Calcium signaling: an emerging player in plant antiviral defense

Abstract

Calcium is a universal messenger in different kingdoms of living organisms and regulates most physiological processes, including defense against pathogens. The threat of viral infections in humans has become very clear in recent years, and this has triggered detailed research into all aspects of host–virus interactions, including the suppression of calcium signaling in infected cells. At the same time, however, the threat of plant viral infections is underestimated in society, and research in the field of calcium signaling during plant viral infections is scarce. Here we highlight an emerging role of calcium signaling for antiviral protection in plants, in parallel with the known evidence from studies of animal cells. Obtaining more knowledge in this domain might open up new perspectives for future crop protection and the improvement of food security.

Recent findings in plant virus research, coupled with comparisons to animal cells, indicate an important role of calcium signaling in plant antiviral responses with high potential for future investigation.

Introduction

The spread of viral infections of crops has increased dramatically all over the world in recent years, causing substantial economic damage (Chauhan et al., 2019; Jones, 2021). Aside from economic loss, these viral diseases pose a significant risk for human food security. The increasing speed of climate change is escalating this problem even further (Trebicki, 2020; Amari et al., 2021). Due to increasing temperature, the virus-transmitting insects are able to expand their geographic distribution and their survival during overwintering, and as a consequence the number of generations of these vectors increases. This also alters the interaction with their natural enemy species (Skendzic et al., 2021). On top of these factors, drought, excessive flooding, the increase of the concentrations of CO₂ and ozone in the atmosphere, as well as UV-B levels, severely affects the host’s physiology and resistance against pathogens (Bastas, 2022). On the other hand, an emerging area of recent research is the mutualistic relationship between plants and viruses, as infected plants sometimes develop increased resistance to abiotic stress (Aguilar and Lozano-Duran, 2022; Gorovits et al., 2022). Climate change may also indirectly affect the efficacy of insecticides. Moreover, adaptation to one environmental stress (e.g. insecticides) causes further increased thermotolerance in whiteflies, a vector for many plant viruses (Aregbesola et al., 2019).

New ways of crop protection must be developed to cope with these inevitable developments. Disease control is mainly based on prophylactic measures to restrain viral spread, for example, using quarantine, certification, the destruction of infected plants, and the use of insecticides to control the virus-transmitting insects (Rubio et al., 2020). The second antiviral protection strategy, which strongly capitalizes on the knowledge of plant–virus interactions, aims to produce genetically resistant varieties that can be exploited in agriculture (Rubio et al., 2020). Therefore, elucidating the molecular mechanisms underlying plant–virus interactions during viral entry, replication, movement, and transmission might contribute to the development of new strategies for effectively combatting viral threats in agricultural settings.

To counteract viral infections, plants defend themselves by using several mechanisms to restrict viral replication and movement. The best-studied defense mechanisms are RNA-mediated gene silencing, innate immune receptor-based signaling, translational repression, ubiquitination- and autophagy-mediated protein degradation, and the resistance genes response. Typical hallmarks of the plant immune response are the generation of the hypersensitive response (HR) and the salicylic acid-induced systemic and acquired resistance (Calil and Fontes, 2017; Wu et al., 2019). Viruses, in turn, have developed their own mechanisms to suppress different plant defense strategies. The primary strategy employed by all viruses to evade the host’s immune defenses is the high mutation rate of their genome during replication, which prevents the recognition of modified viral proteins by the host’s immune system. Moreover, certain viral proteins have the ability to selectively bind to components of various defense pathways. This leads to direct repression of these defenses or to the virus exploiting some of these defense pathways for their own benefit (Wu et al., 2019), both of which ultimately advance viral replication and spreading.

