Supplemental Data. Breuillin-Sessoms et al. Plant Cell (2015) 10.1105/tpc.114.131144 Supplemental Figure 1. M. truncatula Pi transporters of the PHT1 family. (A) Phylogenetic rooted tree of the PHT family of phosphate transporters. The approximately-maximum-likelihood phylogenetic tree was generated using the predicted full length amino acid sequences of phosphate transporters from Arabidopsis thaliana, Solanum lycopersicon, Oryza sativa, Sorghum bicolor, Lotus japonicus, Glycine max and Medicago truncatula. FastTree was used to generate the phylogeny by using a neighbor joining method to create a starting tree and later refining the topology using maximum likelihood nearest-neighbor interchanges and minimum-evolution subtree-pruning-regrafting. Each branch division shows local support values with the Shimodaira-Hasegawa test. Subfamilies 1, 11, 111 and IV are marked. The shaded box highlights the AM induced M. truncatula transporters PT4 and PT8. 1
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Supplemental Data. Breuillin-Sessoms et al. Plant Cell (2015) 10.1105/tpc.114.131144
Supplemental Figure 1. M. truncatula Pi transporters of the PHT1 family.
(A) Phylogenetic rooted tree of the PHT family of phosphate transporters.
The approximately-maximum-likelihood phylogenetic tree was generated using the predicted full
length amino acid sequences of phosphate transporters from Arabidopsis thaliana, Solanum
lycopersicon, Oryza sativa, Sorghum bicolor, Lotus japonicus, Glycine max and Medicago
truncatula. FastTree was used to generate the phylogeny by using a neighbor joining method to
create a starting tree and later refining the topology using maximum likelihood nearest-neighbor
interchanges and minimum-evolution subtree-pruning-regrafting. Each branch division shows
local support values with the Shimodaira-Hasegawa test. Subfamilies 1, 11, 111 and IV are
marked. The shaded box highlights the AM induced M. truncatula transporters PT4 and PT8.
1
Supplemental Data. Breuillin-Sessoms et al. Plant Cell (2015) 10.1105/tpc.114.131144
Supplemental Figure 2. Localization of PT8 and symbiosis phenotype of pt8-1 (A-B) A M. truncatula root expressing MtPT8pro:MtPT8-GFP colonized with G. versiforme. GFP
signal from MtPT8 pro:MtPT8-GFP is observed only in cells with arbuscules and not in the vascular
tissue or in the epidermis. (A) GFP fluorescence and (B) GFP fluorescence overlayed on the
corresponding differential interference contrast image. Bar = 10 µm. (C) Colonization level of R108 and pt8-1 root 4 wpi with G. intraradices (7.5 mM N) grown in pots
(left) or cones (right). Data represent the mean ± the standard error of three independent
replicates. Different letters indicate significant differences (α = 0.01).
(D) G. intraradices in the roots of wildtype (WT) R108 (left) and pt8-1 (right) as visualized by a
morphology of the symbiosis does not differ in these lines. Bars = 50 µm.
(E) Analysis of arbuscule development in wildtype and pt8-1 roots. Graphs show the arbuscule
size distribution in the arbuscule populations at 4 days (left), 6 days (middle) and 8 days (right)
post contact (dpc) with primed G. intraradices spores. Data represent the mean ± the standard
error of arbuscules from multiple infection units from 3 biological replicates. At 4 dpc, arbuscules
were analyzed in R108 (25 infection units (IUs), pt8-1(43 IUs); at 6 dpc, R108 (75 IUs) and pt8-1
(66 IUs); at 8 dpc, R108 (42 IUs) pt8-1 (42 IUs). Different letters indicate significant differences
(α = 0.01).
(F) Shoot biomass and (G) Shoot Pi of R108 (WT), a wild-type segregant from the pt8 mutant
population (WTseg) and two pt8-1 mutants #2 and #8 inoculated with G. intraradices (black bars)
or a mock-inoculated (grey bars) and harvested at seven weeks post inoculation. Data are the
mean of three biological replicates each containing four plants. Error bars represent standard
error. In F, different letters indicate significant differences (p<0.05). In G, different letters indicate
significant differences (p<0.001).
2
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10
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Roo
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WTpt8-1
D
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C
10
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0-25 µm25-35 µm
ControlG.intraradices
35-45 µm45-75 µm
10
20
30
40
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0
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0WT
pt8-1 WTpt8-1
F G
WTpt8-1
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1
2
3
4
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2
4
6
8
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WT #2 #8
pt8-1WT WT #2 #8
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Supplemental figure 2
Supplemental Data. Breuillin-Sessoms et al. Plant Cell (2015) 10.1105/tpc.114.131144
Supplemental Figure 3. M. truncatula Tnt1 lines with insertions in PT4, PT8, AMT2;3 and AMT2;4 Schematic diagram of gene structure and position of Tnt1 insertion. All insertion lines are
predicted to be null alleles. The original numbers of the M. truncatula Tnt1 lines are as follows:
expressing AMT2;3 and AMT2;4 from the GAL1 promoter.
