Introduction

Plants have evolved in spatial proximity to a multitude of different microorganisms in their surroundings. Their constant interaction with commensal, symbiotic, and pathogenic microorganisms shaped highly specialized ecosystems in which plants find their niches and flourish. Since the initial description of the concept of mutual coexistence between dissimilar organisms (de Bary, 1879; Hertig et al., 1937), our knowledge regarding the symbiotic associations of plants with microorganisms has substantially advanced. Numerous studies unambiguously demonstrate that plant-microbe interactions are important to the structure, function, and health of plant communities, and that symbiotic fungi contribute to the adaptations of plants to environmental stresses (Rodriguez et al., 2004).
Serendipita indica (formerly termed Piriformospora indica ) is an axenically cultivable root colonizing plant endophyte of the order Sebacinales with an exceptionally broad host range (Verma et al., 1998; Weiss et al., 2016; Mensah et al., 2020). S. indica promotes plant performance and biomass production (Varma et al., 1999; Peškan-Berghöfer et al., 2004; Vadassery et al., 2009), enhances nutrient assimilation (Bakshi et al., 2017; Prasad et al., 2018), and confers increased biotic and abiotic stress tolerance to its host plants (Waller et al., 2005; Sun et al., 2014; Jogawat et al., 2016). The establishment of the symbiosis between S. indica andArabidopsis thaliana , and the therewith coupled plant growth promotion, involves the perception of conserved microbial components by the plant, generally termed microbial-associated molecular patterns (MAMPs), through specific pattern-recognition receptor proteins. Pattern recognition, in turn, provokes a multitude of downstream events, including MAMP-triggered immunity (Millet et al., 2010) and the induction of early plant defence responses, which comprise the deposition of callose and the production of defence-relates secondary metabolites, e.g. phytoalexins, glucosinolates, and camalexin (Jacobs et al., 2011; Lahrmann et al., 2015). At later stages of the infection, the controlled reduction of plant defence responses becomes paramount to facilitate the establishment of the mutual interaction between the endophyte and its host plant. In this respect, balancing of plant hormone contents and the tight control of indole glucosinolates are reported to play essential roles (Nongbri et al., 2012; Lahrmann et al., 2015; Xu et al., 2018).
The elevation of cytosolic Ca2+ concentrations in Arabidopsis root cells through the influx of Ca2+ via the CYCLIC NUCLEOTIDE GATED CHANNEL 19 (CNGC19) represents a further critical asset in consolidating the plant-fungus interaction (Vadassery et al., 2009; Jogawat et al., 2020). Calcium is an essential plant macronutrient that plays an important role in plant growth and development. At the same time, Ca2+ serves as an important second messenger in plants that is involved in orchestrating adequate responses to external signals, including biotic stresses (Thor, 2019). Cytosolic Ca2+ concentrations show highly dynamic and specific spatiotemporal patterns, which are governed by the type and intensity of the perceived stimulus (Pivato and Ballottari, 2021). Depending on the particular stimulus, plant cells respond by producing specific Ca2+ signatures that differ in their frequency, amplitude, and duration (Batistič and Kudla, 2012). To decipher the different Ca2+ signatures, plants possess a broad set of different receptor molecules that either directly modify target proteins through phosphorylation or act through their physical interaction with specific partner proteins, including protein kinases (Kudla et al., 2018). This diverse set of Ca2+ sensor molecules encompasses Calmodulins (CaMs), Calmodulin-like proteins (CMLs), Ca2+-dependent protein kinases (CDPKs), Calcineurin B-like proteins (CBLs), as well as their interacting kinases (CIPKs). The latter forming a two-component system in which the CBLs act as Ca2+ sensors that relay their stimulation to specific CIPKs, which subsequently interact with downstream target proteins, such as nutrient transporters or ion channels (Liu and Tsay, 2003; Maierhofer et al., 2014; Ragel et al., 2015). In particular, the regulation of the high affinity potassium transporter HAK5 and the highly selective potassium channel AKT1 by CIPK23 is well documented (Lee et al., 2007; Lan et al., 2011; Ragel et al., 2015). However, there is also evidence for an interaction of CIPKs with diverse components of the abscisic acid response pathway, including the co-repressors ABI1 and ABI2 (Ohta et al., 2003). The Arabidopsis genome contains 10 CBLand 26 CIPK genes. Among each other, the CBLs and CIPKs form functional complexes and it is noteworthy that CBLs are not promiscuously interacting with all CIPKs, but show preferences and interact only with a specific subset of CIPKs to facilitate efficient signal transduction and integration (Kudla et al., 2010). Overall, the specific combination of CBL-CIPK modules is the key to provide versatility and flexibility in the regulation of a multitude of external stimuli that marshal ion transport in plants (Tong et al., 2021).
Up to date, the functional role of CBL7 is only partially elucidated. A recent study associates CBL7 with the regulation of plant responses towards low nitrate in Arabidopsis (Ma et al., 2015). The work highlights a substantial expression of CBL7 in root tissues and its induction under nitrogen and nitrate limiting conditions. Moreover, the authors report on the involvement of CBL7 in the transcriptional regulation of two high-affinity nitrate transporter genes, NRT2.4and NRT2.5 , without providing evidence for a molecular mechanism that could explain how the downstream genes are targeted. It is speculated that the localization of CBL7 to the nucleus facilitates its interaction with nitrate-starvation response-related transcription factors, such as NLP7 (Konishi and Yanagisawa, 2013; Marchive et al., 2013; Kiba and Krapp, 2016; Krouk and Kiba, 2020). The experimentally evidenced localization of CBL7 to the nucleus and cytoplasm (Batistič et al., 2010) is, however, inconsistent with the correlation of CBL7 with the plasma membrane localized H+-ATPase AHA2 (Yang et al., 2019). According to this study, CBL7 inactivates AHA2 under normal conditions through the formation of a larger CBL7-CIPK11-AHA2 complex. A direct interaction between CBL7 and CIPK11 is, however, likely to be excluded, as a previous study discarded the interaction of those proteins (Fuglsang et al., 2007). In contrary, this study points towards a possible recruitment of CBL2 to the complex, because CBL2 is the only CBL that showed physical interaction with CIPK11 in the study. Under certain stress conditions, the complex dissolves and AHA2 is released from repression, which consequently translates into the efflux of H+ from the cytoplasm.
In this study, we identified the Ca2+ sensor CBL7 as an essential molecular component in the interaction between the root colonizing endophyte S. indica and A. thaliana .CBL7 is consistently induced upon root infection with the fungus, not only at early stages of the infection, but also at later phases. Furthermore, we were able to demonstrate that the availability of CBL7 is crucial for the development of the fungus-mediated plant growth promotion and for the proper distribution of potassium in the plant. The comprehensive transcriptomics analysis of the cbl7 mutant grown with and without the fungus in comparison to corresponding wild-type plants additionally pinpointed a role of CBL7 in harmonizing plant defense responses for the long-term interaction between the endophyte and Arabidopsis. Taken together, our results establish CBL7 as a novel key component required for the successful establishment of the symbiosis between S. indica and A. thaliana .