Calcium signaling in plant-microbe interaction

Introduction

Calcium ion (Ca²⁺) is an important second messenger involved in many signaling pathways in plants. The concentration of free Ca²⁺ in the cytosol in a plant cell ([Ca²⁺]cyt) connects the extracellular stimuli, including the signal of microbes, to intracellular responses. Since Ca²⁺ is neither synthesized nor degraded by plants, [Ca²⁺]_cyt is completely dependent on the entry of external source or release of Ca²⁺ from its intracellular stores. However, because Ca²⁺ can react with phosphate, the energy source of life, its presence in the cytoplasm will prevent energy metabolism and other cellular activities from taking place. Thus, its concentration is regulated tightly by various proteins. In plant-microbe interaction, different microbes trigger different receptor proteins, causing distinctive Ca²⁺ elevation patterns, referred to as Ca²⁺ signature. Ca²⁺ signature can be different from each other in various aspects: amplitude, duration, frequency, spatial distribution, and times of cycle in [Ca²⁺]_cyt changes. The Ca²⁺ signature produced by microbe signal can be decoded by downstream effectors, changing the expression of defense or symbiosis-related genes, resulting in different responses by plants.

Detection of Microbes

Figure 1. PRRs capable of recognizing MAMPs of bacteria. Featured in Activation of plant pattern-recognition receptors by bacteria (Segonzac et al. 2011).[1].

The first step of a Ca²⁺ signaling event is the detection of microbes performed by pattern-recognition receptors (PRRs), a type of receptor protein located on the plasma membrane of a plant cell. PRRs are capable of recognizing microbe-associated molecular patterns (MAMPs), molecules specific to certain classes of microbes that are present in extracellular space.

Bacteria

All PRRs that can recognize MAMPs of bacteria studied so far are either receptor-like kinase or receptor-like protein. All of them are transmembrane receptors. One example of these PRRs is flagellin-sensitive 2 (FLS2), which recognizes the flagellin protein in bacteria flagellum. MAMPs of symbiotic bacteria can also be recognized by PPRs. A lysin-motif (LysM) receptor-like kinase, nodulation (nod) factor perception (NFP), can recognize lipochitooligosaccharide in the nod factor released by symbiotic bacteria such as rhizobia.

Fungi

An example of PRRs capable of recognizing fungi is the LysM-receptor kinase 5 and chitin elicitor receptor kinase 1 receptor complex, which recognizes chitin in the cell wall of fungi. Lysin-motif (LysM) receptor-like kinases also recognize myc factors released by symbiotic fungi such as mycorrhizal fungi.

Formation of Ca²⁺ Signature

After a PRR detect microbes, it triggers an influx of Ca²⁺ ion from outside the cell, resulting in a unique Ca²⁺ signature. The activated PRRs have two mechanisms: it can either trigger the entry of Ca²⁺ by itself or by activating a downstream receptor. For the first type of mechanism, there are two possibilities. The first possibility is that it can form a complex with the Ca²⁺ ion channels on the membrane physically and regulate the activity of the channels. The second possibility is to produce a signaling molecule by itself to bind to Ca²⁺ permeable channels or Ca²⁺ pumps and activate them. The other mechanism is to activate a downstream receptor-like cytoplasmic kinase (RLCK). Many of the PRRs activate BIK1, a RLCK which activate a NADPH oxidase called RBOHD by phosphorylating it. Activated RBOHD produce reactive oxygen species (ROS) such as H₂O₂, and the ROS interact with Ca²⁺ channels and cause an influx of extracellular Ca²⁺, although the precise mechanism is not known. The increase in [Ca²⁺]_cyt can then trigger the release of Ca²⁺ from internal sources, further increasing [Ca²⁺]cyt.

When PRR detects the presence of nod factors or myc factors, Ca²⁺ signaling in both cytoplasm and nucleus can happen. The influx of Ca²⁺ into nucleus is mediated by both Ca²⁺ channeling proteins and ATP-powered Ca²⁺ pump located on the nucleus membrane, and the influx of Ca²⁺ into cytoplasm is likely to be caused by the interaction of ROS with channel proteins.

