Figure 3. Gene expression analysis of early and late M. truncatula nodulin gene orthologs in A. thaliana roots. (A) Roots of ten days old wild-type or nim1/npr1 seedlings grown on nitrogen poor medium were inoculated in zone 1 with either S. meliloti or treated with nod factor (NF). The expression of Arabidopsis ENOD20 homolog (AtENOD20, At5g57920) was tested two days after inoculation, using quantitative Real-time RT-PCR. The results show the mean of 3 independent repeats for each treatment. Error bars represent SD. (B) Roots of ten days old wild-type and nim1/npr1 seedlings grown on nitrogen poor medium were inoculated with S. meliloti or with P.putida. Late nodulin, (AtMtN21, At5g07050) was assayed after 7 days. Plant roots were frozen in liquid nitrogen and gene expression was analyzed by semiquantitative RT-PCR. The RNA samples were normalized according to actin-2 gene expression. Experiments were repeated 3 times with very similar results. doi:10.1371/journal.pone.0008399.g003 Induction of Oxidative Burst in A. thaliana and M. truncatula Plants Inoculated with S. meliloti or with P. putida or with P. syringae Bacteria A major hallmark of plant interaction with microorganisms, which is particularly characteristic of pathogen attack is generation of reactive oxygen species (ROS), which leads to hypersensitive cell death [36,37]. Recently, however, ROS production was also observed in symbiotic interactions in M. truncatula roots inoculated with S. meliloti [38], or treated with compatible Nod Factor [39]. Moreover, oxidative burst was shown to play an important role in the formation of S. meliloti infection threads [40]. We assayed ROS production in plants inoculated with wild-type or mutants S. meliloti, or with P. putida, or P. syringae bacteria, using 29,79-dichlorofluorescin diacetate, which reports ROS production inside the cells [41,42]. A strong oxidative burst was detected in M. truncatula roots already 5 hours after inoculation with either bacterium (Fig. 4A). In plants inoculated with P. putida or P. syringae bacteria ROS began to decline after the 5 hour peak, and much less ROS were detected after 24 hours, and almost none after 48 hours, particularly in roots inoculated with P. putida. However, in plants inoculated with S. meliloti the accumulation of ROS peaked after 24 hours, and remained high at least for the first 2 days of interaction (Fig. 4A). Npr1 in Symbiotic Interactions To assess the role of intact NF in ROS production, we inoculated S. meliloti, mutated in the nodA gene, which is required for the synthesis of N-acetylglucosamine backbone that is essential for correct NF recognition [43]. The nodA mutant rhizobia evoked a considerably smaller ROS response after 5 hours, which was further decreased by 24 hours (Fig. 5A). In A. thaliana, the 24 hour time point post inoculation was selected for all of the experiments, as preliminary tests established it as peak time in ROS production induced by S. meliloti. Only negligible amounts of ROS were detected after S. meliloti inoculation in the wild-type roots (Fig. 4B, top panel and Fig. 4C). However, a very strong oxidative burst was observed in the nim1/npr1 mutants inoculated with S. meliloti (Fig. 4B, bottom panel and Fig. 4C). To analyze the requirement of intact NF for the recognition of the NF by A. thaliana, the nim1/npr1 mutants were inoculated with S. meliloti mutated in nodA, which resulted in decreased ROS production, in agreement with the M. truncatula data (Fig. 4A). Moreover, strong ROS induction was observed in roots treated with purified wild-type NF, specifically in the nim1/npr1 mutants or NahG transformants (Fig. 5). The ROS results are particularly interesting in view of the studies that showed inhibition of the DMI3 gene (a coordinator of ENODs expression) by diphenyleneiodonium (DPI), implicating activation of NADPH oxidase [38,44]. ROS were also shown to act in the induction of symbiotic peroxidase gene, RIP1 [39]. We were therefore interested to test the involvement of ROS in expression of At5g57920. Ten days-old wild-type and nim1/npr1 Arabidopsis mutants were pretreated with DPI, as described in [44], after which the plants were inoculated with S. meliloti. The expression of At5g57920 was tested by quantitative real-time RT- PCR four days after inoculation (Fig. 6). DPI suppressed the At5g57920 transcription, emphasizing the role of ROS in symbiotic interactions. Our data indicates that ROS induction during the symbiotic interaction is regulated by NF perception. We also show that in legume M. truncatula and in non-legume A. thaliana the response towards S. meliloti is regulated by SA signaling. Suppression of SA signaling, either by decreased SA synthesis, as shown in M. truncatula [10] by a yet unknown mechanism, or by mutation of SA-sensing protein, NPR1 (as shown in A. thaliana) brings out similar responses in both plant species. The NPR1 protein may be involved in reducing intracellular ROS, possibly by inducing antioxidants. This suggestion is supported by analysis of NPR1- dependent expression of multiple genes [45,46]. The intermolec- ular reduction of the NPR1 protein, which follows the oxidative burst, that results in PR1 induction is in agreement with this suggestion [5,47]. The Effect of NPR1 Overexpression and/or Silencing on M. truncatula Root Hair Deformation NPR1 is the founding member of a small gene family that contains several NPR1-related or NIM1-like proteins, all of which share the BTB–POZ and the ankyrin-rich repeats domains [48]. To explore the possible role of NPR1 in symbiotic interactions we identified an NPR1-like homolog of M. truncatula (TC102752) in the public EST database (MtDB2.0). The NPR1-like proteins in M. truncatula also form a family (Fig. S6). The Medicago gene has 40% identity and 58% similarity to the A. thaliana protein and also contains both of the conserved domains that were shown to function in binding and interaction with other proteins, namely the BTB/POZ and ankyrin repeats domains [49]. The M. truncatula protein sequence also contains the conserved cysteines that function in the redox-mediated multimerization [5]. PLoS ONE | www.plosone.org 4 December 2009 | Volume 4 | Issue 12 | e8399