway. SA content and PR1 protein accumulation were altered in CML8 transgenic lines, supporting a role for CML8 in SA-dependent processes. Pathogen-associated molecular pattern (PAMP) treatments with flagellin and elf18 peptides have no effects on CML8 gene expression and do not modify root growth of CML8 knock-down and overexpressing lines compared with wild-type plants. Collectively, our results support a role for CML8 in plant immunity against P. syringae.

Calcium/calmodulin-mediated regulation of plant immunity.

Calcium is a universal messenger involved in the modulation of diverse developmental and adaptive processes in response to various physiological stimuli. Ca(2+) signals are represented by stimulus-specific Ca(2+) signatures that are sensed and translated into proper cellular responses by diverse Ca(2+) binding proteins and their downstream targets. Calmodulin (CaM) and calmodulin-like (CML) proteins are primary Ca(2+) sensors that control diverse cellular functions by regulating the activity of various target proteins. Recent advances in our understanding of Ca(2+)/CaM-mediated signalling in plants have emerged from investigations into plant defence responses against various pathogens. Here, we focus on significant progress made in the identification of CaM/CML-regulated components involved in the generation of Ca(2+) signals and Ca(2+)-dependent regulation of gene expression during plant immune responses. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.

Relationships between calcium and sphingolipid-dependent signalling pathways during the early steps of plant-pathogen interactions.

An increase in cellular calcium ion (Ca(2+)) concentration is now acknowledged to be one of the earliest events occurring during the induction of plant defence responses to a wide variety of pathogens. Sphingoid long-chain bases (LCBs) have also been recently demonstrated to be important mediators of defence-related programmed cell death during pathogen attack. Here, we present recent data highlighting how Ca(2+) and LCBs may be interconnected to regulate cellular processes which lead either to plant susceptibility or to resistance mechanisms. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.

CML9, an Arabidopsis calmodulin-like protein, contributes to plant innate immunity through a flagellin-dependent signalling pathway.

Many stimuli such as hormones and elicitors induce changes in intracellular calcium levels to integrate information and activate appropriate responses. The Ca(2+) signals are perceived by various Ca(2+) sensors, and calmodulin (CaM) is one of the best characterized in eukaryotes. Calmodulin-like (CML) proteins extend the Ca(2+) toolkit in plants; they share sequence similarity with the ubiquitous and highly conserved CaM but their roles at physiological and molecular levels are largely unknown. Knowledge of the contribution of Ca(2+) decoding proteins to plant immunity is emerging, and we report here data on Arabidopsis thaliana CML9, whose expression is rapidly induced by phytopathogenic bacteria, flagellin and salicylic acid. Using a reverse genetic approach, we present evidence that CML9 is involved in plant defence by modulating responses to bacterial strains of Pseudomonas syringae. Compared to wild-type plants, the later responses normally observed upon flagellin application are altered in knockout mutants and over-expressing transgenic lines. Collectively, using PAMP treatment and P. syringae strains, we have established that CML9 participates in plant innate immunity.

RD20, a stress-inducible caleosin, participates in stomatal control, transpiration and drought tolerance in Arabidopsis thaliana.

Plants overcome water deficit conditions by combining molecular, biochemical and morphological changes. At the molecular level, many stress-responsive genes have been isolated, but knowledge of their physiological functions remains fragmentary. Here, we report data for RD20, a stress-inducible Arabidopsis gene that belongs to the caleosin family. As for other caleosins, we showed that RD20 localized to oil bodies. Although caleosins are thought to play a role in the degradation of lipids during seed germination, induction of RD20 by dehydration, salt stress and ABA suggests that RD20 might be involved in processes other than germination. Using plants carrying the promoter RD20::uidA construct, we show that RD20 is expressed in leaves, guard cells and flowers, but not in root or in mature seeds. Water deficit triggers a transient increase in RD20 expression in leaves that appeared predominantly dependent on ABA signaling. To assess the biological significance of these data, a functional analysis using rd20 knock-out and overexpressing complemented lines cultivated either in standard or in water deficit conditions was performed. The rd20 knock-out plants present a higher transpiration rate that correlates with enhanced stomatal opening and a reduced tolerance to drought as compared with the wild type. These results support a role for RD20 in drought tolerance through stomatal control under water deficit conditions.

