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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.