Calcium (Ca²⁺) signaling plays a key role in setting up the innate immunity defense reactions downstream of both cell-surface and intracellular receptor proteins, which are activated by pathogen-associated molecular patterns and effector proteins, respectively (Aldon et al., 2018). The defense reactions include signaling via mitogen-activated protein kinases (MAPKs), reactive oxygen species (ROS) production, salicylic acid (SA) accumulation, and extensive transcriptional reprogramming to induce the local and systemic immune response to various pathogens in plants (Garcia-Brugger et al., 2006; Gilroy et al., 2016; Choi et al., 2017). In recent years it has become clear that functional Ca²⁺ channels are required for immunity against biotrophic and necrotrophic pathogens (Koster et al., 2022; Xu et al., 2022). These channels facilitate a rapid increase of the free Ca²⁺ concentration in the cytoplasm by releasing Ca²⁺ from the intra- and extracellular stores. This activates intracellular Ca²⁺ decoders, which subsequently activate their target proteins, leading to immune responses. Moreover, there is evidence that the immune signaling triggered by elicitor perception can, in turn, regulate the transcription of glutamate receptor-like (GLR) Ca²⁺ channels (Bjornson et al., 2021). Another striking phenomenon is the formation of a resistosome, which consists of several nucleotide-binding leucine-rich repeat receptors (NLRs) that are activated by pathogen-derived effector proteins. The NLR oligomer forms a pore, which is inserted into the plasma membrane (PM) and allows the influx of Ca²⁺ as well as other cations from the apoplast into the cytoplasm. Such ion influx is essential for the HR, which restricts the spread of pathogens including viruses (Forderer and Kourelis, 2023; Ivanov et al., 2023; Shepherd et al., 2023).

The focus of this review is to summarize the emerging evidence that Ca²⁺ signals are essential for setting up most of the antiviral defense mechanisms and that alteration of Ca²⁺ signaling could be a universal strategy used by viruses to suppress plants’ defense responses. Remarkably, the mechanisms that viruses employ to hijack Ca²⁺ signaling of the host cell have been studied very well in animals, but they are clearly underexplored in plants. Since many fundamental molecular mechanisms are similar in all eukaryotes, it seems appropriate to expect that analogous mechanisms occur in plants, as the role of Ca²⁺ signaling is universal in both types of organism (Luan and Wang, 2021). Therefore, we will draw parallels throughout the review between animal and plant viral hosts in order to highlight the crucial aspects that need to be studied in plants in the future.

Viruses manipulate the Ca²⁺ concentration in the cytosol and modify host intracellular Ca²⁺ sensor activity and viral protein stability

Ca²⁺ pumps, channels, and fluxes

Viruses interfere with cellular Ca²⁺ signaling at many levels. In animals, regulation of the host antiviral response includes Ca²⁺-dependent activation of Toll-like receptors and dsRNA-sensing molecules, leading to the production of cytokines and interferons, the activation of immune cells, and inflammation (Qu et al., 2022). In order to counteract host defenses, viruses apply different general strategies to manipulate regulators of Ca²⁺ signaling at intracellular levels to favor viral reproduction (Fig. 1A). First, binding to voltage-gated calcium channels (VGCCs) on the PM promotes fusion of the virion with the cell and virus entry (Qu et al., 2022). Next, specific viral proteins are able to modulate the functions of various Ca²⁺ ion channels and pumps at the PM, such as the store-operated channel (SOC) and receptor-operated channel (ROC), the transient receptor potential channel (TRP), the PM Ca²⁺-ATPase (PMCA), and the Na⁺/Ca²⁺ exchanger (NCX), as well as the pumps and channels at the membranes of the Ca²⁺ internal stores. These include the sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA), the mitochondrial Ca²⁺ uniporter (MCU), the H⁺/Ca²⁺ exchanger (HCX), and the voltage-dependent anion channel (VDAC) or the lysosomal two-pore channel (TPC). Additionally, in animals, Ca²⁺ ions can be released from internal stores through the inositol-1,4,5-triphosphate receptor (IP3R) and ryanodine receptor (RyR). As a result, a temporarily increased concentration of Ca²⁺ in the cytoplasm leads to activation of internal Ca²⁺ sensors such as calmodulins (CaMs) and protein kinases, which change the activity of their target proteins. This facilitates viral replication as well as suppression of the host’s defenses (Zhou et al., 2009; Qu et al., 2022).

Fig. 1.

