Supplemental Figure 1. Gene structure of G. margarita GigmPT. (A) The GigmPT gene contains

three introns. The splicing sites and the UTRs are indicated; TSS indicates the transcriptional start

site. (B) Southern blot analysis of G. margarita genomic DNA digested with BamHI, EcoRI, or

PstI. The blots were hybridized with gene-specific probe. The GigmPT gene does not contain

BamHI or PstI sites.

Supplemental Figure 2. Protein structure of GigmPT in Gigaspora margarita. The putative

membrane-integrated protein contains 543 amino acids and 12 TM domains. The deduced amino

acid sequence of GigmPT was aligned with that of GiPT from Rhizophagus irregularis, GvPT

from Glomus versiforme and GmosPT from Funneliformis mosseae through the MULTIALIN,

and 12 TM domains (TM1-TM12) of GigmPT was predicted by using TopPred (Yadav et al.,

2010).

Supplemental Figure 3. The putative cis-regulatory elements were screened in the promoter of

GigmPT and other mycorrhizal fungal phosphate transporter (PT) genes using the available

Promoter Database of Saccharomyces cerevisiae (SCPD) (http://rulai.cshl.edu/SCPD/). The

PHO2-like DNA binding consensus WWWRTTGAAT, the PHO4-like or NUC-1 transcription

factor binding sites CACGTK (or CACATG), the carbon-response elements CWTCC and

CGGANNA, the stress-response elements (STRE) AGGGG (or CCCCT), and the STE12 DNA

binding consensus TGAAACA were further screened in the promoter region of the PT genes from

mycorrhizal fungi and other fungi using dna-pattern matching analysis (http://rsat.ulb.ac.be/rsat/).

Both PHO4-like DNA binding consensus and carbon-response elements were universally present

in the promoter region of the PT genes from mycorrhizal and filamentous fungi, and yeast. In

contrast, the STRE and STE12 elements were absent in the promoter sequence of GigmPT gene.

The homologous genes and their corresponding accession numbers employed were: GigmPT

(KC887075), GigmPT2 (gi|780761933) and GigmPT5 (gi|780775803) in Gigaspora margarita,

RiPT1 (gi|974129332) and RiPT2 (gi|974129334) in Rhizophagus irregularis DAOM 197198,

HcPT1 (gi|342671945) and HcPT2 (gi|159025254) in Hebeloma cylindrosporum, LbPT1

(gi|170115639), LbPT2 (gi|170115665), LbPT3 (gi|170115669), LbPT4 (gi|170099503) and

LbPT5 (gi|170114113) in Laccaria bicolor, TmPT (gi|296423696) Tuber melanosporum Mel28,

PiPT (gi|281324610) Piriformospora indica, pho-4 (gi|168859) and pho-5 (pho84) (gi|536859) in

Neurospora crassa and pho84 (gi|218454) in Saccharomyces cerevisiae.

Supplemental Figure 4. GigmPT expression depends on phosphate availability and evidence of

phosphate uptake by the extraradical mycelium of G. margarita. (A) expression of GigmPT in

mycorrhizal A. sinicus hairy roots exposed to different Pi concentrations for 14 d. Pi0, Pi3, Pi30,

and Pi300 indicate 0 μM Pi, 3 μM Pi, 30 μM Pi, and 300 μM Pi, respectively. (B) Expression level

of GigmPT in mycorrhizal roots in response to Pi availability. Total RNA was isolated from

mycorrhizal roots (4 wpi) after treatment with 65, 200 and 1000 μM Pi, respectively, for 14 d. (C)

Transcript accumulation of GigmPT in extraradical mycelium exposed to different Pi

concentrations for 14 d. (D) Transcript accumulation of GigmPT in extraradical mycelium in

response to different Pi concentrations for 14 d. Pi65 and Pi1000 indicate 65 μM Pi and 1000 μM

Pi, respectively. The GigmPT genes is expressed as a ratio relative to Gigm-Actin transcripts. The

error bars represent the means of three biological replications with SD values. (E) Uptake of

phosphate by extraradical mycelium. Extraradical mycelium was grown for 28 d in liquid MSR

medium and exposed to 3 μM Pi, 30 μM Pi, or 300 μM Pi after incubation for an additional 14 d

in MSR medium (the left figure); the residual Pi concentrations (the black bars) in liquid MSR

medium were shown after treatment (the right figure). grey: initial Pi concentrations; white:

control Pi. (F) Assessment of polyphosphate and ALP activity in extraradical mycelium of G.

margarita. Polyphosphate accumulation in extraradical mycelium exposed to different Pi

concentrations, and the percentage of ALP activity was increased in extraradical mycelium after Pi

treatment. (Error bars represent SD. n=3)

Supplemental Figure 5. Immunolocalization of GigmPT. The epifluorescence microscopy

images of arbuscular mycorrhizal roots of M. truncatula/G. margarita probed with GigmPT

antibodies visualized with a secondary antibody conjugated with Alexa Fluor 488. The AM roots

were counterstained with WGA-Texas red to visualize G. margarita. (A1) and (B1) Nomarski

views of an arbuscule are shown. (A2) and (B2) Corresponding images showing red

fluorescence from WGA-Texas red staining. (A3) The images showing green fluorescence from

GigmPT immunostaining. (B3) Immunostaining with GigmPT preimmune serum. Merged images

showing both bright field and green fluorescence (A4 and B4), or both red and green fluorescence

(A5 and B5). Overlaps of bright field, red and green fluorescence images are shown (A6 and B6).

GigmPT signal is visible in the arbuscule (a). (B) Immunostaining with GigmPT preimmune

serum to test the specificity of the antibody. The GigmPT preimmune serum does not stain the

arbuscule. a, arbuscule. Scale Bars = 50 μm.

Supplemental Figure 6. Physiological analyses of the mycorrhizal A. sinicus hairy roots in

response to changes in sucrose concentration (0 to 90 mM). (A) 33

P in the hairy roots of G.

margarita plants in response to changes in sucrose concentration at 6 wpi. (B) The significant

effect of Pi availability (300 μM) on allocation of sucrose to hyphae in the fungal compartment

(HC). (C) Long-chained PolyP concentrations in mycorrhizal hairy roots in response to changes in

sucrose concentration, in dpm mg-1

root dry weight. (D) Ratio of short-chain to long-chain polyP

in mycorrhizal hairy roots after addition of sucrose. Asterisks indicate significant differences

within each treatment (* P<0.05). d.wt.: dry weight.

Supplemental Figure 7. Transcription of the G. margarita phosphate transporters was affected by

the sucrose supply. (A-C) Expression patterns of GigmPT, GigmPT1 and GigmPT2 genes in the

intraradical mycelia (IRM) and in the extraradical mycelia (ERM) are sucrose-regulated under low

Pi (3 µM) or high Pi (300 µM) conditions. Transcript levels of GigmPT (A), GigmPT1 (B) and

GigmPT2 (C) in IRM and ERM, and AsPT1 and AsPT4 (D) in mycorrhizal roots of A. sinicus

supplemented with various sucrose levels for 14 days. (E-F) Time-course analysis of GigmPT

expression in the IRM and the ERM of G. margarita grown in low Pi (E) or high Pi (F) media

after the addition of 30 mM sucrose (Suc.) to the root compartment. The equal volume of H2O was

added to the root compartment of control Petri dishes. Relative gene expression was analyzed by

Real-time RT-PCR. The actin genes from G. margarita (Gigm-Actin) and A. sinicus (As-Actin)

were used as reference genes, respectively. Data are from three independent experiments and error

bars represent SD of three biological replicates with three technical replicates (n=3). Asterisks

denoted statistically significant (*P < 0.05) in comparison to the respective control value.

Supplemental Figure 8. (A-B) Mycorrhization level was analyzed in A. sinicus roots inoculated

with G. margarita challenged for 14 days with various sucrose treatments under low Pi (A) or high

Pi (B) conditions. (C-D) Molecular quantification of the fungal colonization of roots challenged

for 14 days with various sucrose treatments under low Pi (C) or high Pi (D) conditions, based on

the expression of the house keeping gene ACTIN from A. sinicus and from G. margarita (BEG34).

Values are the average Ct (Cycle threshold) of three biological and three technical replicates.

(Error bars represent SD; n=3)

Supplemental Figure 9. HIGS of GigmPT gene from G. margarita in A. sinicus hairy roots. (A)

The expression of hairpin construct in hairy roots was validated by RT-PCR. The hairpin loop

waxy-a intron I and GigmPT RNAi targeting region (305bp) were amplified by using specific

primers. (B) GigmPT dsRNA was detected by northern blot analysis. (C) GigmPT siRNAs was

detected by northern blot analysis with specific riboprobe. The rRNA in lines were shown in gel.

EV, empty vector. (D) Expression levels of GigmPT, GigmPT1, AsPT1 and AsPT4 in control (EV)

and RNAi lines were determined by real-time RT-PCR. The actin genes were used as reference

genes. Asterisks indicate a statistically significant difference from respective vector control lines.

(error bars represent SD; **P<0.01; n=3, three technical replicates)

Supplemental Figure 10. HIGS of GigmPT gene from G. margarita in composite plants. (A) The

expression of hairpin construct in lines was confirmed by RT-PCR. The hairpin loop waxy-a

intron I and GigmPT RNAi targeting region (305bp) were amplified by using specific primers.

