ndr1 sid2 plants. Interestingly, only the ssi2 eds1 sid2 plants showed wt-like morphology and did not develop visible or microscopic cell death (Figure 1A and 1B). In contrast, ssi2 sid2, ssi2 ndr1, ssi2 ndr1 sid2 or ssi2 eds1 plants exhibited ssi2-like phenotypes. PR-1 gene expression was restored to wt-like levels in the ssi2 eds1 sid2 and ssi2 ndr1 sid2 plants, due to the sid2-derived reduction in SA levels (Figure 1D). In contrast, expression of the SA-independent PR-2 gene was restored to basal levels only in ssi2 eds1 sid2 [43], but not in ssi2 sid2 or ssi2 ndr1 sid2 plants (Figure 1D, Table S2). Most importantly, ssi2 eds1 sid2 showed basal expression of R genes, unlike ssi2 ndr1 sid2 plants (Figure 1E and 1F; Figure S1A, S1B; Table S1). R gene induction was further confirmed by comparing the transcript profiles of 162 NBS-LRR genes in ssi2 sid2 with that of wt plants using Affymetrix ATH1 GeneChips arrays. Twenty- one NB-LRR genes were specifically expressed at 2-fold or higher levels in ssi2 sid2 plants as compared to wt (Col-0) or eds1 plants (P,0.05) (Table S2). All 21 NB-LRR genes were expressed at low levels in ssi2 eds1 sid2 plants, further confirming the results from the RT-PCR analysis. Transcriptional profiling performed using Affymetrix arrays showed that the induction of several R genes (RPM1, RPS2, RPP5, RPS4) was lower than 2-fold in ssi2 or ssi2 sid2 compared to wt plants (Table S2, data not shown for ssi2). To determine if this low-level induction translated to a significant increase in R protein levels, we analyzed the levels of RPM1 in ssi2 plants. Indeed, ssi2 plants accumulated significantly higher levels of the RPM1-Myc protein (Figure 1G). To rule out the effects of the varied ecotypes of the ssi2 sid2 eds1 (No¨ ssen, Col-0, Ler) plants we introduced eds1-1 (Ws-0 ecotype) and eds1-2 (Ler ecotype) alleles in ssi2 sid2 and ssi2 nahG (No¨ssen ecotype) backgrounds (Table S1). All combinations of ssi2 with eds1-1/eds1-2 and sid2/nahG produced similar phenotypes (data not shown). FA profiling showed that the ssi2 eds1 sid2 plants contained low 18:1 levels, similar to ssi2 plants (Table S3). We thus concluded that EDS1 and SA function downstream of 18:1 levels, but upstream of R gene expression. Furthermore, ssi2 eds1 sid2 plants were wt-like, even though neither ssi2 eds1 nor ssi2 sid2 were restored for defense signaling. Therefore, EDS1 and SA likely fulfill redundant functions in defense signaling induced in response to a reduction in 18:1 levels. To further test the redundancy for EDS1 and SA, ssi2 eds1 sid2 plants were treated with SA or its active analog benzo(1,2,3)thia- diazole-7-carbothioic acid (BTH). Application of SA or BTH induced lesion formation on ssi2 eds1 sid2 plants but not on wt, eds1, sid2, eds1 sid2 or EDS1 SID2 F2 plants (Figure 2A and 2B, data not shown for eds1 sid2 and EDS1 SID2). Also, application of SA or BTH induced R gene expression in ssi2 eds1 sid2 plants (Figure 2C). Thus, application of SA restored ssi2-like phenotypes in ssi2 eds1 sid2 plants. Since glycerol application mimics the effects of the ssi2 mutation, we generated eds1 sid2 plants and evaluated them for their ability to induce R genes in response to glycerol. Exogenous application of glycerol lowered 18:1 levels in all genotypes, but induced the expression of R genes only in wt, eds1, sid2 and EDS1 SID2 F2 plants (Figure 2D, Figure S1C). Only a marginal or no increase in R gene expression was observed in the eds1 sid2 plants (Figure 2D). These results confirmed that EDS1 and SA function redundantly downstream of signaling induced by low 18:1 levels, but upstream of R gene expression. EDS1 and SA function redundantly in pathogen resistance induced in response to reduction in 18:1 levels We next evaluated the effect of simultaneous mutations in EDS1- and SA-signaling pathways on resistance to TCV in the ssi2 background. We reported previously that resistance to TCV is dependent on the R gene, HRT, and a recessive