RT-PCR Assay Total RNA was extracted from roots before and after S. meliloti inoculation. Roots were frozen in liquid nitrogen. RNA was extracted with Tri Reagent (Molecular Research Center, Inc) and transcribed into cDNA using oligo dT as a primer with SuperScript II reverse transcriptase (Invitrogen). cDNA was amplified by PCR using Taq polymerase and the following primers: NPR1: forward TGACTTGTTTTACCTTGAGAA and reverse, AATTATTTTATAGAGAGGAGA. a-dioxygenase: for- ward, GAAGTTTTGGACAAAGTGAGGACT; reverse TGTC- AGTTTTAAGAAGCTCCACAG. At5g57920: forward, TAAC- GAATGGGCTCAAAAGG; reverse CTGGACCGTCGAACT- CAGAT. At5g07050: forward, TGGGATTGTGGCATCAA- GTA; reverse CCCCTTCCGAGATTTTCATT. LRK: forward CAACTCATTTGGTTGGAACTGTAG reverse GGATAAGA- CAAAGGAAAGTCCTCA. ARP: forward TCTTCTCCATT- TCCTCAATTTCA, reverse TTATTAAGAGCAGCCCACT- GAAG. WAK: forward CAGGAGGTTGTCATAAACAAGATG reverse, AAGTGTAACCCGTTGCTAACAAAT. EF1a gene, forward TCACATCAACATTGTGGTCATTGGC; reverse, TTGATCTGGTCAAGAGCCTCAAG. EF1a and Actin2 were used to normalize RNA amounts in M. truncatula and A. thaliana respectively. ROS Production in Plants Seedlings were taken out of agar plates after 3 days, washed, and transferred to new plates with nitrogen-free medium. ROS in Arabidopsis roots were detected by 10 mM 29,79-dichlorofluorescin and ROS levels were quantified with ImagePro Plus analysis package (Media Cybernetics, USA) as described in [38]. Roots were photographed with Nikon Coolpix 4500 camera attached to Olympus IX70 microscope. The fluorescent light pass settings used narrow-band cube (Omega Optical Inc., Brattelboro, VT, USA) 484620 nm excitation and 535610 nm emission filters. The pixels of mean density were collected from representative images for statistical analysis (N = 12). RNAi Cloning Silent sites from the Medicago NPR1 (TC102752) gene were selected, and used to design complementary oligonucleotide primers: forward: 59- ATCTCTGCCGGAATCAACAC-39, and reverse: 59-TCTGATGCACAAGCTCCGTTTTTC-39. The seg- ment was amplified by PCR and cloned into pENTR using Invitrogen TOPO10-cloning kit and transformed at room temperature for 5 min, then on ice for 30 min, 42uC for 45 sec, and spread on LB solid medium with 50 mg/ml kanamycin. Clones were selected one day later, and sequenced. NPR1- pENTR plasmid was used for the LR recombined reaction using Invitrogen’s Gateway LR Clonase II enzyme mix and transformed to TOPO10 competent cells as described above, only the LB contained 100 mg/ml spectomycin and 300 mg/ml streptomycin. Clones were selected one day later, and sequenced using the forward and reverse primers from upstream and downstream sequences of the antisense insertion. Transformation of A. tumefaciens and Root Bombardment NPR1-RNAi cloned plasmids were transfected into A. tumefaciens GV3101 by freezing in liquid Nitrogen for 5 min and spread on TYNG solid medium containing 50 mg/ml rifampicin, 25 mg/ml Gentamicin, 100 mg/ml spectomycin and 300 mg/ml streptomy- cin. Monoclonal colonies were selected two days later, and analyzed by PCR for identification. Bacteria were shot with the Npr1 in Symbiotic Interactions addition of 1:1000 M/V carborundum into 7 day-old M. truncatula roots, using Bim-LAB apparatus (Bio-Oz, Kibbutz Yad-Morde- chai, Israel), essentially, as described in [57]. Plants were taken from plates and the roots were bombarded, using bacterial density of OD600 = 0.5-1 and pressure of 6 Barr, as described in [50]. Plants were then moved to new plates containing N-free medium. Supporting Information Figure S1 Salicylic acid inhibits root hair curling in M. truncatula. (A-F) Three day old M. truncatula seedlings were transferred to plates supplemented with SA (D–F) or replanted on new plates with N-free medium (A–C). The roots of the seedlings were inoculated in zone 1 after 24 h with S. meliloti (C, F) or treated with NF (B, E). SA was applied by dispersing 1 ml of 500 mM SA on top of the plates for 6 days. Pictures were taken 4 days after inoculation, or 2 days after NF treatemnt. At least one hundred root hairs within zone 1 were scored. The percentage of deformed and curled root hairs is indicated on the bottom of each image (upper and lower row, respectively). The 6 number indicated the standard deviation. (G) M. truncatula seedlings were germinated on N-free medium and inoculated with S. meliloti three days later. Total RNA was extracted 24 hours after Rhizobium inoculation. The amount of RNA in the samples was normalized according to the EF1a gene expression. The primers to alpha-dioxygenase gene were selected by BLAST analysis using the A. thaliana gene sequences (At3g01420). All experiments were repeated at least three times with very similar results. Found at: doi:10.1371/journal.pone.0008399.s001 (0.78 MB TIF) Figure S2 The effect of S. meliloti inoculation on A. thaliana dnd1 and nim1/npr1 mutants. A. thaliana seedlings were grown as described in Figure 1. Eight-day old dnd1 or nim1/npr1 mutants were either left untreated (2) or inoculated with S. meliloti (+). Roots were photographed four days after inoculation under bright light. Bar = 25 mm Found at: doi:10.1371/journal.pone.0008399.s002 (0.15 MB TIF) Figure S3 Treatment of M.truncatula with purified nod factor. Five day old M. truncatula seedlings were exposed to nod factor (+NF) or left intact (2NF). Roots were photographed 3 days after treatment under bright light. Roots of six seedlings of each treatment were analyzed. Bar = 35 mm Found at: doi:10.1371/journal.pone.0008399.s003 (0.10 MB TIF) Figure S4 Phylogenetic tree of ENOD20. (A) Phylogenetic N-J tree of the Arabidopsis homologs of the M. truncatula early nodulin ENOD20 (TC114239). The tree was constructed using the Kyoto University ClustalW multiple sequence alignment website, (http:// align.genome.jp/). Right panel shows the unrooted version of the tree. The genes were chosen using the BLAST program: http:// www.arabidopsis.org/wublast/index2.jsp, to the amino acid se- quence of TC114239 (ENOD20) at: http://compbio.dfci.harvard. edu/tgi/cgi-bin/tgi/gireport.pl?gudb = medicago (B) Alignment of the Arabidopsis homologs of the M. truncatula ENOD20 (TC114239). The alignment was done using ClustalW2 multiple sequence alignment website from the European Bioinformatics Institute (http://www.ebi.ac.uk/Tools/clustalw2/index.html). Found at: doi:10.1371/journal.pone.0008399.s004 (0.07 MB PDF) Figure S5 Phylogenetic tree of MtN21. (A) Phylogenetic N-J tree of the Arabidopsis homologs of the M. truncatula MtN21 (TC117774) late nodulin gene. The tree was constructed using the Kyoto University ClustalW multiple sequence alignment website, (http://align.genome.jp/). Right panel shows the un- rooted version of the tree. Genes were selected with the BLAST PLoS ONE | www.plosone.org 8 December 2009 | Volume 4 | Issue 12 | e8399