GigmPT dsRNA was tested by northern blot analysis using the specific probe. (B) GigmPT

siRNAs was detected by northern blot analysis with specific riboprobe. The rRNA in lines were

shown in gel. EV, empty vector; Ri, GigmPT RNAi line.

Supplemental Figure 11. Fluorescence-microscopic characterization of G. margarita infection in

RNAi and EV transgenic roots at early stage (11dpi) of AM symbiosis. (A) The appearance of

WGA488 stained mycorrhizal hyphal structures in transgenic roots of EV (a-d) and RNAi (e-j)

lines. app, appressorium; ih, intraradical hyphae; a, arbuscules. Scale bars=100 µm. (B-C)

Abundance of GigmPT gene transcripts and siRNAs in G. margarita-infected A. sinicus roots. (B)

Relative expression of GigmPT in RNAi and EV transgenic roots after G. margarita infection at

11 days post inoculation by quantitative RT-PCR using AM fungal actin as the reference gene.

The data represent the average and SD of three replicates. (C) Detection of the siRNA

complementary to the GigmPT gene in G. margarita-infected A. sinicus roots at 11 dpi. RNA gel

blot using DIG-labeled GigmPT RNA was performed. No signal was detected in the controls from

EV roots. Ethidium bromide-stained rRNA in the lower panel acted as the loading control. (D)

Diagrams indicate the rates of total infection frequency (F%), appressorium frequency (APP%),

internal hyphae intensity (IH%) as well as the arbuscule abundance (A%) in the whole root system.

Asteriks indicate levels of significance of a Student’s t-test on all values evaluated for EV and

RNAi lines: *** p<0.001; ** p<0.01.

Supplemental Figure 12. Abundance of the GigmPT gene and its paralogs transcripts and

siRNAs in the extraradical mycelium (ERM) from G. margarita. (A-C) The transcript levels of

three PT genes in G. margarita were estimated by qRT-PCR using AM fungal actin as the

housekeeping gene. (A) GigmPT, (B) GigmPT1, and (C) GigmPT2. The total RNA was isolated

from the extraradical mycelium (ERM) grown in hyphae compartment. Error bars represent

average and SD of three technical replicates. The repression in expression of GigmPT in the ERM

from RNAi lines compared with the ERM from EV control line was statistically significant

(*P<0.05; Student’s t-test) (D) Detection of the siRNA complementary to the GigmPT gene in the

ERM from G. margarita at 8 wpi. Northern blot analysis using DIG-labeled GigmPT RNA was

performed. No signal was present in the ERM from the control line. EB-stained rRNA in the lower

panel served as the loading control.

Supplemental Figure 13. Heatmap analysis of phosphate signalling related genes.

DESeq2-normalised expression data for the phosphate signalling related genes, under different

developmental conditions, were plotted in heatmaps for comparison. These genes are possibly

involved in PHO (A), PKA (B), MAPK (C) and TOR (D) signalling pathways. Included

conditions were: germinating spores (germ_spores_mean), strigolactone analogue GR24

(SL)-treated spores (strigo_spores_mean) and symbiotic mycelium thriving inside the roots

(myc_roots_mean) (Salvioli et al., 2015). Heatmaps were generated with the MeV

(MultiExperiment Viewer, v4.9) software. Color scale at the left top of each heatmap indicates the

range of expression values. Descriptions as obtained from GO annotation are shown. PHO,

phosphate signal transduction; PKA, protein kinase A; MAPK, mitogen-activated protein kinase;

TOR, target of rapamycin.

Supplemental Figure 14. Gly3P and PAA are competitive inhibitors for GigmPT Pi uptake. (A)

Uptake of 32

Pi (0.05 mM) in the presence of different Gly3P concentrations. (B) Uptake of 32

Pi in

the presence of different phosphate concentration in the lacking and existing of 0.5 mM, 1 mM,

and 2 mM Gly3P. (C) Uptake of 32

Pi in the existing of different PAA concentrations. (D) Uptake

of 32

Pi in the presence of different phosphate concentration in the lacking (PAA0) and existence of

5 mM PAA (PAA5).

Supplemental Figure 15. Increase in trehalase activity after addition of 1 mM KH2PO4 to the

pho84Δ:pGigmPT(wt) strain and the Ile373C

(A), or Thr374C

(B) mutants without (-M) and with (+M)

pre-addition of 10 mM MTSEA. (The SD were shown. n=3)

Supplemental Figure 16. The view of the phosphate transporter GigmPT model. The 12 TM

helices showed with roman numerals. The targeted residues for SCAM and mutagenesis analysis

are Arg154

, Asp164

, Asp322

, Glu440

and Lys459

.

Supplemental Figure 17. SCAM and mutagenesis analyses of selected residues in TMDs

involved in the recognition and transport of phosphate. (A) to (D) PKA activation after addition of

1 mM KH2PO4 to phosphate-starved cells of pho84Δ:pGigmPT(wt) strain and the Arg154C

(A),

Asp164C

(B), Asp322N

(C) or Lys459E

(D) mutants without (-M) and with (+M) preaddition of 10 mM

MTSEA. (E) Uptake of 1 mM KH2PO4 in phosphate-starved cells of the pho84Δ:pGigmPT (wt)

strain and the Arg154C

, Asp164C

, Asp322N

or Lys459E

mutants without (White) and with (Black)

preaddition of 10 mM MTSEA. (F) Uptake of 1 mM KH2PO4 in the Arg154C

, Asp164C

, Asp322N

or

Lys459E

mutants without (-M) and with (+M) preaddition of 10 mM MTSEA in the lacking or

existence of 50 mM KH2PO4. (The SD were shown. n=3)

Supplemental Figure 18. Signaling and transport via the phosphate-binding site of the GigmPT

Pi transceptor. (A) The GigmPT phosphate transceptor in yeast transports phosphate and mediates

rapid phosphate activation of the protein kinase A (PKA) pathway during growth induction. The

parts with dotted arrows and question marks in the pathway require further evidence. (B)

Postulated mechanism for recognition and H+/Pi co-transport through GigmPT. According to the

previous results, it has been showed the following recognition and transport mechanisms: (i) The

residue Ala146

might couple with Val357

for the initial interaction with substrate; (ii) Arg154

may be

required for the initial transport of substrate; (iii) Asp322

and Lys459

appear to be play a major role

in the recognition and transport of substrate. Substrate is recognized by Lys459

, and then the Asp322

forms a hydrogen bond. Pedersen et al.(2013) proposed that Asp324

(equivalent to Asp322

in

GigmPT) serves as a central proton donor/acceptor in PiPT crystal structure. (iv) Asp322

might be

deprotonated after the release of substrate, then Asp164

is protonated again and interacts with the

substrate. The black circle represents a proton and the blue circle an inorganic phosphate molecule.

Black and blue triangles indicate the concentration gradient of H+ and Pi respectively. occl.,

occluded. Co, outward-facing open; Ci, inward-facing open.

Supplemental Figure 19. Schematic representation of the main nutrient exchange processes and

GigmPT as phosphate sensor in AM symbiosis. The nutrient carriers transport P, N and C at the

soil-fungus and fungus-plant interface. Pi transporter (GigmPT) from AM fungus G. margarita

operates at both the soil-fungus and fungus-plant interfaces, and is essential for AM symbiosis. At

the fungus-plant interface, there also exists a second Pi-transporter AsPT1 (AsPT1 might be a

transceptor) identified from A. sinicus, and also indispensable for the development of AM

symbiosis. In turn, the reciprocal transfer of carbon from plant to AM fungus, and the exchange of

carbon for phosphate are tightly linked.

Supplemental Figure 20. Schematic representation of the dual roles of GigmPT in the regulation

of putative PHO and PKA pathways in arbuscules (A-B) and extraradical hyphae (C-D). (A) In

arbuscules, activation of PHO pathway for sensing and re-absorption of Pi in PAS in the absence

of Pi through GigmPT transporter, while the putative vacuolar Pi transporter Pho91 ortholog of the

yeast (Hürlimann et al., 2007) and AM fungi R. irregularis (Tisserant et al., 2012) and Gigaspora

rosea (Tang et al., 2016) has been proposed to regulate Pi and polyP metabolism. (B) In the

presence of Pi, the low-affinity Pi transporters pho87/90 orthologs of the yeast (Hürlimann et al.,

2009) and AM fungi R. irregularis (Tisserant et al., 2012) and Gigaspora rosea (Tang et al., 2016)

as well as pho1-like proteins Syg1.1/1.2 like the orthologous PHO1 and XPR1 in plant and animal

(Hamburger et al., 2002; Giovannini et al., 2013), respectively, may be responsible for the Pi

homeostasis in arbuscules, whereas the PHO pathway is repressed. Concomitantly, GigmPT

functions as a Pi receptor regarding activation of PKA signaling cascade. (C) In extraradical

hyphae, in the absence of Pi, the PHO signaling subjected to GigmPT-gating is almost similar to

which in arbuscules. (D) In the presence of Pi, the putative pho87 and pho90 may be responsible

for Pi transport. In such a case, a PKA signaling cascade similar to which in arbuscules is also

present in extraradical hyphae. The arrowed and flat-ended lines refer to positive and negative

interactions, respectively. IP7, inositol heptakisphosphate; PolyP, inorganic polyphosphate; Glu.,

glucose; HA1, H+-ATPase (Wang et al., 2014; Krajinski et al., 2014); SPX, Syg1-Pho81-Xpr1

domain; TM, transmembrane domain; EXS, ERD-Xpr1-Syg1 domain; PAM, periarbuscular

membrane; PAS, periarbuscular space; APM, AM fungal plasma membrane. The description of the

depiction is detailed in following Supplemental Figure legend.

