Supporting information – Methods
Pathogenicity assays
Leaves were infected with Pseudomonas syringae pv. tomato DC3000 and Xanthomonas
campestris pv. campestris 8004 as previously reported by infiltrating the leaf mesophyll with
bacterial suspension (5.105 CFU ml-1) using a syringe with no a needle (Katagiri et al., 2002;
Meyer et al., 2005). In each experiment, four leaves per plant and 10 plants per genotype were
inoculated. The plants were then grown under a 9 h light /15 h dark, 22°C, 70% RH regime.
prn1 prn2 and prn2 prn3 double mutants were generated by crossing prn1 (SALK_006939) and
prn3 (SAIL_1243) T-DNA insertion mutants to prn2-1 and prn2-2.
TEM analysis
Plants were grown in growth chambers in short-day conditions (8 h light/16 h dark, 21°C/18°C,
70% RH) for 8 weeks. Transverse hand-sections of hypocotyls were fixed using 2.5% (v/v)
glutaraldehyde in 0.1 M cacodylate buffer overnight at 4 °C. After washing three times in
buffer, the specimens were post-fixed with 1% (v/v) osmium tetroxide in the buffer for 1 h
and washed twice in distilled water. Samples were dehydrated with 50, 70, 95, 100% ethanol
then infiltrated with and embedded in Spurr's resin. Ultrathin (80 nm) sections were made
using a Diatome diamond knife on a Leica EM UC7 (, collected onto copper
grids, then treated with 5% uranyl acetate in water for 20 min followed by Sato's lead staining
for 5 min. Sections were examined using a Jeol 1230 TEM ( Digital images were
captured using a Gatan MSC 600CW (
RNA extraction and RT-PCR
Total RNA was isolated from the 8-day-old seedlings, and leaves, stems, flowers, siliques and
and roots of mature WT (Col-0), prn2 mutants and overexpressors using an RNeasy plant mini
kit (Qiagen, according to the manufacturer’s protocol. All samples were
treated by RNase-Free DNase Set (Qiagen) in columns to remove any remaining DNA. cDNA
was prepared using oligo(dT) and random primers and 1 µg of RNA as template. Semi
quantitative RT-PCR was performed using 1:5 diluted cDNA with Taq polymerase for 30 cycles.
Arabidopsis Actin1 (AT2G37620) was used as a reference gene. Quantitative RT-PCR (qPCR)
was performed using 1:40 diluted cDNA, iQ SYBR Green Supermix (Bio-Rad,, 5 pmol of each primer, an iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad, and the following program: initial denaturation at 95°C for 3 min followed
by 40 cycles of 10 s denaturation at 95°C and 30 s annealing at 55°C. The Arabidopsis SAND
(AT2G28390) gene was used as a housekeeping gene. Three biological replicates were used
for analysis. Primer sequences are listed in Supplemental Table 2.
qPCR after Ralstonia solanacearum infection
Col-0 plants were challenged with the virulent strain GMI1000. Nd-1 plants were challenged
with the virulent strain GMI1000 ΔpopP2 (Deslandes et al., 2003). For qPCR analysis, the aerial
parts of four plants were collected and RNA isolated from frozen tissues using the NucleoSpin
RNAII kit (Macherey-Nagel, according to the manufacturer’s
recommendations. An additional DNase I treatment was performed with Ambion Turbo kit
(Ambion, to eliminate all
remaining DNA. SuperScript II RT kit was used for cDNA synthesis (Invitrogen, qPCR reactions were performed using 1:10 diluted cDNA with a
Lightcycler (Roche, using the LightCycler FastStart DNA
MasterPlus kit and the following program: initial denaturation at 98°C for 9 min followed by
45 cycles of 5 s at 95°C, 10 s at 65°C, and 20 s at 72°C. The Arabidopsis AT5G09810 was used
as a reference gene.
Analysis of subcellular localization of the PRN2:GFP fusion protein
A PRN2:GFP fusion protein was created by amplification of a 3838-bp fragment of the AtPRN2
genomic sequence including promoter and coding regions. The fragment was cloned into
pDONR207 vector by BP Clonase II (Invitrogen, and recombined
into pMDC107 destination vector (Curtis and Grossniklaus, 2003) that was used for
Arabidopsis transformation by floral dipping.
Subcellular localization of the PRN2:GFP fusion protein was determined using a TCS SP2 (Leica, confocal microscope and a 488 nm Ar/Kr laser line. For
plasmolysis, 3 M NaCl in half-strength MS was applied to seedlings on-slide for 5 min prior to
Curtis, M.D. and Grossniklaus, U. (2003) A gateway cloning vector set for high-throughput
functional analysis of genes in planta. Plant Physiol., 133, 462-469.
Deslandes, L., Olivier, J., Peeters, N., Feng, D.X., Khounlotham, M., Boucher, C., Somssich, I.,
Genin, S. and Marco, Y. (2003) Physical interaction between RRS1-R, a protein
conferring resistance to bacterial wilt, and PopP2, a type III effector targeted to the
plant nucleus. Proc. Natl. Acad. Sci. USA, 100, 8024-8029.
Katagiri, F., Thilmony, R. and He, S.Y. (2002) The Arabidopsis thaliana-Pseudomonas syringae
interaction. In The Arabidopsis book. American Society of Plant Biologists, pp. e0039.
Meyer, D., Lauber, E., Roby, D., Arlat, M. and Kroj, T. (2005) Optimization of pathogenicity
assays to study the Arabidopsis thaliana-Xanthomonas campestris pv. campestris
pathosystem. Mol. Plant Pathol., 6, 327-333.
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. and Kumar, S. (2011) MEGA5:
molecular evolutionary genetics analysis using maximum likelihood, evolutionary
distance, and maximum parsimony methods. Mol. Biol. Evol., 28, 2731-2739.