The calcium sensor CBL7 is required for Serendipita indica‐induced growth stimulation in Arabidopsis thaliana, controlling defense against the endophyte and K⁺ homoeostasis in the symbiosis

Abstract

Calcium is an important second messenger in plants. The activation of Ca²⁺ signalling cascades is critical in the activation of adaptive processes in response to environmental stimuli. Root colonization by the growth promoting endophyte Serendipita indica involves the increase of cytosolic Ca²⁺ levels in Arabidopsis thaliana. Here, we investigated transcriptional changes in Arabidopsis roots during symbiosis with S. indica. RNA‐seq profiling disclosed the induction of Calcineurin B‐like 7 (CBL7) during early and later phases of the interaction. Consistently, reverse genetic evidence highlighted the functional relevance of CBL7 and tested the involvement of a CBL7‐CBL‐interacting protein kinase 13 signalling pathway. The loss‐of‐function of CBL7 abolished the growth promoting effect and affected root colonization. The transcriptomics analysis of cbl7 revealed the involvement of this Ca²⁺ sensor in activating plant defense responses. Furthermore, we report on the contribution of CBL7 to potassium transport in Arabidopsis. We analysed K⁺ contents in wild‐type and cbl7 plants and observed a significant increase of K⁺ in roots of cbl7 plants, while shoot tissues demonstrated K⁺ depletion. Taken together, our work associates CBL7 with an important role in the mutual interaction between Arabidopsis and S. indica and links CBL7 to K⁺ transport.

1. INTRODUCTION

Plants have evolved in spatial proximity to a multitude of different microorganisms in their surroundings. Their constant interaction with commensal, symbiotic and pathogenic microorganisms shaped highly specialized ecosystems in which plants find their niches and flourish. Since the initial description of the concept of mutual coexistence between dissimilar organisms (de Bary, ¹⁸⁷⁹; Hertig et al., ¹⁹³⁷), our knowledge regarding the symbiotic associations of plants with microorganisms has substantially advanced. Numerous studies unambiguously demonstrate that plant−microbe interactions are important to the structure, function and health of plant communities, and that symbiotic fungi contribute to the adaptations of plants to environmental stresses (Rodriguez et al., 2004).

Serendipita indica (formerly termed Piriformospora indica) is an axenically cultivable root colonizing plant endophyte of the order Sebacinales with an exceptionally broad host range (Mensah et al., 2020; Verma et al., ¹⁹⁹⁸; Weiss et al., ²⁰¹⁶). S. indica promotes plant performance and biomass production (Peskan‐Berghofer et al., 2004; Vadassery & Ranf, Drzewiecki, et al., ²⁰⁰⁹; Varma et al., ¹⁹⁹⁹), enhances nutrient assimilation (Bakshi et al., 2017; Prasad et al., ²⁰¹⁸), and confers increased biotic and abiotic stress tolerance to its host plants (Jogawat et al., 2016; Sun et al., ²⁰¹⁴; Waller et al., ²⁰⁰⁵). The establishment of the symbiosis between S. indica and 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‐related secondary metabolites, for example, 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 (Lahrmann et al., 2015; Nongbri et al., ²⁰¹²; Xu et al., ²⁰¹⁸).

