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\begin{document}

\title{The calcium sensor CBL7 is required for Serendipita indica -induced
growth stimulation in Arabidopsis thaliana, controlling defense against
the endophyte and K + homeostasis in the symbiosis}



\author[1]{Marta-Marina Pérez-Alonso}%
\author[1]{Carmen Guerrero-Galán}%
\author[1]{Adrián González Ortega-Villaizán}%
\author[1]{Paloma Ortiz-García}%
\author[2]{Sandra Scholz}%
\author[3]{Hitoshi Sakakibara}%
\author[3]{Takatoshi Kiba}%
\author[4]{Jutta Ludwig-Müller}%
\author[5]{Anne Krapp}%
\author[2]{Ralf  Oelmüller}%
\author[1]{Jesús Vicente-Carbajosa}%
\author[1]{Stephan Pollmann}%
\author[6]{Patricio Ramos}%
\affil[1]{Centro de Biotecnologia y Genomica de Plantas}%
\affil[2]{Friedrich Schiller Universitat Jena Institut fur Allgemeine Botanik und Pflanzenphysiologie}%
\affil[3]{Rikagaku Kenkyujo Kankyo Shigen Kagaku Kenkyu Center}%
\affil[4]{Technische Universitat Dresden Institut fur Botanik}%
\affil[5]{Institut Jean-Pierre Bourgin}%
\affil[6]{Universidad de Talca}%


\vspace{-1em}



  
  \date{June 14, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Calcium (Ca \textsuperscript{2+}) is an important second messenger in
plants. The activation of Ca \textsuperscript{2+} signaling cascades is
critical in the activation of adaptive processes in response to
perceived environmental stimuli, including biotic stresses. The
colonization of roots by the plant growth promoting endophyte
\emph{Serendipita~indica} involves the increase of cytosolic Ca
\textsuperscript{2+} levels in \emph{Arabidopsis thaliana}. In this
study, we investigated transcriptional changes in Arabidopsis roots
during symbiosis with \emph{S.~indica}. RNA-seq profiling disclosed the
significant induction of \emph{CALCINEURIN~B-LIKE~7} ( \emph{CBL7})
during early- and later phases of the interaction. Consistent with the
transcriptomics analysis, reverse genetic evidence and yeast two-hybrid
studies highlighted the functional relevance of CBL7 and tested the
involvement of a CBL7-CBL-INTERACTING PROTEIN KINASE~13 (CIPK13)
signaling pathway in the establishment of the mutualistic relationship
that promotes plant growth. The loss-of-function of \emph{CBL7}
abolished the growth promoting effect of \emph{S.~indica} and affected
the colonization of the root by the fungus. The subsequent
transcriptomics analysis of \emph{cbl7} revealed the involvement of this
Ca \textsuperscript{2+} sensor in activating plant defense responses.
Furthermore, we report on the contribution of CBL7 to potassium
transport in Arabidopsis. Triggered by the differential expression of a
small number of K \textsuperscript{+} channels/transporter genes, we
analyzed K \textsuperscript{+} contents in wild-type and \emph{cbl7}
plants and observed a significant accumulation of K \textsuperscript{+}
in root of \emph{cbl7} plants, while shoot tissues demonstrated K
\textsuperscript{+} depletion. Taken together, our work associates CBL7
with an important role in the mutual interaction between Arabidopsis and
\emph{S.~indica} and links the CBL7 Ca \textsuperscript{2+} receptor
protein to K \textsuperscript{+} transport.%
\end{abstract}%



\sloppy


\textbf{Running title: CBL7 contributes to the symbiosis between
Arabidopsis and \emph{S.~indica}}

\textbf{Authors:}

Marta-Marina P\selectlanguage{ngerman}érez-Alonso\textsuperscript{1,2}, Carmen
Guerrero-Galán\textsuperscript{1}, Adrián González
Ortega-Villaizán\textsuperscript{1}, Paloma
Ortiz-García\textsuperscript{1}, Sandra S. Scholz\textsuperscript{3},
Patricio Ramos\textsuperscript{4}, Hitoshi
Sakakibara\textsuperscript{5,6}, Takatoshi Kiba\textsuperscript{5,6},
Jutta Ludwig-Müller\textsuperscript{7}, Anne Krapp\textsuperscript{8},
Ralf Oelmüller\textsuperscript{3}, Jesús
Vicente-Carbajosa\textsuperscript{1,9} and Stephan
Pollmann\textsuperscript{1,9*}

\textsuperscript{1}Centro de Biotecnología y Genómica de Plantas,
Universidad Politécnica de Madrid (UPM)--Instituto Nacional de
Investigación y Tecnología Agraria y Alimentación (INIA), Campus de
Montegancedo, 28223 Pozuelo de Alarcón (Madrid), Spain

\textsuperscript{2}Umeå Plant Science Center, Umeå University, 90736
Umeå, Sweden

\textsuperscript{3}Matthias Schleiden Institute of Genetics,
Bioinformatics and Molecular Botany, Department of Plant Physiology,
Friedrich-Schiller-University Jena, 07743 Jena, Germany

\textsuperscript{4}Centro de Investigación de Estudios Avanzados del
Maule, Universidad Católica del Maule, 3460000 Talca, Chile

\textsuperscript{5}RIKEN Center for Sustainable Resource Science,
1-7-22, Suehiro, Tsurumi, Yokohama, 230-0045, Japan

\textsuperscript{6}Department of Applied Biosciences, Graduate School of
Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601, Japan

\textsuperscript{7}Institute of Botany, Technische Universität Dresden,
01062 Dresden, Germany

\textsuperscript{8}Université Paris-Saclay, INRAE, AgroParisTech,
Institut Jean-Pierre Bourgin, 78000 Versailles, France

\textsuperscript{9}Departamento de Biotecnología-Biología Vegetal,
Escuela Técnica Superior de Ingeniería Agronómica, Alimentaria y de
Biosistemas, Universidad Politécnica de Madrid (UPM), 28040 Madrid,
Spain

\textbf{* Correspondence:} Stephan Pollmann stephan.pollmann@upm.es

Keywords: \emph{Arabidopsis~thaliana} , Ca\textsuperscript{2+}
signaling, Endosymbiosis, Growth promotion, Plant defense, Plant-microbe
interaction, Plant performance, \emph{Serendipita~indica}

Abstract

Calcium (Ca\textsuperscript{2+}) is an important second messenger in
plants. The activation of Ca\textsuperscript{2+} signaling cascades is
critical in the activation of adaptive processes in response to
perceived environmental stimuli, including biotic stresses. The
colonization of roots by the plant growth promoting
endophyte\emph{Serendipita~indica} involves the increase of cytosolic
Ca\textsuperscript{2+} levels in \emph{Arabidopsis thaliana} . In this
study, we investigated transcriptional changes in Arabidopsis roots
during symbiosis with \emph{S.~indica} . RNA-seq profiling disclosed the
significant induction of \emph{CALCINEURIN~B-LIKE~7} (\emph{CBL7} )
during early- and later phases of the interaction. Consistent with the
transcriptomics analysis, reverse genetic evidence and yeast two-hybrid
studies highlighted the functional relevance of CBL7 and tested the
involvement of a CBL7-CBL-INTERACTING PROTEIN KINASE~13 (CIPK13)
signaling pathway in the establishment of the mutualistic relationship
that promotes plant growth. The loss-of-function of \emph{CBL7}abolished
the growth promoting effect of \emph{S.~indica} and affected the
colonization of the root by the fungus. The subsequent transcriptomics
analysis of \emph{cbl7} revealed the involvement of this
Ca\textsuperscript{2+} sensor in activating plant defense responses.
Furthermore, we report on the contribution of CBL7 to potassium
transport in Arabidopsis. Triggered by the differential expression of a
small number of K\textsuperscript{+} channels/transporter genes, we
analyzed K\textsuperscript{+} contents in wild-type and
\emph{cbl7}plants and observed a significant accumulation of
K\textsuperscript{+}in root of \emph{cbl7} plants, while shoot tissues
demonstrated K\textsuperscript{+} depletion. Taken together, our work
associates CBL7 with an important role in the mutual interaction between
Arabidopsis and\emph{S.~indica} and links the CBL7
Ca\textsuperscript{2+} receptor protein to K\textsuperscript{+}
transport.

