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

chitin fungal wall stain, wheat germ agglutinin conjugated to AlexaFluor488. Arbuscule

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

0

10

20

30

40

50

Roo

t len

gth

colo

nize

d (%

)

Roo

t len

gth

colo

nize

d (%

)

WTpt8-1

0

10

20

30

40

50

WTpt8-1

D

a

a a’

a’

C

10

20

30

40

50

0

0-25 µm25-35 µm

ControlG.intraradices

35-45 µm45-75 µm

10

20

30

40

50

0

10

20

30

40

50

0WT

pt8-1 WTpt8-1

F G

WTpt8-1

0

1

2

3

4

5

0

2

4

6

8

10

12

14

WT #2 #8

pt8-1WT WT #2 #8

pt8-1

4 dpc 6 dpc 8 dpc

segWT

seg

E

a a a a a a

a’

a’ a’ a’

a’

a” a” a”a’a”

a”

a”a’’’

a’’’ a’’’a’’’

a’’’ a’’’

a’a’ a’ a’

a’

a’a’

a’ a’

aa

abababb

b b

Arb

uscu

le p

erce

ntag

e

Arb

uscu

le p

erce

ntag

e

Arb

uscu

le p

erce

ntag

e

Sho

ot w

eigh

t(g F

W)

Sho

ot P

hosp

hate

(nm

ol/m

g FW

)

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:

NF5213 (pt8-1), NF3229 (amt2;3), NF4575 (amt2;4), TNK222 (pt4-5).

3

pt4-5

pt8-1

amt2;3

exon

intron

tnt insertion site

392

613 1313 1596 1712 2262

2182

588 1782

1674

(319)

(657)

(465)

1

1

1

amt2;4588 1038 1329 1620

(388)1

Supplemental figure 3

Supplemental Data. Breuillin-Sessoms et al. Plant Cell (2015) 10.1105/tpc.114.131144

Supplemental Figure 4. Arbuscule images and arbuscule population structure in a phosphate

transporter double mutant (R108 background)

(A) Microscopy images of G. versiforme infection units in R108 (WT), pt8-1, pt4-5 and pt4-5 pt8-

1 4 wpi grown with high N (15 mM) fertilization. Arrows = mature arbuscules, arrowheads =

degenerating arbuscules, App = appressorium. Bars =100µm

(B) Arbuscule size distribution and arbuscule populations in pt4-2 and pt4-2 pt8-1 at 9 days post

contact with primed G. intraradices spores grown with low N (1.5mM) fertilization.

Data represent the mean ± the standard error of arbuscules from random infection units (IUs)

from pt4-2 (29 IUs), pt4-2 pt8-1 (14 IUs). Infection units were sampled from 3 independent

biological replicates. Different letters indicate significant differences at α = 0.01.

4

WT (R108) pt8-1 pt4-5 pt4-5 pt8-1App App

App

A High N

B

0-25 µm 25-35 µm 35-45 µm 45-75 µm

pt4-2

pt8-1

pt4-2

0

10

20

30

40

50

60

a

a’a”a’a”

a’’’ a’’’

aArb

uscu

le p

erce

ntag

e

Supplemental Figure 4

Supplemental Data. Breuillin-Sessoms et al. Plant Cell (2015) 10.1105/tpc.114.131144

Supplemental Figure 5. Morphology of AM symbiosis in amt2;3 during growth in varying nitrogen

and phosphate regimes Microscopy images showing roots at 4 wpi with G. versiforme grown with varying levels of N

fertilization. (A) 15 mM N (B), 7.5 mM N (C) 1.5 mM N. Arrows indicate mature arbuscules, App

indicates appressorium. Scale bars = 200 µm.

(D) Arbuscule size distribution in arbuscule populations in the roots of R108 amt2;3 and amt2;4

single mutant grown with high P (1 mM) low N (1.5 mM) fertilization, colonized with Glomus

intraradices and harvested at 4wpi. Data represent the mean ± the standard error of arbuscules

from random infection units from R108 (32 IUs), amt2;3 (95 IUs) and amt2;4 (94 IUs). No

significant difference was observed between the genotype for each arbuscule size class.

5

R108

A 15 mM N

R108

B 7.5 mM N

amt2;3

App

R108

C 1.5 mM N

amt2;3

amt2;3

App

AppApp

1.5 mM N, 1 mM P

0

10

20

30

40

50

60

70

R108

amt2;

4

amt2;

3

D

0-25 µm 25-45 µm 45-75 µm

a

a’

a” a” a”

a’a’

a

a

Arb

uscu

le p

erce

ntag

e

Supplemental figure 5

Supplemental Data. Breuillin-Sessoms et al. Plant Cell (2015) 10.1105/tpc.114.131144

Supplemental Figure 6. Morphology of AM symbiosis in pt4-2 amt2;3 and pt4-5 amt2;3.