The elevation of cytosolic Ca²⁺ concentrations in Arabidopsis root cells through the influx of Ca²⁺ via the Cyclic nucleotide gated channel 19 represents a further critical asset in consolidating the plant−fungus interaction (Jogawat et al., 2020; Vadassery & Ranf, Drzewiecki, et al., ²⁰⁰⁹). Calcium is an essential plant macronutrient that plays an important role in plant growth and development. At the same time, Ca²⁺ serves as an important second messenger in plants that is involved in orchestrating adequate responses to external signals, including biotic stresses (Thor, 2019). Cytosolic Ca²⁺ concentrations show highly dynamic and specific spatiotemporal patterns, which are governed by the type and intensity of the perceived stimulus (Pivato & Ballottari, 2021). Depending on the particular stimulus, plant cells respond by producing specific Ca²⁺ signatures that differ in their frequency, amplitude and duration (Batistič & Kudla, 2012). To decipher the different Ca²⁺ signatures, plants possess a broad set of different sensor molecules that either directly modify target proteins through phosphorylation or act through their physical interaction with specific partner proteins, including protein kinases (Kudla et al., 2018). This diverse set of Ca²⁺ sensor molecules encompasses Calmodulins (CaMs), Calmodulin‐like proteins (CMLs), Ca²⁺‐dependent protein kinases (CDPKs), Calcineurin B‐like proteins (CBLs), as well as their interacting kinases (CIPKs). The latter forming a two‐component system in which the CBLs act as Ca²⁺ sensors that relay their stimulation to specific CIPKs, which subsequently interact with downstream target proteins, such as nutrient transporters or ion channels (Liu & 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 (Lan et al., 2011; Lee et al., ²⁰⁰⁷; 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 corepressors ABI1 and ABI2 (Ohta et al., 2003). The Arabidopsis genome contains 10 CBL and 26 CIPK genes. Among each other, the CBLs and CIPKs form functional complexes and it is noteworthy that CBLs are not promiscuously interacting with all CIPKs, but show preferences and interact only with a specific subset of CIPKs to facilitate efficient signal transduction and integration (Kudla et al., 2010). Overall, the specific combination of CBL‐CIPK modules is the key to provide versatility and flexibility in the regulation of a multitude of external stimuli that marshal ion transport in plants (Tong et al., 2021). In addition to the regulation of K⁺ transporters, CBL‐CIPK interactions have recently been described to control the polarization of plasma membranes (Yang et al., 2019). In this manner, CBL‐CIPKs can also indirectly regulate voltage‐gated K⁺ channels, such as the outward‐rectifying K⁺ channels SKOR and GORK (Dreyer et al., 2004).

Up to date, the functional role of Calcineurin B‐like 7 (CBL7) is only partially elucidated. A recent study associates CBL7 with the regulation of plant responses towards low nitrate in Arabidopsis (Ma et al., 2015). The work highlights a substantial expression of CBL7 in root tissues and its induction under nitrogen and nitrate limiting conditions. Moreover, the authors report on the involvement of CBL7 in the transcriptional regulation of two high‐affinity nitrate transporter genes, NRT2.4 and 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 (TFs), such as NLP7 (Kiba & Krapp, 2016; Konishi & Yanagisawa, ²⁰¹³; Krouk & Kiba, ²⁰²⁰; Marchive et al., ²⁰¹³).

In this study, we identified the Ca²⁺ sensor CBL7 as an essential molecular component in the interaction between the root colonizing endophyte S. indica and A. thaliana. CBL7 is consistently induced upon root infection with the fungus, not only at early stages of the infection, but also at later phases. Furthermore, we were able to demonstrate that the availability of CBL7 is crucial for the development of the fungus‐mediated plant growth promotion and for the proper distribution of potassium in the plant. The comprehensive transcriptomics analysis of the cbl7 mutant grown with and without the fungus in comparison to corresponding wild‐type plants additionally pinpointed a role of CBL7 in harmonizing plant defense responses for the long‐term interaction between the endophyte and Arabidopsis. Taken together, our results establish CBL7 as a novel key component required for the successful establishment of the symbiosis between S. indica and A. thaliana.

