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.