Microscopy images of G. versiforme infection units at 4 wpi in A17 (WT), pt4-2, amt2;3, pt4-2

amt2;3 and pt4-5 amt2;3 grown with high N (15 mM) fertilization. Arrows = mature arbuscules,

arrowheads = degenerating arbuscules, App = an appressorium. Bars =200µm

6

pt4-2

App

A17

pt4-2 amt2;3 pt4-5 amt2;3

App

App

App

High N

amt2;3

App

Supplemental figure 6

Supplemental Data. Breuillin-Sessoms et al. Plant Cell (2015) 10.1105/tpc.114.131144

Supplemental Figure 7. Arbuscule populations in pt4-2 amt2;3 and pt4-5 amt2;3 at 8 days post

contact with G. intraradices

(A) Arbuscule size distribution in arbuscule populations in A17 (WT) and pt4-2 amt2;3 grown with

low-N (1.5mM) fertilization days at 8 days post contact with primed G. intraradices spores. Data

represent the mean ± the standard error of arbuscules from random infection units from A17 (25

IUs), pt4-2 amt2;3 (38 IUs). Infection units were sampled from 3 independent biological replicates.

Different letters indicate significant differences at α = 0.01.

(B) Arbuscule size distribution in arbuscule populations in R108 (WT), amt2;3 and pt4-5 amt2;3

grown with low-N (1.5mM) fertilization days at 8 days post contact with primed G. intraradices

spores. Data represent the mean ± the standard error of arbuscules from random infection units

from R108 (39 IUs), amt2;3 (30 IUs), pt4-5 amt2;3 (30 IUs). Infection units were sampled from 3

independent biological replicates. Different letters indicate significant differences at α = 0.02.

7

a

a’a”

a”

a’’’b

b’’’

0

10

20

30

40

50

60

0

10

20

30

40

50

60

A

rbus

cule

per

cent

age

Arb

uscu

le p

erce

ntag

e

ab b

a’a”a’’’

a’’’

b’’’

a”

b”

a’

a’

a

A B

0-25 µm25-35 µm 35-45 µm 45-75 µm

pt4-2

amt2;

3A17

pt4-5

amt2;

3am

t2;3

R108

a’

Supplemental figure 7

Supplemental Data. Breuillin-Sessoms et al. Plant Cell (2015) 10.1105/tpc.114.131144

Supplemental Figure 8. Arbuscules in a pt4-5 pt8-1 amt2;3 triple mutant

(A) Percentage of mature arbuscules and degenerate arbuscules with septa in the roots of R108

(WT), WTseg (a WT segregant from the F2 population from which the triple mutant was obtained)

and pt4-5 pt8-1 amt2;3 triple mutant grown with high N (15 mM) fertilization, harvested at 4 wpi

with Glomus intraradices. Data represent the mean ± the standard error of arbuscules from

random infection units from R108 (37 IUs), WTseg (19 IUs), pt4-5 pt8-1 amt2;3 (25 IUs). Different

letters indicate significant differences at α = 0.01.

(B) Percentage of mature arbuscules and degenerate arbuscules with septa in the roots of R108

(WT), WTseg and pt4-5 pt8-1 amt2;3 grown with low N (1.5 mM) fertilization, harvested at 4 wpi

with Glomus intraradices. Data represent the mean ± the standard error of arbuscules from

random infection units from R108 (47 IUs), WTseg (15 IUs), pt4-5 pt8-1 amt2;3 (23 IUs). Different

letters indicate significant differences at α = 0.02.

(C) and (D) Microscopy images of G. intraradices infection units at 4 wpi in R108, WTseg and pt4-

5 pt8-1 amt2;3 grown with (C) high-N (15 mM) and (D) low-N (1.5 mM) fertilization. Arrows =

mature arbuscules, arrowheads = degenerating arbuscules, App = an appressorium, Ves =

vesicle, Sp = spore. Bars =200µm

8

0102030405060708090

100 degenerated arbuscules large arbuscules

R108WTseg

a

a’

a

a’b

b’a

a’

a

a’

b

b’

R108WTseg

pt4-5 pt8-1 amt2;3

A High N B Low N

R108

Ves

pt4-5 pt8-1 amt2;3App

App

WTseg

R108 WTseg

C High N

D Low N

pt4-5 pt8-1 amt2;3

Sp

pt4-5 pt8-1 amt2;3

Arb

uscu

le p

erce

ntag

e

0102030405060708090

100

Arb

uscu

le p

erce

ntag

e

Supplemental figure 8

Supplemental Data. Breuillin-Sessoms et al. Plant Cell (2015) 10.1105/tpc.114.131144

