Redundancy in Plant Defense Figure 3. Interaction phenotypes of TCV with HRT ssi2 eds1-1 sid2-1 and HRT eds1-1 sid2-1 plants. (A) Percentage TCV susceptible plants. HRT and hrt indicate resistant and susceptible ecotypes Di-17 and Col-0, respectively. Approximately 70–100 plants were scored for each genotype three- weeks post inoculation and all susceptible plants showed crinkling phenotype and drooping of the bolt [23]. (B) Expression of PR-1 gene in indicated genotypes after mock- or TCV-inoculation. Total RNA was extracted from inoculated leaves at 3 dpi. Ethidium bromide staining of rRNA was used as the loading control. (C) HR formation in indicated genotypes at 3 dpi. The HR response in TCV-inoculated HRT EDS1 SID2 F2 plants was similar to that seen in TCV-inoculated Di-17, HRT sid2-1 or HRT eds1-1 plants. Plants lacking HRT (Col-0, N o¨ ecotypes or EDS1 SID2 F2’s) did not show any HR. (D) Lesion size in indicated genotypes at 3 dpi. Lesion size was determined from ,23 individual leaves from each genotype. Statistical significance was determined using Students t-test. Asterisks indicate data statistically significant from those of HRT, HRT EDS1 SID2, HRT sid2-1 or HRT eds1-1 plants (P,0.05, n = 23). The error bars indicate SD. (E) ELISA showing levels of TCV CP in the inoculated leaves of indicated genotypes at 3 dpi. Asterisks indicate data statistically significant from results for HRT (Di-17 ecotype) plants (P,0.05, n = 4). The error bars indicate SD. (F) Transcript levels of TCV CP in the inoculated leaves of indicated genotypes at 3 dpi. Ethidium bromide staining of rRNA was used as the loading control. (G) Expression of PR-1 gene in indicated genotypes. Total RNA was extracted from inoculated leaves at 3 dpi. Ethidium bromide staining of rRNA was used as the loading control. The PR-1 gene expression in TCV-inoculated HRT EDS1 SID2 F2 plants was similar to that observed in TCV-inoculated HRT, HRT eds1-1 or HRT sid2-1 plants (data not shown). (H) RT– PCR analysis showing HRT and PR-2 transcript levels in indicated genotypes. The plants were inoculated with TCV and leaf samples were harvested 24 h post inoculation. The level of b-tubulin was used as an internal control to normalize the amount of cDNA template. doi:10.1371/journal.pgen.1000545.g003 EDS1- and SA-dependent signals is required to mimic a phenotype produced by the loss of the cognate R gene, RPS2. To determine if the loss of both EDS1- and SA-dependent signaling impaired resistance by affecting the RPS2 protein, we analyzed R protein levels in eds1-1 and sid2 single and eds1-1 sid2 double mutant plants. Analysis of RPS2 tagged with HA epitope at various times did not detect any significant changes in RPS2 levels in response to inoculation with P. syringae expressing AvrRPT2 (Figure 4C). Therefore, RPS2 levels in mutant plants were analyzed at only 12 and 24 h post-pathogen inoculation. The RPS2-HA levels in eds1-1, sid2 or eds1-1 sid2 plants were similar to that in wt plants (Figure 4D). These results suggested that abrogation of resistance in eds1 sid2 double mutants was not due to a defect in the accumulation of the R protein. We next evaluated the effects of mutations in EDS1 and SID2 on RPP8-mediated resistance to Hyalopernospora arabidopsidis biotype Emco5 encoding Atr8. RPP8 (encodes a CC-NBS-LRR type R protein)-mediated resistance signaling was previously reported to be independent of both EDS1 and SA [21,24]. As expected, RPP8 plants (ecotype Ler) inoculated with the Emco5 isolate showed localized HR and did not support growth of the pathogen (Figure 5A). Consistent with earlier reports [21,24], RPP8 eds1-2 plants also did not support the growth of Emco5, although they did develop trailing necrosis (Figure 5A and 5B). The presence of the nahG transgene did not alter HR formation or pathogen response in the RPP8 nahG plants (Ler ecotype). In contrast, eds1-2 nahG plants were affected in both HR as well as resistance; eds1-2 nahG plants not only showed extensive trailing necrosis but also supported growth and sporulation of the pathogen (Figure 