\textbf{Significance statement:} The induction of \emph{CBL7} is
critical for the establishment of the mutual interaction
between\emph{Arabidopsis thaliana} and its root colonizing
endophyte\emph{Serendipita indica} . Moreover, CBL7 plays a vital role
in K\textsuperscript{+} distribution during the plant-fungus
interaction.

\section*{Introduction}

{\label{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).

\emph{Serendipita~indica} (formerly termed \emph{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). \emph{S.~indica}
promotes plant performance and biomass production (Varma et al., 1999;
Pe\selectlanguage{polish}š\selectlanguage{english}kan-Bergh\selectlanguage{ngerman}öfer 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 \emph{S.~indica}
and\emph{Arabidopsis 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 Ca\textsuperscript{2+} concentrations in
Arabidopsis root cells through the influx of Ca\textsuperscript{2+} 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, Ca\textsuperscript{2+} serves as an
important second messenger in plants that is involved in orchestrating
adequate responses to external signals, including biotic stresses (Thor,
2019). Cytosolic Ca\textsuperscript{2+} 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 Ca\textsuperscript{2+} signatures that differ in
their frequency, amplitude, and duration (Batisti\selectlanguage{polish}č \selectlanguage{english}and Kudla, 2012). To
decipher the different Ca\textsuperscript{2+} 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 Ca\textsuperscript{2+} sensor
molecules encompasses Calmodulins (CaMs), Calmodulin-like proteins
(CMLs), Ca\textsuperscript{2+}-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 Ca\textsuperscript{2+} 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
\emph{CBL}and 26 \emph{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 \emph{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,
\emph{NRT2.4}and \emph{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 (Batisti\selectlanguage{polish}č \selectlanguage{english}et al., 2010) is, however, inconsistent with the
correlation of CBL7 with the plasma membrane localized
H\textsuperscript{+}-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\textsuperscript{+} from the cytoplasm.

In this study, we identified the Ca\textsuperscript{2+} sensor CBL7 as
an essential molecular component in the interaction between the root
colonizing endophyte \emph{S.~indica} and \emph{A.~thaliana}
.\emph{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 \emph{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 \emph{S.~indica} and \emph{A.~thaliana} .

\section*{Materials and methods}

{\label{materials-and-methods}}

\subsection*{2.1. Biological material and growth
conditions}

{\label{biological-material-and-growth-conditions}}

In this work, we used the \emph{Arabidopsis~thaliana} Col-0 (N1092) and
Col-3 (N28171) backgrounds as references. We obtained the mutant
alleles\emph{cipk13-1} (SALK\_124748C) and \emph{cipk13-2}
(SALK\_130671), as well as \emph{cbl7-1} (SAIL\_201\_A01) from the
Nottingham Arabidopsis Stock Center (NASC). T-DNA insertion lines were
genotyped as previously described (Alonso et al., 2003), using the
primer pairs listed in\textbf{Supplemental Data Sheet 1} . The T-DNA
insertion mutants\emph{cbl7-2} and \emph{hak5} have previously been
described (Ma et al., 2015; Ragel et al., 2015). Plants were grown on
Petri dishes containing solidified \selectlanguage{ngerman}½ MS medium supplemented with 1\%
sucrose (w/v) (Murashige and Skoog, 1962). Plant growth proceeded in a
growth chamber under strictly controlled environmental conditions (16~h
light, 8~h darkness, constant temperature of 22~ºC,
100~to~105~\selectlanguage{greek}µ\selectlanguage{english}mol~photons~m\textsuperscript{-2}~s\textsuperscript{-1}photosynthetically
active radiation). In addition, we used\emph{Serendipita~indica} strain
DSM 11827, which was obtained from the German Collection of
Microorganisms and Cell Cultures (DSZM) in Braunschweig, Germany. The
fungus was grown at 28~\selectlanguage{ngerman}ºC in darkness on solidified arginine phosphate
(AP) medium (Rodríguez-Navarro and Ramos, 1984) and refreshed weekly.

\subsection*{2.2. Root growth promotion
assay}

{\label{root-growth-promotion-assay}}

Surface-sterilized Arabidopsis seeds were plated on vertical ½ MS
plates. After stratification (2 days at 4~ºC), the plates were
transferred to the growth chamber and the seedlings were grown
vertically for one week. Thereafter, four to five seedlings were
transferred to Petri dishes containing solidified Plant Nutrition Medium
(PNM) supplemented with 50~mM NaCl (Johnson et al., 2013). Each seedling
was then associated with a 5~mm Ø medium plug extracted from either
sterile AP plates (control) or from AP plates harboring a
one-week-old\emph{S.~indica} mycelium (co-cultivation). The PMN plates
with the control seedlings and the seedlings co-cultivated with the
fungus were further kept in a growth chamber maintained at 23.5~ºC,
16/8~h photoperiod, 100
\selectlanguage{greek}µ\selectlanguage{english}mol~photons~m\textsuperscript{-2}~s\textsuperscript{-1} light intensity
for another fourteen days. After that time, the plants were photographed
for the further analysis of the root system and the plant material was
either used for RNA extraction or the determination of the fresh weight
(fw).

\subsection*{2.3. Quantitative analysis of root system architecture
traits}

{\label{quantitative-analysis-of-root-system-architecture-traits}}

The stimulation of root growth is a well-described trait in the
interaction of \emph{S.~indica} with its host plant. With the aim to
quantify the effect of \emph{S.~indica} in the different genotypes and
treatments, respectively, photographs of the plates were captured with a
digital camera at a fixed distance of 29~cm. Using Adobe Photoshop CC,
the images were cropped to a height of 14~cm maintaining only the part
containing the root system and converted the pictures to black and white
images. The root network traits of the plants in the prepared images
were then analyzed using the GiA Roots software (Galkovskyi et al.,
2012). Further processing of the images included their segmentation
employing global thresholding (Binary\_inverted) and Gaussian adaptive
thresholding. For the comparative analysis of alterations of the root
system architecture, the total network area and total network length was
used as readout. Taking the biological variability of the system into
account, 24 individual plants per genotype and growth condition were
analyzed, respectively.

\subsection*{2.4. Total RNA extraction, library construction, and RNA-seq
analysis}

{\label{total-rna-extraction-library-construction-and-rna-seq-analysis}}

To study transcriptional alterations provoked by either the
co-cultivation of Arabidopsis roots with \emph{S.~indica} or the
functional knockout of \emph{CBL7} , total RNA from 100~mg plant roots
was extracted as previously described (O\selectlanguage{ngerman}ñate-Sánchez and
Vicente-Carbajosa, 2008). The quality and concentration of the extracted
RNA was tested by absorbance analysis using a Nanodrop® ND-1000
spectrophotometer (ThermoFisher). After an additional confirmation of
the RNA sample integrity on a Bioanalyzer 2100 (Agilent) by the Novogene
Genomics Service (Cambridge, UK), the service laboratory proceeded with
the library construction and RNA sequencing (PE150) on Illumina NovaSeq\selectlanguage{english}
6000 platforms. The Novogene Genomics Service also provided basic data
analysis applying their RNAseq pipeline. This included data filtering
and sequence alignment using HISAT2 v2.0.5 with default parameters (Kim
et al., 2019), transcript quantification with HTSeq v0.6.1 with -m union
parameter (Anders et al., 2014), and differential gene expression
analysis employing the DESeq2 v1.22.2 algorithm with a cut-off value of
an adjusted \emph{p} -value of \textless{} 0.05 (Love et al., 2014). For
each genotype and treatment, respectively, three biological replicates
were processed. The resulting \emph{p} -values were adjusted for
multiple testing using the Benjamini--Hochberg correction (Benjamini and
Hochberg, 1995). Along with the adjusted \emph{p} -value (FDR) of
\textless{} 0.05 an absolute differential expression of
log\textsubscript{2} fold change (FC)~\selectlanguage{english}[?]~1.25 was chosen to select
differentially expressed genes (DEGs). The functional classification of
DEGs was performed using the MapMan v3.6 software (Thimm et al., 2004),
paying special attention to DEGs related with plant defense and nutrient
assimilation. Furthermore, functional relationships between the DEGs
were investigated using the applications stringApp v1.7 (Doncheva et
al., 2019), MCODE v2.0 (Bader and Hogue, 2003), EnrichmentMaps v3.3.3
(Merico et al., 2010), and ClueGO v2.5.8 (Bindea et al., 2009) in
Cytoscape v3.9.0 (Shannon et al., 2003).