Interaction of a plant pseudo-response regulator with a calmodulin-like protein.

Calmodulin (CaM) plays a crucial role in the regulation of diverse cellular processes by modulating the activities of numerous target proteins. Plants possess an extended CaM family including numerous CaM-like proteins (CMLs), most of which appear to be unique to plants. We previously demonstrated a role for CML9 in abiotic stress tolerance and seed germination in Arabidopsis thaliana. We report here the isolation of PRR2, a pseudo-response regulator as a CML9 interacting protein by screening an expression library prepared from Arabidopsis seedlings with CML9 as bait in a yeast two-hybrid system. PRR2 is similar to the response regulators of the two-component system, but lacks the invariant residue required for phosphorylation by which response regulators switch their output response, suggesting the existence of alternative regulatory mechanisms. PRR2 was found to bind CML9 and closely related CMLs but not a canonical CaM. Mapping analyses indicate that an almost complete form of PRR2 is required for interaction with CML9, suggesting a recognition mode different from the classical CaM-target peptide complex. PRR2 contains several features that are typical of transcription factors, including a GARP DNA recognition domain, a Pro-rich region and a Golden C-terminal box. PRR2 and CML9 as fusion proteins with fluorescent tags co-localized in the nucleus of plant cells, and their interaction in the nuclear compartment was validated in planta by using a fluorophore-tagged protein interaction assay. These findings suggest that binding of PRR2 to CML9 may be an important mechanism to modulate the physiological role of this transcription factor in plants.

GATEWAY technology and E. coli recombinant system produce a properly folded and functional recombinant allergen of the lipid transfer protein of apple (Mal d 3).

The lipid transfer protein of apple fruit, Mal d 3, has been produced as a soluble recombinant protein in transformed Escherichia coli cells using the GATEWAY technology. Circular dichroism spectra showing the protein essentially consists of alpha-helices indicate that the rMal d 3 is properly folded. The (1)H NMR spectra also indicates a correct fold for the recombinant allergen. The reactivity of rMal d 3 towards IgE from apple allergic patients and in vitro degranulation activity measured on transformed rat basophil leukemia cells expressing the human Fc epsilon RI alpha-subunit of rMal d 3 is similar to those of the native allergen purified from apple fruits. The expression of active rMal d 3 in E. coli is readily feasible and offers an interesting alternative to the production of recombinant allergen in the yeast Pichia pastoris. This expression in E. coli open the way to the modification of Mal d 3 by site-directed mutagenesis for immunotherapy purposes.

Mutations in AtCML9, a calmodulin-like protein from Arabidopsis thaliana, alter plant responses to abiotic stress and abscisic acid.

Many stimuli, such as hormones and abiotic stress factors, elicit changes in intracellular calcium levels that serve to convey information and activate appropriate responses. The Ca2+ signals are perceived by different Ca2+ receptors, and calmodulin (CaM) is one of the best-characterized Ca2+ sensors in eukaryotes. Calmodulin-like (CML) proteins also exist in plants; they share sequence similarity with the ubiquitous and highly conserved CaM, but their roles at the physiological and molecular levels are largely unknown. We present data on Arabidopsis thaliana CML9 (AtCML9) that exhibits 46% amino acid sequence identity with CaM. AtCML9 transcripts are found in all major organs, and a putative AtCML9 regulatory region confers reporter gene expression at various sites, including root apex, stomata, hydathodes and trichomes. AtCML9 expression is rapidly induced by abiotic stress and abscisic acid (ABA) in young seedlings, and by using cml9 knock-out mutants we present evidence that AtCML9 plays essential roles in modulating responses to salt stress and ABA. Seed germination and seedling growth for the mutant lines present a hypersensitive response to ABA that could be correlated with enhanced tolerance to salt stress and water deficit. Mutations of the AtCML9 gene also alter the expression of several stress-regulated genes, suggesting that AtCML9 is involved in salt stress tolerance through its effects on the ABA-mediated pathways.

