Npr1 in Symbiotic Interactions Figure 7. The effect of NPR1 overexpression or silencing by RNAi on the root hair deformation/curling. (A, B) Roots of seven days-old M. truncatula were bombarded with A. tumefaciens containing an empty vector, RNAi silencing (NPR1-RNAi), or with NPR1 overexpressing (NPR1-Overexp) vectors. After transformation the seedlings were transferred to new plates and either left intact (A), or inoculated with S. meliloti after one hour (B). Seedlings were observed for root hair deformation or curling by bright light microscopy two days after inoculation. Roots of six seedlings of each treatment were analyzed. Note the root hair curling already 2 days after S. meliloti inoculation in the NPR1-RNAi transformed roots, while the empty vector transformed roots (control) show only moderate root hair deformation. Bars = 50 mm. (C) NPR1 gene expression following transformation, normalized according to EF1a gene expression. (D) Quantification of the root hair deformation and curling in M. truncatula seedlings overexpressing the NPR1 gene (NPR1-OE) or transformed with RNAi construct (NPR1-RNAi) to suppress NPR1 gene expression. doi:10.1371/journal.pone.0008399.g007 Figure 8. Semiquantitative RT-PCR analysis of pathogenesis- associated gene expression in M. truncatula. M. truncatula seeds were germinated on an N-free medium and after 6 days inoculated with 107 cells of S. meliloti. Total RNA was extracted 24 hours after Rhizobium inoculation. The amount of RNA in the samples was normalized according to EF1a gene expression. Primers to the NPR1-dependent genes in M. truncatula: LRK (TC107689), ARP (TC104767) and WAK (TC109689) were identified by BLAST analysis, using the M. truncatula homologues to A. thaliana gene sequences [59]. M. truncatula seedlings were germinated on N-free medium and inoculated with S. meliloti three days later. Total RNA was extracted 0, 4, 9, 24, and 72 hours post inoculation (h.p.i.) and analyzed by semiquantitative RT-PCR, using the M. truncatula EF1a expression to normalize the amounts of RNA. All experiments were repeated at least three times, independently, with very similar results. doi:10.1371/journal.pone.0008399.g008 Materials and Methods Biological Material and Plant Treatment S. meliloti and M. truncatula were grown as described in [54], except the M. truncatula seeds were scarified for 5 min by exposure to concentrated sulfuric acid. The GFP-labeled fluorescent bacteria was a gift from M. Crespi (CNRS, Gif sur Yvette, France). A. thaliana NPR1 construct was a gift from Xinnian Dong (Duke University, North Carolina). P. syringae were grown as described in [55]. Mutant A. thaliana seeds were grown on agar plates containing 1/60 MS medium. S. meliloti were inoculated on the roots in zone 1 at a concentration of 107 cells. NF was prepared from the S. meliloti strain 1021, according to [56]. Bioinformatics Phylogenetic N-J tree of the plant genes was constructed by using the Kyoto University ClustalW multiple sequence alignment website, (http://align.genome.jp/). Protein sequences. The A. thaliana genes were downloaded from The Arabidopsis Informa- tion Resource (TAIR) website (http://www.arabidopsis.org/) and were uploaded to the Kyoto ClustalW website. The M. truncatula genes were from public EST database (MtDB2.0). PLoS ONE | www.plosone.org 7 December 2009 | Volume 4 | Issue 12 | e8399