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. indicaand Arabidopsis, we took a comprehensive transcriptomics approach analyzing 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 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) (padj. ≤ 0.05) and setting the fold-change 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 ten days of co-cultivation (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 (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 (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 (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+ homeostasis and Ca2+ signaling processes, which attracted our attention and on which we followed up in this study.
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 calcium signaling-related genes from the non-filtered RNA-seq datasets and performed a hierarchical cluster analysis (Supplemental 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 ),CBL-INTERACTING PROTEIN KINASE 13 (CIPK13 ), andCALCINEURIN B-LIKE PROTEIN 7 (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.