Pinus pinaster seedling

Watering tube

Parafilm

Hebeloma cylindrosporum

plug

Filter paper

Liquid medium (N1+P or N1+P-K)

Leaves

Stem

Roots

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.

Supporting Information Figs S1-S4 and Table S1

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T0 T24 -K T48 -K Resup24 +K05

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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 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.

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).

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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 RT-qPCR 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).

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Amplification Name 5' - 3‘ Sequence

HcTrk1- Translational fusion vector

PromTrk1-SpeI-F CCTTACTAGTTTACCTAGAACCAAGCTCATCGACGFT-Trk1-SpeI-R CCTTACTAGTAATTCTAAGAGGCGGTAAATTAGGAG

PromTrk1-Trk1-F TTATACCTATCCTGATACCCATGGTGGACCAGCCCGCAGAPromTrk1-Trk1-R TCTGCGGGCTGGTCCACCATGGGTATCAGGATAGGTATAA

HcTrk1 cDNATrk1-SpeI-F ACTAGTATGGTGGACCAGCCCGCAGATTTA

OETrk1-SpeI-R ACTAGTCTAAATTCTAAGAGGCGGTAAATTAGG

HcTrk1- In situ hybridization

ISHTrk1 T7-F‑ GCGAAATTAATACGACTCACTATAGGGCGAAGGATTGCTTCAAGGATTCTTCCGGAISHTrk1-R GCGAAATTAATACGACTCACTATAGGGCGAAGGGTGCAAGGCTCGCAACTGGAAC

ISHT7-Prom GCGAAATTAATACGACTCACTATAGGGCGAA

PCR verificationspPZP-219-F TCATTAGGCACCCCAGGCTTTAC pPZP-492-R ATTGTAAAGCGGGGTGCCTGA

Pgpd-F GAGCTCGGTACCCGGGGATCAGCTTTA

Tubuline-qPCRqPCR-Tub-F GTCTTCAAGGCTTCTTCGTCTTCqPCR-Tub-R ACAGTCAGAGTGCTCCAAGGTAGT

HcTrk1-qPCRqPCR-Trk1-F TGGAACCCAACTCATTGCTGqPCR-Trk1-R GAAACCAAGCGAGGTTGAGC

HcTrk2-qPCRqPCR-Trk2-F GAGTTCAAGCAGGACGAAGGqPCR-Trk2-R CACACCATTCCCGTTCTCTT

HcHAK-qPCRqPCR-HAK-F GTGCGGTTTTCCACAAGATTqPCR-HAK-R GCGAACCTTGGTTACGACAT

HcSKC-qPCRqPCR-SKC-F GGCCAGATTGGGAAAGGCCGqPCR-SKC-R CCGGCCAACTCCTCTATCTG

HcTOK1-qPCRqPCR-TOK1-F GGAACCAGGGGATGGTCTATqPCR-TOK1-R ATGGGTTGTATGCGGAAGAG

HcTOK2.1-qPCRqPCR-TOK2.1-F GGTGCACCACTATCCGAACTqPCR-TOK2.1-R CACCCATGCTTACGTGTGTC

HcPT1.1-qPCRqPCR-PT1.1-F CACAAATAAATTCGTCAAGCATATTCTCGqPCR-PT1.1-R GCGTTCTCGCACACCTCTG

HcPT2-qPCRqPCR-PT2-F CTTCGGTTGCTGTATCGCTGqPCR-PT2-R TACGCACACGGATTTCCTCC

Table S1 Primer list. Restriction enzyme sites indicated in primer name, are underlined in the corresponding 5’-3’ sequence.