Supplemental Table 1. Summary of amino acids identity (%) between G. margarita GigmPT and other

fungal and plant phosphate transporters.

Name of the Organism Phosphate transporter Accession Number Identity with GigmPT (%)

Gigaspora margarita GigmPT (543) KC887075 100

Rhizophagus irregularis GintPT (521) AF359112 56

Rhizophagus irregularis GiPT2(297) remain_c8224 17

Rhizophagus irregularis GiPT4(217) remain_c6893 18

Glomus versiforme GvPT (521) U38650 55

Saccharomyces cerevisiae PHO84 (596) D90346 46

Lotus japonicas LjPT3 (544) AB257214 40

Medicago truncatula MtPT4 (528) AY116210 39

Astragalus sinicus AsPT1(559) JQ956415 38

Astragalus sinicus AsPT4(527) JQ956418 39

Arabidopsis thaliana AtPT2 (534) U62331 39

Solenum tuberosum StPT3 (535) AJ318822 37

Supplemental Table 2. The maximal increase of trehalase activity after supply of inorganic

phosphate or different organic phosphate esters to phosphate-starved yeast cells.

Stain

Compound WT GigmPT GiPT2 PHO84 GiPT4 pho84Δ

Agonists

Phosphate 65.90±8.49 74.55±16.05 63.20±21.21 80.45±6.29 17.45±3.04 23.10±5.52

Gly-3-P 62.34±16.64 55.45±6.58 55.90±7.78 56.30±3.54 10.40±5.37 27.55±1.91

Glu-6-P 55.70±14.85 70.46±7.80 50.90±6.36 48.70±13.72 25.90±11.31 13.80±4.53

disodium β-glycerol 49.20±15.56 62.45±13.79 54.60±9.33 44.00±14.14 20.90±3.96 23.45±6.01

Non-agonists

Phosphonoacetic acid 28.00±6.22 28.50±13.73 25.40±4.67 22.95±8.56 12.65±6.43 15.85±6.29

Phosphonpropionic acid 21.25±4.95 30.00±12.49 15.55±3.18 31.65±24.11 2.85±1.63 14.00±3.54

Triethyl phosphonoacetate 7.10±2.97 17.10±4.24 20.90±7.64 15.25±3.04 9.45±2.05 3.30±2.12

Background trehalase activity before supply of inorganic phosphate or different organic phosphate esters was

74.3±13.60 for the WT strain, 76.80±6.92 for the pho84Δ+pGigmPT strain, 64.30±7.39 for the pho84Δ+pGiPT2

strain, 67.10±4.07 for the pho84Δ+pPHO84 strain, 13.28±3.13 for the pho84Δ+pGiPT4 strain, 15.85±1.75 for

pho84Δ strain with empty vector112A1NE. This background activity has been subtracted from the peak value after

addition of the compound. (SD are shown in this table. n=3)

Supplemental Table 3. PCR primers used for GigmPT phosphate transporter assays.

Gene Primer name Primer sequences (from 5’ to 3’) Used for

GigmPT GigmPT7 GGAGGMGAYTAYCCHCTDKC

Clone gene GigmPT8 GATCKGTAACGWGTBGGGAA

GigmPT

GigmPT-A1 TTGCGGAGATAGAAGAATGGAATGC Inverse PCR

GigmPT-A2 GCATAGCGAAAACAGCAGCAATC Inverse PCR

GigmPT-A3 GCATACCCTTTTACGACCCACG Inverse PCR

GigmPT-A4 CAAGGCAGATAGCAAATGTAGA Inverse PCR

GigmPT-S1 CGTTACTACTTTCGTTGTGCCTG Inverse PCR

GigmPT-S2 GGTTTTGCTGTTCTCACCATAC Inverse PCR

GigmPT-S3 TTACTATACCTGAGAGTCCACG Inverse PCR

GigmPT-S4 TTGGTAAATGGAAAAATGGAAGG Inverse PCR

GigmPT

GigmPT5R1 GGTATATTGGCTGATCTCGTGGG 5’RACE

GigmPT5R2 CCATAATACACGTAGCCTAACATG 5’RACE

GigmPT3F CATGGATTAAGTGCAGCCTCCGG 3’RACE

GigmPT GigmPT-T1 TACAGTAATCCAATAACCCG Southern blot

GigmPT-T2 TTCCATTCTTCTATCTCCGC Southern blot

GigmPT GigmPTPF GGCAAGCTTGTCACCGATGAGACTATTCCG Promoter

GigmPTPR ACGGATCCGGTTATTAACTATTTTTATTTATAT Promoter

GigmPT GigmPT-Y1 TCATGGATTAAGTGCAGCCTC Real-time PCR

GigmPT-Y2 AGAAAACATAAAGATAGCGAAT Real-time PCR

Gigactin Gigm-actinF TGTTCTTGACTCTGGTGATGG Real-time PCR

Gigm-actinR CAAATCACGACCAGCCAAATC Real-time PCR

ALP GigmALPF ACCGAATATCTTAACTTGGATCCTG Real-time PCR

GigmALPR AAGTTCGGCGAGGTATATGACC Real-time PCR

AsPT1 AsPT1QF TAATCAGAAAACAAGAGGAGG Real-time PCR

AsPT1QR GGTTGAGTAGATAGCACAGGA Real-time PCR

AsPT4 AsPT4QF1 CAGAAACAAAGGGAAGATCATTGG Real-time PCR

AsPT4QR1 CATGTAATTCAGATCCTTCACACTG Real-time PCR

β-actin AsactinF GTTCTTTTCCAGCCTTCTATGA Real-time PCR

AsactinR ATGTTTCCGTACAGATCCTTTC Real-time PCR

GigmPT

GigmPTF ATGACCGAAGAAAATATAGTAATTGAAG ORF

GigmPTR TTAGTTGACTTGATTGTTATCATTGACTTC ORF

GigmPTFF AACTGCAGATGACCGAAGAAAATATAGTAATTGAAG GFP fusion

GigmPTFR CATGCCATGGTGTTGACTTGATTGTTATCATTGACTTC GFP fusion

GigmPTCF AAACTGCAGATGACCGAAGAAAATATAGTAATTGAAG Complementation

GigmPTCR GCGGATCCTTAGTTGACTTGATTGTTATCATTGACTTC Complementation

GigmPTdsF TAACTAGTGGTACCATGACCGAAGAAAATATAGTAATTGAAG HIGS

GigmPTdsR TAGAGCTCGGATCCCACGAGATCAGCCAATATACC HIGS

GigmPTsiRP GTTTCATATCAGGACATGCATC siRNA detection

Waxy-a W-a-intronIF TCTGATCTGCTCAAAGCTCTG Waxy-a intron I

W-a-intronIR AGGAGCAGTTTCTTGGGTG Waxy-a intron I

GiPT2 GiPT2F1 CCGGAATTCATGGAACCATATAGTTATTCTTGGC Complementation

GiPT2R1 CGCGGATCCTTAAACTAGATGACACATCTTCGT Complementation

GiPT4 GiPT4F1 CCGGAATTCATGGCGTCTCCGATATCATCACC Complementation

GiPT4R1 CGCGGATCCTCACTGAATGATACTAACC Complementation

PHO84 PHO84F CCACGAATAGAATTCAAATGAGTTCCGTC Complementation

PHO84R GGGGGATCCTTATGCTTCATGTTGAAGTTGAGATGGG Complementation

Supplemental Table 4. Oligonucleotides used for site-directed mutagenesis of the GigmPT gene

Amino acid substitution Mutated oligonucleotide (5’- to -3’)

