Supplemental figure 1. Effect of ectopic cytokinin application on Ckx3 mRNA levels. Expression

relative relative to mock treatment levels 0-12 h after BAP (10-8 M) treatment are shown. Values

indicate mean ± 95% CI for n=3. P-values were calculated using Wilcoxon rank-sum testing between

mock and treatment groups and are indicated by *<0.05

0 3 6 120

5

10

Time after treatment (h)

Relativeexpression

10-8 BAP

*

*

Supplemental figure 2. Nodulation phenotypes of ckx3 mutants grown in open pot conditions. Total

nodule numbers for Gifu, ckx3-1 and ckx3-2 5 w after inoculation with M. loti R7A and grown in

nitrate-free open vermiculite pots. Values are mean ± 95% CI for n=65-69. P-values were calculated

using Student’s T-test between Gifu wild-type and mutants and are indicated by *<0.05, ***<0.001.

Supplemental figure 3. BAP induced spontaneous nodules. A, Gifu wild-type. B, ckx3-1. Plants were

grown in nitrate free conditions on agar slopes containing 10-8 M BAP. Spontaneous nodules are

shown 4 weeks after transfer to plates containing BAP. Scale bar 0.5 mm.

A B

0.5 mm

Supplemental figure 4. Depiction of quantification used for root development. Measurements for

root tip length and root hair development angle relevant to Figure 7 are indicated.

Supplemental figure 5. Cytokinin base and riboside concentrations in Ljckx3-2 relative to Gifu in the

same treatment conditions. Values represent mean ± 95% CI for n=4 biological replicates. P-values as

determined by Wilcoxon rank-sum testing are indicated by *<0.05.

mock24h

nodC24h

R7A24h

mock72h

nodC72h

R7A72h

mock24h

nodC24h

R7A24h

mock72h

nodC72h

R7A72h

mock24h

nodC24h

R7A24h

mock72h

nodC72h

R7A72h

mock24h

nodC24h

R7A24h

mock72h

nodC72h

R7A72h

0

1

2

3

4

ckx3-2relativeCK(ckx3-2/WT)

* **

**

**

*

*

*

*

*

tZ cZ DHZ iP

Supplemental figure 6. Effect of ectopic cytokinin application on pCkx3::tYFPnls as determined

with confocal microscopy. Roots were treated with BAP (10-8 M) for 3 h. A, Untreated roots showed

expression in the central root vasculature. B, BAP treated roots showed enhanced cortical YFP

activity while maintaining vascular activity. C, Cortex; V. Vascular cylinder. Images are whole

mounts.

A B

C C

V

V

 

Supplemental Figure 7. Activity of LjCkx3 2kb promoter during root and nodule development. Spatio-temporal expression driven by the Ckx3 promoter was determined in hairy roots using confocal microscopy with a pLjCkx3::tYFPnls reporter (nuclear-localised, green). A to C are uninoculated while D-H are inoculated with M. loti expressing DsRED (Magenta) A, The meristematic zone at the root tip independent of inoculation. B, Expression in uninoculated roots and C, Root cross-section showing expression in pericycle adjacent to xylem poles. D, Expression in the central vascular cylinder of inoculated roots. E and F, Nodule primordia at 5 d after inoculation with M. loti. Note the expression associated with dividing cells in the cortex and pericycle. G, Cortex of growing nodules. H, Periphery of fixing nodules. A, B and D are whole mounts while others show sections (80-100 µM). C, cortex; E, endodermis; P, pericycle; X, xylem; Ep, epidermis; V, Vascular cylinder. Scale bar 100 µM.

 

Supplemental Figure 8. Absence of lateral root phenotypes of Ljckx3 mutants. The lateral root

density of 3 week-old Gifu, ckx3-1 and ckx3-2 plants grown on agar slants was not significantly

altered between WT and mutant plants. Values represent mean ± 95% CI for n=18-20. Statistical

comparisons were conducted between WT and ckx3 mutants using ANOVA and Tukey post-hoc

testing.

0"

0.02"

0.04"

0.06"

0.08"

0.1"

0.12"

Gifu" ckx311" ckx312"

Lateral'roo

t'den

sity'(LR/mm)'

Lateral'root'density'

Supplemental table 2. Oligonucleotide primer sequences used in this study

Primer name Purpose Sequence

Annealing temperature

qPCR efficiency

CKX3 F real-time catgaacagaaacaagtggga 60 1.830

CKX3 R real-time ttctgagcatcataagccttc 60 1.830

UBC F real-time atgtgcattttaagacaggg 61 1.874

UBC R real-time gaacgtagaagattgcctgaa 61 1.874

PP2A F real-time GTAAATGCGTCTAAAGATAGGGTCC 60 1.873

PP2A R real-time ACTAGACTGTAGTGCTTGAGAGGC 60 1.873 ATP synth F real-time CAATGTCGCCAAGGCCCATGGTG

60 1.869

ATP synth R real-time AACACCACTCTCGATCATTTCTCTG

60 1.869

5497 LORE1 F ckx3-1 genotyping CAAAAGATTCTTCCTCCCCTC

5497 LORE1 R ckx3-1 genotyping GATTCCAACACCATTCCCTC

17827 LORE1 F ckx3-2 genotyping CCTTGTCCACATTGACTTCGGATTGG

17827 LORE1 R ckx3-2 genotyping

TCCTTCTAACTTGGATGTGACACGTTGAAA

LORE1 P2

LORE1 insert primer CCATGGCGGTTCCGTGAATCTTAGG

pCkx3 F promoter cloning CACCATTCCAACTGCCATCAACAT

pCkx3 R promoter cloning TTTTTGCTATGAAGAAAGAGAGTGATTG

tYFPnls F YFP CATGGGCTAGAAGCTTGGAT

tYFPnls R YFP including nls TTAATCCTCAACCTTTCTCTTCTTCTTAGGAATGTACAGCTCGTCCAT

 

Supplemental table 3. CKX gene ID and accession numbers for L. japonicus, M. truncatula and A.

thaliana.

Gene Gene ID/Accession no.

AtCKX5 AT1G75450.1

AtCKX2 AT2G19500.1

AtCKX1 AT2G41510.1

AtCKX6 AT3G63440.1

AtCKX4 AT4G29740.2

AtCKX7 AT5G21482.1

AtCKX3 AT5G56970.1

MtCKX1 Medtr1g015410

Medtr2g039340

MtCKX3 Medtr2g039410

Medtr3g036100

Medtr3g112350

Medtr4g044110

MtCKX2 Medtr4g126150

Medtr4g126160

Medtr7g090920

LjCKX1 KR296932

LjCKX2 KR296933

LjCKX3 KR296934

LjCKX4 KR296935

LjCKX5 KR296936

LjCKX6 KR296937

LjCKX7 KR296938

LjCKX8 KR296939

LjCKX9 KR296940