Ca²⁺ signaling perturbations caused by viral infection in animal and plant cells. (A) In animal cells, viral proteins interfere with the functions of Ca²⁺ pumps and/or channels at the plasma membrane (PM) and at the membranes of Ca²⁺ internal stores, including the endoplasmic reticulum (ER), Golgi apparatus, and mitochondria. Viral proteins interfere with Ca²⁺ release from internal stores accomplished by ryanodine receptors (RyR) and inositol-1,4,5-triphosphate receptors (IP3R). In animals and plants, viral proteins interact with intracellular Ca²⁺ sensors and buffers to remodel the Ca²⁺ signaling network or to activate or repress Ca²⁺-responsive transcription. (B) Plants have additional Ca²⁺ stores with the maximal concentration of Ca²⁺ in the cell: the central vacuole, the apoplast, and chloroplasts. In chloroplasts, a viral protein interacts with the chloroplast Ca²⁺ sensor (CAS) protein, suppressing CAS-dependent immune defense. The size-exclusion limit of the plasmodesmata regulates viral spread. Viral proteins regulate the size of plasmodesmata, facilitating cell-to-cell movement by competing for interaction with the Ca²⁺-binding protein pCaP1 and its interactor remorin. The gradient color bar at the bottom of the figure indicates the total Ca²⁺ concentration in the different subcellular compartments. Calcium ions are shown as blue dots. ACA, autoinhibited Ca²⁺-ATPase; BICAT 1, 2, bivalent cation transporter 1 and 2; CALR, calreticulin; CaM, Calmodulin; CAMTA, calmodulin-binding transcription activator; CAX, Ca²⁺-ATPases and Ca²⁺/H⁺ exchangers; cMCU, chloroplast-localized mitochondrial Ca²⁺ uniporter; CNGC, cyclic nucleotide-gated channel; ECA, ER-type Ca²⁺-ATPase; GLR, glutamate receptor-like channel; HCX, H⁺/Ca²⁺ exchanger; MCA, Mid1-complementing activity channel; MCU, mitochondrial Ca²⁺ uniporter; MSL, mechanosensitive-like channel; NCX, Na⁺/Ca²⁺ exchanger; OSCA, reduced hyperosmolarity-induced [Ca²⁺] cytosolic increase channel; PEC1/2, plastid envelope ion channel 1 and 2; PMCA, plasma membrane Ca²⁺-ATPase; ROC, receptor-operated channel; SERCA, sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase; SOC, store-operated channel; TF, transcription factor; TPC, two-pore channel; TR, transient receptor potential channel; VDAC, voltage-dependent anion channel; VGCC, voltage-gated Ca²⁺ channel. Figure created with Biorender.com. The template of the animal cell is redrawn from Zhou et al. (2009), with permission from Elsevier.

Based on the observations of Ca²⁺-sensitive processes, it therefore seems likely that, similar to animal viral proteins, plant viral proteins would also directly target the channels and pumps that are responsible for the Ca²⁺ fluxes in and out of the cytoplasm (Fig. 1B). There are three plant-specific Ca²⁺ storage pools: the central vacuole, the extracellular space between the PM and the cell wall (the apoplast), and the chloroplast. Regulation of Ca²⁺ influx from the apoplast must be accomplished by PM-localized Ca²⁺ channels and pumps, such as GLRs, cyclic nucleotide-gated channels (CNGCs), mechanosensitive-like channels (MSLs), Mid1-complementing activity channels (MCAs), reduced hyperosmolarity-induced cytosolic [Ca²⁺] increase channels (OSCAs), autoinhibited Ca²⁺-ATPases (ACA, an analogue of animal PMCA) and NCXs (Luan and Wang, 2021). Some of these channels, being also present at the membranes of the different organelles, are responsible for the regulation of Ca²⁺ release from internal stores (Fig. 1B). Some Ca²⁺ channels and pumps are unique to specific plant organelles. The vacuolar membrane harbors the TPC, the Ca²⁺/H⁺ exchangers (CAXs), and PIEZO ion channels (Schonknecht, 2013). Mitochondrial membranes contain the MCUs and VDACs (Pirayesh et al., 2021). In chloroplast membranes, the chloroplast-localized MCUs (cMCUs), the bivalent cation transporter 1 and 2 (BICAT 1, 2) (Frank et al., 2019), and the POLLUX plastid envelope ion channel 1 and 2 (PEC1/2) have been reported (Volkner et al., 2021). The endoplasmic reticulum (ER) membrane harbors the ER-type Ca^2+-ATPases (ECAs), which are the analogs of animal SERCAs at the ER and presumably also at the Golgi apparatus (Edel et al., 2017). Finally, the peroxisomal membrane also contains porins, which have been implicated in Ca²⁺ transport (Fig. 1B) (Pirayesh et al., 2021).