Decoding of Ca²⁺ Signature

Figure 2. Ca²⁺-modified CaM bounding to other proteins and activating them, an important step in the decoding of Ca²⁺ signal. Featured in Calcium Signaling Mechanisms Across Kingdoms (Luan et al. 2021).[2].

As [Ca²⁺]_cyt change, the Ca²⁺ signature is decoded by Ca²⁺ sensor proteins. There are various types of sensor proteins in plants that can bind to Ca²⁺ ions, and they respond to the rise in [Ca²⁺]_cyt in two different ways. The first possible type are Calmodulin (CaM) and CaM-like proteins (CMLs). Binding to Ca²⁺ changes their structures and enable them to bind to their targets, calcineurin b-like proteins (CBLs). CBLs then interact with CBL-interacting protein kinases (CIPKs), enzymes that are capable of phosphorylation. The second possible way a sensor can decode the Ca²⁺ signature is that the sensor has a kinase domain of its own and can phosphorylate other molecules directly. An example of this type of sensor is Ca²⁺-dependent protein kinases (CDPKs or CPKs).

This can cause changes at both transcriptional and post-translational levels. At post-translational level, the phosphorylation events by CIPKs or CPKs activate RBOHD in the cell membrane and lead to a further increase in the level of ROS production. The ROS then further promotes the influx of Ca²⁺, forming a positive-feedback loop. CCaMK, a sensor protein involved in decoding the Ca²⁺ signal at post-translational level, is involved in plant’s response to symbiotic signal. It is found in a complex that activates a transcription factor called RAM1, which regulates arbuscular branching, a process in the symbiosis of plant with arbuscular mycorrhizae. At transcriptional level, Ca²⁺-modified CaM can bind to transcription factors that promote the transcription of genes related to plant immunity, for example, the gene for synthesis of salicylic acid in plants, causing the whole plant to be ready for pathogen attack. However, more research is needed to understand how the transcription factors lead to plants’ defense against pathogens.

Ca²⁺ signal as a negative regulator

Although Ca²⁺ have been evolved to be utilized as a ubiquitous secondary messenger in plants, it is, by its nature, still a toxin, and long-term exposure to high Ca²⁺ level can eventually lead to harm to plant cells, even cell death. So, Ca²⁺ ions’ presence in the cytoplasm needs to be removed after the signaling event. In fact, Ca²⁺ signal can also act as a negative regulator that down regulates the influx of Ca²⁺. It can down regulate it from two processes: formation of Ca²⁺ signature and decoding of Ca²⁺ signature.

In the formation of Ca²⁺ signature, some CPKs can bind to BIK1, phosphorylate it, thus promoting its degradation. Because BIK1 is directly downstream of many of the PRRs and that it promotes ROS production, the degradation of BIK1 basically stops ROS-dependent defense signaling in plants. What’s more, some Ca²⁺-modified CaM can bind to Ca²⁺ channel proteins, for example, Cyclic nucleotide-gated ion channels (CNGCs), leading to its inactivation, preventing the Ca²⁺ influx and preventing the Ca²⁺ signature from forming.

In the decoding of Ca²⁺ signature, some transcription factors bound by Ca²⁺-modified CaM actually suppress the expression of defense related genes of plants, for example, CaM-binding transcriptional factor 3 (CaMTA3) suppress the expression of both EDS1 and NDR1, proteins required for the activation of plant defenses, by binding to their promotors.

Conclusion

Ca²⁺ signaling plays an important role in the plant-microbe interaction and plant immunity. PRRs are capable of detecting different classes of microbes, leading to a series of phosphorylation events by themselves or downstream effectors, causing the influx of Ca²⁺, and generating the Ca²⁺ signature. The Ca²⁺ signature is then decoded at both post-translational and transcriptional level. It also negatively regulates the influx of Ca²⁺. However, little is known about how different patterns of Ca²⁺ signature correlate with external stimuli and how they lead to effective immune responses to pathogen infection, and there are still plenty of areas for further research.

References

Edited by Yueqi Song, student of Joan Slonczewski for BIOL 116 Information in Living Systems, 2021, Kenyon College.