2. MATERIALS AND METHODS

2.1. Biological material and growth conditions

In this work, we used the A. thaliana Col‐0 (N1092) and Col‐3 (N28171) backgrounds as references. We obtained the mutant alleles CBL‐interacting protein kinase 13 (cipk13‐1) (SALK_124748C) and cipk13‐2 (SALK_130671), as well as cbl7‐1 (SAIL_201_A01) from the Nottingham Arabidopsis Stock Centre (NASC). T‐DNA insertion lines were genotyped as previously described (Alonso et al., 2003), using the primer pairs listed in Supporting Information: Data Sheet 1. The T‐DNA insertion mutants cbl7‐2 and hak5 have previously been described (Ma et al., 2015; Ragel et al., ²⁰¹⁵). Plants were grown on Petri dishes containing solidified ½ MS medium supplemented with 1% sucrose (w/v) (Murashige & Skoog, 1962). Plant growth proceeded in a growth chamber under strictly controlled environmental conditions (16 h light, 8 h darkness, constant temperature of 22°C, 100−105 µmol photons m⁻² s⁻¹ photosynthetically active radiation). In addition, we used S. indica strain DSM 11827, which was obtained from the German Collection of Microorganisms and Cell Cultures (DSZM) in Braunschweig, Germany, and the plant growth‐promoting fungus Penicillium minioluteum that has been extracted from roots of Chenopodium quinoa grown in the Atacama Desert (González‐Teuber et al., 2018). The fungi were grown at 28°C in darkness on solidified arginine phosphate (AP) medium (Rodríguez‐Navarro & Ramos, 1984) and refreshed weekly.

2.2. Root growth promotion assay

Surface‐sterilized Arabidopsis seeds were plated on vertical ½ MS plates. After stratification (2 days at 4°C), the plates were transferred to the growth chamber and the seedlings were grown vertically for 1 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 harbouring a 1‐week‐old S. indica mycelium (cocultivation). The PMN plates with the control seedlings and the seedlings cocultivated with the fungus were further kept in a growth chamber maintained at 23.5°C, 16/8 h photoperiod, 100 µmol photons m⁻² s⁻¹ light intensity for another 10 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.

2.3. Quantitative analysis of root system architecture traits

The stimulation of root growth is a well‐described trait in the interaction of S. indica with its host plant. With the aim to quantify the effect of 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 to black and white images. The root network traits of the plants in the prepared images were then analysed 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 analysed, respectively.

2.4. Total RNA extraction, library construction and RNA‐seq analysis

To study transcriptional alterations provoked by either the cocultivation of Arabidopsis roots with S. indica or the functional knockout of CBL7, total RNA from 100 mg plant roots was extracted as previously described (Oñate‐Sánchez & Vicente‐Carbajosa, 2008). The quality and concentration of the extracted RNA was tested by absorbance analysis using a Nanodrop® ND‐1000 spectrophotometer (Thermo Fisher Scientific). After an additional confirmation of the RNA sample integrity on a Bioanalyzer 2100 (Agilent) by the Novogene Genomics Service, the service laboratory proceeded with the library construction and RNA sequencing (PE150) on Illumina NovaSeq™ 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 DESeq. 2 v1.22.2 algorithm with a cut‐off value of an adjusted p< 0.05 (Love et al., 2014). For each genotype and treatment, respectively, three biological replicates were processed. The resulting p values were adjusted for multiple testing using the Benjamini−Hochberg correction (Benjamini & Hochberg, 1995). Along with the adjusted p value [false discovery rate (FDR)] of <0.05 an absolute differential expression of log₂ fold change (FC) ≥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 & 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).

2.5. qPCR analysis

Real‐time quantitative RT‐PCR was conducted as previously described (Pérez‐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)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 Supporting Information: Data Sheet 1. The reactions were monitored on a Lightcycler 480 Real‐time PCR system (Roche Diagnostics). Differential gene expression in Arabidopsis was analysed by using the comparative $2^{‐\mathrm{\Delta \Delta }{C}_{\mathrm{t}}}$method (Livak & Schmittgen, 2001) with Adenine phosphoribosyl transferase 1 (APT1, At1g27450) as reference gene (Jost et al., 2007). Root colonization with S. indica was monitored with a primer pair for fungal translation elongation factor EF‐1α (TEF1) (Bütehorn et al., 2000). The fungal TEF1 cDNA levels were expressed relative to the plant Glycerinaldehyde‐3‐phosphate dehydrogenase C2 (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.

2.6. Trypan blue staining of fungal hyphae and spores

To visually inspect root colonization, 10−12 small root samples from control and cocolonized 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 five times with sterile H₂O, 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 10 min. Before the specimen were mounted on glass slides and examined by microscopy, they were washed once with 100% ethanol and three times with sterile H₂O and stored in 60% glycerol (v/v).