Herbivory-responsive calmodulin-like protein CML9 does not guide jasmonate-mediated defenses in Arabidopsis thaliana.

Calcium is an important second messenger in plants that is released into the cytosol early after recognition of various environmental stimuli. Decoding of such calcium signals by calcium sensors is the key for the plant to react appropriately to each stimulus. Several members of Calmodulin-like proteins (CMLs) act as calcium sensors and some are known to mediate both abiotic and biotic stress responses. Here, we study the role of the Arabidopsis thaliana CML9 in different stress responses. CML9 was reported earlier as defense regulator against Pseudomonas syringae. In contrast to salicylic acid-mediated defense against biotrophic pathogens such as P. syringae, defenses against herbivores and necrotrophic fungi are mediated by jasmonates. We demonstrate that CML9 is induced upon wounding and feeding of the insect herbivore Spodoptera littoralis. However, neither different CML9 loss-of-function mutant lines nor overexpression lines were impaired upon insect feeding. No difference in herbivore-induced phytohormone elevation was detected in cml9 lines. The defense against the spider mite Tetranychus urticae was also unaffected. In addition, cml9 mutant lines showed a wild type-like reaction to the necrotrophic fungus Alternaria brassicicola. Thus, our data suggest that CML9 might be a regulator involved only in the defense against biotrophic pathogens, independent of jasmonates. In addition, our data challenge the involvement of CML9 in plant drought stress response. Taken together, we suggest that CML9 is a specialized rather than a general regulator of stress responses in Arabidopsis.

Respective contribution of CML8 and CML9, two arabidopsis calmodulin-like proteins, to plant stress responses.

In their natural environment, plants have to continuously face constraints such as biotic and abiotic stresses. To achieve their life cycle, plants have to perceive and interpret the nature, but also the strength of environmental stimuli to activate appropriate physiological responses. Nowadays, it is well established that signaling pathways are crucial steps in the implementation of rapid and efficient plant responses such as genetic reprogramming. It is also reported that rapid raises in calcium (Ca2+) levels within plant cells participate in these early signaling steps and are essential to coordinate adaptive responses. However, to be informative, calcium increases need to be decoded and relayed by calcium-binding proteins also referred as calcium sensors to carry-out the appropriate responses. In a recent study, we showed that CML8, an Arabidopsis calcium sensor belonging to the calmodulin-like (CML) protein family, promotes plant immunity against the phytopathogenic bacteria Pseudomonas syringae pv tomato (strain DC3000). Interestingly, other CML proteins such as CML9 were also reported to contribute to plant immunity using the same pathosystem. In this addendum, we propose to discuss about the specific contribution of these 2 CMLs in stress responses.

Calcium Sensors as Key Hubs in Plant Responses to Biotic and Abiotic Stresses.

The Ca(2+) ion is recognized as a crucial second messenger in signaling pathways coupling the perception of environmental stimuli to plant adaptive responses. Indeed, one of the earliest events following the perception of environmental changes (temperature, salt stress, drought, pathogen, or herbivore attack) is intracellular variation of free calcium concentrations. These calcium variations differ in their spatio-temporal characteristics (subcellular location, amplitude, kinetics) with the nature and strength of the stimulus and, for this reason, they are considered as signatures encrypting information from the initial stimulus. This information is believed to drive a specific response by decoding via calcium-binding proteins. Based on recent examples, we illustrate how individual calcium sensors from the calcium-dependent protein kinase and calmodulin-like protein families can integrate inputs from various environmental changes. Focusing on members of these two families, shown to be involved in plant responses to both abiotic and biotic stimuli, we discuss their role as key hubs and we put forward hypotheses explaining how they can drive the signaling pathways toward the appropriate plant responses.

A signal transfer system through three compartments transduces the plant cell contact-dependent signal controlling Ralstonia solanacearum hrp genes.

