P. syringae and TCV, even though only the genotypes used for TCV were of mixed ecotypic backgrounds. Fourth, F2 plants containing wild-type alleles behaved like wild-type parents. Finally, the effects of various mutant backgrounds on ssi2 phenotypes were also confirmed by glycerol application on individual mutants. Although glycerol treatment failed to induce R gene expression in eds1 sid2 plants, it did induce cell death. This is in contrast to the absence of a cell death phenotype in ssi2 eds1 sid2 leaves. One possibility is that the glycerol-triggered cell death is not due to a reduction in 18:1 levels. However, significant overlap between ssi2- and exogenous glycerol-triggered signaling pathways lessens such a possibility [40]. An alternate possibility is that, while EDS1 affects a majority of the responses induced by low 18:1 levels, the cell death phenotype is also governed by some additional molecular factor(s). This is supported by the fact that ssi2 pad4 sid2 plants exhibit improved morphology and reduced cell death even though they are not restored for other defense-related phenotypes. Since the overexpression of R genes can initiate defense signaling in the absence of a pathogen [48,52], it is possible that the induced defense responses in ssi2 plants are the result of increased R gene expression. This idea is supported by the fact that ssi2-related phenotypes can be normalized by restoring R gene expression to wt- like levels, irrespective of their 18:1 levels. Thus, wt-like defense phenotypes are restored in suppressors containing high 18:1 levels, such as ssi2 act1, ssi2 gly1 or ssi2 acp4 [30,31,35], as well as in suppressor containing low 18:1 levels, such as ssi2 eds1 sid2 (this work) and restored in defective crosstalk (rdc) 2 (unpublished data) (Figure 9A). We have also characterized additional ssi2 suppressors that show wt-like phenotypes even though they contain low 18:1 levels and express R genes constitutively (rdc3, rdc4). Together, these results suggest that the ssi2-associated phenotypes can be restored by normalizing R gene expression to wt-like levels either by increasing 18:1 levels, impairing factors downstream of signaling induced by low 18:1 levels, or impairing events downstream of R gene expression induced by low 18:1 levels. In addition to 18:1 levels or R gene expression, ssi2-related defense signaling could also be normalized by altering some factor(s) that function downstream of R gene induction. Indeed, our preliminary characterizations have identified additional ssi2 suppressors that yield wt-like phenotypes with regards to defense signaling but continue to express R genes at high levels. Reduced 18:1 levels may induce defense signaling by directly regulating the transcription of activators or suppressors of defense gene expression. This is supported by the fact that 18:1-mediated activation of a transcription factor induces the expression of genes required for neuronal differentiation [53]. Similarly, in Sacharro- myces cerevisiae as well as mammalian cells, binding of 18:1 to specific transcription factors induces the transcription of genes carrying 18:1 responsive elements in their promoters [54,55]. On the other hand, expression of the oncogene HER2 is inhibited via the 18:1-upregulated expression of its transcriptional repressor [56]. Reduced 18:1 might also directly activate/inhibit/alter protein activities. For example, 18:1 is known to activate the Arabidopsis phospholipase D [57] and inhibit glucose-6-phosphate transporter activity in Brassica embryos [58]. Indeed, we have also identified several Arabidopsis proteins for which enzymatic activities are inhibited upon binding to 18:1 (unpublished data). In conclusion, results presented here redefine the currently accepted pathway for SA-mediated signaling by showing that EDS1 and SA play a redundant role in plant defense mediated by R proteins and in signaling induced by low 18:1 fatty acid levels. Further biochemical characterization should help determine if 18:1 binds to EDS1 and if cellular levels of 18:1 modulate the as yet undetected lipase activity of EDS1. Redundancy in Plant Defense Materials and Methods Plant growth conditions and genetic analysis Plants were grown in MTPS 144 Conviron (Winnipeg, MB, Canada) walk-in-chambers at 22uC, 65% relative humidity and 14 hour photoperiod. The photon flux density of the day period was 106.9 mmoles m22 s21 and was measured using a digital light meter (Phytotronic Inc, Earth city, MO). All crosses were performed by emasculating the flowers of the recipient genotype and pollinatng with the pollen from the donor. All the genotypes and crosses analyzed in this work, their genetic background and number of single, double, or triple mutant plants studied are listed in Table S1. In most cases, single, double, or triple mutant plants were obtained from more than one combination of crosses and showed similar morphological, molecular and biochemical phenotypes. F2 plants showing the wt genotype at the mutant locus were used as controls in all experiments. The wt and mutant alleles were identified by PCR, CAPS, or dCAPS analysis and/or based on the FA profile [30,31,38,40]. The EDS1 KO mutant in At3g48090 was, isolated by screening SALK_071051 insertion line, obtained from ABRC. The EDS1 KO was designated eds1-22, based on the previous designation assigned to SALK_071051 T-DNA KO line [48]. The At3g48090 gene showed 98.8% identity at amino acid level to EDS1 allele from Ler ecotype. The homozygous insertion lines were verified by sequencing PCR products obtained with primers specific for the T- DNA left border in combination with an EDS1-specific primer. The eds1-22 lines did not show any detectable expression of EDS1. RNA extraction and northern analyses Small-scale extraction of RNA from one or two leaves was performed with the TRIzol reagent (Invitrogen, CA), following the manufacturer’s instructions. Northern blot analysis and synthesis of random-primed probes for PR-1 and PR-2 were carried out as described previously [29]. Reverse Transcription–PCR RNA quality and concentration were determined by gel electrophoresis and determination of A260. Reverse transcription (RT) and first strand cDNA synthesis were carried out using Superscript II (Invitrogen, CA). Two-to-three independent RNA preparations were used for RT-PCR and each of these were analyzed at least twice by RT–PCR. The RT–PCR was carried out for 35 cycles in order to determine absolute levels of transcripts. The number of amplification cycles was reduced to 21–25 in order to evaluate and quantify differences among transcript levels before they reached saturation. The amplified products were quantified using ImageQuant TL image analysis software (GE, USA). Gene-specific primers used for RT–PCR analysis are described in Table S5. Trypan-blue staining The leaves were vacuum-infiltrated with trypan-blue stain prepared in 10 mL acidic phenol, 10 mL glycerol, and 20 mL sterile water with 10 mg of trypan blue. The samples were placed in a heated water bath (90uC) for 2 min and incubated at room temperature for 2–12 h. The samples were destained using chloral hydrate (25 g/10 mL sterile water; Sigma), mounted on slides and observed for cell death with a compound microscope. The samples were photographed using an AxioCam camera (Zeiss, Germany) and images were analyzed using Openlab 3.5.2 (Improvision) software. Pathogen infections The asexual conidiospores of H. arabidopsidis Emco5 expressing Atr8 were maintained on the susceptible host No¨ ssen (No¨) or No¨ PLoS Genetics | www.plosgenetics.org 14 July 2009 | Volume 5 | Issue 7 | e1000545