\subsection*{2.5. qPCR analysis}

{\label{qpcr-analysis}}

Real-time quantitative RT-PCR was conducted as previously described
(Perez-Alonso et al., 2021). In brief, total RNA from three different
biological samples was converted into cDNA using M-MLV reverse
transcriptase and oligo(dT)\textsubscript{15} primer. Two nanograms of
cDNA was then used as template for the qPCR reactions, which were
conducted in triplicate (technical replicates). The oligonucleotide
pairs used in the experiments are given in \textbf{Supplementary Data
Sheet 1} . The reactions were monitored on a Lightcycler 480 Real-time
PCR system (Roche Diagnostics). Differential gene expression in
Arabidopsis was analyzed by using the comparative
2\selectlanguage{english}\textsuperscript{-[?][?]CT} method (Livak and Schmittgen, 2001)
with\emph{ADENINE PHOSPHORIBOSYL TRANSFERASE~1} (\emph{APT1} ,
At1g27450) as reference gene (Jost et al., 2007). Root colonization
with\emph{S.~indica} was monitored with a primer pair for fungal
translation elongation factor EF-1\selectlanguage{greek}α \selectlanguage{english}(\emph{SiTEF1} ) (B\selectlanguage{ngerman}ütehorn et al.,
2000). The fungal \emph{SiTEF1} cDNA levels were expressed relative to
the plant\emph{GLYCERINALDEHYDE-3-PHOSPHATE DEHYDROGENASE~C2}
(\emph{GAPC2} , At1g13440) cDNA levels. To exclude that the amplified
DNA fragments stem from DNA of dead fungal tissues within the roots, all
data presented here derived from cDNA libraries generated from RNA of
colonized roots.

\subsection*{2.6. Trypan blue staining of fungal hyphae and
spores}

{\label{trypan-blue-staining-of-fungal-hyphae-and-spores}}

To visually inspect root colonization, 10-12 small root samples from
control and co-colonized plants were employed. First, the root samples
were thoroughly washed with deionized water. Next, the root samples were
cut in 1~cm long pieces and incubated overnight in 10~N KOH. The root
samples were then rinsed 5 times with sterile H\textsubscript{2}O,
before they were incubated for 5~min in 0.1~N HCl. Finally, the samples
were incubated in a 0.05\% trypan blue solution (w/v), before they were
partially decolorized with lactophenol over ten minutes. Before the
specimen were mounted on glass slides and examined by microscopy, they
were washed once with 100\% ethanol and thrice with sterile
H\textsubscript{2}O and stored in 60\% glycerol (v/v).

\subsection*{2.7. Yeast two-hybrid
analysis}

{\label{yeast-two-hybrid-analysis}}

In order to examine the physical interaction between CBL7 and CIPK13,
total RNA from four weeks-old Arabidopsis seedlings (Col-0) was
extracted (Oñate-Sánchez and Vicente-Carbajosa, 2008) and first-strand
synthesis was performed according to the supplier's instructions, using
M-MLV reverse transcriptase and oligo(dT)\textsubscript{15} primer
(Promega). For the cloning of \emph{CBL7} and \emph{CIPK13} , the
corresponding cDNA fragments were amplified by PCR using specific primer
pairs listed in \textbf{Supplementary Data Sheet 1} . The resulting PCR
fragments were inserted into the vector pGEM® -T Easy (Promega).
Sequence integrity of the obtained products was confirmed by commercial
sequencing (StabVida). Subsequently, the fragments were introduced into
the vector pENTR-3C (Thermo Fisher) using the \emph{Eco} RI
and\emph{Sal} I restriction sites included in the primer sequences. The
obtained pENTR-CBL7 and pENTR-CIPK13 vectors were used in Gateway
LR-recombination reactions with the destination vectors pDEST-22 and
pDEST-32 according to the manufacturer's instructions. The resulting
destination vectors were then used to
transform\emph{Saccharomyces~cerevisiae} strain HF7c. Transformants were
plated on SD (simple drop-out) medium /-Leu/-Trp/+Ade and incubated at
28ºC for 2 days. Protein interaction was tested by growing transformants
on SD medium /-Leu/-Trp/-His/+Ade. The plates were incubated at 28ºC for
up to one week. To investigate the interaction of CBL7 with two
additional putative interaction partners, CIPK9 and CIPK24, the yeast
two-hybrid vectors PDEST-AD092F08 and PDEST-AD107D03 for CIPK9 and
CIPK24, respectively, were obtained from the Arabidopsis Biological
Resource Center (ABRC).

\subsection*{\texorpdfstring{2.8. K\textsuperscript{+} ion content
quantification}{2.8. K+ ion content quantification}}

{\label{k-ion-content-quantification}}

The analysis of endogenous cation contents of infected and control
plants was performed in fractions of root and shoot samples. To avoid
carry overs from the medium, roots were thoroughly rinsed with 10~mM
MES-Ca\textsuperscript{2+} pH~6.5. Next, root and shoot samples were
dried, weight and extracted with 1~M HNO\textsubscript{3}. The
K\textsuperscript{+} contents of the supernatants were then determined
by atomic emission spectroscopy. The results are given as the means and
their standard errors of three independent experiments.

\subsection*{2.9. Statistical analysis}

{\label{statistical-analysis}}

For statistical data assessment and the generation of box plots, JASP
v0.16 was employed. The box plots show the median, quartiles, and
extremes of the compared experimental values. One-way anova followed by
Tukey's post-hoc test or Student's \emph{t} -test were performed to
statistically analyze the data. Sample sizes (n) for each experiment are
given in the respective figure legends. Hierarchical clustering and
heatmaps of selected gene expression levels across the different
experiments was conducted using the Instant Clue software v0.10.10
(Nolte et al., 2018).

\section*{Results}

{\label{results}}

\subsection*{\texorpdfstring{3.1. \emph{CBL7} is induced in the symbioses
between Arabidopsis and \emph{S.
indica}}{3.1. CBL7 is induced in the symbioses between Arabidopsis and S. indica}}

{\label{cbl7-is-induced-in-the-symbioses-between-arabidopsis-and-s.-indica}}

The growth promoting effect of \emph{S.~indica} on multiple host plant
species has already been well-characterized (Mensah et al., 2020).
However, the precise molecular mechanism by which the fungal root
endophyte induces plant growth remains largely elusive. To shed further
light on the molecular basis of the interaction between
\emph{S.~indica}and Arabidopsis, we took a comprehensive transcriptomics
approach analyzing the transcriptional differences between control
plants and plants challenged with \emph{S.~indica} at both early (2 days
post infection (dpi)) and later (10 dpi) stages of co-cultivation. At
the indicated time points, total RNA was isolated and, after library
construction, subjected to mRNA-seq analysis. The application of a
Benjamini-Hochberg false discovery rate (FDR)
(p\textsubscript{adj.}~\selectlanguage{english}[?]~0.05) and setting the fold-change cut-off to
log\textsubscript{2}FC~\selectlanguage{english}[?]~1.25, identified 138 induced and 10 repressed
genes, respectively, at 2 dpi, while we identified 411 induced and 26
repressed genes after ten days of co-cultivation (\textbf{Supplemental
Data Sheet 2} ). The expression data revealed that 9.8\% of the
differentially expressed genes (DEGs) were induced both after two days
and ten days of co-cultivation, while 16.2\% and 67.3\% of the DEGs were
only induced in the early and later phases of the interaction,
respectively (\textbf{Figure 1A} ). Notably, only 6.8\% of the DEGs were
repressed under the tested conditions, and only one
gene,\emph{SENESCENCE-ASSOCIATED GENE~13} (\emph{SAG13} ), turned out to
be repressed during the establishment of the plant-fungus interaction,
while being induced at later stages of the symbiosis. To gain further
insight into the affected biological processes triggered by the fungus
during the studied interaction, we performed gene ontology (GO) analyses
of the induced DEG groups (\textbf{Supplemental Image 1A} ). The
analysis revealed a substantial enrichment of genes associated with
stress and defense-related GO terms. To decipher possible molecular
mechanisms, we further employed a functionally enriched network analysis
to identify biological interpretations and interrelations of functional
groups in biological networks (\textbf{Figure 1B} ) (Bindea et al.,
2009). The network analysis largely confirmed the obtained enriched GO
term classifications. In addition, it provided evidence for the
enrichment of genes related to a group of GO terms associated with
cellular Ca\textsuperscript{2+} homeostasis and Ca\textsuperscript{2+}
signaling processes, which attracted our attention and on which we
followed up in this study.