2.7. K⁺ quantification

The analysis of endogenous cation contents of infected and control plants was performed according to Conchillo et al. (2021) in fractions of root and shoot samples. In brief, the fungi were first K⁺ depleted buy inoculating 100 ml AP medium containing 1 mM K⁺ with 1.5 × 10³ of the corresponding spores and incubating it for 1 week at 28°C. Next, the mycelia were harvested by centrifugation and transferred to fresh AP medium without K⁺. After another week of incubation, the K⁺ starved spores were harvested. At the same time, Arabidopsis seedlings were germinated on ½ MS plates for 1 week. Thereafter, the seedlings were transferred to square Petri dishes containing modified Hoagland agar medium adjusted to pH5.7 (Álvarez‐Aragón et al., 2016) without glucose and either no KCl or 1 mM KCl. After 1 week, the roots of 20 Arabidopsis seedlings were inoculated with 250 µl of a solution containing 1.5 × 10⁶ spores ml⁻¹. Noninfected plants were grown under similar conditions. K⁺ quantification was performed after 1 week of cocolonization with the fungi. To avoid carry overs from the medium, roots were thoroughly rinsed with 10 mM MES‐Ca²⁺ pH 6.5. Next, root and shoot samples were dried, weight and extracted with 1 M HNO₃ at ambient temperature over 36 h. The K⁺ 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.

2.8. 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 t‐test were performed to statistically analyse 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).

3. RESULTS

3.1. CBL7 is induced in the symbioses between Arabidopsis and S. indica

The growth promoting effect of 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 S. indica and Arabidopsis, we took a comprehensive transcriptomics approach analysing the transcriptional differences between control plants and plants challenged with S. indica at both early [2 days post infection (dpi)] and later (10 dpi) stages of cocultivation. 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 _adj. ≤ 0.05) and setting the FC cut‐off to log₂FC ≥ 1.25, identified 138 induced and 10 repressed genes, respectively, at 2 dpi, while we identified 411 induced and 26 repressed genes after 10 days of cocultivation (Supporting Information:Data Sheet 2). The expression data revealed that 9.8% of the DEGs were induced both after 2 and 10 days of cocultivation on PMN medium, while 16.2% and 67.3% of the DEGs were only induced in the early and later phases of the interaction, respectively (Figure 1a). Notably, only 6.8% of the DEGs were repressed under the tested conditions, and only one gene, Senescence‐associated gene 13 (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 (Supporting Information: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 (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 Ca²⁺ homoeostasis and Ca²⁺ signalling processes, which attracted our attention and on which we followed up in this study.

Figure 1.

Transcriptional analysis of Arabidopsis seedlings cocultivated with Serendipita indica. (a) Venn diagram showing the numbers of differentially expressed genes in Arabidopsis plants 2‐ and 10‐days post infection with S. indica compared to control plants that were mock infected. (b) ClueGo analysis of induced DEGs. The figure shows the representative molecular function interaction among the targets. (c) qPCR analysis of transcriptional responses of identified target genes in plants that were cocultivated for 2 and 10 days, respectively, with S. indica compared to mock treated plants. The data represent means ± SE (n = 3). DEGs, differentially expressed genes; dpi, days post infection. [Color figure can be viewed at wileyonlinelibrary.com]

To gain a better understanding of the role of Ca²⁺‐related processes in the mutual interaction between S. indica and Arabidopsis, we extracted the relative expression profiles of 88 Ca²⁺ signalling‐related genes from the nonfiltered RNA‐seq data sets and performed a hierarchical cluster analysis (Supporting Information: Image 1B). We have been particularly intrigued by the expression profile of the genes in Cluster 1, which contained the four genes High affinity K ⁺ transporter 5 (HAK5), Calmodulin‐like 4 (CML4), CIPK13 and 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 (Figure 1c). Activation of these genes might therefore be required both for the establishment and preservation of the mutual plant−fungus interaction.