locus rrt [23]. Redundancy in Plant Defense However, the ssi2 mutation overcomes the requirement for rrt in HRT-containing plants [23,37]. Furthermore, the ssi2 mutation only confers resistance to TCV when HRT is present (Figure 3A). The ssi2 mutation also overrides a requirement for EDS1 and SA and consequently ssi2 HRT eds1 as well as ssi2 HRT sid2 plants exhibit resistance to TCV [37] (Figure 3A). Unlike HRT ssi2, HRT ssi2 eds1 or HRT ssi2 sid2 plants, the HRT ssi2 eds1 sid2 plants showed susceptibility to TCV; ,85% HRT ssi2 eds1 sid2 plants were susceptible to TCV as against ,2–4% of HRT ssi2 sid2 or HRT ssi2 eds1 plants (Figure 3A). TCV-induced expression of PR-1 is also independent of EDS1 and SA. However, TCV inoculation failed to induce PR-1 expression in HRT ssi2 eds1 sid2 plants, unlike in HRT ssi2 sid2 plants (Figure 3B). These results showed that both EDS1 and SA have redundant functions in ssi2-mediated resistance to TCV in HRT plants. EDS1 and SA function redundantly in signaling mediated by HRT, RPS2, and RPP8 genes that encode CC-NBS-LRR proteins To determine the redundancy of EDS1 and SA in signaling mediated by CC-NBS-LRR R proteins, we tested the effects of mutations in EDS1- and/or SID2 on HR to TCV. Earlier, we showed that HRT-mediated HR to TCV and PR-1 gene expression is not affected by mutations in the EDS1 or SID2 genes [23]. Consistent with previous results, Di-17 (HRT-containing resistant ecotype), HRT sid2 and HRT eds1 plants revealed discrete and similar-sized HR lesions on TCV-inoculated leaves (Figure 3C and 3D). In comparison, HR in HRT eds1 sid2 plants was diffused and formed larger lesions (Figure 3C and 3D). Increased lesion size in HRT eds1 sid2 plants correlated with increased accumulation of the TCV coat protein (CP) and TCV CP transcript (Figure 3E and 3F). Analysis of PR-1 and PR-2 gene expression indicated that TCV-inoculated HRT eds1 sid2 plants accumulated lower levels of PR-1 and PR-2 transcripts, unlike Di-17, HRT eds1 or HRT sid2 plants (Figure 3G and 3H). In contrast to PR, HRT expression remained unaltered in HRT eds1 sid2 plants (Figure 3H). Together, these results suggested that EDS1 and SA function redundantly in HRT-mediated signaling leading to HR formation and expression of PR-1. The functional redundancy with SA was specific to EDS1 and did not extend to PAD4; HRT pad4 sid2 plants showed normal replication of the virus and wt-like HR and PR-1 gene expression (Figure 3C–3G). A majority of CC-domain containing R proteins, including RPS2, have been reported as not requiring EDS1 for resistance signaling [21]. To determine the effect of simultaneous mutations in EDS1 and SID2 on RPS2-mediated resistance, we compared defense phenotypes produced in single or double mutant plants with that of plants lacking a functional RPS2 gene. Since different alleles of RPS2 confer varying levels of resistance to Pseudomonas syringae (containing AvrRPT2) [44], we screened and isolated an EDS1 knockout (KO) mutant (designated eds1-22) in the Col-0 background and crossed it into the sid2 background (Col-0 ecotype). Inoculation with P. syringae expressing AvrRPT2 induced severe chlorosis on eds1-22 sid2 leaves (Figure 4A). Similar results were obtained when P. syringae expressing AvrRPT2 was inoculated into eds1-1 sid2 double mutant plants (Figure S2A). Interestingly, these phenotypes were very similar to those produced on plants lacking a functional RPS2 (rsp2-101c), while eds1 and sid2 showed no or very mild symptoms, respectively (Figure 4A, Figure S2A). The appearance of symptoms correlated with bacterial growth; eds1-22 sid2 plants and the rps2 mutant supported maximum growth of the pathogen, followed by sid2 plants (Figure 4B). Similarly, the eds1-1 sid2 double mutant plants supported more pathogen growth compared to eds1-1 or sid2 plants (data not shown). Together, these data suggest that the simultaneous loss of PLoS Genetics | www.plosgenetics.org 4 July 2009 | Volume 5 | Issue 7 | e1000545