5A–5C). Although RPP8 EDS1 nahG and RPP8 eds1-2 nahG plants showed contrasting phenotypes (Figure 5A–5C), we still wanted to rule out the possibility that susceptibility of eds1 nahG plants was not due to the accumulation of catechol, which is formed upon degradation of SA by NAHG. Estimation of SA levels in Emco5 inoculated RPP8 (Ler) plants showed marginal increase in SA and no significant increase in SAG levels compared to mock-inoculated plants (data not shown). This suggests that Emco5 inoculated nahG plants are unlikely to show a significant increase in catechol levels. In addition to this, we tested two independent lines of RPP8 eds1-2 sid2 (in the ssi2 background) plants and both showed increased susceptibility to Emco5 (Figure 5D). In comparison, RPP8 eds1-2 or RPP8 sid2 genotypes did not support any growth or sporulation of the pathogen (Figure 5D). Taken together, these results show that EDS1 and SA have redundant functions in RPP8-mediated resistance to H. arabidopsidis Emco5. Exogenous SA and overexpression of EDS1 have additive effects on pathogen resistance in wild-type plants To determine the relation between EDS1- and SA-derived signaling, we compared PR-1 gene expression and resistance in plants that were either overexpressing EDS1 or were pretreated with SA. EDS1 overexpression was achieved by expressing EDS1 (At3g48090 from the Col-0 ecotype) under control of the CaMV 35S promoter in Col-0 plants (Figure 6A). The 35S-EDS1 plants analyzed in the T2 and T3 generations showed wt-like morphology (data not shown), wt-like expression of the PR-1 gene (Figure 6A) and accumulated wt-like levels of SA/SAG (data not shown). In comparison, exogenous application of SA induced PR-1 and EDS1 gene expression [data not shown; 16]. Analysis of RPS4 (encodes a TIR-NBS-LRR type R protein)- mediated resistance showed that exogenous application of SA enhanced resistance to P. syringae (expressing AvrRPS4) in wt as well as eds1-22 plants, although wt plants were more resistant to AvrRPS4 bacteria than the eds1-22 plants (Figure 6B). Overexpres- sion of EDS1, on the other hand, did not alter the response to AvrRPS4 bacteria. Strikingly, exogenous application of SA on 35S- EDS1 plants enhanced resistance even more than in the SA- treated wt or eds1-22 plants. Together, these results suggest that EDS1- and SA-derived signaling contribute additively towards pathogen resistance. Simultaneous defects in EDS1 and SA biosynthesis do not additively lower basal defense We next evaluated the effect of the eds1 sid2 mutations on basal resistance to virulent P. syringae, since both EDS1 and SID2 are known to contribute to basal defense as well. The eds1-1, eds1-22, sid2 and eds1 sid2 plants all showed enhanced susceptibility to virulent bacteria as compared to the respective wt ecotypes (Figure 7A). Interestingly, unlike in the case of the avirulent bacteria, growth of virulent bacteria was similar in eds1 sid2 double mutant plants as compared to that in eds1 or sid2 single mutant plants. These results suggested that loss-of-function mutations in EDS1 and SID2 do not additively reduce basal resistance to virulent P. syringae. Similar to the results obtained with the bacterial pathogen, the loss of both EDS1- and SA-dependent signals did not additively lower basal resistance to TCV either (Figure 7B). This further suggested that the redundant functions of EDS1 and SA might be relevant only for R gene-mediated signaling. Mutations in FAD7 FAD8 and EDS5 restore altered defense signaling in ssi2 eds1 plants Besides SID2, mutations in FAD7 FAD8, which catalyze desaturation of 18:2 to 18:3 on membrane glycerolipids, also lower the SA levels in ssi2 plants [40]. To test if fad7 or fad7 fad8 mutations produced a similar effect as sid2, these mutations were mobilized into the ssi2 eds1 background. The ssi2 eds1 fad7 and ssi2 eds1 fad7 fad8 plants were bigger in size compared to ssi2 fad7 or ssi2 fad7 fad8 plants (Figure S3A). The ssi2 eds1 fad7 fad8 were wt- PLoS Genetics | www.plosgenetics.org 7 July 2009 | Volume 5 | Issue 7 | e1000545