To gain a better understanding of the role of
Ca\textsuperscript{2+}-related processes in the mutual interaction
between \emph{S.~indica} and Arabidopsis, we extracted the relative
expression profiles of 88 calcium signaling-related genes from the
non-filtered RNA-seq datasets and performed a hierarchical cluster
analysis (\textbf{Supplemental Image 1B} ). We have been particularly
intrigued by the expression profile of the genes in Cluster~1, which
contained the four genes \emph{HIGH AFFINITY
K\textsuperscript{+}TRANSPORTER~5} (\emph{HAK5} ),
\emph{CALMODULIN-LIKE~4} (\emph{CML4} ),\emph{CBL-INTERACTING PROTEIN
KINASE~13} (\emph{CIPK13} ), and\emph{CALCINEURIN B-LIKE PROTEIN~7}
(\emph{CBL7} ). The genes were consistently induced both at early and
later stages of the plant-fungus interaction, which could be confirmed
by qRT-PCR analysis (\textbf{Figure 1C} ). Activation of these genes
might therefore be required both for the establishment and preservation
of the mutual plant-fungus interaction.

\section*{3.2. CBL7 is required for fungus-mediated growth
promotion}

{\label{cbl7-is-required-for-fungus-mediated-growth-promotion}}

CBL proteins are plant-specific Ca\textsuperscript{2+} sensors that
decode transient calcium fluctuations through the interaction with
CBL-interacting protein kinases (CIPKs) (Tang et al., 2020). It has
previously been shown that several CBL proteins, i.e. CBL1, CBL8, CBL9,
and CBL10, interact with CIPK23 to regulate the transport activity of
HAK5 (Ragel et al., 2015). Hence, we speculate that CBL7 and CIPK13 may
physically interact with each other to control HAK5 activity as their
downstream target, because of their shared expression profiles in
response to the infection of Arabidopsis seedlings with\emph{S.~indica}
. To test this hypothesis, we first conducted a yeast two-hybrid
analysis (\textbf{Figure 2} ). The corresponding full-length sequences
of CBL7 and CIPK13 were included into the pDEST-22/pDEST-32 yeast
2-hybrid system vectors. The direct interaction of the two proteins was
monitored by the ability of transformed \emph{Saccharomyces cerevisiae}
HF7c cells to grow on selection medium (SD/-Trp/-Leu/-His) containing 5
mM 3-amino-1,2,4-triazole. \textbf{Figure 2A} provides evidence of a
weak interaction between CBL7 and the protein kinase CIPK13. A GeneMANIA
protein-protein interaction query (Warde-Farley et al., 2010), provided
additional evidence for the direct interaction of CBL7 with two other
CIPK proteins (\textbf{Figure 2B} ), CIPK9 and CIPK24. However, we were
not able to verify the predicted interaction of CBL7 with these two
CIPKs by yeast two-hybrid studies (\textbf{Supplemental Image 2} ).

Next, we took a reverse genetics approach to assess the role of CBL7,
CIPK13, and HAK5 in the fungus-mediated promotion of plant growth. To do
so, we used two independent T-DNA insertion mutants for both
\emph{CBL7}and \emph{CIPK13} , as well as the previously described
\emph{hak5}mutant. The mutants were grown for seven days on 1/2 MS medium
alongside with corresponding wild-type control plants, before the
seedlings were transferred to PNM medium, where they were either
co-cultivated with\emph{S.~indica} or a mock control. After ten days,
the vertically grown plants were photographed, and the root system
architecture was analyzed using the GiA Roots software. The statistical
assessment of the monitored parameters facilitated a quantitative
comparison of the growth promoting effect elicited by \emph{S.~indica}
in the different genotypes. As shown in \textbf{Figure 3A} , the
functional knockout of\emph{CIPK13} had no impact on the total root
network length. Both\emph{cipk13} alleles exhibited a significant growth
promotion through the co-cultivation with \emph{S.~indica} , similar to
the response of the wild-type control plants. The same observation was
made for the\emph{hak5} mutant (\textbf{Figure 3C} ). In contrast, the
loss-of-function mutants of \emph{CBL7} , \emph{cbl7-1} and\emph{cbl7-2}
, are characterized by a substantial reduction of the growth promoting
effect observed in Col-3 control plants (\textbf{Figure 3B} ). The
additional analysis of the total network area of the different groups of
seedlings confirmed the obtained results (\textbf{Table 1} ). Taken
together, the results suggest that CBL7 plays an important role in the
establishment/maintenance of the symbiosis, because one the one hand the
functional inactivation of CBL7 coincides with a loss of the growth
promoting effect normally exerted by the fungus. CIPK13 and HAK5, on the
other hand, are seemingly likely not vital or replaceable in the
plant-fungus interaction, given that the knockout mutants showed no
significant alteration of their growth behavior when infected
with\emph{S.~indica} .

\subsection*{\texorpdfstring{3.3. A loss of \emph{CBL7} interferes with
potassium distribution in
Arabidopsis}{3.3. A loss of CBL7 interferes with potassium distribution in Arabidopsis}}