A146CF CGAATGCTAAAGTTATCTCCTGTCAAGGCCTTATGTTATTCTTG

A146CR CAAGAATAACATAAGGCCTTACACGGAGATAACTTTAGCATTCG

M150CF GTTATCTCCGCTCAAGGCCTTTGTTTATTCTTGCGTATTCTCTTG

M150CR CAAGAGAATACGCAAGAATAAACAAAGGCCTTGAGCGGAGATAAC

R154CF GGCCTTATGTTATTCTTGTGTATTCTCTTGGGTATCGGAATTGG

R154CR CCAATTCCGATACCCAAGAGAATACACAAGAATAACATAAGGCC

G158CF GTTATTCTTGCGTATTCTCTTGTGTATCGGAATTGGTGGTGATTATC

G158CR GATAATCACCACCAATTCCGATACACAAGAGAATACGCAAGAATAAC

G160CF CTTGCGTATTCTCTTGGGTATCTGTATTGGTGGTGATTATCCCCT

G160CR AGGGGATAATCACCACCAATACAGATACCCAAGAGAATACGCAAG

G162CF GTATTCTCTTGGGTATCGGAATTTGTGGTGATTATCCCCTTTCCGC

G162CR GCGGAAAGGGGATAATCACCACAAATTCCGATACCCAAGAGAATAC

D164CF CTTGGGTATCGGAATTGGTGGTTGTTATCCCCTTTCCGCAATTATTG

D164CR CAATAATTGCGGAAAGGGGATAACAACCACCAATTCCGATACCCAAG

P166CF GTATCGGAATTGGTGGTGATTATTGCCTTTCCGCAATTATTGCTAGTG

P166CR CACTAGCAATAATTGCGGAAAGGCAATAATCACCACCAATTCCGATAC

S168CF GAATTGGTGGTGATTATCCCCTTTGCGCAATTATTGCTAGTGAGTTC

S168CR GAACTCACTAGCAATAATTGCGCAAAGGGGATAATCACCACCAATTC

I171CF GATTATCCCCTTTCCGCAATTTGTGCTAGTGAGTTCGCTACAACG

I171CR CGTTGTAGCGAACTCACTAGCACAAATTGCGGAAAGGGGATAATC

A172CF GATTATCCCCTTTCCGCAATTATTTGTAGTGAGTTCGCTACAACGAAAC

A172CR GTTTCGTTGTAGCGAACTCACTACAAATAATTGCGGAAAGGGGATAATC

D322NF GCTTTTTCATGGTTCGCTTTAAATATAGCTTTTTATGGTATAGGT

D322NR ACCTATACCATAAAAAGCTATATTTAAAGCGAACCATGAAAAAGC

V357CF GAAACTTTATATAAAGCTTCTTGTGGTAATATTATCATCGCAATG

V357CR CATTGCGATGATAATATTACCACAAGAAGCTTTATATAAAGTTTC

I373CF GGTACTGTACCGGGTTATTGGTGTACTGTATTCCTCATTGATATTTG

I373CR CAAATATCAATGAGGAATACAGTACACCAATAACCCGGTACAGTACC

T374CF GTACTGTACCGGGTTATTGGATTTGTGTATTCCTCATTGATATTTGG

T374CR CCAAATATCAATGAGGAATACACAAATCCAATAACCCGGTACAGTAC

E440QF ACTACTTTCGTTGTGCCTGGTCAAGTTTTCCCAACTCGTTACCGT

E440QR ACGGTAACGAGTTGGGAAAACTTGACCAGGCACAACGAAAGTAGT

K459EF GATTAAGTGCAGCCTCCGGTGAATTAGGTGCAATTATTTCACAAG

K459ER CTTGTGAAATAATTGCACCTAATTCACCGGAGGCTGCACTTAATC

Supplemental Table 5. List of the primers used in quantitative RT-PCR experiments.

Gene name Gene ID Forward (5’- 3’) Reverse (5’- 3’)

GigmPT1 gi|780761939 ACTTGCATTCATAGACAAAATGTTTG CACCTTTAACAAAACTATCTTGATCTTC

GigmPT2 gi|780761933 AAGTTTTCCCCACGAGATACC AACGACTGCTCCTAACTTTCC

GigmPT3 gi|780730427 AGTATTAGGGTTTGGAGTGGTTC GTTGCTTGTTCGATGTCTTGC

GigmPT4 gi|780736522 TGCGTGTGGGACTTTGATAG AGACATTGCAGAACCGAAGG

GigmPT5 gi|780775815 TGGTTGAAACGCCAAAAGC AAGACCATAGAACGCCACATC

Pho87 gi|780762093 AGGGAATTTATAGTCTGGGAAAGG CTAATTGCTCAACTGCCACAG

Pho90 gi|780762106 GGATTAATGCTACTTATTGGC CCGATGATTCAATAACCAACAG

Pho91 gi|780762097 ATGAAATTCTCTCACTCACTTC GCTTTTTGAGGTTTGAGTATGC

Pho88 gi|780859215 CCTCAGGGGCTCATGATAATT CCCCATTATGCAATGTATCGC

Syg1.1 gi|780698274 CTCTTAGAAATTTCGAGGTAATT GCGACGTGTTCAGTTTCAAC

Syg1.2 gi|780698250 CTCTTAGAAATTTCGAGACGTTG GCGACGTGTTCAGTTTCAAC

HA5 gi|780684722 CGCAACACTTGTCATAGCTG GTGAGCAAAAGAAACACCGG

VIP1 gi|780725165 TTCCCACAAGAATTCCCAGG TCCTTTTCACCAGATATACCACG

Pho81 gi|780739152 TCCCCTTTACATTTAGCATCTCG TCTATGTTCCCTTCACTTGCAG

Pho80 gi|780702733 AGTCGATCAGCCGATTTTGAG TCATTATATCGAAGGGCTGTTACC

Pho85 gi|780730893 CTCCTGTGACTACGTTACCTTC ACTTAAGTTACGTGGCAGTGG

Pho2 gi|780738370 TGGTATCACTGTTCCGCAAG GGATCACCAGCACCTAAAGAG

NUC-1 gi|780924604 TCCCCAAACACTAAAACCTACG CCAAGTGATTTCGATGTTCCC

Msn5 gi|780681364 AGTCAAGCATCCGAAGGTAAAG AAACACCAGCAACTACCCTAC

Pho5 gi|780662882 TTGCTGGTCATACTCATGTTCTAG CATTCCCCAATCTTTGCCAG

Pho12 gi|780746361 TCAAGAACGGGTCAATCTGG TCATGTCACTCAACGATCCG

ACP5 gi|780603269 CTCGTCTTCGACCACTCTTTG TCAAATTTACCTCCGGCACC

ALP1 gi|780859265 TTGATCATATATTCAGTGTGCC CCGTGTTTTCGTCTAAATCG

ALP2 gi|780859271 GAAATAACAGTAACGATACCG CCGTGTTTTCGTCTAAATCG

Vtc1 gi|780757476 GCAAACGAGCGAACATTTCTC ATCTCCAAAGTTGAGCAGTCC

Vtc2 gi|780702490 CCCTTGTGGAAGAGAATCGAG TTCATGGGACGGTGTATCAAG

Vtc4 gi|780878247 TCTATTCACTTCCCAAGCACC CCCTTCGTCATAATCTTCCTCG

Ppn1 gi|780939511 CGGTGATAACGCTAGGCATG TGTTTTAGTTCTTGTGGTTCTTGG

Ppx1 gi|780928403 TCCCTGGTTAATCATGCAGTG GCATAAGATACTGATCCCACGATAG

Gde1-1 gi|780871058 TCACTTTGCTTACAGGATCGG TGATTCAATTCCTTCGGCTCC

Gde1-2 gi|780934162 CAACAGGCAAAGGTGAAGC ACAGCCCAAACCTTAGAATCTT

Vma10 gi|780931383 AGAGGCTGAAAAGGATGCTG GTTCTGCTTCGAATGATTGAAATTC

TPK1 gi|780760266 CACATACCCAATCTACTCACCC ACTATAGGAAGACGGAGGTGAG

BCY1 gi|780932770 ACCCACTACAGATTACTTTCCAG TGATTCGGCACTTACAGACG

Sch9 gi|780711113 GTCCTTATTGTGTCGTTGAATTCG CAGCCTTTTGTTTCCAGACG

NTH1 gi|780895131 AAACCCCTAACTTGTCTAGCG TTCTGGAAATGGGAGACACAG

TPS2 gi|780740247 GATGGCACACTTACACCAATTG CACGCCCAGATACTATCCATAC

TSL1 gi|780931993 TCTAACCGTTTGCCAGTCAC GCCACCCAATCCAAGTAAATG

Rim15 gi|780657393 TCCTTTTGTGCCTAACCCTG CTACGATCCACATAGGTAGCAAC

Msn4 gi|780720225 GGGATCTTTACCTTCACCACC TCGTCCTCTTGATTTGCCAG

Gene name Gene ID Forward (5’- 3’) Reverse (5’- 3’)

