Author Summary Salicylic acid and enhanced disease susceptibility 1 are important components of resistance gene-mediated de- fense signaling against diverse pathogens in a variety of plants. Present understanding of plant defense signaling pathways places salicylic acid and enhanced disease susceptibility 1 downstream of resistant protein activation. In addition, enhanced disease susceptibility 1 is primarily thought to function in the signaling initiated via Toll- interleukin 1-receptor type of resistance proteins. Here, we show that salicylic acid and enhanced disease susceptibil- ity 1 serve redundant functions in defense signaling mediated by coiled-coil-domain containing resistance proteins that were thought to function independent of enhanced disease susceptibility 1. Furthermore, resistance signaling induced under low oleic acid conditions also requires enhanced disease susceptibility 1 and salicylic acid in a redundant manner, but these components are required upstream of resistance gene expression. Togeth- er, these results show that the functional redundancy between salicylic acid and enhanced disease susceptibility 1 has precluded their detection as required components of many resistance protein–signaling pathways. However, RPW8 is not a typical NBS-LRR type of R protein; it contains an N-terminal transmembrane domain in addition to the CC domain. Although several components contributing to resistance against pathogens have been identified, the molecular signaling underlying R gene-mediated resistance still remains obscure. Furthermore, potential relationship(s) among different downstream components and how they relay information leading to resistance remains unknown. The EDS1 and PAD4 proteins are structurally related to lipase/ esterase-like proteins although their lipase-like biochemical functions have not been demonstrated [16,19]. EDS1 interacts with PAD4 and SAG (senescence associated gene) 101 and the combined activities of these proteins are required for HR formation and to restrict the growth of virulent bacterial strains [26]. PAD4 and SAG101 also restrict the post-invasive growth of non-pathogenic fungi in Arabidopsis [27]. In addition to the major phytohormone-mediated defense pathways, fatty acid (FA)-derived signals have emerged as important mediators of defense signaling [28–35]. The Arabidop- sis SSI2/FAB2-encoded stearoyl-acyl carrier protein-desaturase (SACPD) converts stearic acid (18:0) to oleic acid (18:1). A mutation in SSI2 results in the accumulation of 18:0 and a reduction in 18:1 levels. The mutant plants show stunting, spontaneous lesion formation, constitutive PR gene expression, and enhanced resistance to bacterial and oomycete pathogens [29,36]. Characterization of ssi2 suppressor mutants has shown that the altered defense-related phenotypes are the result of the reduction in the levels of the unsaturated FA, 18:1 [30,31,35,37– 40]. The altered defense-related phenotypes in ssi2 plants can be rescued by restoring the 18:1 levels via second site mutations in genes encoding a glycerol-3-phosphate (G3P) acyltransferase [ACT1, 30], a G3P dehydrogenase [GLY1, 31], and an acyl carrier protein [ACP4, 35]. A mutation in act1 disrupts the acylation of G3P with 18:1 resulting in the increased accumulation of 18:1, thereby restoring wild-type (wt) phenotypes in ssi2 plants. ACT1 preferentially utilizes 18:1 conjugated to the ACP4 isoform in Arabidopsis [35]. Thus, a mutation in acp4 produces similar phenotypes as the act1 mutant and suppresses ssi2-mediated signaling by increasing 18:1 levels [35]. A mutation in GLY1 also Redundancy in Plant Defense restores 18:1 levels in ssi2 gly1 plants because it disrupts the formation of G3P from dihydroxyacetone phosphate [31]. Reduced availability of G3P in turn impairs the ACT1-catalyzed reaction resulting in accumulation of 18:1 in ssi2 gly1 plants. Concurrently, increasing the endogenous G3P levels via exoge- nous application of glycerol reduces 18:1 levels and induces ssi2- like phenotypes in wt plants [31,40]. This effect of glycerol is highly specific because ssi2-associated phenotypes are not induced upon glycerol treatment of act1 (defective in the acylation of G3P with 18:1) or gli1 (defective in the phosphorylation of glycerol to G3P) mutants [40]. Recently, we showed that a reduction in 18:1 levels upregulates the expression of several R genes in an SA-independent manner [37]. Furthermore, we showed that pathogen resistance induced via this mode bypasses the requirement for components that are normally required for signaling downstream of R protein activation. For example, resistance to TCV mediated by the R gene HRT (HR to TCV), requires the recessive locus rrt (regulates resistance to TCV), SA, EDS1 and PAD4 [23]. Exogenous application of SA induces the expression of HRT and overcomes the requirement for rrt. However, exogenous SA is unable to induce HRT or confer resistance in pad4 background [23]. Interestingly, even though a reduction in 18:1 levels also upregulates HRT expression to confer resistance to TCV, this mode of resistance is independent of PAD4, SA, EDS1 and EDS5, which are required for HRT-mediated resistance to TCV [37]. Remarkably, induction of R genes in response to reduced 18:1 is conserved in plants as diverse as Arabidopsis and soybean [41]. Furthermore, this low 18:1-mediated induction of defense responses was also demonstrated in rice recently [42]. Together, these studies strengthen the conserved role of 18:1 in plant defense signaling. Here, we show that R gene expression induced in response to a reduction in 18:1 levels and the associated defense signaling can be suppressed by simultaneous mutations in EDS1 and the genes governing synthesis of SA. We also show that EDS1 and SA function redundantly in R gene-mediated resistance against bacterial, viral and oomycete pathogens and that EDS1 also regulates signaling mediated by CC domain containing R proteins. Results EDS1 and SA are essential but redundant components required for R gene expression induced in response to a reduction in 18:1 levels Signaling mediated by many R genes is known to require EDS1 and/or NDR1. Previously, we have shown that ssi2 eds1 plants continue to express R genes at high levels, including those that are dependent on EDS1 for their signaling [37]. To determine if NDR1 played a role in ssi2-triggered phenotypes, we generated ssi2 ndr1 plants. The double-recessive plants segregated in a Mendelian fashion and all ssi2 ndr1 plants showed ssi2-like morphology in the F2, F3 and F4 generations (Figure 1A; Table S1). Although the ssi2 ndr1 plants accumulated significantly less SA/SAG (Figure 1C), compared to ssi2 plants, they showed ssi2-like PR-1 and R gene expression (Figure 1D and 1E, Figure S1A). Exogenous glycerol application, which reduces 18:1 levels, also induced R gene expression in eds1 and ndr1 plants (data not shown). Together, these results suggest that R gene expression induced by low 18:1 levels does not require EDS1 or NDR1. The SA/SAG levels in ssi2 eds1 and ssi2 ndr1 plants were significantly higher compared to those in wt plants (Figure 1C). To determine whether high SA in these genotypes was responsible for increased R gene expression, we generated ssi2 eds1 sid2 and ssi2 PLoS Genetics | www.plosgenetics.org 2 July 2009 | Volume 5 | Issue 7 | e1000545