It has been shown that the Arabidopsis putative ortholog of the animal PIEZO protein becomes activated upon viral infection and inhibits the systemic movement of the silencing suppressor-deficient cucumber mosaic virus (CMV) and the turnip mosaic virus (TuMV) (Zhang et al., 2019). According to the authors of that study, the animal ortholog has a mechanosensitive cation channel activity. However, the cation transport function and the subcellular localization of this particular Arabidopsis PIEZO ortholog have not been determined. For the rest of the channels mentioned above, it is still unclear whether any of them contribute to the release of Ca²⁺ into the cytoplasm or to organelle-specific defense responses during viral infection. Thus, the question of whether they might be potential viral targets for the regulation of plant–virus interactions is still open, although available transcriptomics data would support this hypothesis. Chiu et al. (2021) identified by RNA-seq analysis that the transgenic expression in Arabidopsis of the TuMV protein P1/HcPro results in a significant change of genes that are responsible for the regulation of Ca²⁺ signaling at all levels. This included several calcium uniporters, calcium- and CaM-binding proteins, as well as several calcium-dependent protein kinases (CPKs). Then et al. (2021) demonstrated that several genes encoding Ca²⁺ channels such as GLRs and CNGCs were misregulated in cauliflower mosaic virus- and TuMV-infected plants. Still, the abundance of these channels at the protein level, as well as their functionality, remains to be studied. Importantly, the authors state that the plant viruses can somewhat slow down the aphid-triggered elevations of Ca²⁺ in infected leaves (Then et al., 2021). Mechanical wounding triggers a massive influx of Ca²⁺ into phloem cells, which leads to the activation of plant defense by protein plugging and callose sealing of the sieve elements. However, the gel saliva of aphids is able to limit this Ca²⁺ influx by sealing the site of penetration, which additionally reduces the loss of turgor pressure and prevents the activation of the mechanosensitive Ca²⁺ channels on the PM and subsequent occlusion of sieve elements, therefore attenuating the host plant’s defense and improving aphid feeding efficiency (van Bel et al., 2014). The phloem is a major route for viral infection to spread systemically throughout the plant (Vuorinen et al., 2011). Since clogging of the phloem would also restrict the long-distance transport of viruses, it can be assumed that viruses could indeed contribute to suppression of the Ca²⁺ influx caused by insect feeding. On the other hand, the rice gall dwarf virus reduces the secretion of its insect vector’s specific Ca²⁺-binding saliva protein, which leads to increased Ca²⁺ influx and subsequent callose deposition in the phloem. The insect vector encounters a stronger barrier to feeding and changes its behaviour, probing more frequently and secreting more saliva into rice plants, thus ultimately enhancing transmission of the virus between plants (Wu et al., 2022). Based on these data, Ca²⁺ signaling likely plays a significant role in the horizontal transmission of these viruses. However, further research is required to provide more clarity about the regulatory mechanisms.

Internal Ca²⁺-sensor proteins

The regulation of plant immune responses, similar to those in animals, depends highly on the activation of intracellular Ca²⁺ receptors and Ca²⁺-dependent changes in protein–protein interactions. The protein families of CPKs and CaMs/calmodulin-like proteins (CMLs), as well as calcineurin B-like proteins (CBLs) together with CBL/CBL-interacting protein kinases (CIPKs), control plant defense gene expression by altering the activity of their target proteins, including transcription factors (Yuan et al., 2021). Therefore, manipulating the Ca²⁺ concentration in the cytoplasm and targeting intracellular Ca²⁺ sensors were shown to be the common strategies of host immunity suppression used by plant viruses. It has been reported that tobacco mosaic virus (TMV) uses several strategies to manipulate cytoplasmic Ca²⁺ signaling and the function of intracellular Ca²⁺ sensors during infection. Initially, TMV causes an increase of the cytosolic and nuclear Ca²⁺ concentrations in the systemic tissue of root tips, which favors plant defense. Here, the authors suggest an interesting model that elevated cytosolic Ca²⁺ concentration could trigger increased ROS production, which in turn would increase the Ca²⁺ concentration in the nucleus, presumably triggering programmed cell death and therefore restricting viral spread (Li et al., 2018). It is important to elucidate the signal triggering such an effect in the systemic tissues and to know whether such a defense mechanism occurs in the primary infected tissue as well. Another study demonstrated a counter-defense mechanism of TMV by regulating the abundance of CML30 in Nicotiana benthamiana. TMV up-regulates the expression of the host coat protein (CP)-interacting protein L (IP-L), which binds to CML30 in a Ca²⁺-dependent manner. Such an interaction would lead to CML30 degradation and, at the same time, down-regulate its own expression. Presumably, the down-regulation of CML30 would reduce Ca²⁺-activated oxidative stress, providing a replication advantage for the virus (Liu et al., 2022).

