plants. Similarly, HRT-mediated signaling leading to HR formation and PR-1 gene expression is only affected in eds1 sid2 plants, while eds1 or sid2 plants behave similar to wt plants. Furthermore, RPP8-mediated resistance, which was previously reported not to require EDS1 or SA [21,24], is compromised in plants lacking both EDS1 and SA. In contrast to their effect on R gene-mediated resistance, loss of both EDS1- and SA-dependent signals did not additively lower basal resistance to P. syringae or TCV. Together, these data suggests that the redundant functions of EDS1 and SA might be relevant only for R gene-mediated signaling. In contrast to SA application, overexpression of EDS1 was unable to confer increased resistance to the avirulent pathogen P. syringae. Furthermore, unlike SA, overexpression of EDS1 was not associated with the induction of PR-1 gene expression. These findings, together with the observation that SA was able to induce EDS1 expression and that SA application on wt plants resulted in higher resistance than that in eds1, suggests that SA feedback regulates EDS1-derived signaling in a unidirectional manner (Figure 9B). Thus, SA application induces both SA- and EDS1- derived signaling, the additive effects of which enhance resistance in wt plants much more than in eds1-22 plants. Furthermore, the combined effects of SA pretreatment and EDS1 overexpression induced much better resistance than the individual effects of each. This is consistent with a previous report that 35S-EDS1 plants induce rapid and stronger expression of PR-1 in response to pathogen inoculation [47]. The additive effects of EDS1 and SA was also supported by the observation that eds1 sid2 plants showed pronounced chlorosis upon inoculation with AvrRPS4 expressing pathogen, which is recognized by a TIR-NBS-LRR protein RPS4 (Figure S2B). Since mutations in SA-independent branch of EDS1 pathway and sid2 have additive effects on R gene-mediated resistance [46], it is possible that overexpression of EDS1 triggers signaling via both SA-dependent and/or -independent branches of EDS1 pathway. Although the Col-0 ecotype is thought to contain two functional alleles of EDS1 [26], a KO mutation in At3g48090 was sufficient to compromise both basal and R gene (RPS4)-mediated resistance. However, the Col-0 eds1-22 mutant consistently supported less growth of virulent or avirulent pathogens compared to eds1-1 or eds1-2 plants. This suggests that the second EDS1 allele in the Col- 0 ecotype might also contribute towards the resistance response. This is consistent with another study where constitutive defense phenotypes due to the overexpression of the SNC1 gene, encoding a TIR-NBS-LRR R protein, are not completely suppressed by a mutation in eds1 in the Col-0 background but restored by the eds1 mutation in the Ws background [48]. The inability to accumulate SA together with a mutation in EDS1 was also required to suppress constitutive defense signaling resulting from the overexpression of R genes induced in response to reduced 18:1 levels. Although eds1 or sid2 plants were entirely competent in inducing R gene expression in response to a reduction in 18:1, eds1 sid2 plants were not. Thus, ssi2 eds1 sid2 as well as glycerol-treated eds1 sid2 plants showed wt-like expression of R genes while ssi2 eds1, ssi2 sid2 and glycerol-treated eds1 or sid2 plants showed increased expression of R genes. Moreover, treatment of ssi2 eds1 sid2 plants with exogenous SA restored R transcript induction and cell death in these plants. The fact that glycerol treatment is unable to induce R gene expression in eds1 sid2 plants supports the possibility that EDS1 and SA function upstream of, and not merely serve as a feedback loop in, R gene induction. Signaling induced by low 18:1 levels continues to function in the absence of SA, suggesting a novel SA-independent role for EDS1 in defense signaling. Redundancy in Plant Defense Figure 9. Models for signaling induced by low 18:1 fatty acid levels and R genes. (A) EDS1 and SA function upstream of R genes and regulate expression of R genes induced by low 18:1 fatty acid levels. Mutations in EDS1 and SA-synthesizing enzyme, encoded by SID2, abolish constitutive upregulation of R genes and associated enhanced resistance in genetic backgrounds containing low 18:1 levels. Similar to EDS1/SA, restored in defective crosstalk (RDC) 2 acts downstream of signaling induced by low levels of 18:1 but upstream of R gene expression. Signaling induced by low 18:1 fatty acid levels can also be suppressed by mutations in ACT1-encoded G3P acyltransferase [30], GLY1-encoded G3P dehydrogenase [31], or ACP4-encoded acyl carrier protein 4 [35], which normalize 18:1 levels, or by blocking steps downstream of R gene expression (rdc3 and rdc4). Upregulation of R genes induced by low 18:1 fatty acid levels does not require PAD4, SAG101, or EDS5, which are components of the resistance signaling pathway(s) initiated upon R-Avr interaction. (B) Direct or indirect interaction between host-encoded R and pathogen-encoded Avr products initiate resistance signaling, which requires EDS1 and SA. Exogenous application of SA induces expression of PR-1 and EDS1 genes but overexpression of EDS1 does not induce PR-1 expression or increase SA levels. The EDS1– and SA–dependent pathways have additive effects. doi:10.1371/journal.pgen.1000545.g009 Since ssi2 eds1 sid2 plants contain a mixed ecotypic background (No¨ , Ws/Ler, Col-0, ecotypes), it is possible that ecotypic variations in various genetic backgrounds resulted in the restoration of ssi2- triggered defense phenotypes. Indeed, phenotypic variations amongst different Arabidopsis ecotypes have been associated with many physiological processes [48–51]. Moreover, certain alleles can express themselves only in specific ecotypic backgrounds [48,51]. However, since ssi2 EDS1 SID2, ssi2 EDS1 sid2 or ssi2 eds1 SID2 plants (F2 population) always exhibited ssi2-like phenotypes, it is highly unlikely that ecotypic variations resulted in the restoration of phenotypes in ssi2 eds1 sid2 plants. The effect of ecotypic variations on the observed phenotypes can be further ruled out for the following reasons. First, the effects of different mutations were assessed in multiple backgrounds. For example, we used both eds1-1 (Ws-0 ecotype) and eds1-2 (Ler ecotype) alleles in ssi2 sid2 (No¨ , Col-0 ecotypes) and ssi2 nahG (No¨ ecotype) backgrounds and all combinations of ssi2 with eds1-1/eds1-2 and sid2/nahG produced similar phenotypes (Table S1). Second, all defense phenotypes were assessed over three generations using multiple progeny. Third, similar results were obtained when different ecotypic backgrounds were evaluated for their response to different pathogens. For example, eds1 nahG or eds1 sid2 backgrounds conferred increased susceptibility to H. arabidopsidis, PLoS Genetics | www.plosgenetics.org 13 July 2009 | Volume 5 | Issue 7 | e1000545