Supplemental Figure 9. Arbuscules in a pt4-5 amt2;4 double mutant

(A) Arbuscule size distribution in arbuscule populations in the roots of two pt4-5, amt2;3 double

mutants (plants #157 and #159), a pt4-5 amt2;4 double mutant, pt4-5 single mutant, a wildtype

segregant from the pt4-5 amt2;4 population and R108 (WT), grown with low N (1.5 mM)

fertilization, harvested at 4 wpi with Glomus versiforme. Data represent the mean ± the standard

error of arbuscules from random infection units from pt4-5 amt2;3 plant 157 (25 IUs), pt4-5 amt2;3

plant 159 (26 IUs), pt4-5 amt2;4 (21 IUs), pt4-5 (26 IUs), WTseg (23 IUs) and R108 (25 IUs).

Different letters indicate significant differences at α = 0.05.

9

0-25 µm 25-45 µm 45-75 µm

Low N

pt4-5

amt2;

3 #15

7

pt4-5

amt2;

3 #15

9

pt4

-5 am

t2;4

WT

pt4-5

R108

Arb

uscu

le p

erce

ntag

e

0

10

20

30

40

50

60

70

seg

ab a

bc ab

cdd

a’a’b’ a’

a’b’a’

b’

a” a”a”b” b”

c” b”

a”c”

Supplemental figure 9

Supplemental Data. Breuillin-Sessoms et al. Plant Cell (2015) 10.1105/tpc.114.131144

Supplemental Figure 10. Analysis of yeast 31019b (∆mep1; ∆mep2; ∆mep3) mep cells

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.

10

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.

Genotype

Shoot fresh weight

(g)

Shoot P content (nmoles.mg-1

dry weight)

Shoot N content (%)

A17 pt4-2 pt4-2 pt8-1

1.21 ±0.11a 1.04 ±0.09ab 0.75 ±0.13b

120.57 ±16.15 111.61 ±11.18 112.83 ±17.23

4.57 ±0.33 4.92 ±0.34 5.53 ±0.31

R108 pt8-1 pt4-5 pt4-5 pt8-1

1.40 ±0.12a’ 1.36 ±0.06a’ 1.19 ±0.13a’ 1.43 ±0.11a’

84.38 ±6.37 76.54 ±6.93 92.56 ±16.81 78.25 ±10.22

3.40 ±0.06 3.57 ±0.21 3.50 ±0.18 4.16 ±0.10

1

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.

Genotype

Shoot fresh weight (g)

Shoot P content (nmoles.mg-1

dry weight)

Shoot N content (%)

Infection unit length (mm)

A17 pt4-2 pt4-2 pt8-1

0.29 ±0.03a 0.28 ±0.01a 0.34 ±0.03a

246.96 ±39.38 242.16 ±6.30

175.50 ±21.79

1.40 ±0.07 1.41 ±0.08 1.83 ±0.17

3.35 ±0.22a 3.05 ±0.25ab 2.77 ±0.26b

R108 pt8-1 pt4-5 pt4-5 pt8-1

0.64 ±0.17a’ 0.69 ±0.14a’ 0.77 ±0.26a’ 0.76 ±0.28a’

128.56 ±4.75 123.34 ±11.67 112.55 ±8.73 120.83 ±5.94

1.23 ±0.18 1.37 ±0.12

1.48 ±0.12

2.03 ±0.06

2.19 ±0.15a’ 2.05 ±0.15a’ 1.47 ±0.09b’ 1.43 ±0.10b’

2

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.

Genotype

Shoot fresh weight

(g)

Shoot P content (nmoles.mg-1

dry weight)

Shoot N content (%)

A17 pt4-2 pt4-2 amt2;3

1.34 ±0.12a 1.26 ±0.09a

1.05 ±0.17a

153.89 ±17.05 185.38 ±27.17 152.45 ±10.66

3.30 ±0.18 3.45 ±0.33 3.26 ±0.19

R108 amt2;3 pt4-5 pt4-5 amt2;3

1.50 ±0.19a’ 1.34 ±0.15a’ 1.59 ±0.15a’ 1.26 ±0.21a’

91.75 ±5.43 96.74 ±18.78 113.10 ±7.33

111.68 ±33.66

2.93 ±0.15 3.79 ±0.64 2.81 ±0.16 3.36 ±0.31

3

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.