3.2. CBL7 is required for fungus‐mediated growth promotion

CBL proteins are plant‐specific Ca²⁺ sensors that are predicted to decode calcium transients through interaction with CIPKs (Tang et al., 2020). It has previously been shown that several CBL proteins, that is, CBL1, CBL8, CBL9 and CBL10, interact with CIPK23 to regulate HAK5 transport activity (Ragel et al., 2015). Hence, we speculated 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 S. indica. To test this hypothesis, 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 CBL7 and CIPK13, as well as the previously described hak5 mutant. The mutants were grown for 7 days on ½ MS medium alongside with corresponding wild‐type control plants, before the seedlings were transferred to PNM medium, where they were either cocultivated with S. indica or a mock control. After 10 days, the vertically grown plants were photographed (Supporting Information: Figure S2), and the root system architecture was analysed 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 2a, the functional knockout of CIPK13 had no impact on the total root network length. Both cipk13 alleles exhibited a significant growth promotion through the cocultivation with S. indica, similar to the response of the wild‐type control plants. The same observation was made for the hak5 mutant (Figure 2b). In contrast, the loss‐of‐function mutants of CBL7, cbl7‐1 and cbl7‐2, are characterized by a substantial reduction of the growth promoting effect observed in Col‐3 control plants (Figure 2c). The additional analysis of the total network area and biomass 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 on 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 behaviour when infected with S. indica.

Figure 2.

Total root network length of Arabidopsis wild‐type and mutant plants after 14 days of cocultivation with Serendipita indica or mock treatment. (a) Comparison of two cipk13 T‐DNA insertion mutant alleles with Col‐0. (b) The growth promoting effect of S. indica is not affected in the hak5 mutant. (c) Analysis of the fungus‐triggered growth promoting effect in Col‐3 and cbl7 mutant plants. The box plots show the median, quartiles and extremes of the compared data sets (n = 24). Asterisks indicate significant differences between S. indica and mock treated samples. Student's t‐test: **p ≤ 0.01, ***p ≤ 0.001. cbl7, Calcineurin B‐like; cipk13, CBL‐interacting protein kinase 13. [Color figure can be viewed at wileyonlinelibrary.com]

Table 1.

Growth promoting effects on Arabidopsis plants mediated by the endophytic fungus

Genotype	Network area (cm2)			Root fresh weight (mg)
	+ Serendipita indica	– S. indica	∆ ± S. indica	+ S. indica	– S. indica	∆ ± S. indica
Col‐3	7.3 ± 0.3	4.0 ± 0.3	3.3 ± 0.4***	126 ± 15	85 ± 15	41 ± 6**
cbl7‐1	6.0 ± 0.3	5.0 ± 0.4	1.0 ± 0.5	84 ± 11	64 ± 7	20 ± 4
cbl7‐2	5.4 ± 0.3	4.4 ± 0.2	1.0 ± 0.3	97 ± 15	73 ± 9	25 ± 5
Col‐0	3.8 ± 0.2	2.6 ± 0.2	1.2 ± 0.3**	96 ± 16	61 ± 12	36 ± 5**
cipk13‐1	4.3 ± 0.3	3.0 ± 0.2	1.3 ± 0.3***	112 ± 8	70 ± 6	42 ± 4**
cipk13‐2	3.3 ± 0.1	2.2 ± 0.2	1.1 ± 0.2***	105 ± 9	73 ± 10	31 ± 5**
hak‐5	4.7 ± 0.3	2.5 ± 0.2	2.2 ± 0.4***	100 ± 10	70 ± 13	31 ± 5*

Note: Differences in total root network area and root fresh weight were used as molecular marker of the growth promoting effect of S. indica on its host plants. Data represent means ± SE (n = 24). Asterisks indicate significant differences between S. indica and mock treated samples. Student's t‐test: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Abbreviations: cbl7, Calcineurin B‐like; cipk13, CBL‐interacting protein kinase 13.