Gis1 gi|780742112 GAGTTTCTTGTTTCGCCTTCTG TCGCCCTCTCGTTGAATAAC

SSA3 gi|780619294 GTGTGGGTGTATGGCAAAATG ATAGGGATTCATGGCGACTTG

Hsp30 gi|780641595 CGCTCGCACAAGATTTTAATCG TCAGAAACATCAACAGACGGAC

Hsp20 gi|780763221 GGAAATACCCACATTCAAGAACG CCTTTGGAAGTTTCACCTCAAG

SOD1 gi|780604800 CTCAAAAGTCTCCAACGAAAGC AAATCCATGATCTCCGTCTGTG

SOD2 gi|780861972 TGACACTTCCGAACCAAGATC ACCACATTCCAAATAGCTTTCAAG

RPS13 gi|780768075 GGTAAAGGTATCTCAAGCTCGG CGTCTTCGGGTGTAGTCTTTAAC

Mapk2 gi|780932989 AGACGATGTATTTGGGATGTGG GCAGTAAACTTGAAACTCCGC

mek-2 gi|780693002 CAGCACCAAAACTACCACAAG AGCCGAAGCCTTAACATATGG

NRC-1 gi|780604878 TGGACTTAATTCTGCCACCG ATGTGCAGAAGTTCCAGTAGG

os-4 gi|780861357 CCTGTTGAACGATAGACTGTCC CGCAGTAAAGTTGGTATGTGTATG

os-5 gi|780886967 TGCTTCACTGCTATGTATCCG TTGTAAATAGAGCAGGCGAGG

os-2 gi|780670055 TTAGCTCGTGTTCAAGATCCTC TCTACCCTCCAACATTTCAGC

mik-1 gi|780769471 TCCTCGAACATCCTCAATTGG TGTGACTGCTCCGGTAAATG

mek-1 gi|780666060 AAGAAATGTAGGCTCTAGCGC GAGATGGAGAAAGAGACGAAGG

mkc-1 gi|780645193 CAGCGAACCTACCCTATCAAG TGGCCTTTTCTGCATACGAG

Tor2.1 gi|780692220 GACGTCACAATTTACATCATTCCTC GTAAGCATAGACAACTTGTGGTGCTGC

Tor2.2 gi|780692216 GAAAGAGTGCACCCATCGATTTTAG TAACATAATTCAACAATATGCCGCC

Tor2.3 gi|780692208 GAAAGAGTGCACCCATCGATTTTAG ATCAGGTATAGTTTCCGGTTCCC

Gad8 gi|780924017 CTACGCCCATTCTAGCTTCTG CATGCTATCTCTGTTACCCCG

Ste20 gi|780729226 CTGATGCTGTGGGTGAGAAG TCTTCGTCCCTTTCAACATCC

Sin1 gi|780603739 AAGGTGAACAGTCTAAGGCG CGTGTATGGGAGCTTAAGTGAG

Mip1 gi|780604082 GTGCGTATCCAACCTAGAGATG CCAGATTTACTCCCAGACACC

Supplemental Results

Sequence analysis of GigmPT promoter

To obtain further insights into the promoter sequence of the GigmPT gene, the 5’

flanking region of GigmPT gene (1488 bp upstream of the translation initiator ATG)

was isolated using inverse PCR to search for putative cis-regulatory elements exist in

the promoter regions of phosphate-regulated genes of yeast, filamentous and

mycorrhizal fungi (Supplemental Figure 3). One CTCATG motif functioning as the

possible DNA binding site of the PHO4-like or NUC-1 transcription factors, key

components of the phosphate signal transduction (PHO) pathway in fungal species

identifies so far, was found at position -394 (relative to the translation initiator ATG)

(Supplemental Figure 3). In addition, two CWTCC and three CGGANNA

consensuses of the carbon-response elements that in yeast recognize Gcr1p, a

transcription factor of genes involved in glycolysis (Huie & Baker, 1996), and

transcriptional activator Rtg1 which plays a major role in the glucose-induction of

glucose transporter genes (Kim & Johnston, 2006), were found at the positions

-462/-501 and -869/-893/-1418 (Supplemental Figure 3), respectively.

Knock-down of GigmPT affects the arbuscule development.

Asynchronization nature of AM fungal growth within the roots determines

non-synchronous arbuscule morphology. Consequently, in AM roots, arbuscule

formation, arbuscules with low-order or dichotomous branches and arbuscule

senescence continue to occur in cortical cells, and the host roots harboring a

population of developing, mature, degenerating, collapsed or dead arbuscules.

Therefore, we measured the arbuscule size and investigate the arbuscule population

by analysis of arbuscule morphology classes.

To evaluate the arbuscule size and the arbuscule population representing the

arbuscule development in GigmPT RNAi and EV roots, the arbuscule size in RNAi-2

and EV-1 roots were classified into eight classes. In the EV roots, a significant

increase in comparison to RNAi-2 line in arbuscule size in the 65-75 and 75-85 μm

classes was detected, while a significant decrease in comparison to RNAi-2 line in

arbuscules in the 15-25 μm class was observed; however, in RNAi-2 roots, the

observed arbuscule classes showed the opposite size distribution (main text, Figure

5C). As expected, RNAi-2 and control roots also contained a considerable proportion

of arbuscules in the 35-45 and 45-55 μm classes as well as in the 55-65 μm class. In

contrast, fewer arbuscules in the 0-15 m formed in both RNA-2 and control roots.

In order to unravel the alterations in the proportion of arbuscule developmental

stages, confocal images of arbuscules were analyzed after acid fuchsin staining. we

next qualitatively classified the arbuscule developmental stages based on the

magnitude of arbuscule branching and the formation of septa on arbuscule branches.

The control roots harbored almost 60% mature arbuscules without septa on the

branches or hyphae and contained about 20% of observed arbuscules with septa on

arbuscule branches, and less than 18% of the collapsed arbuscules were detected in

the same samples (main text, Figure 5D). In contrast, we found a significant shift of

mature arbuscules towards degenerating and collapsed or dead arbuscules in the

RNAi-2 roots (main text, Figure 5D), indicating that activation of GigmPT delayed

the progression to senescence stages of arbuscule development.

To further investigate if GigmPT silencing would affect arbuscule development also

in composite plants with hairy roots, in which mycorrhiza was well-established and

the synchronicity of arbuscule development was better than in hairy roots, we

transformed roots with GigmPT RNAi vector carrying a hairpin RNAi-silencing

cassette. As expected, these transformed roots strongly expressed the hairpin construct

and the siRNAs (Supplemental Figures 10A and 10B). We then inoculated GigmPT

RNAi and control composite plants with G. margarita and detected the colonization

28 dpi. All the GigmPT RNAi roots except for RNAi-13 line showed lower

abundance of arbuscules than the control roots (main text, Figure 6). Relative to the

controls (main text, Figures 6 A and 6B), GigmPT RNAi-7,-8,-9,-14 and -15 roots

showed a dramatically reduced arbuscule development (main text, Figures 6C-6E and

6G-6H). Similar to the GigmPT RNAi-1 and-2 in hairy roots (main text, Figure 5), the

arbuscule morphology was abnormal: arbuscules were smaller with less hyphal

branches and contained many septa as compared to control arbuscules (main text,

Figure 6). The observed results suggested that the arbuscule development in those

RNAi roots progressed to degenerating, collapsed and dead stages. Relative to the

EV-5 and-9, in those RNAi lines, the transcript levels of GigmPT in these lines was

much lower, indicating that GigmPT was effectively silenced (main text, Figure 6J).

By contrast, the expression levels of AsPT1 and AsPT4 was not significantly reduced

in comparison with the control roots (main text, Figures 6K and 6L), while the

transcription of AsPT4 was slightly but significantly down-regulated (P<0.5) in

GigmPT RNAi-8 and -9 lines (main text, Figure 6L). These observed phenotypes

suggested that GigmPT is required for AM symbiosis.

GigmPT is not involved in the growth of intraradical hyphae at early stage of

symbiosis

To determine whether GigmPT function is essential for the hyphal growth of AM

fungus within roots, we have examined the effect of GigmPT RNAi lines on

intraradical hyphae, the infection of G. margarita and the growth of intraradical

hyphae have been investigated in RNAi and EV transgenic roots at early stage of AM

symbiosis. The results show that down-regulation of GigmPT has no effects on the

infection of G. margarita and intraradical hyphae thriving inside the roots, but leads

to a lower arbuscule numbers (Supplemental Figures 11A and 11D). This is consistent

with the results shown in Figure 5B of the main text.

To detect the expression levels of the GigmPT gene in RNAi lines, gene expression

of GigmPT is examined by qRT-PCR in mycorrhizal transgenic roots. Relative to the

EV-3 and-4 lines, in those RNAi lines, the transcription of GigmPT is strongly

down-regulated, indicating that GigmPT is effectively silenced (Supplemental Figure

10B). As expected, these transformed roots obviously express the siRNAs

(Supplemental Figure 11C).

This finding suggests that down-regulation of GigmPT has no effects on the hyphal

growth of G. margarita within roots at early stage of AM symbiosis.

Disruption of GigmPT affects the intraradical hyphae status of G. margarita

We further wondered to know whether the disruption of GigmPT gene could alter

the numbers of septa in intraradical hyphae. The number of septa (per unit root length)

was counted in intraradical hyphae in all RNAi and control roots. Relative to the EV-1

and-2 roots, the intercellular hyphae containing more septa was observed in RNAi-1

and-2 roots rather than in RNAi-3 roots (main text, Figure 5E), suggesting that

disruption of GigmPT gene also affects intraradical hyphae status of G. margarita.

GigmPT gene is down-regulated in extraradical mycelium of HIGS mutants

To determine the transcript profiling of GigmPT gene in extraradical mycelium of

G. margarita in HIGS mutants, abundance of the GigmPT gene and its paralogs

transcripts and siRNAs have been estimated in RNAi and control lines by qRT-PCR

and Northern blot analysis, respectively. The repression in expression of GigmPT in

the extraradical mycelium from RNAi lines compared with the extraradical mycelium

from EV control is statistically significant (P<0.05), while the transcription of other

two fungal PT genes GigmPT1 and GigmPT2 in RNAi lines is remained as high as in

the EV control (Supplemental Figures 12A-12C), suggesting that GigmPT is

specifically down-regulated in the extraradical mycelium of G. margarita. As

expected, the siRNA complementary to the GigmPT gene in the extraradical

mycelium is also detectable in RNAi lines by Northern blot analysis (Supplemental

Figure 12D).

