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
Oelmüller, 2009). Ca2+ signaling depends on the
comprehensive interaction between membrane-located receptors that sense
external signals, which then trigger transient changes in intracellular
Ca2+ levels through the activation of
Ca2+ permeable channels that release
Ca2+ from internal stores, such as the endomembrane
system, or transport Ca2+ across the plasma membrane.
Within the cytoplasm, a wide array of different
Ca2+-binding proteins are responsible for deciphering
the incoming transient Ca2+ signatures and translating
them into appropriate molecular responses (Pirayesh et al., 2021). These
Ca2+ sensors include calmodulins (CaMs),
calmodulin-like-proteins (CMLs), Ca2+-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 S. indica , both at early and later
stages of the interaction (Figure 1 ). Our studies also
demonstrated that CIPK13 is a target of CBL7 in yeast two-hybrid studies
(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 S. indicaon its host plants, which particularly manifests in a considerable
induction of root growth (Pérez-Alonso et al., 2020). In addition, the
co-expression of CBL7 , CIPK13 , and 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
(Figure 3 ). While both 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 in vivo(Schliebner et al., 2008).
Previous work on the role of CBL7 highlighted its interaction with theA. 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 Ca2+-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 Shaker -like
K+ channels in the root. SKOR, an outward-rectifying
K+ channel, is reported to be crucial for the loading
of K+ into the xylem and, thus, the long-distant
transport of K+ within the plant (Gaymard et al.,
1998). Moreover, SKOR is known to form heteromeric outward-rectifying
K+ channel units with a second Shaker -like
channel, GORK (Dreyer et al., 2004). SKOR and GORK facilitate
K+ 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+ would accumulate in the
roots, and only reduced amounts of K+ would reach the
shoot. In fact, our analysis of K+ levels in wild-type
and cbl7 mutants corroborates this hypothesis (Figure
4 ). The cbl7 mutant plants showed a pronounced accumulation of
K+ in the roots, which we attribute to possible
impairment of K+ xylem transport. Potassium depletion
in the shoot could trigger the induction of HAK5 in cbl7 ,
as multiple independent studies demonstrated that HAK5 is induced
under K+ starvation (Ahn et al., 2004; Armengaud et
al., 2004; Shin and Schachtman, 2004; Gierth et al., 2005). A recent
study demonstrated the induction of HAK5 in the host plant as a
general consequence of the symbiotic interaction (Conchillo et al.,
2021), which would also explain why we found HAK5 under the
consistently induced genes in wild-type plants (Figure 1). Furthermore,
the latter study demonstrated that the inoculation of Arabidopsis withS. indica does not improve the K+ nutrition of
the host plant under K+ limiting conditions.
Intriguingly, the authors showed that root colonization is stimulated
under K+ limiting conditions, leading to the
assumption that the endophyte may benefit from the relatively high
cellular K+ concentrations in plant cells or apoplast.
The K+ content in plant tissue was reported to be
significantly reduced when plants were inoculated with S. indica .
Our own work confirms this finding of reduced K+levels in wt plants co-cultivated with the fungus. On the contrary, the
reduction of K+ levels upon infection with the fungus
was largely absent in cbl7 mutant plants, possibly because the
flux of K+ 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 S. indica -triggered growth
promotion in inoculated 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 cbl7-2 mutant to additional RNA-seq analyses. In a first
analysis, we compared the transcriptional pattern of the cbl7-2mutant with that of the corresponding wild type, Col-3, under control
conditions. After applying an arbitrary cut-off value of
log2FC ≥ 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 SUPPRESSOR-OF-WHITE-APRICOT/SURP DOMAIN-CONTAINING
PROTEIN (SWAP ) gene (Lorkovic et al., 2005). This suggests a
possible impairment of alternative splicing processes in cbl7 . It
is, however, noteworthy that the gene that showed the strongest
repression with a log2FC of –2.34,MOS4-ASSOCIATED COMPLEX SUBUNIT 5C (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 HYDROXYPROLINE-RICH GLYCOPROTEIN 1 (HRGP1 ) and the
proline- and leucine-rich extensin-like family protein genesEXT3 , EXT4 , EXT6 , EXT10 , and LRX1 ,
respectively, which are moderately induced in 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 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 cbl7-2 . When comparing the transcriptional responses
of cbl7-2 plants infected with S. indica versus uninfectedcbl7-2 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 cbl7-2 . This group
contains the TF genes NAC042 , WRK33 , and WRK51 , as
well as the cytochrome P450 genes CYP71B3 , CYP71A12 ,CYP71A13 , and 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 S. indica(Nongbri et al., 2012). The authors provide conclusive evidence that the
infection of Arabidopsis with 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-deficientcyp79b2/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 S. indicaand Arabidopsis into a physiological burden for the host plant. In case
of the 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 cbl7 mutant roots withS. indica relative to those of wild-type controls.
In summary, our work provides functional analyses of the cytoplasmic
Ca2+ sensor CBL7 in the context of plant-fungus
interactions. Based on the presented results, we propose thatCBL7 is induced during root infection of Arabidopsis withS. indica and contributes to the control of the classical plant
defense. The loss of CBL7 unequivocally blocks the normally
observed promotion of 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, Ca2+ 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+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 S. indicaand 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 cbl7 are the cause of the observed reduced root
colonization and missing fungus-triggered plant growth promotion.