Npr1 in Symbiotic Interactions Figure 1. Induction of root hair deformation and attachment of S. meliloti to A. thaliana hairs. (A–C) Wild-type (A), nim1/npr1 mutant (B) and NahG transformed (C) A. thaliana seedlings were grown on nitrogen poor medium (1/60 strength MS) for eight days. Plants were left untreated (A–C, cont), inoculated with S. meliloti in zone 1 region (lower-mid root) (S.m), inoculated with P. putida (P.p), inoculated with S. meliloti nodA [58] or nodH mutants (not shown) or treated with purified nod factors (NF). Roots were photographed 4 days after inoculation under bright light. Bar = 25 mm. Arrows point to deformed root tips. (D) Quantization of root hair deformation response in A. thaliana plants inoculated with S. meliloti producing intact nod factor (grey) or nodA2 mutant (black), P. putida (black stipes), or left uninoculated (white). One hundred root hairs from 10 seedlings of wild-type and nim1/npr1 mutants in zone 1 were scored in each treatment. Error bars represent SD. The experiment was repeated at least 3 times with similar results. doi:10.1371/journal.pone.0008399.g001 medium almost completely removed the bacteria from wild type and from dnd1 mutants, but not from the nim1/npr1 seedlings. Substantial amount of attached S. meliloti in the nim1/npr1 mutants were observed even after further wash with 200 mM NaCl, which completely removed all bacteria from the wild-type roots (Fig. 2A). These results suggest that the attachment of S. meliloti to its host is regulated by SA-dependent signaling in the host. Induction of nodulin Gene Expression in Arabidopsis following Rhizobium Inoculation The symbiotic interaction between legumes and Rhizobium is characterized by induction of nodulin gene expression. Nodulins Figure 2. Attachment of Rhizobium to A. thaliana root hairs. (A) Wild-type nim1/npr1 and dnd1 mutants were inoculated with S. meliloti. Four days after inoculation plant roots were rinsed with phosphate buffer, as described by [31], followed by buffer supplemented with 100, 200, or 500 mM NaCl. S. meliloti in 2500 mm2 area in the vicinity of A. thaliana root hairs were vewed at 600X magnification with Olympus IX70 microscope and quantified from six roots of each treatment. Wild-type (white), nim1/npr1 (grey) or dnd1 (black). The ndr1 mutants showed similar results to dnd1, and therefore were not included. Error bars represent SD. (B) Confocal image of GFP-expressing S. meliloti bacteria in the vicinity of wild- type and nim1/npr1 root hairs after washing with 150 mM NaCl as described in (A). doi:10.1371/journal.pone.0008399.g002 are divided according to their expression time into early (called ENODs) that act in accommodating the rhizobial bacteria, and late nodulins that are thought to be involved in the nodule functioning. ENODs are induced within one or few days after inoculation, while late nodulins take several days [32]. Genomic sequencing has identified nodulin homologs in Arabidopsis and other non-legume genomes of higher plants [33]. The homologs of related gene families in A. thaliana are shown in Fig. S4 and Fig. S5. We analyzed the expression of two ENOD homologs, representing early (AtENOD20, At5g57920) and a late (AtMtN21, At5g07050) nodulins, that are expressed 2–5 and 7 days post infection, respectively, in M. truncatula [34,35]. The A. thaliana ENOD20 homolog, At5g57920, also called early nodulin-like protein in the Arabidopsis TAIR database, shares 38% identity and 56% similarity with the M. truncatula protein, while the At5g07050, also called nodulin-related protein, contains 64% identity and 78% similarity. Both genes were induced by the inoculation of S. meliloti in the nim1/npr1 background, but not in wild-type plants (Fig. 3B). The induction was specific for inoculation of S. meliloti, but not of P. putida bacteria. Moreover, we tested the induction of At5g57920 following NF treatment. Strong induction was detected only in the nim1/npr1 background (Fig. 3A). PLoS ONE | www.plosone.org 3 December 2009 | Volume 4 | Issue 12 | e8399