Gly3P and PAA are competitive inhibitors of Pi uptake through GigmPT

We found that the PKA signaling agonist Gly3P inhibited Pi transport in the strain

pho84Δ expressing GigmPT (Supplemental Figures 14A and 14B). This indicates that

Gly3P served as a competitive inhibitor of Pi uptake via GigmPT, meaning that Gly3P

and Pi could directly interact with the phosphate-binding site of GigmPT. The

nonagonist PAA also inhibited Pi uptake of GigmPT (Supplemental Figure 14C), but

this inhibition by PAA was offset by an increased phosphate concentration

(Supplemental Figure 14D). The PAA also served as a competitive inhibitor in a

similar manner as phosphate and Gly3P. However, the only interaction between PAA

with GigmPT is still not enough to activate the PKA pathway (Supplemental Table 2).

The conserved residues are required for the GigmPT transport activity

The conserved residues Arg154

, Asp164

, Asp322

, and Lys459

were also predicted to be

localized near the phosphate binding site on the GigmPT 3D model (Supplemental

Figure 16 and Supplemental Methods). The newest report indicated the residues of

Asp164

, Asp322

and Lys459

in GigmPT are equivalent to Asp149

, Asp324

and Lys459

in

PiPT (Pedersen et al., 2013). Thus, we also tested whether the R154C

, D164C

, D322N

and

K459E

mutants are involved in the signaling and transport of GigmPT. The results

indicated that all of them were still capable of activating the PKA signaling at nearly

the normal level (Supplemental Figures 17A-17D), in spite of the significantly

reduced transport activity of these mutant proteins (Supplemental Figure 17E).

Addition of phosphate offseted the binding of MTSEA, resulting in recovery of

phosphate transport of the R154C

, D322N

and K459E

mutants except the D164C

(Supplemental Figure 17F), thus suggesting that these residues also is required for the

GigmPT transport activity.

The proposed working model of GigmPT in G. margarita

In the first version of picture for the Pi sensing and signaling networks, it has been

proposed that GigmPT protein, which is predominantly located in extraradical

mycelium, arbuscules and intraradical hyphae in G. margarita (main text, Figure 9), is

functional in PHO pathway in the absence of Pi (Supplemental Figures 20A and C)

and is inactive under abundant Pi conditions regarding transcription of the

Pi-repressible genes, while GigmPT is active in PKA signaling cascade in the

presence of Pi regarding a response in PKA targets (Supplemental Figures 20B and

D). In arbuscules, up-regulation of PHO pathway for sensing and re-absorption of Pi

in PAS through GigmPT transporter under Pi limitation (Supplemental Figure 20A).

Pi depletion-induced VIP1 might increase the levels of IP7, then IP7 could

allosterically interact with the pho81 (Lee et al., 2008; Secco et al., 2012) to inactivate

the complex pho81-pho80-pho85, alternatively, IP7 activates pho81-MAPK-2 to

negatively interact with pho80-pho85, the inactive complex hypo-phosphorylates

Neurospora crassa homologue of transcription factor NUC-1, resulting in the

activation of NUC-1, pho2 is also proposed to facilitate the transcriptional outputs,

and then activation of PHO responsive genes, including high-affinity phosphate

transporters (GigmPT and GigmPT1/2) and secretory phosphatases (pho5, pho12 and

ALP2), thus activating the PHO pathway. Consequently, the putative pho87/90 could

be endocytosed and targeted to the vacuole, dependently of the existence of their SPX

domains. Simultaneously, the Vtc1/2/4 complex is also activated and may be involved

in polyP accumulation in the vacuoles. In contrast, the induced Ppx1 and Ppn1 genes

have been proposed to participate in polyP degradation. The putative Gde1-1 and

Gde1-2 may be responsible for scavenging Pi through hydrolysis of GroPcho (Secco

et al., 2012). On the other hand, at the symbiotic interface, the Pi released in PAS is

also competitively perceived and transported by the AM-specific Pi transporters

AsPT1/4. In the presence of Pi, the active ternary complex pho81-pho80-pho85 might

hyper-phosphorylate the NUC-1, which is removed to cytoplasm via Msn5, and thus

the PHO pathway is repressed, whereas the low-affinity Pi transporters pho87/90 as

well as pho1-like proteins Syg1.1/Syg1.2 may be responsible for the Pi homeostasis in

arbuscules (Supplemental Figure 20B). Concomitantly, the Pi sensor GigmPT

functions as Pi receptor to rapidly trigger the activation of PKA signaling regarding

the response in PKA targets. Pi/GigmPT-dependent activation of PKA could trigger

the trehalase activity for mobilization of reserve carbohydrates and modulate the

activator Rap1 for induction of ribosomal protein gene RPS13, while the PKA

signaling negatively controls STRE -driven gene expression via Msn4 and Rim15

(Supplemental Figure 20B). As a result, GigmPT and GigmPT1/2 are down-regulated

by an unknown negative feedback loop. In extraradical hyphae, in the absence of Pi,

the PHO signaling subjected to GigmPT-gating is almost similar to which in the

arbuscules (Supplemental Figure 20C). In the presence of Pi, the putative low-affinity

Pi transporters pho87/90/91 are responsible for Pi uptake and transport, whereas the

PHO pathway is inactive. In such a case, a PKA signalling cascade similar to which in

arbuscules is also present (Supplemental Figure 20D). Thus, GigmPT is proposed to

be the main Pi sensor in G. margarita, being involved in Pi acquisition from

rhizosphere (or Pi re-absorption from PAS) via up-regulation of PHO pathway as well

as sensing extracellular Pi changes through the activation of the PKA signaling

cascade.

Supplemental Materials and Methods. Detailed procedures and Accession numbers

Plant materials and growth conditions

A. sinicus L. was used in this study. Seeds were surface sterilized and then sown on

water plates (0.8% agar) for germination. In pot system, the plantlets grown in sand

were adapted to glasshouse conditions (16: 8h light: dark cycle at 24: 20°C,

respectively) for one week first, and then, an equal number of plants were transferred

either to pots containing a soil: sand mixture (v/v,1:2), or to pots containing the same

mixture supplemented with roots of A. sinicus colonized with G. margarita (BEG34).

The inoculated plants were supplemented once a week with Hoagland’s nutrient

solution (Hoagland & Arnon, 1950) containing 30 μM KH2PO4, twice a week with

water, and kept in a growth chamber for approximately 2 months with a photoperiod

of 16 h of light and 8 h of darkness at 24 and 20 °C, respectively. Mycorrhization

levels were estimated using the method described by Trouvelot et al. (1986).

Medicago truncatula A17 was used in this study for immunolocalization and gene

expression experiments. Plants were grown in a growth chamber under a 16h light

(24°C)/8h dark (21°C) regime. M.truncatula/G.margarita mycorrhizal association was

established as described above with minor modifications. In brief, seedlings were

grown in Petri dishes containing 0.6 % plant agar until a trifoliate leaf was opened,

and then were transplanted to pots containing sterile sands and inoculated with 100

spores per pot. The plants were fertilized once a weekly with a modified Long-Ashton

solution (Hewitt, 1966) containing a high (1 mM NaH2PO4), control (300 μM

NaH2PO4), or low (3 μM NaH2PO4) phosphate concentration. The plants were

harvested at 35 days after inoculation. Mycorrhizal roots were firstly selected under a

binocular microscope on the basis of the presence of external mycelia. The roots from

the same line were mixed together and then divided into three parts: one to check the

colonization levels; another parts was embedded, and then fixed in FAA solution to

prepare for immunolocalization experiment; the remaining materials was frozen in

liquid nitrogen and stored at -70℃ for subsequent RNA extraction. The colonization

levels was estimated according to Trouvelot et al. (1986) using MYCOCALC

(http://www2.dijon.inra.fr/mychintec / Mycocalc- prg/download.html).

Sucrose treatment of mycorrhizal roots

AM monoxenic cultures contained Agrobacterium rhizogenes K599-transformed A.

sinicus roots colonized with the AM fungus G. margarita BEG34. Cultures were

established in the split-petri dishes to well separate the mycorrhizal roots from the

extraradical mycelia.

For the sucrose- and phosphate-treatment experiments, several different treatments

were prepared: (1) The IRM and ERM from the two compartments of control plates

containing MSR medium treated with 3 or 300 μM KH2PO4 in the both compartments,

(2) The IRM and ERM growing in the two compartments of plates containing 3 μM

KH2PO4 treated with the availability of sucrose (0, 3, 30 or 90 mM) in the root

compartment, and (3) The IRM and ERM growing in the two compartments of plates

containing 300 μM KH2PO4 supplemented with the availability of sucrose (0, 3, 30 or

90 mM) in the root compartment. The IRM and ERM were harvested 1, 2, 7 and 14

days after treatments.

To collect the IRM within roots, extraradical hyphae attached to the mycorrhizal

roots were removed with forceps under the microscope, and the ERM was collected

with dissecting forceps, all the materials were then rinsed in sterile dH2O and dried

with filter paper, immediately frozen in liquid nitrogen and stored at -70°C until use.

Real-time quantitative RT-PCR

To validate the data derived from the RNA-seq experiment (Salvioli et al., 2016), we

qRT-PCR analysis of a batch of selected genes. In brief, total RNA extracted as

previously described was used for cDNA synthesis using Superscript II Reverse

Transcriptase (Life Technologies, Carlsbad, CA, USA). qRT-PCR experiments were

carried out using the StepOne Real-time PCR System (Applied Biosystems).

qRT-PCR reactions were performed in a final volume of 20 µl containing 10 µl of

SYBR Green Supermix 2 × (Life Technologies), 0.2 µM of each primer and 1 µl of a

1:3 dilution of cDNA template. The PCR program included an initial incubation at

95 °C for 5 min, followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s and 72 °C for

30 s. The specificity of PCR amplification was detected with a heat dissociation from

60 to 95 °C after the PCR reactions. qRT-PCR estimations were performed on three

independent biological samples from three replicate experiments. The relative

expression of the 2－△△Ct

method was applied to compute the fold change of each gene,

using the actin gene of G. margarita as a reference gene. Data analysis was carried

out using the StepOne Software v2.0. The gene-specific primers and corresponding

sequences are listed in Supplemental Table 5.

