Npr1 in Symbiotic Interactions Figure 4. Accumulation of reactive oxygen species (ROS) in roots of Medicago and of wild-type and nim1/npr1 Arabidopsis seedlings, inoculated with S. meliloti, P. putida, or P. syringae. (A) Roots of 6 day-old M. truncatula seedlings were inoculated with wild- type or nodA mutant S. meliloti, or with P. putida. ROS production was assayed 5, 24 and 48 hours after inoculation by epi-fluorescent microscopy with 29,79-dichlorodihydrofluorescein diacetate and narrow-band GFP filter (Ex 485610 nm/Em 525610 nm). ROS production in uninoculated roots was below detection level (left bar space in each group). P. syringae inoculation produced similar result as P. putida (not shown). Error bars indicate standard deviation of the mean (N = 12). (B) Roots of nine day-old wild-type or nim1/npr1 A. thaliana seedlings were inoculated with wild-type or nodA, or nodH (not shown) mutant S. meliloti, P. putida, or P. syringae. ROS production was assayed 24 hours after inoculation by epi-fluorescent microscopy with 29,79-dichlorodihydrofluorescein diacetate and a narrow-band GFP filter (Ex 485610 nm/Em 525610 nm). All samples were analyzed using identical exposure conditions. Fluorescence from npr1 plants inoculated with S. meliloti nodH2 mutant was even below uninoculated control (data not shown). Shown are representative A. thaliana roots images from four similar experiments 24 hours after inoculation. Bar = 125 mm. (C) Quantitation of ROS production in A. thaliana roots, 24 hours after inoculation with wild-type S. meliloti (S.mel), nodA mutants (nodA2), P. syringae (P.syr) or P. putida (P. put). ROS were quantified using ImagePro Plus software package. Error bars indicate standard deviation of the mean (N = 12). doi:10.1371/journal.pone.0008399.g004 To test the role of NPR1 in symbiotic interaction, we bombarded the M. truncatula roots with Arabidopsis NPR1 gene, attached to a constitutive CaMV 35S promoter, in the zone 1 region, using the BIM-LAB-mediated high pressure air-gun apparatus [50]. Such in planta application of Agrobacterium vectors has been shown to efficiently deliver the transgenes to different plants, other than Arabidopsis [51]. Expression of the NPR1 gene was tested two days after the bombardment by RT-PCR, and showed increased expression in transformed roots (Fig. 7C). Plants were analyzed two days after S. meliloti inoculation, when root hairs stop elongating and begin to show swelling of the tip [52]. Overexpression of the NPR1 gene in M. truncatula resulted in a strikingly long and straight root hair phenotype (Fig. 7B, compare the NPR1-Overexp and empty vector control root hairs; Fig. 7D shows quantification of the above results from 12 seedlings). To further analyze the role of NPR1 in root hair curling, we silenced the NPR1 expression in M. truncatula roots, prior to Rhizobium inoculation, by using the RNAi technique. In order to assure the blocking of interaction between NPR1 and TGA transcription factors, which is essential for induction of PR genes transcription, RNAi was targeted to the NPR1 ankyrin repeats domain that is present in all members of the NPR1-like protein family [53]. Transformation of the NPR1-RNAi almost completely blocked the NPR1 gene expression (Fig. 7C), and resulted in strongly curled root hairs already two days after the PLoS ONE | www.plosone.org 5 December 2009 | Volume 4 | Issue 12 | e8399