mutations that reduce SA production (pad4 or eds1) suppressed the edr phenotype in all of the edr mutants [8]. Furthermore, the edr phenotype was also suppressed by expression of SA hydroxylase NahG transgene, which converts SA to catechol, resulting in rapid SA decomposition [9]. Compared to the pathogenic interactions only a few studies addressed the involvement of SA in symbiotic interactions. SA measurements in M. truncatula during the first stages of symbiotic interaction with Rhizobium showed a reduction in the amount of SA [10]. Moreover, reduction of endogenous SA levels in M. truncatula by the NahG transgene resulted in increased rhizobial infection and nodulation. Furthermore, inoculation of incompat- ible strains of S. meliloti on alfalfa (Medicago sativa) roots led to accumulation of SA, and exogenous application of SA to alfalfa plants inhibited nodule formation [10]. Aborted infections were also shown to be accompanied by an HR-like defense response, including necrosis and accumulation of PR proteins, suggesting activation of plant defense responses in aborted S. meliloti infection threads [11,12]. Exogenous SA addition was also shown to inhibit indeterminate nodulation (e.g., in vetch, with a persistent meristem), but not in determinate nodulation (e.g., in Lotus japonicus with no persistent meristem) [13]. Interestingly, ROS production has been recently shown to occur not only in pathogenic interactions but also during symbiotic interactions [14]. Physiological concentrations of SA were also shown to markedly increase defense gene induction and H2O2 accumulation in soybean infected with avirulent pathogens [15]. Thus, redox and SA signaling, both may have direct effects during symbiotic, as well as pathogenic interaction. Here, we show that SA and NPR1 negatively affect the symbiotic interactions between M. truncatula and Rhizobium. We also show that npr1 mutants in non-legume A. thaliana respond to S. meliloti by activating root hair deformation and induction of early and late nodulin genes. Interestingly, both M. truncatula and npr1 mutant A. thaliana responded with an extremely strong oxidative burst to S. meliloti inoculation, which lasted beyond the restoration of redox after inoculation of Pseudomonas putida or Pseudomonas syringae. Results/Discussion The Effect of Salicylic Acid on Root Hair Deformation Following Sinorhizobium meliloti Inoculation Salicylic acid is a major regulator of plant defenses to pathogenic microorganisms, and was shown to adversely affect plant symbiotic interactions [12]. We analyzed the early steps in legume-Rhizobium interaction, involving root hair deformation that precedes hair curling in legumes. Root hair deformation is one of the first steps in interaction with compatible rhizobacteria [16]. To examine the effect of SA on Rhizobium-induced root hair deformation we first pretreated M. truncatula seedlings with SA prior to inoculation of S. meliloti, which resulted in the inhibition of root hair deformation (compare Fig. S1F with S1C). SA pre- treatment also inhibited the root hair deformation by NF (compare Fig. S1E with S1B). To analyze the SA-mediated signaling during symbiotic interactions in M. truncatula, we tested the expression of the alpha-Dioxygenase (a-Dox) gene that is regulated by SA in tomato, tobacco and Arabidopsis thaliana [17,18,19,20]. The alpha-Dox gene expression in M. truncatula roots was reduced during the first day after S. meliloti inoculation (Fig. S1G), which is in agreement with the reduced amount of SA seen in Medicago sativa during the first stage of rhizobial infection [10]. To explore if the root hair deformation is a typical legume response to compatible Rhizobium species, or a general plant Npr1 in Symbiotic Interactions response to rhizobacteria, we examined the root hair responses in a non-legume Arabidopsis thaliana. Seedlings were inoculated with S. meliloti, or with mutualistic P. putida or with pathogenic P. syringae bacteria [21]. We chose several mutants that are compromised in pathogenesis responses to avoid possible activation of pathogen- esis-associated hypersensitive reaction (HR) that may obscure other physiological responses. We focused on the major pathogen resistance pathway that is mediated by SA: dnd1 (defense no death), which are mutated in cyclic nucleotide-gated ion channel [22], ndr1-1 (nonrace-specific disease resistance), which encodes a plasma membrane protein with unknown function [23], and nim1 (noninducible immunity) [24], also called npr1 (nonexpresser of PR genes) [25]. The list of mutants and their putative signaling pathways is summarized in Table S1. A. thaliana seeds were germinated on nitrogen poor medium, and inoculated in the root elongation zone with S. meliloti eight days later. No difference in root hair behavior was seen in dnd1 (Fig. S2), or in ndr1 mutants, that do not show HR (data not shown) [22,23]. However, a very strong root hair deformation and even hair bending were detected in the nim1/npr1 seedlings (Fig. 1B, and Fig. S2). Root hair deformation was specific for S. meliloti that produced intact NFs, as no such effect was seen in plants inoculated with mutant S. meliloti, in the nod factor genes, nodA (Fig. 1D), or nodH (data not shown). No deformation was seen also in plants inoculated with P. putida (Fig. 1A-C, P.p). Interestingly, quantitative analysis of A. thaliana nim1/npr1 mutants showed a similar percentage of deformed root hairs (Fig. 1D), as in M. truncatula dmi2 mutants after rough NF application/treatment [26]. Importantly, no root hair deformation was seen in plants inoculated with Pseudomonas syringae or P. putida, or following medium refreshment, as in the case described by Esseling et al. It should be noted that in our experiments the seedlings were left intact in the Petri dish throughout the whole experiment, and were not manipulated before or during the microscopic observation. Thus, in our case the root hair phenotype is not related to touch response during the experimental handling [26]. In addition, we inoculated SA-deficient A. thaliana with S. meliloti that were transformed with NahG [27]. The NahG-transformats mimicked the root hair deformation of nim1/npr1 (Fig. 1C). Also, in the case of NahG plants, the effect was a specific response to S. meliloti, since no deformation was seen upon inoculation of P. putida or P. syringae (not shown). To substantiate the NF-dependent early signaling in non-legume A. thaliana, we applied purified nod factor from the S. meliloti strain used above to seedlings’ roots, without the bacteria. The NF treatment induced root hair deformation exclusively in the SA- insensitive nim1/npr1 mutants, or in SA-deficient NahG transfor- mants, in agreement with data observed in plants treated with intact S. meliloti (Fig. 1, NF). It should be noted that although root hair deformation in A. thaliana was significant, we did not observe branching that was seen in M. truncatula (Fig. S3). The attachment of S. meliloti to legume root hairs involves a specific activation of a plant-dependent process, which requires more than just inherent adhesiveness of bacteria to plant cell walls [28,29]. The attachment process involves secretion of specific glycoprotein lectin-polysaccharides by the host symbiont, which induces formation of biofilm in zone 1 of legume roots [29,30]. We used GFP-labeled S. meliloti to observe the bacteria plant interaction. Strong adherence of S. meliloti to the A. thaliana root hairs was seen in nim1/npr1 mutants, but not in wild-type or in dnd1, or in ndr1 roots (Fig. 2A and data not shown). The bacteria remained attached to the nim1/npr1 roots even after extensive washing (Fig. 2B), as described by [31]. Moreover, increasing the washing stringency by addition of 100 mM NaCl to the wash PLoS ONE | www.plosone.org 2 December 2009 | Volume 4 | Issue 12 | e8399