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 mRNA‐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 p _adj. ≤ 0.05, log₂FC ≥ 1.25), we identified only 20 induced and 53 repressed DEGs (Figure 3a,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 _adj. ≤ 0.05, log₂FC ≥ 0.35), giving 119 induced and 947 repressed DEGs, respectively (Figure 3a,b). Subsequent GO and KEGG enrichment analysis revealed the repression of 22 genes associated with the spliceosome (Figure 3c). Furthermore, we were able to identify the induction of 18 (p _adj. = 3.34 × 10⁻⁹) and 12 (p _adj. = 2 × 10⁻³) genes related to cell wall organization and ion transport GO terms, respectively (Supporting Information:Data Sheet 2). 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, p _adj. = 0.022). Although our previous results questioned the role of HAK5 in the plant−fungus interaction, the reiterated appearance of HAK5 led us to analyse the potassium content in roots and shoots of cbl7‐2 and Col‐3 to determine whether CBL7 could be involved in the regulation of potassium homoeostasis in Arabidopsis. For this, the K⁺ contents of fungus and mock‐treated mutant and wt seedlings were quantified by atomic emission spectroscopy. As shown in Figure 4, 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 in cbl7‐2. 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 4b), only wild‐type Col‐3 plants showed the expected decrease in K⁺ in response to the S. indica infection. Furthermore, we found a significant increase of K⁺ in cbl7‐2 roots, while the K⁺ content in cbl7‐2 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 increase 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 cocultivated with S. indica.

Figure 3.

Analysis of transcriptional differences between cbl7 mutant and wild‐type Col‐3 plants. (a) DEGs statistics for the cbl7 mutant compared to Col‐3 control plants under control conditions. (b) Volcano plot of the distribution of all DEGs, mapping the 20 upregulated genes (red) and 53 down‐regulated genes (blue). The figure shows the relation between the significance [−log₁₀ (q value)] and the strength (log₂FC) of the differential expression. (c) Bar plot of the KEGG enrichment analysis of induced DEGs in 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 ‐log₁₀ (q value). cbl7, Calcineurin B‐like; DEGs, differentially expressed genes; FC, fold change. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4.

K⁺ contents of root and shoot tissues of Serendipita indica infected and noninfected control plants. The plants were grown in modified Hoagland medium under K⁺ starvation (0 mM KCl) (a) or at 1 mM KCl (b). The box plots show the median, quartiles and extremes of the compared data sets (n= 15). Different letters indicate significant differences between the compared conditions and genotypes analysed by ANOVA and a Tukey−Kramer post hoc test (p < 0.05). [Color figure can be viewed at wileyonlinelibrary.com]

3.4. The cbl7 mutant shows increased plant defense responses

The above results highlighted the transcriptional regulation of CBL7 in response to the infection of Arabidopsis roots with S. 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 mRNA‐seq data surveyed from cbl7‐2 knockout plants and corresponding Col‐3 control plants infected with S. indica and mock treated, respectively (Supporting Information: Data Sheet 2). Quantitative analysis of differential gene expression revealed a substantial increase in induced genes in the cbl7 mutant relative to wt (Figure 5a). Furthermore, the examination of the possible logical relations between the two data sets revealed four nonoverlapping and two overlapping groups for DEGs in cbl7‐2 and Col‐3 (S. indica vs. mock) (Figure 5c). 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 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 (Figure 5b,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 (Frerigmann et al., 2016; Glawischnig, ²⁰⁰⁷). Furthermore, we found an induction of the myrosinase genes BGLU34 and BGLU35 that are involved in the turnover of glucosinolates (Wittstock & Burow, 2010). The observation of an induction of plant defense‐related compounds is further supported by the induction of TFs NAC042, WRK33 and WRK51, which have been linked with the regulation of camalexin and indole glucosinolate biosynthesis (Birkenbihl et al., 2012; Frerigmann & Gigolashvili, ²⁰¹⁴; Saga et al., ²⁰¹²; 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). Moreover, 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 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 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 (Chen et al., 2010, ²⁰¹²; Ding et al., ²⁰¹⁵), or the repression of bHLH100 and MYB72 mediated metal ion homoeostasis (Sivitz et al., 2012; Zamioudis et al., ²⁰¹⁵), are likely to contribute to the significant difference in growth promotion between cbl7 and wt triggered by the fungus.

Figure 5.

