Supporting Information Figs S1-S4 and Table S1
Pinus pinaster
seedling
Leaves
Watering tube
Stem
Parafilm
Roots
Hebeloma
cylindrosporum
plug
Filter paper
Liquid medium
(N1+P or N1+P-K)
Fig. S1 In vitro method of co-culture of the Pinus pinaster host plant and Hebeloma
cylindrosporum ectomycorrhizal fungus. Plant seedlings were co-cultivated with three to five
plugs of agarose containing the fungus in a glass tube. Filter paper was placed in the tube to
maintain the plant-fungus system against the tube wall and to let the culture media travel
up. N1+P or N1+P-K media were provided regularly using the watering tube. The system was
covered with parafilm, with the aerial part of the plant outside the tube.
Relative expression of
HcTrk1
(a)
45
40
35
30
25
20
15
10
5
0
T0
T24 -K
T48 -K
Resup24 +K
T0
-Na
T24 -K
T48 -Na
-K
Resup24 -Na
+K
Relative expression of
Hctrk1
(b)
45
40
35
30
25
20
15
10
5
0
Fig. S2 Quantification of the expression level of the potassium transporter HcTrk1 under K+
and Na+ deprivation in Hebeloma cylindrosporum h7. Expression levels of HcTrk1 were
quantified by RT-qPCR after (a) K+ or (b) Na+ removal for 24 and 48 h, followed by 24 h of resupply. Relative expression levels were normalized using the α-tubulin housekeeping gene
from H. cylindrosporum. Mean values are given with standard errors (n = 4) for each data
point.
Normalized relative expression
200
E.V.
**
180
OE-Trk1-7
OE-Trk1-9
160
140
120
100
80
*
**
60
40
20
0
HcTrk2
HcKUP
HcHAK
HcTOK1
HcTOK2.1
HcTOK2.2
HcSKC
Fig. S3 Expression of putative potassium transport systems of Hebeloma cylindrosporum in
HcTrk1 over-expressed fungal lines. Expression levels of HcTrk2 and HcHAK transporters
and HcTOK1, HcTOK2.1, HcTOK2.2 and HcSKC channels were quantified using RT-qPCR in
the empty vector (E.V.) and over-expressing (OE-Trk1-7 and OE-Trk1-9) fungal strains.
Mean values are given with the standard error (n = 4 to 6) for each data point. Statistical
tests were made with the Student’s test, with respect to the empty vector control (*: p <
0.05; **: p < 0.01).
Relative expression of
HcTrk1
(a)
**
45
40
35
30
25
20
15
10
5
0
T0
T24 -P
T48 -P
Resup24 +P
(b)
**
Relative expression of
HcPT1.1
60
50
40
30
20
10
0
T0
T24 -K
T48 -K
Resup24 +K
Fig. S4 Quantification of the expression level of the potassium transporter HcTrk1 and
phosphate transporter HcPT1.1 under Pi and K+ deprivation respectively in H.
cylindrosporum h7. Expression levels of HcTrk1 (a) and HcPT1.1 (b) were quantified by RTqPCR after Pi (a) or K+ (b) restriction for 24 and 48 h, followed by 24 h of re-supply. Relative
expression levels were normalized using the α-tubulin housekeeping gene from H.
cylindrosporum. Mean values are given with standard errors (n = 4) for each data point.
Statistic tests were made with Student’s test with respect to the T0 point (p < 0.01). (**:
p < 0.01).
Amplification
HcTrk1Translational
fusion vector
HcTrk1 cDNA
HcTrk1- In situ
hybridization
PCR verifications
Tubuline-qPCR
HcTrk1-qPCR
HcTrk2-qPCR
HcHAK-qPCR
HcSKC-qPCR
HcTOK1-qPCR
HcTOK2.1-qPCR
HcPT1.1-qPCR
HcPT2-qPCR
Name
PromTrk1-SpeI-F
FT-Trk1-SpeI-R
PromTrk1-Trk1-F
PromTrk1-Trk1-R
Trk1-SpeI-F
OETrk1-SpeI-R
ISHTrk1-T7-F
ISHTrk1-R
ISHT7-Prom
pPZP-219-F
pPZP-492-R
Pgpd-F
qPCR-Tub-F
qPCR-Tub-R
qPCR-Trk1-F
qPCR-Trk1-R
qPCR-Trk2-F
qPCR-Trk2-R
qPCR-HAK-F
qPCR-HAK-R
qPCR-SKC-F
qPCR-SKC-R
qPCR-TOK1-F
qPCR-TOK1-R
qPCR-TOK2.1-F
qPCR-TOK2.1-R
qPCR-PT1.1-F
qPCR-PT1.1-R
qPCR-PT2-F
qPCR-PT2-R
5' - 3‘ Sequence
CCTTACTAGTTTACCTAGAACCAAGCTCATCGACG
CCTTACTAGTAATTCTAAGAGGCGGTAAATTAGGAG
TTATACCTATCCTGATACCCATGGTGGACCAGCCCGCAGA
TCTGCGGGCTGGTCCACCATGGGTATCAGGATAGGTATAA
ACTAGTATGGTGGACCAGCCCGCAGATTTA
ACTAGTCTAAATTCTAAGAGGCGGTAAATTAGG
GCGAAATTAATACGACTCACTATAGGGCGAAGGATTGCTTCAAGGATTCTTCCGGA
GCGAAATTAATACGACTCACTATAGGGCGAAGGGTGCAAGGCTCGCAACTGGAAC
GCGAAATTAATACGACTCACTATAGGGCGAA
TCATTAGGCACCCCAGGCTTTAC
ATTGTAAAGCGGGGTGCCTGA
GAGCTCGGTACCCGGGGATCAGCTTTA
GTCTTCAAGGCTTCTTCGTCTTC
ACAGTCAGAGTGCTCCAAGGTAGT
TGGAACCCAACTCATTGCTG
GAAACCAAGCGAGGTTGAGC
GAGTTCAAGCAGGACGAAGG
CACACCATTCCCGTTCTCTT
GTGCGGTTTTCCACAAGATT
GCGAACCTTGGTTACGACAT
GGCCAGATTGGGAAAGGCCG
CCGGCCAACTCCTCTATCTG
GGAACCAGGGGATGGTCTAT
ATGGGTTGTATGCGGAAGAG
GGTGCACCACTATCCGAACT
CACCCATGCTTACGTGTGTC
CACAAATAAATTCGTCAAGCATATTCTCG
GCGTTCTCGCACACCTCTG
CTTCGGTTGCTGTATCGCTG
TACGCACACGGATTTCCTCC
Table S1 Primer list. Restriction enzyme sites indicated in primer name, are underlined in
the corresponding 5’-3’ sequence.