{\label{a-loss-of-cbl7-interferes-with-potassium-distribution-in-arabidopsis}}

To further investigate the biological functions of CBL7, we conducted
another set of RNA-seq analyses comparing the transcriptional profiles
of \emph{cbl7-2} and Col-3 (wt) plants under control conditions. After
applying the same threshold as before (FDR p\textsubscript{adj.}~\selectlanguage{english}[?]~0.05,
log\textsubscript{2}FC~\selectlanguage{english}[?]~1.25), we identified only 20 induced and 53
repressed DEGs (\textbf{Figure 4A,B} ), respectively. The GO analysis of
this reduced number of DEGs did not provide evidence for the significant
enrichment of any GO term. As the arbitrary chosen expression threshold
provided no significant results, we repeated the analysis with less
stringent threshold values (FDR p\textsubscript{adj.}~\selectlanguage{english}[?]~0.05,
log\textsubscript{2}FC~\selectlanguage{english}[?]~0.35), giving 119 induced and 947 repressed
DEGs, respectively (\textbf{Figure 4A} ,\textbf{B} ). Subsequent GO and
KEGG enrichment analysis revealed the repression of 33 genes associated
with the spliceosome (\textbf{Figure 4C, Supplementary Data Sheet 3} ).
Furthermore, we were able to identify the induction of 23
(p\textsubscript{adj.}~=~1*13\textsuperscript{-12}) and
11~(p\textsubscript{adj.}~=~3.45*10\textsuperscript{-2}) genes related
to cell wall organization and ion transport GO terms, respectively
(\textbf{Supplemental Data Sheet 3} ). Among the latter genes, we found
the high-affinity nitrate transporter \emph{NRT2.4} as well as the
low-affinity nitrate transporter \emph{NPF2.9} . The involvement of CBL7
in the transcriptional regulation of nitrate transporters has previously
been reported (Ma et al., 2015). Additionally, we again found that the
high-affinity potassium transporter gene \emph{HAK5} was significantly
induced (log\textsubscript{2}FC~=~1.85, p\textsubscript{adj.}~=~0.022).
Although our previous results questioned the role of HAK5 in the
plant-fungus interaction, the reiterated appearance of \emph{HAK5} led
us to analyze the potassium content in roots and shoots of
\emph{cbl7-2}and Col-3 to determine whether CBL7 could be involved in
the regulation of potassium homeostasis in Arabidopsis. To quantify the
K\textsuperscript{+} content in Arabidopsis seedlings, mutant and wt
seeds were germinated on 1/2 MS and then transferred to square plates with
modified Hoagland agar medium lacking glucose and containing either no
KCl or 1~mM KCl. After three days of acclimatization, the seedling roots
were inoculated with \emph{S.~indica} chlamydospores and mock treated,
respectively. After another week of co-cultivation, the potassium
contents were quantified by atomic emission spectroscopy. As shown
in\textbf{Figure 5} , the potassium content of the \emph{cbl7-2} roots
grown at 0~mM KCl was similar to that of the Col-3 control plants.
However, the previously described decrease in
K\textsuperscript{+}contents in response to the inoculation with
\emph{S.~indica} (Conchillo et al., 2021) was only observed in control
plants, but not in\emph{cbl7-2} . Interestingly, the
K\textsuperscript{+} level in the shoots of \emph{cbl7-2} plants was
significantly lower than in the mock-treated wild type, equal to the
level observed for Col-3 treated with the fungus. Although
K\textsuperscript{+} levels showed a tendency to decrease in
\emph{cbl7-2} shoots, there was no significant difference between mock
and fungus-treated \emph{cbl7-2} plants. Plants grown on plates
containing 1~mM KCl showed a similar picture (\textbf{Figure 5B} ), only
wild-type Col-3 plants showed the expected decrease in
K\textsuperscript{+} in response to the \emph{S.~indica} infection.
Furthermore, we found a significant accumulation of
K\textsuperscript{+}in \emph{cbl7-2} roots, while the
K\textsuperscript{+} content in\emph{cbl7-2} shoots appeared to be
significantly reduced. We have therefore concluded that CBL7 contributes
to the regulation of the distribution of K\textsuperscript{+} in the
plant. The functional loss of CBL7 is unequivocally linked to an altered
K\textsuperscript{+}distribution profile, which is characterized by an
accumulation of K\textsuperscript{+} in the roots, most likely due to a
lack of transport of K\textsuperscript{+} to areal plant tissues. At the
same time, our data suggest that CBL7 could also play a key role in the
observed decrease in K\textsuperscript{+} levels in plants challenged
with \emph{S.~indica} , as \emph{cbl7} mutants present a reduced
reduction of K\textsuperscript{+} when co-cultivated
with\emph{S.~indica} .

\subsection*{\texorpdfstring{3.4. The \emph{cbl7} mutant shows increased
plant defense
responses}{3.4. The cbl7 mutant shows increased plant defense responses}}

{\label{the-cbl7-mutant-shows-increased-plant-defense-responses}}

The above results highlighted the transcriptional regulation
of\emph{CBL7} in response to the infection of Arabidopsis roots
with\emph{S.~indica} and its involvement in
K\textsuperscript{+}partitioning in the plant. With the goal of further
exploring the role of CBL7 in the establishment and maintenance of the
symbiosis between Arabidopsis and \emph{S.~indica} , we compared the
RNA-seq data surveyed from \emph{cbl7-2} knockout plants and
corresponding Col-3 control plants infected with \emph{S.~indica} and
mock treated, respectively (\textbf{Supplementary Data Sheet 3} ).
Quantitative analysis of differential gene expression revealed a
substantial increase in induced genes in the \emph{cbl7} mutant relative
to wt, while the number of repressed genes in \emph{cbl7} is clearly
diminished (\textbf{Figure 6A} ). Furthermore, the examination of the
possible logical relations between the two data sets revealed four
non-overlapping and two overlapping groups for DEGs in \emph{cbl7-2} and
Col-3 (\emph{S.~indica}vs mock) (\textbf{Figure 6C} ). Next, we
performed a GO and functional network analysis to identify biological
processes significantly affected by the loss of \emph{CBL7} . The main
alterations in the \emph{cbl7}mutant could be associated with plant
defense-related processes, especially with metabolic pathways that are
involved in the biosynthesis of secondary metabolites related to plant
defense (\textbf{Figure 6B,D} ). The enriched genes in these GO terms
included a substantial number of cytochrome P450 enzymes, such as
\emph{CYP71B3} ,\emph{CYP71A12} , \emph{CYP71A13} , and \emph{CYP71B15}
(PAD3), which are known to participate in glucosinolate and camalexin
biosynthesis (Glawischnig, 2007; Frerigmann et al., 2016). Furthermore,
we found an induction of the myrosinase genes \emph{BGLU34} and
\emph{BGLU35} that are involved in the turnover of glucosinolates
(Wittstock and Burow, 2010). The observation of an induction of plant
defense-related compounds is further supported by the induction of
transcription factors (TFs) \emph{NAC042} , \emph{WRK33} , and
\emph{WRK51} , which have been linked with the regulation of camalexin
and indole glucosinolate biosynthesis (Birkenbihl et al., 2012; Saga et
al., 2012; Frerigmann and Gigolashvili, 2014; Zhou et al., 2020).
Furthermore, we also observed an induction of \emph{WRKY70} , a TF
involved in modulating cell wall-related defense responses (Li et al.,
2017). In addition, the functional analysis of the transcriptomics data
revealed an enrichment of glutathione S-transferases among the induced
genes in \emph{cbl7-2} , which included the genes \emph{GSTU10} ,
\emph{GSTU12} , \emph{GSTF3} ,\emph{GSTF6} , and \emph{GSTF7} .
Glutathione S-transferases are readily induced by a wide range of stress
conditions, including bacterial and fungal infections (Dixon et al.,
2002; Gullner et al., 2018). Considering that all these processes are
observed in the \emph{cbl7}loss-of-function mutant, it must be concluded
that CBL7 is involved in the suppression of multilayered defense
responses to facilitate the establishment of \emph{S.~indica} in the
root apoplast. However, other processes that appear enriched in
\emph{S.~indica} challenged wt plants but not in \emph{cbl7} , such as
the induction of sucrose transporter genes and WRKY46 orchestrated
abiotic stress responses, or the repression of bHLH100 and MYB72
mediated metal ion homeostasis, are likely to contribute to the
significant difference in growth promotion between \emph{cbl7} and wt
triggered by the fungus.

Based on our hypothesis of an increased defense response in
the\emph{cbl7} knockout mutant, it must be expected that the roots of
mutant plants are less well colonized by \emph{S.~indica} than
comparable wt roots. To prove this, we analyzed root colonization
in\emph{cbl7} and Col-3 by qPCR and trypan blue staining.
Indeed\textbf{,}root colonization of the \emph{cbl7} mutant was
significantly reduced (\textbf{Figure 7} ). Our experiments have shown
that CBL7 plays a critical role in maintaining the equilibrium between
symbiotic interaction and plant defense that keeps the endophytic fungus
load under control.