Transcriptomics analysis of mock and Serendipita indica treated the cbl7 mutant plants to wild‐type Arabidopsis. (a) DEGs statistics for the cbl7 mutant and Col‐3 control plants after cocultivation with S. indica for 14 days. (b) KEGG enrichment analysis of induced DEGs in cbl7 cocultivated 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 colour gradient referring to the adjusted p value (padj). (c) Venn diagram showing the numbers of DEGs in Col‐3 and cbl7 14‐days post infection with S. indica compared to control plants that were mock infected. (d) Functional network analysis of DEGs in cbl7 and Col‐3. Up‐ and down‐regulated genes are differentiated by colour and shape. The shape size refers to the different levels of differential expression according to the log₂FC levels observed in the RNAseq analysis. Functional associations are highlighted in the figure. cbl7, Calcineurin B‐like; DEGs, differentially expressed genes; FC, fold change. [Color figure can be viewed at wileyonlinelibrary.com]

Based on our hypothesis of an increased defense response in the cbl7 knockout mutant, it must be expected that the roots of mutant plants are less well colonized by plant growth promoting fungi than comparable wt roots. To prove this, we analysed root colonization in cbl7 and Col‐3 by qPCR and trypan blue staining. Indeed, root colonization of the cbl7 mutant was significantly reduced, both for S. indica and P. minioluteum (Figure 6). Our experiments have shown that CBL7 plays a critical role in maintaining the equilibrium between symbiotic interaction and plant defense that keeps the load of endophyte fungi under control.

Figure 6.

Analysis of the colonization of Col‐3 and cbl7 roots with Serendipita indica. (a) Trypan blue stain of root segments at 1−2 cm distance from the root tip. The figure shows representative pictures for the three studied genotypes after 14 days of cocultivation with S. indica. (b) Quantitative assessment of root colonization by S. indica and Penicillium minioluteum using qPCR analysis of the corresponding TEF1 genes of the two fungi. Depicted are means ± SE of three independent biological experiments performed in triplicate. The Arabidopsis GAPC2 gene served as endogenous control. Different letters indicate significant differences between the compared conditions and genotypes analysed by ANOVA and a Tukey−Kramer post hoc test (p < 0.05). cbl7, Calcineurin B‐like; GAPC2, glycerinaldehyde‐3‐phosphate dehydrogenase C2. [Color figure can be viewed at wileyonlinelibrary.com]

4. DISCUSSION

Calcium signalling 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 & Oelmüller, 2009). 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). The coexpression 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 (Amo et al., 2021; Ragel et al., ²⁰¹⁵). Hence, an interaction cascade between the three components appeared possible. However, when we analysed 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 2). 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 large 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 the 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‐like 5 (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 & 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 3). The cbl7 mutant plants showed a pronounced increase of K⁺ contents 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; Gierth et al., ²⁰⁰⁵; Shin & Schachtman, ²⁰⁰⁴). 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 with S. 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 cocultivated 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 potassium channels/transporters. 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 cbl7‐2 mutant 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 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 genes EXT3, 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 uninfected 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 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 & 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‐deficient 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 S. indica and 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 cocultivation. 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 with S. 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 that CBL7 is induced during root infection of Arabidopsis with S. 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 increase of potassium in the roots. In this context, it needs to be remarked that the expression of neither SKOR and NPF7.3/NRT1.5 nor SGN3, which have previously been associated with K⁺ transport in Arabidopsis (Drechsler et al., 2015; Pfister et al., ²⁰¹⁴), appeared to be significantly altered in the cbl7‐2 mutant. 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.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Supporting information

Supporting information.

Supporting information.

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Supporting information.

Pérez‐Alonso, M.‐M. , Guerrero‐Galán, C. , González Ortega‐Villaizán, A. , Ortiz‐García, P. , Scholz, S.S. , Ramos, P. et al. (2022) The calcium sensor CBL7 is required for Serendipita indica‐induced growth stimulation in Arabidopsis thaliana, controlling defense against the endophyte and K⁺ homoeostasis in the symbiosis. Plant, Cell & Environment, 45, 3367–3382. 10.1111/pce.14420

DATA AVAILABILITY STATEMENT

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