There is some indirect evidence suggesting the importance of Ca²⁺ signaling in the antiviral response, but further research is needed to establish the exact interconnection and mechanisms involved. For example, the transgenic expression of soybean CaMs in tobacco plants led to enhanced resistance against TMV, with plants demonstrating fewer and smaller HR lesions that also appeared earlier (Heo et al., 1999). Interestingly, the TMV movement protein (MP) binds the Ca²⁺-binding ER chaperone calreticulin to accomplish cell-to-cell movement of the virus, and overexpression of calreticulin hinders normal interaction with MP and delays cell-to-cell movement (Fig. 1) (Chen et al., 2005). There is one more piece of evidence that the Ca²⁺ concentration in the cytoplasm is a regulator of plant defense against TMV. It has been shown that polysaccharides may act as elicitors of the plant immune system by triggering Ca²⁺ influx into the cytoplasm, which presumably regulates calreticulin activity as well as ROS and SA production (Menard et al., 2004; Yin et al., 2010; Zhao et al., 2018).

One important player in antiviral defense is the regulator of gene silencing-calmodulin-like protein (rgs-CaM), which acts as an activator of plant defense and is therefore often targeted by viruses. First, rgs-CaM was found to be involved in the development of both local and systemic acquired resistance against CMV in tobacco plants (Jeon et al., 2017). It binds to a viral RNA silencing suppressor and directs it to be degraded via autophagy. The authors also stated that the interaction of rgs-CaM with the viral suppressor of RNA silencing occurs concurrently with the activation of Ca²⁺ influx into the cytoplasm. This induces defense reactions such as SA signaling, ROS generation, and cell death. Thus, rgs-CaM acts as an immune receptor. Surprisingly, in the case of tobacco etch virus (TEV) infection, rgs-CaM performs a fundamentally different function: it interacts with the viral RNA silencing suppressor protein HC-Pro and itself acts as a silencing suppressor (Anandalakshmi et al., 2000). Nevertheless, the direct demonstration of a connection to Ca²⁺ signaling for this case has not been established, and it remains a matter of speculation whether HC-Pro stimulates an endogenous mechanism of Ca²⁺ regulation to suppress RNA silencing efficiently. A similar mode of RNA silencing suppression by rgs-CaM has been shown for tomato yellow leaf curl China virus (TYLCCNV) (Li et al., 2014). The authors demonstrated that the viral RNA silencing suppressor βC1 up-regulates the expression of rgs-CaM in N. benthamiana. This leads to the suppression of secondary siRNAs production, likely through down-regulation of the expression of RNA-dependent RNA polymerase 6. However, the mechanism of rgs-CaM up-regulation by βC1, and whether Ca²⁺ signaling is affected, remains unclear.

Another remarkable mechanism of CaM-dependent activation of antiviral RNA silencing and its suppression by a viral protein has been described by Wang et al. (2021). Insect-mediated leaf wounding triggers the influx of Ca²⁺ into the cell and activates the interaction of the CaM CAM3 with the calmodulin-binding transcription activator 3 (CAMTA3) (Fig. 1). Upon binding, CAMTA3 activates the transcription of RNA interference (RNAi) machinery components, therefore alleviating the viral infection. The protein V2 from cotton leaf curl Multan virus (CLCuMuV) is able to disrupt the CaM–CAMTA3 interaction, providing an advantage for the virus (Wang et al., 2021). All these examples support the foundation of a whole new perspective for the study of Ca²⁺-dependent regulation of RNAi.

Interestingly, some changes in transcriptional regulation happen due to the activation of transcription factors that are themselves Ca²⁺ sensitive. In animals, such a regulation may not only activate the expression of defense genes, but also have a counter-defense effect, promoting viral replication or establishing persistent infection (Zhou et al., 2009; Qu et al., 2022). The existence of such a type of transcriptional regulation has to be considered in plants as well.

Binding Ca²⁺ ions

Importantly, both animal (Zhou et al., 2009) and plant viruses use the strategy of binding Ca²⁺ ions in order to stabilize their capsids. Such data are available for the turnip crinkle virus (TCV) (Laakso and Heaton, 1993; Lin and Heaton, 1999), TMV (Pattanayek et al., 1992), tomato bushy stunt virus (Llauro et al., 2015), and sesbania mosaic virus (Gopinath et al., 1994). Mutation of the Ca²⁺-binding sites of TCV can affect cell-to-cell or long-distance movement and induce delayed mild systemic symptoms (Laakso and Heaton, 1993; Lin and Heaton, 1999). In animals, Ca²⁺ binding is also required for the optimal enzymatic activity and stability of some viral protein oligomers (Zhou et al., 2009). Whether plant viral proteins require Ca²⁺ binding for their activity remains unclear.