\section*{Discussion}

{\label{discussion}}

Calcium signaling plays an important role in the regulation of a wide
array of biological processes in eukaryotic physiology, including
pathogenic and beneficial plant-microbe interactions (Vadassery and
Oelmuller, 2009). Ca\textsuperscript{2+} signaling depends on the
comprehensive interaction between membrane-located receptors that sense
external signals, which then trigger transient changes in intracellular
Ca\textsuperscript{2+} levels through the activation of
Ca\textsuperscript{2+} permeable channels that release
Ca\textsuperscript{2+} from internal stores, such as the endomembrane
system, or transport Ca\textsuperscript{2+} across the plasma membrane.
Within the cytoplasm, a wide array of different
Ca\textsuperscript{2+}-binding proteins are responsible for deciphering
the incoming transient Ca\textsuperscript{2+} signatures and translating
them into appropriate molecular responses (Pirayesh et al., 2021). These
Ca\textsuperscript{2+} sensors include calmodulins (CaMs),
calmodulin-like-proteins (CMLs), Ca\textsuperscript{2+}-dependent
protein kinases (CDPKs) and calcineurin~B-like (CBL) proteins. The
latter proteins generally interact with protein kinases (CIPKs) to form
two-component systems. Together, these sensor systems orchestrate a
multitude of downstream responses, including the control of various ion
channels and transporters (Tang et al., 2020). In this study we showed
that CBL7 is among the very few genes that display a consistent positive
transcriptional response in Arabidopsis wild-type plants infected with
the beneficial root endophyte \emph{S.~indica} , both at early and later
stages of the interaction (\textbf{Figure 1} ). Our studies also
demonstrated that CIPK13 is a target of CBL7 in yeast two-hybrid studies
(\textbf{Figure 2} ), while the interaction with other CIPKs, i.e. CIPK9
and CIPK24, could not be experimentally confirmed. The physical
interaction of CBL7 with CIPK13 attracted our attention because previous
studies reported their expression in roots (Schliebner et al., 2008; Ma
et al., 2015), and the protein-protein interaction of CIPK13 with the
auxin transporter PIN5 (Jones et al., 2014). Although PIN5 is not
involved in the directional cell to cell transport of auxin, but rather
in maintaining the intracellular auxin homeostasis through shuttling
auxin from the cytosol to the lumen of the endoplasmic reticulum (Mravec
et al., 2009), this interaction could possibly represent an important
link that could explain the growth promoting effect of
\emph{S.~indica}on its host plants, which particularly manifests in a
considerable induction of root growth (Perez-Alonso et al., 2020). In
addition, the co-expression of \emph{CBL7} , \emph{CIPK13} , and
\emph{HAK5} prompted us to speculate that there might be a relevant
connection between these components, which could regulate the uptake of
potassium into host plants and, thus, their nutrition with this
essential macronutrient. The activation of HAK5 through phosphorylation
by CIPK23 has previously been described for Arabidopsis and tomato
plants (Ragel et al., 2015; Amo et al., 2021). Hence, an interaction
cascade between the three components appeared possible. However, when we
analyzed the relevance of the different components with respect to their
impact on the growth promoting effect on Arabidopsis roots, we had to
realize that only CBL7, but neither CIPK13 nor HAK5 interfered with
growth promotion (\textbf{Figure 3} ). While both \emph{cbl7} mutant
alleles showed a severe loss of growth promotion, none of the other
investigated mutants demonstrated significant differences to wt.
Although CIPKs and potassium transporters form bigger gene families,
which opens the possibility that their loss might be compensated by
other family members, it must be concluded that CIPK13 and HAK5 are not
critical for the fungus-triggered plant growth promotion. The possible
plastid localization of CIPK13 further argues against an interaction
with CBL7 \emph{in vivo}(Schliebner et al., 2008).

Previous work on the role of CBL7 highlighted its interaction with
the\emph{A.~thaliana} PLASMA MEMBRANE PROTON ATPASE~2 (AHA2) (Yang et
al., 2019). In their model, the CBL7/AHA2 complex is further stabilized
by the interaction with PROTEIN KINASE SOS2-LIKE5 (PKS5), also referred
to as CIPK11. The authors suggest that the
Ca\textsuperscript{2+}-mediated dissociation of the CBL7/CIPK11/AHA2
complex under saline-alkali stress conditions translates into the
activation of AHA2. Activation of AHA2, in turn, leads to
hyperpolarization of the plasma membrane, which is likely to affect the
transport activity of \emph{Shaker} -like K\textsuperscript{+} channels
in the root. SKOR, an outward-rectifying K\textsuperscript{+} channel,
is reported to be crucial for the loading of K\textsuperscript{+} into
the xylem and, thus, the long-distant transport of K\textsuperscript{+}
within the plant (Gaymard et al., 1998). Moreover, SKOR is known to form
heteromeric outward-rectifying K\textsuperscript{+} channel units with a
second \emph{Shaker} -like channel, GORK (Dreyer et al., 2004). SKOR and
GORK facilitate K\textsuperscript{+} transport only when the plasma
membrane is depolarized (Dreyer and Blatt, 2009). A recent publication
further pinpointed the importance of the complex interplay between
different nutrient transporter systems and proton pumps in nutrient
cycling in plants (Dreyer, 2021). Based on these observations, it must
be assumed that a loss of CBL7 will likely result in a disrupted
regulation of AHA2 activity and, consequently, a hyperpolarization of
the plasma membrane, which entails a reduced transport activity of SKOR
and GORK. As a result, the assimilated K\textsuperscript{+} would
accumulate in the roots, and only reduced amounts of
K\textsuperscript{+} would reach the shoot. In fact, our analysis of
K\textsuperscript{+} levels in wild-type and \emph{cbl7} mutants
corroborates this hypothesis (\textbf{Figure 4} ). The \emph{cbl7}
mutant plants showed a pronounced accumulation of K\textsuperscript{+}
in the roots, which we attribute to possible impairment of
K\textsuperscript{+} xylem transport. Potassium depletion in the shoot
could trigger the induction of \emph{HAK5} in \emph{cbl7} , as multiple
independent studies demonstrated that \emph{HAK5} is induced under
K\textsuperscript{+} starvation (Ahn et al., 2004; Armengaud et al.,
2004; Shin and Schachtman, 2004; Gierth et al., 2005). A recent study
demonstrated the induction of \emph{HAK5} in the host plant as a general
consequence of the symbiotic interaction (Conchillo et al., 2021), which
would also explain why we found \emph{HAK5} under the consistently
induced genes in wild-type plants (Figure 1). Furthermore, the latter
study demonstrated that the inoculation of Arabidopsis
with\emph{S.~indica} does not improve the K\textsuperscript{+} nutrition
of the host plant under K\textsuperscript{+} limiting conditions.
Intriguingly, the authors showed that root colonization is stimulated
under K\textsuperscript{+} limiting conditions, leading to the
assumption that the endophyte may benefit from the relatively high
cellular K\textsuperscript{+} concentrations in plant cells or apoplast.
The K\textsuperscript{+} content in plant tissue was reported to be
significantly reduced when plants were inoculated with \emph{S.~indica}
. Our own work confirms this finding of reduced
K\textsuperscript{+}levels in wt plants co-cultivated with the fungus.
On the contrary, the reduction of K\textsuperscript{+} levels upon
infection with the fungus was largely absent in \emph{cbl7} mutant
plants, possibly because the flux of K\textsuperscript{+} from the host
plant to the fungus is hampered by inactivated voltage-gated potassium
channels. However, to validate this hypothesis further studies are
needed. Nevertheless, together with the widely missing \emph{S.~indica}
-triggered growth promotion in inoculated \emph{cbl7} mutants, this
observation further underlined the vital role of CBL7 in the symbiotic
plant-fungus interaction.

To further investigate the role of CBL7 in this context, we subjected
the \emph{cbl7-2} mutant to additional RNA-seq analyses. In a first
analysis, we compared the transcriptional pattern of the
\emph{cbl7-2}mutant with that of the corresponding wild type, Col-3,
under control conditions. After applying an arbitrary cut-off value of
log\textsubscript{2}FC~\selectlanguage{english}[?]~1.25, we identified only 73 DEGs that did not
show enrichment of any biological process or function. Lowering the
stringency of our analysis disclosed the repression of genes that are
associated with the KEGG spliceosome term, including various splicing
factors and the \emph{SUPPRESSOR-OF-WHITE-APRICOT/SURP DOMAIN-CONTAINING
PROTEIN} (\emph{SWAP} ) gene (Lorkovic et al., 2005). This suggests a
possible impairment of alternative splicing processes in \emph{cbl7} .
It is, however, noteworthy that the gene that showed the strongest
repression with a log\textsubscript{2}FC of --2.34,\emph{MOS4-ASSOCIATED
COMPLEX SUBUNIT 5C} (\emph{MAC5C} ), does not appear to form part of the
spliceosome associated MAC complex (Monaghan et al., 2010), but is more
closely linked with processes related with secondary cell wall synthesis
(Taylor-Teeples et al., 2015). This possible relation of MAC5C with cell
wall synthesis is consistent with the observed enrichment of cell wall
biogenesis-related genes, including the \emph{HYDROXYPROLINE-RICH
GLYCOPROTEIN~1} (\emph{HRGP1} ) and the proline- and leucine-rich
extensin-like family protein genes\emph{EXT3} , \emph{EXT4} ,
\emph{EXT6} , \emph{EXT10} , and \emph{LRX1} , respectively, which are
moderately induced in \emph{cbl7} . Extensin proteins play an important
role in cell wall sensing. They are insoluble cell wall components that
act as protein-protein interaction platforms to which peptide hormones
and transmembrane receptors can bind, thereby relaying the perception of
extracellular stimuli to the cytoplasm (Herger et al., 2019). These
transcriptomic alterations possibly make the \emph{cbl7} mutant more
responsive to changes in its environment, including the perception of
beneficial and pathogenic microbes on the root surface and in the
apoplast.