Ralstonia solanacearum hrp genes encode a type III secretion system required for disease development in host plants and for hypersensitive response elicitation on non-hosts. hrp genes are expressed in the presence of plant cells through the HrpB regulator. This activation, which requires physical interaction between the bacteria and the plant cell, is sensed by the outer membrane receptor PrhA. PrhA transduces the plant cell contact-dependent signal through a complex regulatory cascade integrated by the PrhJ, HrpG, and HrpB regulators. In this study, we have identified two genes, named prhI and prhR, that belong to the hrp gene cluster and whose predicted products show homology with extracytoplasmic function sigma factors and transmembrane proteins, respectively. Strains carrying a mutation in prhIR show a delayed pathogenic phenotype toward host plants. PrhIR control the plant cell contact-dependent activation of hrp genes. prhIR gene expression is induced by a signal present in the plant cell coculture that is not PrhA-dependent. Genetic evidence shows that PrhIR act upstream of PrhJ in the regulatory cascade, likely transducing the signal sensed by PrhA through the periplasm as described for signal transfer systems through three compartments. This is the first report of such a surface signaling mechanism activating pathogenicity determinants in response to a nondiffusible plant cell wall signal.

CML9, a multifunctional Arabidopsis thaliana calmodulin-like protein involved in stress responses and plant growth?

Plants have evolved complex signaling networks to respond to their fluctuating environment and adapt their growth and development. Calcium-dependent signaling pathways play key role in the onset of these adaptive responses. In plant cells, the intracellular calcium transients are triggered by numerous stimuli and it is supposed that the large repertory of calcium sensors present in higher plants could contribute to integrate these signals in physiological responses. Here, we present data on CML9, a calmodulin-like protein that appears to be involved in plant responses to both biotic and abiotic stress. Using a reverse genetic approach based on gain and loss of function mutants, we present here data indicating that this CML might also be involved in root growth control in response to the flagellin, a pathogen-associated molecular pattern (PAMP) also involved in plant immunity.

Calmodulin and calmodulin-like proteins in plant calcium signaling.

Calmodulin (CaM) is a primary calcium sensor in all eukaryotes. It binds calcium and regulates the activity of a wide range of effector proteins in response to calcium signals. The list of CaM targets includes plant-specific proteins whose functions are progressively being elucidated. Plants also possess numerous calmodulin-like proteins (CMLs) that appear to have evolved unique functions. Functional studies of CaM and CMLs in plants highlight the importance of this protein family in the regulation of plant development and stress responses by converting calcium signals into transcriptional responses, protein phosphorylation or metabolic changes. This review summarizes some of the significant progress made by biochemical and genetic studies in identifying the properties and physiological functions of plant CaMs and CMLs. We discuss emerging paradigms in the field and highlight the areas that need further investigation.

Involvement of RD20, a member of caleosin family, in ABA-mediated regulation of germination in Arabidopsis thaliana.

The RD20 gene encodes a member of the caleosin family, which is primarily known to function in the mobilization of seed storage lipids during germination. In contrast to other caleosins, RD20 expression is early-induced by water deficit conditions and we recently provided genetic evidence for its positive role in drought tolerance in Arabidopsis. RD20 is also responsive to pathogen infection and is constitutively expressed in diverse tissues and organs during development suggesting additional roles for this caleosin. This addendum describes further exploration of phenotypic alterations in T-DNA insertional rd20 mutant and knock-out complemented transgenic plants in the context of early development and susceptibility to a phytopathogenic bacteria. We show that the RD20 gene is involved in ABA-mediated inhibition of germination and does not play a significant role in plant defense against Pseudomonas syringae.

Plant calmodulins and calmodulin-related proteins: multifaceted relays to decode calcium signals.

The calmodulin (CaM) family is a major class of calcium sensor proteins which collectively play a crucial role in cellular signaling cascades through the regulation of numerous target proteins. Although CaM is one of the most conserved proteins in all eukaryotes, several features of CaM and its downstream effector proteins are unique to plants. The continuously growing repertoire of CaM-binding proteins includes several plant-specific proteins. Plants also possess a particular set of CaM isoforms and CaM-like proteins (CMLs) whose functions have just begun to be elucidated. This review summarizes recent insights that help to understand the role of this multigene family in plant development and adaptation to environmental stimuli.