RNA sequencing data analyses: functional annotation and transcript profiling

The G. margarita BEG34 transcriptome shotgun assembly (TSA) data were

deposited at GenBank in the NCBI (Salvioli et al., 2016). Functional annotation

followed the Gene Ontology (Ashburner et al., 2000) and G. margarita Transcriptome

Database (Salvioli et al., 2016). Briefly, TSA sequences were firstly searched against

the homologs from the draft genome and transcriptome in R. irregularis DAOM

197198 (Tisserant et al., 2013;Tisserant et al., 2012) using TBLASTN (e-value <1e-5).

The obtained sequences based on the best BLAST hits (e-value <1e-5, identity >50%,

score >150) were also compered with the proteins of Basidiomycota (Laccaria

bicolor) and Ascomycota (S.cerevisiae, Neurospora crassa and Tuber melanosporum)

using BLASTX (e-value <1e-5). To identify conserved protein domains in TSA

sequences, the predicted protein sequences were compared with the Eukaryotic

Orthologous Groups database. GO annotations had been perfermed by Salvioli et al.

(2016). To predict metabolic pathways in G. margarita, sequences were queried for

KO assignments using the KAAS tool, and KEGG pathway maps were obtained from

KAAS (Tisserant et al., 2012; Salvioli et al., 2016). The selected TSA sequences

based on the BLAST hits after gene annotations were employed to subsequent gene

expression analysis.

In the second RNA-sequencing experiment, DESeq2-normalised expression data

for the phosphate signalling related genes, at three developmental stages of

G.margarita (germinating spores, GR24 treated spores and symbiotic mycelium

thriving inside roots) (Salvioli et al., 2016), were plotted in heatmaps for comparison.

The visualization of data was performed using the MeV (MultiExperiment Viewer,

v4.9) software.

Colocalization of GigmPT and PHO84 in yeast

To generate the GigmPT-eGFP and PHO84-mCherry fusions, GigmPT and PHO84

transcripts were PCR amplified using G.margarita and yeast cDNAs as templates,

respectively, and then genes were recombined into an appropriate yeast expression

vector, the eGFP-and mCherry-tagged pESC-lue, with Gibson Assembly Cloning

technology (Gibson et al., 2009). The GigmPT-eGFP and PHO84-mCherry fusions

were driven under the opposite directional promoters GAL10 and GAL1, respectively.

The coexpression of the eGFP tagged GigmPT construct with the plasma

membrane-integrated protein PHO84-mCherry as PM marker was performed in yeast

cells through the LiOAc/PEG transformation as described by previously analyzed

(Gietz & Schiestl, 1991). A confocal laser scanning microscope (Zeiss LSM 510) was

used for imaging.

Antibody preparation and immunolocalization of GigmPT

Antibodies specific for the GigmPT-drived peptides were produced in rabbits. The

antibodies were against a peptide corresponding to the N-terminal 15 amino acids of

the GigmPT protein (5’-NIVIEDNDYDKRRRE-3’) predicted to a nonconserved

region in the protein. An additional Cys residue was added to the C-terminal of the

peptide to enable coupling to protein carrier KLH (Garcia et al., 2013). The

preimmune serum was collected before immunolocalization and serum was analyzed

for the presence of anti-GigmPT antibodies that recognized the peptide by ELISA

analysis. A dilution (1:500) of the antibodies was prepared for immunolocalization

analysis.

Immunolocalization experiment was performed as described in Garcia et al. (2013),

Pérez-Tienda et al. (2011), Harrison et al. (2002) and Blancaflor et al. (2001) with

some modifications. Arbuscular mycorrhizal roots colonized by G.margarita were

embedded in 8% low melting agarose (electrophoresis grade) and cut into 100 μm

longitudinal sections with a vibratome (Leica VT1000S). The agarose slices were then

fixed with FAA fixative (50% absolute ethanol, 5% acetic acid and 4% formaldehyde)

at 4°C in 1×phosphate-buffered saline (PBS buffer) (137 mM NaCl, 2.7 mM KCl, 10

mM Na2HPO4 and 2 mM KH2PO4, pH7.4). The root segments were fixed under

vacuum at room temperature (RT) for 2 h. After fixation, root sections were washed

in 96 well plates two times for 5 min each with 1×PBS buffer and then incubated in

4% BSA containing 0.2% (v/v) Tween 20 in 1×PBS buffer at RT for 1 h. The BSA

was removed and root sections were rinsed twice in 1×PBS buffer by changing the

solution in the wells, the root segments were incubated overnight at 4°C with

anti-GigmPT antibody (dilution 1:500) in 4% BSA in PBS solution, and the

pre-immune serum was added as negative control. The sections were washed three

times in 1×PBS buffer and incubated in 4% BSA in PBS buffer containing secondary

antibody: a dilution of 1:100 for the goat anti-rabbit IgG-Alexa Fluor 488 conjugate

(Garcia et al., 2013; Harrison et al., 2002) or the goat anti-rabbit IgG-Alexa Fluor 568

(Pérez-Tienda et al., 2011) (Molecular Probes, Invitrogen, Carlsbad, CA, USA), at RT

in the dark for 2 h. After two washes in 1×PBS buffer, the root sections were

counterstained with 0.1 mg/ml WGA-Texas red in PBS buffer to visualize the fungus

G.margarita (Genre & Bonfante, 1997). A zeiss LSM 510 confocal laser scanning

microscope was used for imaging. The excitation and emission wavelengths for the

Alexa Fluar 488 were 488 and 500 to 530 nm, respectively, while fluorescence from

Alexa Fluor 568 or WGA-Texas red was excited at 568 nm and emission detected at

590 nm wavelength. The image J software was used to image analysis and merging of

images.

Selection of transmembrane domains (TMD) for substituted cysteine accessibility

method (SCAM) and mutagenesis analysis

The TMD IV of GigmPT was selected for SCAM analysis for the following reasons:

(i) This region contains the strongly conserved R154

residue, as in Pho84 (Popova et

al., 2010), which may be involved in phosphate binding; (ii) This region carries the

phosphate signature (GGDYPLSATIXSE) found in Pht1 phosphate transporters

(Karandashov & Bucher, 2005); (iii) GigmPT contains the motif conserved in Pi

transporters from plants and fungi (TLCFFRFWLGFGIGGDYPLSATIMSE)

(Harrison et al., 2002; Popova et al., 2010); (iv) This region contains the

phosphate-binding signature sequence GXGXGG; (v) Because of the 3D structure of

Pho84 in yeast (Lagerstedt et al., 2004) and the crystal structure of GlPT in

Escherichia coli (Huang et al., 2003).

To identify residues contributing to phosphate binding and translocation, the

multiple amino acid sequence alignment of GigmPT with selected phosphate

transporters from plants and fungi was performed (data not shown). For the MFS

members, helices I, II, IV, V, VII, VIII, X and XI are predicted to be channel-lining

domains (Supplemental Figure 16). The helices IV, VII, VIII, X and XI harbored

several highly conserved residues. GlpT or Pho84 structure as a template for

modeling GigmPT is that the putative substrate binding site might be located at a

similar position (Huang et al., 2003; Samyn et al., 2012). The amino acid residues

Arg154

and Asp164

in helice IV, Asp322

in helice VII, Lys459

in helice XI, which are

conserved in all phosphate transporters. The conserved residues were mapped on the

GigmPT 3D model (Supplemental Figure 16), showing their predicted localization.

Arg154

(helice IV) seems to be located towards the periplasmic side. The acidic

residues Asp164

(helice IV) and Asp322

(helice VII) may interact with protons or form

hydrogen-bond with phosphate. Both residues are located in the proposed putative

binding site (Supplemental Figure 16). On the GigmPT model, Lys459

(helice XI) is

located in a similar position as Lys492

(helice XI) in Pho84 (Samyn et al., 2012), this

residue is located in the putative binding or translocation site.

