3.2. CBL7 is required for fungus-mediated growth
promotion
CBL proteins are plant-specific Ca2+ 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 withS. indica . To test this hypothesis, we first conducted a yeast
two-hybrid analysis (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 Saccharomyces
cerevisiae HF7c cells to grow on selection medium (SD/-Trp/-Leu/-His)
containing 5 mM 3-amino-1,2,4-triazole. 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 (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
(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 CBL7and CIPK13 , as well as the previously described hak5mutant. The mutants were grown for seven days on ½ MS medium alongside
with corresponding wild-type control plants, before the seedlings were
transferred to PNM medium, where they were either co-cultivated withS. 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 S. indica in the different
genotypes. As shown in Figure 3A , the functional knockout ofCIPK13 had no impact on the total root network length. Bothcipk13 alleles exhibited a significant growth promotion through
the co-cultivation with S. indica , similar to the response of the
wild-type control plants. The same observation was made for thehak5 mutant (Figure 3C ). In contrast, the
loss-of-function mutants of CBL7 , cbl7-1 andcbl7-2 , are characterized by a substantial reduction of the
growth promoting effect observed in Col-3 control plants (Figure
3B ). The additional analysis of the total network area of the different
groups of seedlings confirmed the obtained results (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 withS. indica .
3.3. 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 cbl7-2 and Col-3 (wt) plants under control conditions. After
applying the same threshold as before (FDR padj. ≤ 0.05,
log2FC ≥ 1.25), we identified only 20 induced and 53
repressed DEGs (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 padj. ≤ 0.05,
log2FC ≥ 0.35), giving 119 induced and 947 repressed
DEGs, respectively (Figure 4A ,B ). Subsequent GO and
KEGG enrichment analysis revealed the repression of 33 genes associated
with the spliceosome (Figure 4C, Supplementary Data Sheet 3 ).
Furthermore, we were able to identify the induction of 23
(padj. = 1·13-12) and
11 (padj. = 3.45·10-2) genes related
to cell wall organization and ion transport GO terms, respectively
(Supplemental Data Sheet 3 ). Among the latter genes, we found
the high-affinity nitrate transporter NRT2.4 as well as the
low-affinity nitrate transporter 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 HAK5 was significantly
induced (log2FC = 1.85, padj. = 0.022).
Although our previous results questioned the role of HAK5 in the
plant-fungus interaction, the reiterated appearance of HAK5 led
us to analyze the potassium content in roots and shoots of cbl7-2and Col-3 to determine whether CBL7 could be involved in the regulation
of potassium homeostasis in Arabidopsis. To quantify the
K+ content in Arabidopsis seedlings, mutant and wt
seeds were germinated on ½ 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 S. indica chlamydospores and mock treated,
respectively. After another week of co-cultivation, the potassium
contents were quantified by atomic emission spectroscopy. As shown inFigure 5 , the potassium content of the 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+contents in response to the inoculation with S. indica (Conchillo
et al., 2021) was only observed in control plants, but not incbl7-2 . Interestingly, the K+ level in the
shoots of 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+ levels showed a tendency
to decrease in cbl7-2 shoots, there was no significant difference
between mock and fungus-treated cbl7-2 plants. Plants grown on
plates containing 1 mM KCl showed a similar picture (Figure
5B ), only wild-type Col-3 plants showed the expected decrease in
K+ in response to the S. indica infection.
Furthermore, we found a significant accumulation of K+in cbl7-2 roots, while the K+ content incbl7-2 shoots appeared to be significantly reduced. We have
therefore concluded that CBL7 contributes to the regulation of the
distribution of K+ in the plant. The functional loss
of CBL7 is unequivocally linked to an altered K+distribution profile, which is characterized by an accumulation of
K+ in the roots, most likely due to a lack of
transport of K+ 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+ levels in plants challenged
with S. indica , as cbl7 mutants present a reduced
reduction of K+ when co-cultivated withS. indica .
3.4. The cbl7 mutant shows increased
plant defense
responses
The above results highlighted the transcriptional regulation ofCBL7 in response to the infection of Arabidopsis roots withS. indica and its involvement in K+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 S. indica , we compared the RNA-seq data surveyed
from cbl7-2 knockout plants and corresponding Col-3 control
plants infected with S. indica and mock treated, respectively
(Supplementary Data Sheet 3 ). Quantitative analysis of
differential gene expression revealed a substantial increase in induced
genes in the cbl7 mutant relative to wt, while the number of
repressed genes in cbl7 is clearly diminished (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 cbl7-2 and Col-3 (S. indicavs mock) (Figure 6C ). Next, we performed a GO and functional
network analysis to identify biological processes significantly affected
by the loss of CBL7 . The main alterations in the cbl7mutant 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 (Figure
6B,D ). The enriched genes in these GO terms included a substantial
number of cytochrome P450 enzymes, such as CYP71B3 ,CYP71A12 , CYP71A13 , and 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 BGLU34 and 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) NAC042 , WRK33 , and 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 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 cbl7-2 ,
which included the genes GSTU10 , GSTU12 , GSTF3 ,GSTF6 , and 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 cbl7loss-of-function mutant, it must be concluded that CBL7 is involved in
the suppression of multilayered defense responses to facilitate the
establishment of S. indica in the root apoplast. However, other
processes that appear enriched in S. indica challenged wt plants
but not in 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 cbl7 and wt triggered by the fungus.
Based on our hypothesis of an increased defense response in thecbl7 knockout mutant, it must be expected that the roots of
mutant plants are less well colonized by S. indica than
comparable wt roots. To prove this, we analyzed root colonization incbl7 and Col-3 by qPCR and trypan blue staining. Indeed,root colonization of the cbl7 mutant was significantly reduced
(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.