Figure 5. Accumulation of ROS in wild-type, nim1/npr1, and NahG A. thaliana roots, treated with nod factor. Roots of nine day- old wild-type, nim1/npr1, or NahG transformants A. thaliana seedlings were either left intact or treated with nod factor (NF) and 24 hours later were assayed for ROS production by epi-fluorescent microscopy using 29,79-dichlorodihydrofluorescein diacetate and a narrow-band GFP filter (Ex 485610 nm/Em 525610 nm). All samples were analyzed using identical exposure conditions. Bars = 50 mm. doi:10.1371/journal.pone.0008399.g005 Rhizobium inoculation (Fig. 7B, compare the NPR1-RNAi and empty vector control root hairs). Since normally after two days root hairs show only swelling, and root hair curling occurs around 4 days after S. meliloti inoculation [52], this data demonstrate accelerated root hair response in the antisense transformants. These results suggest an inhibitory function of NPR1 on root hair curling. Expression of NPR1-Dependent Gene Homologs in M. truncatula To compare the NPR1-dependent gene expression in M. truncatula following S. meliloti inoculation with gene expression in A. thaliana infected with pathogenic bacteria, we selected several defense genes that were shown to be regulated by NPR1 in A. thaliana [45]. The M. truncatula orthologs of the Arabidopsis LRK (Lectin Receptor Kinase), ARP (Ankyrin repeat-containing protein) and WAK (Wall Associated Kinase) genes were identified by BLAST analysis of the M. truncatula genome project database (http://www.medicago.org/genome/). All of the genes showed Npr1 in Symbiotic Interactions Figure 6. The effect of diphenyleneiodonium on S.meliloti- induced ENOD expression. Roots of ten days old wild-type or nim1/ npr1 seedlings were transferred to plates supplemented with 8 mM diphenyleneiodonium (DPI) or replanted on the same (nitrogen poor) medium. After four hours the roots of all plants were inoculated in zone 1 with S. meliloti. The expression of Arabidopsis ENOD20 homolog (At5g57920) was tested two days after inoculation, using quantitative Real-time RT-PCR. The RNA samples were normalized according to actin-2 gene expression. The results show the mean of 3 independent repeats for each treatment. Error bars represent SD. doi:10.1371/journal.pone.0008399.g006 constitutive expression in M. truncatula roots. However, inoculation of S. meliloti caused a window of transcriptional downregulation, starting at 4 hours post inoculation (p.i.) and culminated at 9 hours p.i. (Fig. 8). The gene expression began to recover 24 hours p.i., and resumed to normal levels after 48–72 hours (Fig. 8). These results are in line with the observed reduction in SA accumulation in M. truncatula during first 24 hours after S. meliloti inoculation [10,12]. Concluding Remarks True symbiotic interactions in plants are thought to be limited to legume family. Our results show that the early basic responses to Rhizobium inoculation, such as root hair deforma- tion and induction of early and late nodulin-like genes are conserved between lugume M. truncatula and a non-legume A. thaliana. However, in A. thaliana these responses were observed only in nim1/npr1 mutant background, suggesting that the NPR1 protein suppresses the plant responses to Rhizobium. This suggestion is supported by transient overexpression of the NPR1 in M. truncatula roots, which suppressed the root hair deformation, resulting in straight root hairs (Fig. 7). On the other hand, silencing of the NPR1 expression by RNAi accelerated the root hair deformation after inoculation of S. meliloti. In legumes, the levels of SA are reduced during first days of Rhizobium infection, which may result in reduced NPR1- dependent gene expression [10]. In non-legume the symbiotic- like responses were observed only in nim1/npr1 mutants, or in NahG transformants, both of which suppress the SA signaling (Fig. 1). Inhibition of the default SA-mediated defense pathway in legumes during Rhizobium infection is probably necessary to allow bacterial entry into the host. Interestingly, inoculation of S. meliloti caused a strong oxidative burst in M. truncatula and in A. thaliana nim1/npr1 mutants (Fig. 4, 5), suggesting that the NPR1 protein activates antioxidant responses. It is possible that one or more of the NPR1- dependent genes have antioxidant activity. Alternatively, NF signaling may be less active in the presence of NPR1. PLoS ONE | www.plosone.org 6 December 2009 | Volume 4 | Issue 12 | e8399