Accession numbers

Fungi and plant Pi transport protein names are followed by GenBank accession

numbers: GigmPT (KC887075) from G. margarita, GmPT (DQ074452) from

Funneliformis mosseae; GiPT (AF359112) from R. irregularis; GvPT (U38650) from

Glomus verisforme; GsPT1 (ADG27910.1) from Glomus sp. DAOM 212150; GsPT2

(ADG27908.1) from Glomus sp. DAOM 211637;GsPT3 (ADG27907.1) from Glomus

sp. DAOM 240410; GcPT(ADG27915.1) from Glomus custos; GpPT (ADG27916.1)

from Glomus proliferum DAOM 226389; GaPT(ADG27911.1) from Glomus

aggregatum DAOM 240163; RiPT (ADG27901.1) from Rhizophagus irregularis

DAOM 240721; RcPT(ADG27914.1) from Rhizophagus clarus DAOM 234281;

GdPT(ADG27912.1) from Glomus diaphanum DAOM 229456; FcPT(ADG27892.1)

from Funneliformis coronatum DAOM 240746; PHO84(D90346) from

Saccharomyces cerevisiae; LbPT1(XP_001889013), LbPT2 (XP_001889026), LbPT3

(XP_001889028), LbPT4 (XP_001880970), LbPT5 (XP_001888254) from Laccaria

bicolor, PiPT(DQ899728) from Piriformospora indica; AfPT(XP_746548.1) from

Aspergillus fumigatus Af293; AnPT(CAK46483.1) from Aspergillus niger;

NfPT(XP_001262447.1) from Neosartorya fischeri NRRL 181;

AtPT(XP_001217524.1) from Aspergillus terreus NIH2624; TsPT (XP_002477856.1)

from Talaromyces stipitatus ATCC 10500; CiPT(XP_001246568.1) Coccidioides

immitis RS; EdPT(EHY60034.1) from Exophiala dermatitidis NIH/UT8656;

TmPT(XP_002145650.1) from Talaromyces marneffei ATCC 18224;

GlPT(EHK97789.1) from Glarea lozoyensis 74030; MpPT(EKG12188.1) from

Macrophomina phaseolina MS6; NpPT(EOD52934.1) from Neofusicoccum parvum

UCRNP2; RdPT(EIE76964.1) from Rhizopus delemar RA 99-880;

HcPT(CAI94746.1) from Hebeloma cylindrosporum; FmPT(EJD06711.1) from

Fomitiporia mediterranea MF3/22; DsPT(EJF64691.1) from Dichomitus squalens

LYAD-421 SS1; PnPT (AB060641) from Pholiota nameko, and MgPT (EHA50344)

from Magnaporthe grisea. LePT1 (AF022873), LePT2 (AF022874) from

Lycopersicon esculentum; AtPT1 (U62330), AtPT2 (U62331) from Arabidopsis

thaliana; StPT1 (X98890), StPT2 (X98891), StPT4 (AY793559) from Solanum

tuberosum; MtPT1.1(XP_003615445.1), MtPT1 (AF000354), MtPT2 (AF000355),

and MtPT4 (AY116210) from Medicago truncatula; SrPT1 (AJ286743) from

Sesbania rostrata; AsPT1 (JQ956415), AsPT2 (JQ956416), AsPT3(JQ956417),

AsPT4 (JQ956418), AsPT5 (JQ956419), AsPT6 (JQ956420) from Astralegus sinicus;

GmPT7(ACY74622) and GmPT10 (ACP19346) from Glycine max;

LjPT1.1(AP010556.1) and LjPT4 (BAE93354) from Lotus japonicus;

VvPT1(XP_002275526) from Vitis vinifera; EcPT4(BAE94386) from Eucalyptus

camaldulensis; PtPT8(JGI ID: 784338) from Populus trichocarpa.

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Supplemental Figure legend in main text

Figure 4. Immunolocalization of GigmPT. (A-C) Immunolocalization of GigmPT in

the arbuscules of G. margarita depends on external Pi availability. The

epifluorescence microscopy images of arbuscular mycorrhizal roots of M.

truncatula/G. margarita (A3, B3 and C1) probed with GigmPT antibodies visualized

with a secondary antibody conjugated with Alexa Fluor 488. The AM roots were

counterstained with WGA-Texas red to visualize G. margarita. Corresponding

images in bright field (A1 and B1). (A2), (B2), and (C2) Corresponding images

showing red fluorescence from WGA-Texas red staining. Merged images showing

both bright field and green fluorescence (A4, B4), or both red and green fluorescence

(A5, B5 and C3). Overlaps of bright field, red and green fluorescence images are

shown (A6 and B6). (A-C) The arbuscules in root cortical cells of Medicago

truncatula colonized by AM fungus G. margarita from plants grown in pots in the

presence of either 300 µM (A) or 3 µM (B-C) phosphate. In arbuscules, green

fluorescence signals are more intense in low Pi than in high Pi treatments. (D-G)

Laser scanning confocal microscopy images of AM roots of M. truncatula/G.

margarita probed with GigmPT antibodies. Nomarski (bright field) views of an

arbuscule trunk (D1), a mature arbuscule (D4), a degenerating arbuscule (D7), a

collapsed arbuscule (D10), and the intraradical hyphae (E1) are shown. (D2), (D5),

(D8), (D10) and (E2) Corresponding images showing green fluorescence from

GigmPT immunostaining. (D3), (D6), (D8), (D12) and (E3) Merged images showing

both bright field and green fluorescence. The GigmPT signals are visible at the

periphery of the arbuscule trunks (D2 and D8), of the first branch (D2) of the young

arbuscule, of the branches of mature (D5) and degenerating (D8) arbuscules and of

the intraradical hyphae (E2), whereas weak GigmPT immunostaining is detectable at

the periphery of the collapsed arbuscule (D11) and the dead arbuscule (D12) does not

show the GigmPT signal. (F) Localization of the GigmPT protein in the arbuscules

and intraradical hyphae of G. margarita. Antibodies to GigmPT were detected by

indirect immunofluorescence with secondary antibodies (anti-rabbit IgG-Alexa Fluor

568). Strong GigmPT signal is visible in the mature arbuscules (ma) and at the

periphery of the intraradical hyphae (ih). (G) Immunostaining with GigmPT

preimmune serum to test the specificity of the antibody. The GigmPT preimmune

serum does not stain the arbuscules and intraradical hyphae. t, arbuscule trunks; b,

branches of the arbuscules; ma, mature arbuscule; ca, collapsed arbuscule; da, dead

arbuscule; ih, intraradical hyphae. Scale bars = 50 μm.

Supplemental Figure legend in Supplemental data

Supplemental Figure 20. Proposed Working Model of GigmPT in Gigaspora

margarita.

(A-D) Schematic representation of the dual roles of GigmPT in the regulation of

putative phosphate signaling (PHO) pathway and PKA signaling cascade in

arbuscules (A-B) and intraradical hyphae (C-D). (A) In arbuscules, activation of the

PHO pathway for sensing and competitive re-absorption of phosphate in PAS in the

absence of Pi through GigmPT transporter. Pi depletion is proposed to induce the

expression of VIP1, which positively regulates the IP7 concentration, and IP7 could

inactivate the ternary complex pho81-pho80-pho85 (Lee et al., 2008; Secco et al.,

2012) or activate pho81-MAPK-2 to negatively interact with pho80 and pho85, the

inactive complex prevents the phosphorylation of NUC-1, and then activation of PHO

responsive genes; like S. cerevisiae, pho2 could facilitate the expression of PHO

responsive genes, and thus activating the PHO pathway. Consequently, the putative

low-affinity Pi transporters pho87 and pho90 are non-functional. Simultaneously, the

putative Vtc1/2/4 complex is also activated and is involved in polyP accumulation in

the vacuoles. In contrast, the induced Ppx1 and Ppn1 genes have been proposed to

participate in polyP degradation. At the symbiotic interface, the Pi released in PAS is

also competitively perceived by the host, and is transported across the PAM into plant

cells via the AM-specific AsPT1/4. (B) In the presence of Pi, the low-affinity Pi

transporters pho87/90 orthologs of the yeast (Hürlimann et al., 2009) and AM fungi R.

irregularis (Tisserant et al., 2012) and Gigaspora rosea (Tang et al., 2016) as well as

pho1-like proteins Syg1.1/1.2 like the orthologous PHO1 and XPR1 in plant and

animal (Hamburger et al., 2002; Giovannini et al., 2013), respectively, may be

responsible for the Pi homeostasis in arbuscules, whereas GigmPT1/2/5 are

down-regulated in response to high Pi. The excess of Pi may be stored in form of

polyP in vacuoles via the putative pho91 (Hürlimann et al., 2007) and Vtc complex

(data not shown; Secco et al., 2012). In this case, the PHO pathway is down-regulated.

Concomitantly, the Pi sensor GigmPT functions as Pi receptor for activation of the

PKA signalling. Pi/GigmPT-induced activation of PKA is proposed to trigger the

trehalase activity for mobilization of reserve carbohydrates and to modulate the Rap1

activity for induction of ribosomal protein gene RPS13, while the PKA negatively

controls STRE-driven gene expression via Msn4 and Rim15. The inhibition of the

nuclear localization of Msn4 and Rim15 by the PKA is not shown in this picture. (C)

In extraradical hyphae, in the absence of Pi, the PHO signalling subjected to

GigmPT-gating is almost similar to which in the arbuscules. (D) In the presence of Pi,

the putative low-affinity Pi transporters pho87 and pho90 may be responsible for Pi

uptake and transport. In such a case, a PKA signalling cascade similar to which in

arbuscules is also present. The MAPK signalling in which the MAPK-2 is possibly

involved are not shown in the proposed model. The arrowed and flat-ended lines refer

to positive and negative interactions, respectively. IP7, inositol heptakisphosphate;

PolyP, inorganic polyphosphate; Glu., glucose; HA1, H+-ATPase (Wang et al., 2014;

Krajinski et al., 2014); SPX, Syg1-Pho81-Xpr1 domain; TM, transmembrane

domain; EXS, ERD-Xpr1-Syg1 domain; PAM, periarbuscular membrane; PAS,

periarbuscular space; APM, AM fungal plasma membrane.