Organelle-specific regulation of Ca²⁺ signaling during viral infections

ER, mitochondria, and chloroplasts

Another mechanism leading to attenuation of the host immune response in animals is caused by the modification of protein-trafficking pathways due to decreased Ca²⁺ concentrations in the ER and Golgi complex. Interestingly, Ca²⁺ released from the ER can be taken up by mitochondria, resulting in the so-called ER–mitochondria Ca²⁺ flux (Fig. 1). In the mitochondria, Ca²⁺ can regulate ATP synthesis, to meet the increased energy demand in the infected cell (Zhou et al., 2009). However, the resulting excess of Ca²⁺ in the mitochondrial matrix causes the loss of mitochondrial potential and the release of cytochrome c, which in turn activates caspase 9 and leads to apoptosis. At the early and middle stages of infection, apoptosis serves as a host defense mechanism to decrease the number of infected cells and therefore restrict the infection. In order to counteract this type of defense and thereby promote viral replication, viruses employ proteins that suppress the ER–mitochondrial Ca²⁺ fluxes and, therefore, apoptosis. On the other hand, at the late stages of infection, apoptosis might be beneficial, to facilitate the release of the virions from infected cells. It has been shown that some viral proteins are able to alter the mitochondrial membrane potential. As a result, the Ca²⁺ concentration increases in the mitochondrial matrix, triggering apoptosis that might benefit the virus (Zhou et al., 2009; Qu et al., 2022).

We assume that in plants a similar mechanism might also involve the chloroplasts, which are one of the major Ca²⁺ depots in plant cells. Chloroplasts play an important role in the induction of cell death. It has been suggested that ROS production and the release of cytochrome f from chloroplasts may lead to apoptosis as a protective mechanism that limits viral spread (Van Aken and Van Breusegem, 2015). Chloroplasts are often targeted by viruses to promote viral reproduction. Accordingly, viral infections often affect chlorophyll fluorescence, photosystem efficiency, and photoassimilate accumulation; they alter the chloroplast ultrastructure as well as the expression of nuclear-encoded photosynthetic genes (Bhattacharyya and Chakraborty, 2018). Furthermore, direct binding of viral components to chloroplast proteins has been shown (Zhao et al., 2016). Moreover, chloroplasts provide the required energy for defense responses and are the source of ROS, SA and jasmonic acid, and defense compounds such as phenylpropanoids (Serrano et al., 2016; Bittner et al., 2022).

It has been reported that the C4 protein of tomato yellow leaf curl virus (TYLCV) is relocated from the PM to the chloroplast in the course of viral infection. There, C4 interacts with the chloroplast Ca²⁺ sensor (CAS) protein, suppressing CAS-dependent immune defense, including SA biosynthesis and the cytoplasmic flg22-triggered Ca²⁺ burst (Fig. 1). Interestingly, the Ca²⁺-dependent protein kinase CPK16 is relocated in a similar manner in the plant from the PM to the chloroplast to promote a chloroplast-mediated defense response (Medina-Puche et al., 2020). The localization of CPK16 to the PM is dependent on its N-terminal acylation, and suppressing only the N-terminal myristoylation was shown to be sufficient to lead to localization of CPK16 to the chloroplast (Stael et al., 2011). Thus, the mechanisms of correct subcellular targeting also present effective targets for plant viruses, as these would also impair the effective function of host proteins that regulate Ca²⁺-dependent immune responses in the chloroplast. Therefore, suppressing defense processes in chloroplasts might be a key target for viruses in order to establish a successful infection.

