Materials and methods
2.1. Biological material and growth
conditions
In this work, we used the Arabidopsis thaliana Col-0 (N1092) and
Col-3 (N28171) backgrounds as references. We obtained the mutant allelescipk13-1 (SALK_124748C) and cipk13-2 (SALK_130671), as
well as cbl7-1 (SAIL_201_A01) from the Nottingham Arabidopsis
Stock Center (NASC). T-DNA insertion lines were genotyped as previously
described (Alonso et al., 2003), using the primer pairs listed inSupplemental Data Sheet 1 . The T-DNA insertion mutantscbl7-2 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
and Skoog, 1962). Plant growth proceeded in a growth chamber under
strictly controlled environmental conditions (16 h light, 8 h darkness,
constant temperature of 22 ºC,
100 to 105 µmol photons m-2 s-1photosynthetically active radiation). In addition, we usedSerendipita indica strain DSM 11827, which was obtained from the
German Collection of Microorganisms and Cell Cultures (DSZM) in
Braunschweig, Germany. The fungus was grown at 28 ºC in darkness on
solidified arginine phosphate (AP) medium (Rodríguez-Navarro and 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 ºC), the plates were
transferred to the growth chamber and the seedlings were grown
vertically for one 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 harboring a one-week-oldS. indica mycelium (co-cultivation). The PMN plates with the
control seedlings and the seedlings co-cultivated with the fungus were
further kept in a growth chamber maintained at 23.5 ºC, 16/8 h
photoperiod, 100
µmol photons m-2 s-1 light intensity
for another fourteen 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
(fw).
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 the pictures to black and white
images. The root network traits of the plants in the prepared images
were then analyzed 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
analyzed, respectively.
2.4. Total RNA extraction, library construction, and RNA-seq
analysis
To study transcriptional alterations provoked by either the
co-cultivation 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 and
Vicente-Carbajosa, 2008). The quality and concentration of the extracted
RNA was tested by absorbance analysis using a Nanodrop® ND-1000
spectrophotometer (ThermoFisher). After an additional confirmation of
the RNA sample integrity on a Bioanalyzer 2100 (Agilent) by the Novogene
Genomics Service (Cambridge, UK), 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 DESeq2 v1.22.2 algorithm with a cut-off value of
an adjusted p -value of < 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 and
Hochberg, 1995). Along with the adjusted p -value (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 and 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 Supplementary Data
Sheet 1 . The reactions were monitored on a Lightcycler 480 Real-time
PCR system (Roche Diagnostics). Differential gene expression in
Arabidopsis was analyzed by using the comparative
2-∆∆CT method (Livak and Schmittgen, 2001) withADENINE PHOSPHORIBOSYL TRANSFERASE 1 (APT1 , At1g27450) as
reference gene (Jost et al., 2007). Root colonization withS. indica was monitored with a primer pair for fungal translation
elongation factor EF-1α (SiTEF1 ) (Bütehorn et al., 2000). The
fungal SiTEF1 cDNA levels were expressed relative to the plantGLYCERINALDEHYDE-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 co-colonized 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 5 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 ten minutes. Before the
specimen were mounted on glass slides and examined by microscopy, they
were washed once with 100% ethanol and thrice with sterile
H2O and stored in 60% glycerol (v/v).
2.7. Yeast two-hybrid
analysis
In order to examine the physical interaction between CBL7 and CIPK13,
total RNA from four weeks-old Arabidopsis seedlings (Col-0) was
extracted (Oñate-Sánchez and Vicente-Carbajosa, 2008) and first-strand
synthesis was performed according to the supplier’s instructions, using
M-MLV reverse transcriptase and oligo(dT)15 primer
(Promega). For the cloning of CBL7 and CIPK13 , the
corresponding cDNA fragments were amplified by PCR using specific primer
pairs listed in Supplementary Data Sheet 1 . The resulting PCR
fragments were inserted into the vector pGEM® -T Easy (Promega).
Sequence integrity of the obtained products was confirmed by commercial
sequencing (StabVida). Subsequently, the fragments were introduced into
the vector pENTR-3C (Thermo Fisher) using the Eco RI andSal I restriction sites included in the primer sequences. The
obtained pENTR-CBL7 and pENTR-CIPK13 vectors were used in Gateway
LR-recombination reactions with the destination vectors pDEST-22 and
pDEST-32 according to the manufacturer’s instructions. The resulting
destination vectors were then used to transformSaccharomyces cerevisiae strain HF7c. Transformants were plated
on SD (simple drop-out) medium /-Leu/-Trp/+Ade and incubated at 28ºC for
2 days. Protein interaction was tested by growing transformants on SD
medium /-Leu/-Trp/-His/+Ade. The plates were incubated at 28ºC for up to
one week. To investigate the interaction of CBL7 with two additional
putative interaction partners, CIPK9 and CIPK24, the yeast two-hybrid
vectors PDEST-AD092F08 and PDEST-AD107D03 for CIPK9 and CIPK24,
respectively, were obtained from the Arabidopsis Biological Resource
Center (ABRC).
2.8. K+ ion content
quantification
The analysis of endogenous cation contents of infected and control
plants was performed in fractions of root and shoot samples. 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. 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.
2.9. 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 analyze 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).