(A) Confocal microscope image of the yeast 31019b (∆mep1; ∆mep2; ∆mep3) strain transformed
with pYEUra3-GFP-MtAMT2;4 showing that some of the transformed cells show a fluorescent
signal. Left panel, Fluorescence detected between 505nm to 545nm; middle panel, corresponding
bright field image; right panel, overlay of fluorescence and bright field images. This fluorescent
signal is not GFP as determined by evaluation of the emission spectrum (triangles and dashed
line) in comparison with the emission spectrum of GFP (squares and solid line). Similar results
were obtained for GFP-MtAMT2;4 and also for the empty pYEUra3 vector transformants, further
confirming that this signal is not GFP. Although transcripts could be detected, GFP-tagged
proteins were not detected. The signal detected is autofluorescence and the proportion of cells
showing such a signal increases with the age of the culture.
(B) Yeast 31019b (∆mep1; ∆mep2; ∆mep3) mep cells transformed with either pYEUra3-
MtAMT2;3 or pYEUra3-MtAMT2;4 accumulate AMT2;3 or AMT2;4 transcripts respectively.
RT-PCR indicates the presence of AMT2;3, AMT2;4 and yeast α-tubulin (Sc-Tub1) transcripts
in yeast cells transformed with either pYEUra3-MtAMT2;3 or pYEUra3-MtAMT2;4. Control RT-
PCR reactions lacking reverse transcriptase, pYEUra3-MtAMT2;3(-RT) and pYEUra3-
MtAMT2;4(-RT), are shown also. Yeast cells were grown on medium prepared with nitrogen-
free YNB, supplemented with 2.5mM ammonium sulphate and with galactose as a carbon
source.
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Supplemental Data. Breuillin-Sessoms et al. Plant Cell (2015) 10.1105/tpc.114.131144
Supplemental Table 1. Shoot fresh weight, P and N content of the pt4-2 pt8-1 and pt4-5 pt8-1 Pi transporter double mutants in high-N growth conditions Shoot fresh weight, shoot phosphorus (P) and shoot nitrogen (N) content of the Pi transporter double mutants in the inter-ecotype background (A17 control) and the R108 background. Plants were harvested 4 weeks post inoculation with G. versiforme. High-N fertilizer contains 15 mM N. Experiments in the two backgrounds were carried out separately. For each genotype there were two biological replicates each containing 4 plants. The mean shoot biomass and standard error were calculated from 8 individual plants. Different letters indicate significant differences between genotypes (α =0.01). The shoot P and N contents were measured on pools of 4 plants with two biological replicates per genotype. Data shown are the mean ± standard deviation.
Supplemental Data. Breuillin-Sessoms et al. Plant Cell (2015) 10.1105/tpc.114.131144
Supplemental Table 2. Shoot fresh weight, P and N content and infection unit length of the pt4-2 pt8-1 and pt4-5 pt8-1 Pi transporter double mutants in low-N growth conditions Shoot fresh weight, shoot phosphorus (P), shoot nitrogen (N) and infection unit length of the Pi transporter double mutants in the inter-ecotype background (A17 control) and the R108 background. Experiments in the two backgrounds were carried out separately. Plants harvested at either 4 weeks post inoculation (A17) or 5 weeks post inoculation (R108) with G. versiforme. Low-N fertilizer contains 1.5 mM N. For each genotype there were two biological replicates each containing 4 plants. The mean shoot biomass and standard error were calculated from 8 individual plants. Different letters indicate significant differences between genotypes (α =0.01). The shoot P and N contents were measured on pools of 4 plants with two biological replicates per genotype. Data shown are the mean ± standard deviation. The mean infection unit length ± standard error were calculated from the same random infection units sampled in Figure 2C-D. Different letters indicate significant differences at α of 0.01.
Supplemental Data. Breuillin-Sessoms et al. Plant Cell (2015) 10.1105/tpc.114.131144
Supplemental Table 3. Shoot fresh weight, P and N content of the pt4-2 amt2;3 and pt4-5 amt2;3 double mutants in high-N growth conditions Shoot fresh weight, shoot phosphorus (P) and shoot nitrogen (N) of the phosphate transporter and ammonium transporter double mutants in the inter-ecotype (A17 control) and R108 backgrounds. Experiments in the A17 and R108 backgrounds were carried out separately. In both experiments, plants harvested at 4 weeks post inoculation with G. versiforme. High-N fertilizer contains 15 mM N. For each genotype there were two biological replicates each containing 4 plants. The mean shoot biomass and standard error were calculated from 8 individual plants. Different letters indicate significant differences between genotypes (α =0.01). The shoot P and N contents were measured on pools of 4 plants with two biological replicates per genotype. Data shown are the mean ± standard deviation.
