1. 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)

2. 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). Fold change Log2 RT-qPCR Fold change Log2 Affymetrix

3. Figure S3 Control HyPer 66 kD 45 kD (a)(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 (—).

4. 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 H 2 O 2 treatment in two biological replicates. The baseline corresponds to MtHap2-1 expression, the gene least strongly expressed in our conditions.

5. 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.

6. 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