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 .