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.