This notion is further supported by the strongly induced plant defense
response in \emph{cbl7-2} . When comparing the transcriptional responses
of \emph{cbl7-2} plants infected with \emph{S.~indica} versus
uninfected\emph{cbl7-2} control plants with the corresponding responses
of Col-3 plants, we found a significant enrichment of genes that fall
into the secondary metabolite biosynthesis GO term in \emph{cbl7-2} .
This group contains the TF genes \emph{NAC042} , \emph{WRK33} , and
\emph{WRK51} , as well as the cytochrome P450 genes \emph{CYP71B3} ,
\emph{CYP71A12} ,\emph{CYP71A13} , and \emph{CYP71B15} (PAD3) encoding
proteins involved in the biosynthesis of glucosinolates and camalexin,
two secondary metabolites involved in the defense against pathogens
(Glawischnig, 2007; Malka and Cheng, 2017). A previous study has already
investigated the role of compounds derived from indole-3-acetaldoxime
(IAOx) in the interaction of Arabidopsis with its root endophyte
\emph{S.~indica}(Nongbri et al., 2012). The authors provide conclusive
evidence that the infection of Arabidopsis with \emph{S.~indica}
includes the induction of the formation of IAOx derived compounds during
early stages of the interaction as a general defense response of the
host plant. After the establishment of the symbiosis, the formation of
indole glucosinolates and camalexin was reported to decrease. However, a
certain wild-type level of these compounds appears to be required to
avoid excessive root colonization, as the lack of these compounds in the
IAOx-deficient\emph{cyp79b2/cyp79b3} double mutant results in
significantly increased root colonization, which is accompanied by a
complete loss of fungus-conferred plant growth promotion. Excessive root
colonization is suggested to convert the beneficial interaction between
\emph{S.~indica}and Arabidopsis into a physiological burden for the host
plant. In case of the \emph{cbl7-2} mutant, the limitation of the
formation of IAOx-derived secondary metabolites is not working properly,
because we still found these plant defense-related genes induced after
14 days of co-cultivation. Consequently, it must be concluded that the
mutant overreacts to the biotic stress caused by the fungus, which
results in a stronger defense response and a less efficient
establishment of the symbiosis. Both the visual inspection of root
colonization by Trypan blue staining and the quantification of root
colonization by qPCR confirmed the reduced colonization of \emph{cbl7}
mutant roots with\emph{S.~indica} relative to those of wild-type
controls.

In summary, our work provides functional analyses of the cytoplasmic
Ca\textsuperscript{2+} sensor CBL7 in the context of plant-fungus
interactions. Based on the presented results, we propose that\emph{CBL7}
is induced during root infection of Arabidopsis with\emph{S.~indica} and
contributes to the control of the classical plant defense. The loss of
\emph{CBL7} unequivocally blocks the normally observed promotion of
\emph{S.~indica} -triggered plant growth, likely through a reduced root
colonization, as the long-term harmony between the two symbionts is out
of balance in the mutant. Furthermore, our work shed additional light on
the so far undisclosed role of CBL7 in controlling potassium
translocation in the plant body, most probably through its interaction
with the plasma membrane ATPase AHA2. Under normal conditions,
Ca\textsuperscript{2+} signals elicited by the root infection trigger
the dissociation of the CBL7/CIPK11/AHA2 complex (Yang et al., 2019),
which subsequently results in the hyperpolarization of the membrane.
Consequently, this leads to the inactivation of outward rectifying
potassium channels and a reduced flux of K\textsuperscript{+}into the
xylem, which in turn results in an accumulation of potassium in the
roots. This increase in potassium levels in the root is possibly an
important asset in establishing the interaction between
\emph{S.~indica}and Arabidopsis, as a very recent study demonstrated
that the fungus may benefit not only from plant-derived
photoassimilates, but also from the relatively high potassium contents
of plant tissues (Conchillo et al., 2021). It will be a thrilling future
task to decipher whether impaired repression of plant defense responses
or out-of-control potassium translocation in \emph{cbl7} are the cause
of the observed reduced root colonization and missing fungus-triggered
plant growth promotion.

\section*{Funding}

{\label{funding}}

This work was financially supported by the collaborative IPSC research
project executed in the framework of the EIG CONCERT-Japan joint call on
Food Crops and Biomass Production Technologies and the related national
funding agencies: grant PCIN-2016--037 from the Ministry of Economy and
Competitiveness (MINECO), Spain, to SP and JVC; grants 01DR17007A and
01DR17007B from the Federal Ministry of Education and Research (BMBF),
Germany, to JL-M and RO, respectively; grant JPMJSC16C3 from the Japan
Science and Technology Agency (JST) to HS; and grant EIG\_JC1JAPAN-045
from the Centre National de la Recherche Scientifique (CNRS), France, to
AK. In addition, the project obtained financial support by grant
PID2020-119441RB-I00 funded by MCIN/AEI/10.13039/501100011033 and, as
appropriate, by ``ERDF A way of making Europe'', by the ``European
Union'' or by the ``European Union NextGenerationEU/PRTR'' to SP, as
well as by grant REDES \#190078 and Fondecyt 1211057 to PR from the
Agencia Nacional de Investigacion y Desarrollo (ANID, Chile). CGG was
supported by the Severo Ochoa Program for Centers of Excellence in R\&D
from the Agencia Estatal de Investigacion, Spain, grant SEV-2016-0672
(2017--2021) to the Centro de Biotecnologia y Genomica de Plantas.

\section*{Acknowledgments}

{\label{acknowledgments}}

The authors are grateful to Mar Gonzalez Ceballos for her excellent
technical support. Moreover, we acknowledge the critical discussion of
our work with experts in the field, including Ingo Dreyer (Universidad
de Talca, Chile) and Begona Benito (CBGP), who also kindly shared mutant
seeds with us. In addition, the authors thank the NASC and ABRC for
providing T-DNA insertion mutant seeds and plasmids for yeast two-hybrid
studies.

\section*{Conflict of Interest}

{\label{conflict-of-interest}}

The authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a
potential conflict of interest.

\section*{Author Contributions}

{\label{author-contributions}}

SP, RO, JLM, JCV, AK and HS conceived and planned the study. MMPA, CGG,
AGOV, POG, SSS and TK performed experimental procedures and collected
data. MMPA, CGG, AGOV, POG, SSS, PR and SP performed analysis and SP
wrote the manuscript.

\section*{Data Availability Statement}

{\label{data-availability-statement}}

All data supporting the findings of this study are available within the
paper and within its supplementary data published online.

\section*{Short legends for Supporting
Information}

{\label{short-legends-for-supporting-information}}

Supplemental Data Sheet 1: Primers used for genotyping, cloning and
expression

analysis

Supplemental Data Sheet 2: RNA-seq analysis of \emph{S.~indica} and mock
treated Arabidopsis wild-type seedlings.

Supplementary Data Sheet 3: RNA-seq analysis of \emph{S.~indica} and
mock treated Arabidopsis \emph{cbl7-2} and Col-3 seedlings

Supplemental Image 1: GO and cluster analysis of induced DEGs
in\emph{S.~indica} challenged wt plants compared to mock treated control
plants.

Supplemental Image 2: Yeast two-hybrid analysis of the interaction
between CBL and CIPK9 and CIPK24.

