Figure S1

Enod11

Mtc27

MtGshs cDNA

gDNA

S. meliloti

DPI

- - ++

- + +-

Supporting Information Fig. S1. Validation of the selected biological conditions for MtEnod11 gene expression and ROS levels. (a) After root treatment (see methods), the level of expression of Enod11 and Mtc27 in the biological samples used for transcriptome analysis was evaluated by RT-PCR. We checked that there was no genomic DNA in RNA samples with primers spanning the intron of MtGshs1. Representative colorimetric detection of the superoxide anion (NBT; b) and hydrogen peroxide (DAB; c) in root hairs. Scale bar = 50 µm. n > 10

(a)

(c)(b)

R2 = 0.9348

-10

-8

-6

-4

-2

0

2

4

6

8

10

-10 -8 -6 -4 -2 0 2 4 6 8 10

Figure S2

Supporting Information Fig. 2. Comparison of expression ratios of probesets in RT-qPCR and Affymetrix gene array analyses. Analysis, RT-qPCR, of the differential accumulation of transcripts, initially identified by Affymetrix GeneChip analysis (Table S2), on two new biological replicates (data from Table 1).

Fol

d ch

ange

Log

2 R

T-q

PC

R

Fold change Log2 Affymetrix

Figure S3

Con

trol

HyP

er

66 kD

45 kD

(a)

0

25

50

75

100

450 500 550 600 650

Wavelength (nm)

Arbi

trary

uni

ts

(b)

Supporting Information Fig. 3. HyPer protein production in composite plants of Medicago truncatula. Western-blot analysis of HyPer in root cultures expressing a control or HyPer construct. (b) Representative emission spectra after 405-nm excitation () and 488-nm excitation (—).

Rip1 bHlhSpk1Spk2AblL Srl1 Nin Pri1

10

Fo

ld c

ha

ng

e

Figure S4

Supporting Information Fig. 4. Relative expression of selected genes in untreated Medicago truncatula roots. The relative expression level of the selected genes was evaluated before H2O2 treatment in two biological replicates. The baseline corresponds to MtHap2-1 expression, the gene least strongly expressed in our conditions.

Figure S5

Control amiSpk1

(a) (b)

(c) (d)

Supporting Information Fig. 5. Phenotypes of infection threads in amiRNA MtSpk1 plants. Composite plants expressing either an empty vector (control; a, c) or the amiRNA against MtSpk1 (amiSpk1; b, d) were inoculated with LacZ (a, b) and DsRed (c, d) S. meliloti strains. IT were observed 4 dai (a, b) and 10 dai (c, d), as described in Figure 5. n > 10. Arrows show ITs. Scale bar = 100 µm.

Figure S6

Supporting Information Fig. 6. Subcellular distribution of MtSPK1. Composite plants expressing a cytosolic GFP (a), a BUBR1::GFP (b; nuclear protein) or an SPK1::GFP translational fusion (c, d) were obtained and GFP fluorescence was monitored. Scale bar = 100 µm.

(a)

(b)

(c) (d)

GFP

BUB1::GFP

SPK1::GFP