Figure 6. Effect of SA pretreatment and EDS1 overexpression on pathogen resistance. (A) Expression of EDS1 and PR-1 in EDS1 (Col-0) and 35S-EDS1 (Col-0) plants. Total RNA was extracted from 4- week-old plants and ethidium bromide staining of rRNA was used as the loading control. (B) Growth of P. syrinage AvrRPS4 on indicated genotypes (all in Col-0 background). Single asterisks indicate data statistically significant from results for water-treated wt (Col-0) (P,0.05, n = 4). Two asterisks indicate data statistically significant from results for SA–treated wt (Col-0) (P,0.05, n = 4). The error bars indicate SD. doi:10.1371/journal.pgen.1000545.g006 like in morphology and showed no or greatly reduced cell death lesions (Figure S3A, S3B). PR-1 expression was greatly reduced or abolished in ssi2 eds1 fad7 and ssi2 eds1 fad7 fad8 plants, respectively (Figure S3C) and correlated with their endogenous SA/SAG levels; the ssi2 eds1 fad7 and ssi2 eds1 fad7 fad8 plants showed greatly reduced or basal levels of SA and SAG, respectively (Figure S3D, S3E). Expression of some R genes (SSI4, RPS2, RPP5) was nominally or moderately reduced in ssi2 eds1 fad7 plants (Figure S3F). By comparison, all R genes tested were expressed at basal levels in ssi2 eds1 fad7 fad8 plants (Figure S3F). These results showed that presence of fad7 fad8 mutations restored the altered defense phenotypes of ssi2 eds1 plants. FA profiling did not detect any significant increase in 18:1 levels in ssi2 eds1 fad7 and ssi2 eds1 fad7 fad8 plants, compared to ssi2 fad7 and ssi2 fad7 fad8, respectively (Table S4). This suggested that restoration of defense phenotypes in ssi2 eds1 fad7 fad8 was not the result of restored 18:1 levels, but rather the reduction of SA levels in the eds1 background. Redundancy in Plant Defense Mutations in EDS5 and PAD4 also lower SA/SAG levels in ssi2 plants [40]. To determine if mutations in these can substitute for sid2 triple mutants containing ssi2 eds1 pad4 and ssi2 eds1 eds5 were generated. The ssi2 eds1 pad4 plants were morphologically similar to ssi2 eds1 or ssi2 pad4 plants and showed spontaneous cell death and increased expression of PR-1 gene (Figure 8A–8C). In comparison, ssi2 eds1 eds5 showed wt-like morphology, greatly reduced cell death and basal expression of PR-1 gene (Figure 8A– 8C). Quantification of endogenous SA levels showed that both ssi2 eds1 eds5 and ssi2 eds1 pad4 accumulated lower SA/SAG levels compared to ssi2 eds5 and ssi2 pad4, respectively (Figure 8D and 8E). However, while ssi2 eds1 eds5 plants accumulated basal levels of SA/SAG, the ssi2 eds1 pad4 accumulated significantly higher levels of SA/SAG compared to wt, ssi2 sid2 and ssi2 eds1 eds5 plants (Figure 8D and 8E). Analysis of R gene expression showed greatly reduced levels in ssi2 eds1 eds5 plants but the ssi2 eds1 pad4 expressed ssi2-like levels of R genes (Figure 8F, Figure S1D). Taken together, these results suggest that the suppression of SA levels was required for the normalization of defense phenotypes in the ssi2 eds1 background. PAD4, SAG101, and EDS5 are not functionally redundant with SA in low 18:1-mediated signaling Besides EDS1, the SA signaling pathway is also regulated by PAD4 and EDS5 and via the physical association of EDS1 with SAG101 and PAD4 [17,19,45]. To determine if PAD4, SAG101 or EDS5 also function redundantly with SA, we introduced the pad4, sag101 and eds5 mutations in the ssi2 and ssi2 sid2 backgrounds. The ssi2 sag101, ssi2 pad4 and ssi2 eds5 plants showed ssi2-like morphology, visible and microscopic cell death and constitutive PR-1 gene expression (Figure S4A, S4B, S4C and Figure S5A, S5B, S5C). Consistent with these phenotypes, the ssi2 sag101, ssi2 pad4, ssi2 eds5 plants showed increased expression of R genes (Figure S4D and Figure S5D) and accumulated elevated levels of SA and SAG (Figure S4E, S4F and Figure S5E, S5F). Notably, the SA levels in ssi2 sag101 plants were ,6-fold lower than in ssi2 plants, suggesting that SAG101 contributed to the accumulation of SA in ssi2 plants. To determine if the reduced SA in the sag101 background could restore wt-like phenotypes in ssi2 eds1 plants, triple mutant ssi2 eds1 sag101 plants were generated. Although the ssi2 eds1 sag101 plants accumulated significantly lower levels of SA/SAG (Figure S4E, S4F), these plants were only slightly bigger than ssi2 eds1 or ssi2 sid2 plants (Figure S4A), showed spontaneous cell death (Figure S4B) and expressed PR-1 (Figure S4C) and R genes constitutively (Figure S4D). We next analyzed the triple mutant ssi2 sag101 sid2, ssi2 pad4 sid2 and ssi2 eds5 sid2 plants. All the triple mutants contained wt-like levels of SA and SAG (Figure S4E, S4F and Figure S5E, S5F). The ssi2 sag101 sid2 plants were morphologically similar to ssi2 plants, showed spontaneous cell death and expressed R genes constitutively (Figure S4A, S4B, S4C, S4D). In comparison, the ssi2 pad4 sid2 and ssi2 eds5 sid2 plants were bigger in morphology. However, plants of both genotypes showed cell death (Figure S5A, S5B) and expressed R genes constitutively (Figure S5D). Together, these data suggest that the functional redundancy with SA was specific only to EDS1 and did not extend to PAD4, SAG101 or EDS5. Discussion SA is long known as an essential modulator of R gene-derived signaling in pathogen defense. Several molecular components, including EDS1, have been identified as essential effectors of SA- derived signaling [23,26,45]. Since SA upregulates expression of EDS1, both SA and EDS1 are thought to function in a positive PLoS Genetics | www.plosgenetics.org 10 July 2009 | Volume 5 | Issue 7 | e1000545