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\section*{Tables}

{\label{tables}}

\textbf{Table 1. Growth promoting effects on Arabidopsis plants mediated
by the endophytic fungus.} Differences in total root network area were
used as molecular marker of the growth promoting effect
of\emph{S.~indica} on its host plants. Data represent means ± SE
(n~=~24). Asterisks indicate significant differences between
\emph{S.~indica} and mock treated samples. Student's \emph{t} -test:
**P\selectlanguage{english}[?]0.01, ***P\selectlanguage{english}[?]0.001.\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
Genotype & Network area (cm\textsuperscript{2}) & Network area
(cm\textsuperscript{2}) & Network area
(cm\textsuperscript{2})\tabularnewline
\midrule
\endhead
& + \emph{S. indica} & -- \emph{S. indica} & [?] +/-- \emph{S.
indica}\tabularnewline
Col-3 & 7.31~\selectlanguage{ngerman}±~0.27 & 4.01~\selectlanguage{ngerman}±~0.28 & 3.30~\selectlanguage{ngerman}±~0.39***\tabularnewline
\emph{cbl7-1} & 6.01~\selectlanguage{ngerman}±~0.29 & 4.97~\selectlanguage{ngerman}±~0.35 & 1.04~\selectlanguage{ngerman}±~0.45\tabularnewline
\emph{cbl7-2} & 5.42~\selectlanguage{ngerman}±~0.26 & 4.41~\selectlanguage{ngerman}±~0.22 & 1.01~\selectlanguage{ngerman}±~0.34\tabularnewline
Col-0 & 3.75~\selectlanguage{ngerman}±~0.24 & 2.58~\selectlanguage{ngerman}±~0.22 & 1.16~\selectlanguage{ngerman}±~0.33**\tabularnewline
\emph{cipk13-1} & 4.31~\selectlanguage{ngerman}±~0.25 & 2.99~\selectlanguage{ngerman}±~0.15 &
1.32~\selectlanguage{ngerman}±~0.29***\tabularnewline
\emph{cipk13-2} & 3.31~\selectlanguage{ngerman}±~0.14 & 2.24~\selectlanguage{ngerman}±~0.15 &
1.07~\selectlanguage{ngerman}±~0.21***\tabularnewline
\emph{hak-5} & 4.65~\selectlanguage{ngerman}±~0.31 & 2.46~\selectlanguage{ngerman}±~0.24 & 2.19~\selectlanguage{ngerman}±~0.39***\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}



\section*{Figure Legends}

{\label{figure-legends}}

\textbf{Figure 1. Transcriptional analysis of Arabidopsis seedlings
co-cultivated with \emph{S.~indica}.} \textbf{A} ) Venn diagram showing
the numbers of differentially expressed genes in Arabidopsis plants 2-
and 14-days post infection with \emph{S.~indica} compared to control
plants that were mock infected. \textbf{B} ) ClueGo analysis of induced
DEGs. The figure shows the representative molecular function interaction
among the targets. \textbf{C} ) qPCR analysis of transcriptional
responses of identified target genes in plants that were co-cultivated
for 2 and 14 days, respectively, with \emph{S.~indica} compared to mock
treated plants. The data represent means ± SE (n~=~3).

\textbf{Figure 2. Protein-protein interaction (PPI) studies to identify
binding partners of CBL7. A} ) Yeast cells co-expressing the indicated
combinations of constructs were grown on non-selective (-LW) or
selective (--LWH) media. Colony growth on the selective media indicated
protein--protein interactions. The empty activation domain (AD) vector
was used as a negative control. Fivefold diluted cell cultures were
inoculated on each spot as indicated by the triangle widths. A
representative data set of an experiment replicated three times on
different biological samples is shown. BD, binding domain. \textbf{B} )
Interaction network for CBL7 inferred from 58 publicly available
datasets included in GeneMANIA v3.5.2. The PPI network indicates the
protein set that either directly (diamonds) or indirectly (circles)
interact with CBL7 (triangle). The different shape size refers to the
calculated interaction score, while the thickness of the edges indicates
the normalized maximal weight of the interaction.

\textbf{Figure 3. Total root network length of Arabidopsis wild-type and
mutant plants after 14 days of co-cultivation with \emph{S.~indica} or
mock treatment} . \textbf{A} ) Comparison of two \emph{cipk13} T-DNA
insertion mutant alleles with Col-0. \textbf{B} ) Analysis of the
fungus-triggered growth promoting effect in Col-3 and \emph{cbl7} mutant
plants. \textbf{C} ) The growth promoting effect of \emph{S.~indica} is
not affected in the \emph{hak5} mutant. The box plots show the median,
quartiles, and extremes of the compared datasets (n~=~24). Asterisks
indicate significant differences between S.~indica and mock treated
samples. Student's \emph{t} -test: **P\selectlanguage{english}[?]0.01, ***P\selectlanguage{english}[?]0.001.

\textbf{Figure 4. Analysis of transcriptional differences
between\emph{cbl7} mutant and wild-type Col-3 plants.} \textbf{A} ) DEGs
statistics for the \emph{cbl7} mutant compared to Col-3 control plants
under control conditions. \textbf{B} ) Volcano plot of the distribution
of all DEGs, mapping the 20 upregulated genes (red) and 53 downregulated
genes (blue). The figure shows the relation between the significance
(-log\textsubscript{10}(q-value)) and the strength
(log\textsubscript{2}FC) of the differential expression. \textbf{C} )
Bar plot of the KEGG enrichment analysis of induced DEGs in
\emph{cbl7}compared to Col-3 control plants under control conditions.
Only the spliceosome KEGG term appeared to contain significantly
enriched genes. Gene enrichment is given by
-log\textsubscript{10}(q-value).

\textbf{Figure 5. K\textsuperscript{+} contents of root and shoot
tissues of \emph{S.~indica} infected and non-infected control plants} .
The plants were grown in modified Hoagland medium under
K\textsuperscript{+} starvation (0 mM KCl) (\textbf{A} ) or at 1 mM KCl
(\textbf{B} ). The box plots show the median, quartiles, and extremes of
the compared datasets (n~=~15). Different letters indicate significant
differences between the compared conditions and genotypes analyzed by
ANOVA and a Tukey-Kramer post hoc test (P\textless{}0.05).

\textbf{Figure 6. Transcriptomics analysis of mock and
\emph{S.~indica}treated the \emph{cbl7} mutant plants to wild-type
Arabidopsis.A} ) DEGs statistics for the \emph{cbl7} mutant and Col-3
control plants after co-cultivation with \emph{S.~indica} for 14
days.\textbf{B} ) KEGG enrichment analysis of induced DEGs in
\emph{cbl7}co-cultivated with the fungus versus similarly treated Col-3
plants. Each circle in the figure represents a distinct KEGG pathway,
and the circle size indicates the number of genes enriched in the
corresponding metabolic pathway. The significance of the observed gene
enrichment is represented by a color gradient referring to the adjusted
p-value (padj). \textbf{C} ) Venn diagram showing the numbers of DEGs in
Col-3 and \emph{cbl7} 14-days post infection with \emph{S.~indica}
compared to control plants that were mock infected. \textbf{D} )
Functional network analysis of DEGs in \emph{cbl7} and Col-3. Up- and
down-regulated genes are differentiated by color and shape. The shape
size refers to the different levels of differential expression according
to the\emph{log} \textsubscript{2}FC levels observed in the RNAseq
analysis. Functional associations are highlighted in the figure.

\textbf{Figure 7. Analysis of the colonization of Col-3 and
\emph{cbl7}roots with \emph{S.~indica}} . \textbf{A} ) Trypan blue stain
of root segments at 1 to 2 cm distance from the root tip. The figure
shows representative pictures for the three studied genotypes after 14
days of co-cultivation with S.~indica. B) Quantitative assessment of
root colonization by qPCR analysis of \emph{SiTEF1} . Depicted are
means\selectlanguage{english} +-\selectlanguage{english} SE of three independent biological experiments performed in
triplicate. The Arabidopsis \emph{GAPC2} gene served as endogenous
control. Asterisks indicate significant differences (Student's \emph{t}
-test: ***P\selectlanguage{english}[?]0.001).\selectlanguage{english}
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