Plasmodesmata

An important aspect in plant immunity to viruses is the restriction of the size-exclusion limit of the plasmodesmata (PD). To facilitate cell-to-cell movement, viruses often aim to increase the size of the PD, for instance, by decreasing the amount of callose deposition in PD. This process is regulated by the activity of the PM nanodomain-associated proteins, the remorins. There is evidence that the recognition of potato virus X (PVX) CP and triple gene block 1 (TGB1) proteins trigger changes in the distribution of the remorin REM1.3 within the PM. This will increase the association of REM1.3 with PD, which leads to restriction of the movement of the virus from cell to cell (Perraki et al., 2018). Previously, a partial membrane localization, dependent on N-terminal myristoylation, was shown for the Ca²⁺-dependent protein kinase CPK3 and, among others, remorin was suggested as a potential target of CPK3 (Mehlmer et al., 2010). Perraki et al. (2018) demonstrated that REM1.3 is directly phosphorylated by CPK3 and proposed that this will increase its association with PD, which leads to restriction of the cell-to-cell movement of viruses. Similarly, the same researchers show further that the infection of Arabidopsis with plantago asiatica mosaic virus induced CPK3-dependent increase of REM1.2 diffusion in the PM, which affected the spread of infection (Jolivet et al., 2023). Notably, the accumulation of AtCPK3 transcripts is significantly increased in response to various viral infections (Valmonte-Cortes et al., 2022), further suggesting an important role of CPK3 in antiviral response regulation. Remorin-related regulation of cell-to-cell transport has also been shown for TuMV. The viral P3N-PIPO protein recruits the PM-associated Ca²⁺-binding host protein plasma membrane-associated cation-binding protein 1 (pCaP1) to PD. There, pCaP1 is able to depolymerize actin filaments, thereby increasing the size of PD and facilitating viral cell-to-cell movement. The remorin REM1.2 competes with pCaP1 for binding actin filaments and, therefore, restricting cell-to-cell movement. To counteract this host defense mechanism and to establish systemic infection, another TuMV protein, VPg, binds to remorin, which leads to proteasome-mediated degradation of remorin, giving an advantage to the virus (Cheng et al., 2020). Nonetheless, the definitive association between remorin-dependent regulation of PD functions and Ca²⁺ signaling remains not fully confirmed in the described studies and requires further investigation.

Outlook

In animal cells, viral proteins are able to modulate Ca²⁺ signaling to modify energy turnover, the transcriptional regulation of defense genes, and vesicle trafficking in order to promote viral replication. Furthermore, these viruses can establish latent infections to evade host defenses, to direct the infected cell into pro-survival pathways in order to inhibit the host innate immune response or, at a later stage of infection, into apoptosis to facilitate the release of new virions and further spread the infection. Interestingly, often one viral protein can affect different stages of Ca²⁺ regulation, steering it in a pro- or counter-defense direction. At the same time, the role of Ca²⁺ signaling during the plant–virus interaction is an emerging topic, which might become useful for developing new plant protection strategies. In this review, we summarized that, similar to animal viruses, plant viruses make use of their proteins to target important components of Ca²⁺ signaling in order to counteract plant defenses. As follows from Table 1, often the viral RNAi suppressor proteins have to interact with the Ca²⁺-regulated host proteins. Presumably, the hijacking of host Ca²⁺ signaling by the viral proteins has great importance for establishing efficient suppression of the major antiviral mechanism, RNAi. The detailed mechanisms for this process still have to be studied in more detail, but work carried out to date has already opened a new horizon for investigating the role of Ca²⁺signaling in developing other types of antiviral immune responses.

Table 1.

Regulation of the Ca²⁺-dependent defense response by plant viruses

Virus	Viral Protein	Host interacting/affected protein	Effect on host immunity		Reference
			Activation	Suppression
TMV	CP	CML30 via CP-IP-L		CML30 down-regulation Suppression of oxidative stress	Liu et al. (2022)
	MP	Calreticulin		Facilitation of cell-to-cell movement	Chen et al. (2005)
CMV	2b	rgs-CaM	Degradation of 2b by autophagy		Jeon et al. (2017)
TEV	HC-Pro			Suppression of RNAi	Anandalakshmi et al. (2000)
TYLCCNV	βC1			Suppression of RNAi	(Li et al., 2014)
CLCuMuV TYLCCNV	V2	CaM–CAMTA3		Disruption of CaM–CAMTA3 interaction, suppression of RNAi	Wang et al. (2021)
TYLCV	C4	CAS		Suppression of SA biosynthesis and flg22-triggered Ca2+ burst	Medina-Puche et al. (2020)
PVX	CP, TGB1	CPK3 Remorin 1.3	Restriction of cell-to-cell movement		Perraki et al. (2018)
TuMV	P3N-PIPO	pCaP1		Facilitation of cell-to-cell movement	Cheng et al. (2020)
	VPg	Remorin 1.2 Binding to pCaP1 restricts cell-to-cell movement		Proteasome-mediated degradation of remorin