Supplemental Data. Breuillin-Sessoms et al. Plant Cell (2015) 10.1105/tpc.114.131144
Supplemental Table 4. Shoot fresh weight, P and N content of the pt4-2 amt2;3 and pt4-5 amt2;3 double mutants in low-N growth conditions Shoot fresh weight, shoot phosphorus (P) and shoot nitrogen (N) of the phosphate transporter and ammonium transporter double mutants in the inter-ecotype (A17 control) and R108 backgrounds. Experiments in the A17 and R108 backgrounds were carried out separately. In both experiments, plants harvested at 4 weeks post inoculation with G. versiforme. Low-N fertilizer contains 1.5 mM N. For each genotype there were two biological replicates each containing 4 plants. The mean shoot biomass and standard error were calculated from 8 individual plants. Different letters indicate significant differences between genotypes (α =0.01). The shoot P and N contents were measured on pools of 4 plants with two biological replicates per genotype. Data shown are the mean ± standard deviation.
Supplemental Data. Breuillin-Sessoms et al. Plant Cell (2015) 10.1105/tpc.114.131144
Supplemental Table 5. Shoot fresh weight, P and N content of the pt4-5 pt8-1 amt2;3 triple mutant in low-N growth conditions Shoot fresh weight, shoot phosphorus (P) and shoot nitrogen (N) of the pt4-5 pt8-1 amt2;3 triple mutant and a wildtype segregant from the triple mutant population F2 (WTseg). Plants harvested at 4 weeks post inoculation with G. intraradices. Low-N fertilizer contains 1.5 mM N. For each genotype there were two biological replicates each containing 4 plants. The mean shoot biomass and standard error were calculated from 8 individual plants. Different letters indicate significant differences between genotypes (α =0.01). The shoot P and N contents were measured on pools of 4 plants with two biological replicates per genotype. Data shown are the mean ± standard deviation.
Genotype
Shoot fresh weight
(g)
Shoot P content
(nmoles.mg-1
dry weight)
Shoot N content
(%)
WTseg pt4-5 pt8-1 amt2;3 0.57 ±0.11a
0.78 ±0.09a 105.07 ±18.88 114.38 ±14.63
1.62 ±0.51 1.23 ±0.14
5
Supplemental Data. Breuillin-Sessoms et al. Plant Cell (2015) 10.1105/tpc.114.131144
Supplemental Table 6. Shoot fresh weight, P and N content of the pt4-5 pt8-1 amt2;3 triple mutant, a wildtype segregant and R108 in high-N growth conditions Shoot fresh weight and shoot nitrogen (N) of the pt4-5 pt8-1 amt2;3 triple mutant, a wildtype segregant from the triple mutant population F2 (WTseg) and R108. Plants harvested at 4 weeks post inoculation with G. intraradices. High-N fertilizer contains 15 mM N. For each genotype there were two biological replicates each containing 4 plants. The mean shoot biomass and standard error were calculated from 8 individual plants. Different letters indicate significant differences between genotypes (α =0.01). The shoot N contents were measured on pools of 4 plants with two biological replicates per genotype. Data shown are the mean ± standard deviation.
Genotype
Shoot fresh weight
(g)
Shoot N content
(%)
R108 WTseg pt4-5 pt8-1 amt2;3
1.65 ±0.12a 0.98 ±0.15b 1.18 ±0.20ab
2.77 ±0.10 3.52 ±0.75 3.43 ±0.16
6
Supplemental Data. Breuillin-Sessoms et al. Plant Cell (2015) 10.1105/tpc.114.131144
Supplemental Table 7. Shoot fresh weight and N content of the pt4-5 pt8-1 amt2;3 triple mutant, a wildtype segregant and R108 in low-N growth conditions Shoot fresh weight, shoot phosphorus (P), shoot nitrogen (N) and infection unit length of the pt4-5 pt8-1 amt2;3 triple mutant, a wildtype segregant from the triple mutant population F2 (WTseg) and R108. Plants harvested at 4 weeks post inoculation with G. intraradices. Low-N fertilizer contains 1.5 mM N. For each genotype there were two biological replicates each containing 4 plants. The mean shoot biomass and standard error were calculated from 8 individual plants. Different letters indicate significant differences between genotypes (α =0.01). The shoot N contents were measured on pools of 4 plants with two biological replicates per genotype. Data shown are the mean ± standard deviation.
Supplemental Data. Breuillin-Sessoms et al. Plant Cell (2015) 10.1105/tpc.114.131144 Color shading indicates primer use as follows: blue - Q-RT-PCR, orange -genotyping insertion lines, purple – cloning into yeast expression vector, green – cloning GFP fusions into yeast expression vector, grey – RT-PCR in yeast.