Genotype

Shoot fresh weight

(g)

Shoot P content (nmoles.mg-1

dry weight)

Shoot N content (%)

A17 pt4-2 pt4-2 amt2;3

0.29 ±0.02a 0.28 ±0.02a 0.33 ±0.02a

272.22 ±36.04 299.69 ±15.25 210.42 ±33.25

1.25 ±0.11 1.41 ±0.02 1.41 ±0.11

R108 amt2;3 pt4-5 pt4-5 amt2;3

0.64 ±0.04a’ 0.67 ±0.08a’ 0.76 ±0.05a’ 0.55 ±0.08a’

118.50 ±12.65 106.71 ±18.49 93.93 ±12.12

106.86 ±18.75

1.35 ±0.02 1.34 ±0.08 1.31 ±0.04 1.32 ±0.18

4

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.

Genotype

Shoot fresh weight

(g)

Shoot P content (nmoles.mg-1

dry weight)

Shoot N content

(%)

Infection unit length (mm)

R108 WTseg pt4-5 pt8-1 amt2;3

0.63 ±0.02a 0.53 ±0.08a 0.63 ±0.09a

112.45 ±14.07 120.40 ±35.60 135.40 ±43.81

1.16 ±0.04 1.33 ±0.25 1.45 ±0.16

1.25 ±0.15a 0.83 ±0.09a 0.58 ±0.06b

7

Supplemental Data. Breuillin-Sessoms et al. Plant Cell (2015) 10.1105/tpc.114.131144 Supplemental Table 8

Genes* Primer name Sequence (5'-3') α-tubulin (G.v) Gv α-Tubulin F TGTCCAACCGGTTTTAAAGT Gv α-Tubulin-R AAAGCACGTTTGGCGTACAT EF1α EF-1α-F TGACAGGCGATCTGGTAAGG EF-1α-R TCAGCGAAGGTCTCAACCAC PT4 qMtPT4-F GACACGAGGCGCTTTCATAGCAGC qMtPT4-R GTCATCGCAGCTGGAACAGCACCG PT8 MtPT8-F TATGGCCCTTGCGGTTGTG MtPT8-R CGACACTAGCGTTTCTTGTTCC AMT2;1 AMT2;1-F ACCAGCATACCAAGCACATC AMT2;1-R TGTTGCTGCTGTCATTTGCC AMT2;2 AMT2;2-F CCAAAGGAGGTGTCTATGGAGGC AMT2;2-R GTGACACCACTTGAAGCCATGCC AMT2;3 AMT2;3-F TGTCCGGTTCAATTCCATGG AMT2;3-R TGGCAAACACACCAGAAAGG AMT2;4 AMT2;4-F ACAAGGCAATTCATGGAGAG AMT2;4-R GGGAATGGACGAGAAATCTT AMT2;5 AMT2;5-F ATGGAAATCGGTGACGAGGC AMT2;5-R CTCAATCTGGCTACCACGCT AMT2;6 AMT2;6-F GAAGCTTCAACAACACAACCACT AMT2;6-R AGCCATCCAAGCCCTAATGTT Tnt1 TNT1 GCATTCAAACTAGAAGACAGTGCTACC AMT2;3 AMT2;3-F2 AACCACACTCCACCAACCAG AMT2;4 AMT2;4prom-F AAGTGCCAACGAATTAGCCG PT8 MtPT8 R2 CGACACTAGCGTTTCTTGTTCC PT4 MtPT4-F2 CTGGCATGGGATTCTTCACT AMT2;3 AMT2;3EcoRI-F ACAGAATTCATGAATTTTAATTCATCTAAGTATATATCCC AMT2;3XhoI-R ACACTCGAGTTACTCATCAATTTTATTGATAGG AMT2;4 AMT2;4EcoRI-F ACAGAATTCATGGAGCTACCTTCAAACC AMT2;4BamHI-R ACAGGATCCTCATACCATTTGAAGTTCACC AMT2;3 AMT2;3BsrGI-F TATTGTACAAGATGAATTTTAATTCATCTAAGTATATATCCCATTTACC AMT2;3XhoI/NotI-R TATGCGGCCGCCTCGAGTTACTCATCAATTTTATTG AMT2;4 AMT2;4BsrGI-F TATTGTACAAGATGGAGCTACCTTCAAACCTGTTGCCTG AMT2;4SalI/NotI-R TATGCGGCCGCGTCGACTCATACCATTTGAAGTTCAC α-tubulin (yeast) yeast α-tubulin-F AGAAACTTGGATATCCCAAGACCAA Yeast α-tubulin-R GCAATTAGGTTGTTTAAGTTTGCAA G.V – Glomus versiforme

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.