It will be important to show whether any of the viral proteins directly bind to Ca²⁺ channels or pumps, as well as to find new mechanisms that regulate the activity of the intracellular Ca²⁺ sensors and therefore their downstream targets, for example, a potential phosphorylation of Ca²⁺ channels. Most of the Ca²⁺ channels that regulate plant immunity against bacteria and fungi are located on the PM and play a role in Ca²⁺ release from the apoplast (Xu et al., 2022). However, unlike bacteria and fungi, and unlike animal viruses, most plant viruses are directly injected into the cell cytoplasm by their insect vectors. Therefore, it is more likely that they would need to interact with the intracellular Ca²⁺ sensors, as well as to regulate Ca²⁺ signaling inside the cell and across the organelles. Aside from the apoplast, plants have two other main Ca²⁺ storage pools: the central vacuole and, to a lesser extent, the chloroplasts (Stael et al., 2012). Special attention must be paid to investigating whether Ca²⁺ signaling across these storage pools might contribute to antiviral defense (Xu et al., 2022). Another important aspect to be studied is the existence of Ca²⁺-mediated signaling between organelles, similar to the ER–mitochondria Ca²⁺ flux in animal cells. The advance in genetically encoded calcium indicators (GECIs) and the development of high-resolution imaging systems will allow us to obtain more important information concerning the functions of Ca²⁺ signaling in antiviral immunity (Resentini et al., 2021).

Thus, in all eukaryotes Ca²⁺ signaling regulates both the activation and the suppression of immune responses to various pathogens. We believe that revealing the novel mechanisms of such regulation might become valuable for the development of future antiviral strategies in crops. For instance, the use of chemicals that specifically suppress certain Ca²⁺channels might be considered as a potential antiviral treatment in plants.

Glossary

Abbreviations

ACA

autoinhibited Ca²⁺-ATPase

BICAT 1, 2

bivalent cation transporter 1 and 2

CAMTA3

calmodulin-binding transcription activator-3

CaM

calmodulin

CAS

chloroplast Ca²⁺ sensor protein

CAX

Ca²⁺ ATPases and Ca²⁺/H⁺ exchanger

CBL

calcineurin B-like protein

CLCuMuV

cotton leaf curl Multan virus

cMCU

chloroplast-localized mitochondrial Ca²⁺ uniporter

CML

calmodulin-like protein

CMV

cucumber mosaic virus

CNGC

cyclic nucleotide-gated channel

CPK

calcium-dependent protein kinase

ECA

ER-type Ca^2+-ATPase

GLR

glutamate receptor-like Ca²⁺ channel

HCX

H⁺/Ca²⁺ exchanger

IP-L

CP-interacting protein L

IP3R

inositol-1,4,5-triphosphate receptor

MCA

Mid1-complementing activity channel

MCU

mitochondrial Ca²⁺ uniporter

MP

movement protein

MSL

mechanosensitive-like channel

NCX

Na⁺/Ca²⁺ exchanger

NLR

nucleotide-binding leucine-rich repeat receptor

OSCA

reduced hyperosmolarity-induced cytosolic [Ca²⁺] increase channel

pCaP1

plasma membrane-associated cation-binding protein 1

PEC 1/2

POLLUX plastid envelope ion channel 1 and 2

PM

plasma membrane

PMCA

plasma membrane Ca²⁺-ATPase

PVX

potato virus X

rgs-CaM

regulator of gene silencing-calmodulin-like protein

RNAi

RNA interference

ROS

reactive oxygen species

RyR

ryanodine receptor

SA

salicylic acid

SERCA

sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase

TCV

turnip crinkle virus

TEV

tobacco etch virus

TGB1

triple gene block 1 protein

TMV

tobacco mosaic virus

TPC

two-pore channel

TuMV

turnip mosaic virus

TYLCCNV

tomato yellow leaf curl China virus

TYLCV

tomato yellow leaf curl virus

VDAC

voltage-dependent anion channel

VGCC

voltage-gated calcium channel.

Author contributions

ASZ: conceptualization, writing – original draft, writing – review & editing, visualization; MK: writing – review & editing, visualization; MT: conceptualization, writing – review & editing.

Conflict of interest

The authors declare no conflict of interest.

Funding

This work is supported by The Austrian Science Fund (FWF) under the Lise Meitner Program (grant number M-2745-B) to ASZ and the EU Horizon 2020 research and innovation project ADAPT (GA 2020 862-858) to MT.