NahG. The spores were removed by agitating the infected leaves in water and suspended to a final concentration of 105 spores/mL. Two-week-old seedlings were sprayed with spore suspension and transferred to a MTR30 reach-in chamber (Conviron, Canada) maintained at 17uC, 98% relative humidity and 8 h photoperiod. Plants were scored at ,10–14 dpi and the conidiophores were counted under a dissecting microscope. The bacterial strain DC3000 derivatives containing pVSP61 (empty vector), AvrRpt2 or AvrRps4 were grown overnight in King’s B medium containing rifampicin (Sigma, MO). The bacterial cells were harvested, washed and suspended in10 mM MgCl2. The cells were diluted to a final density of 105 to 107 CFU/mL (A600) and used for infiltration. The bacterial suspension was injected into the abaxial surface of the leaf using a needle-less syringae. Three leaf discs from the inoculated leaves were collected at 0 and 3 dpi. The leaf discs were homogenized in 10 mM MgCl2, diluted 103 or 104 fold and plated on King’s B medium. Transcripts synthesized in vitro from a cloned cDNA of TCV using T7 RNA polymerase were used for viral infections [59,60]. For inoculations, the viral transcript was suspended at a concentration of 0.05 mg/mL in inoculation buffer, and the inoculation was performed as described earlier [56]. After viral inoculations, the plants were transferred to a Conviron MTR30 reach-in chamber maintained at 22uC, 65% relative humidity and 14 hour photoperiod. HR was determined visually three-to-four days post-inoculation (dpi). Resis- tance and susceptibility was scored at 14 to 21 dpi and confirmed by northern gel blot analysis. Susceptible plants showed stunted growth, crinkling of leaves and drooping of the bolt. Transcriptional profiling Total RNA isolated from four-week-old plants using TRIZOL as outlined above. The experiment was carried out in triplicate and a separate group of plants was used for each set. RNA was processed and hybridized to the Affimetric Arabidopsis ATH1 genome array GeneChip following the manufacturers instructions (http://www.affymetrix.com/Auth/support/downloads/manuals/ expression_analysis_technical_manual.pdf). All probe sets on the Genechips were assigned hybridization signal above back- ground using Affymetrix Expression Console Software v1.0 (http://www.affymetrix.com/Auth/support/downloads/manuals/ expression_console_userguide.pdf). Data was analyzed by one-way Anova followed by post hoc two sample t-tests. The P values were calculated individually and in pair-wise combination for each probe set. The identities of 162 NBS-LRR genes were obtained from the Arabidopsis information resource (TAIR; www.arabidopsis.org) and disease resistance gene homolog databases (http://niblrrs. ucdavis.edu/). Fatty acid profiling FA analysis was carried out as described previously [61]. For FA profiling, one or few leaves of four-week-old plants were placed in 2 ml of 3% H2SO4 in methanol containing 0.001% butylated hydroxytoluene (BHT). After 30 minutes incubation at 80uC, 1 mL of hexane with 0.001% BHT was added. The hexane phase was then transferred to vials for gas chromatography (GC). One-microliter samples were analyzed by GC on a Varian FAME 0.25 mm650 m column and quantified with flame ionization detection. The identities of the peaks were determined by comparing the retention times with known FA standards. Mole values were calculated by dividing peak area by molecular weight of the FA. SA and SAG quantification SA and SAG quantifications were carried out from ,300 mg of leaf tissue as described before [23]. Redundancy in Plant Defense Chemical treatment of plants SA treatments were carried out by spraying or subirrigating 3- week-old plants with 500 mM SA or 100 mM BTH. For glycerol treatment, plants were sprayed with 50 mM solution prepared in sterile water. Enzyme linked immuno-sorbent assay and western analysis Total protein was extracted in buffer containing 50 mM Tris pH 8.0, 1 mM EDTA, 12 mM b-mercaptoethanol and 10 mg ml21 phenylmethylsulfonyl fluoride. Proteins were fraction- ated on a 10–12% SDS-PAGE to confirm the quality. An antigen- coated enzyme-linked immunosorbent assay was used to determine levels of TCV CP in the infected plants as described before [62]. For protein gel blot analysis, leaf tissue from 4-week-old plants was extracted with a buffer containing 50 mM Tris-HCl, pH 7.5, 10% glycerol, 150 mM NaCl, 10 mM MgCl2, 5 mM EDTA, 5 mM DTT, and 16 proteinase inhibitor (Sigma). Protein concentrations were determined by the Bradford assay (Bio-Rad, CA). For immunodetection, 10–50-mg protein samples were electrophoresed on 10–15% polyacrylamide gels and run in the presence of 0.38 M Tris and 0.1% SDS. Proteins were transferred from the gels to polyvinylidene difluoride membranes by electroblotting, incubated with primary anti-HA antibody (Sigma) and alkaline phosphatase-conjugated secondary antibody (Sigma). Immunoblots were developed using color detection. Supporting Information Figure S1 Relative expression levels of R genes in indicated genotypes. One representative quantification is shown for each Figure (noted above the graph) showing RT-PCR results. The R gene transcript levels were normalized for b-tubulin and relative differences in expression levels were quantified using ImageQuant TL image analysis software (GE, USA). Two-to-three independent RNA preparations were used for RT-PCR and each of these were analyzed at least twice by RT-PCR. The fold differences in expression levels were consistent between experiments and between repeats within an experiment. Found at: doi:10.1371/journal.pgen.1000545.s001 (0.16 MB TIF) Figure S2 Interaction phenotypes of AvrRPT2 or AvrRPS4 expressing P. syringae with eds1 sid2 plants. (A) Photograph showing phenotypes produced upon infiltration of 105 CFU/ml bacteria (AvrRPT2). The leaves were photographed at 3 days post inoculation (dpi). The mock- or pathogen-inoculated EDS1 SID2 F2 plants showed absence of any visible symptoms in response to bacterial inoculations, similar to Col-0 or Ws-0 plants (data not shown). (B) Photograph showing phenotypes produced upon infiltration of 105 CFU/mL bacteria. The leaves were photo- graphed at 3 dpi. The phenotypes seen on pathogen inoculated eds1-1 sid2-1 leaves were comparable to those seen on RLD (ecotype) plants, which lack a functional RPS4 gene (data not shown). The mock- or pathogen -inoculated EDS1 SID2 F2 plants showed absence of any visible symptoms in response to bacterial inoculations, similar to Col-0 or Ws-0 plants (data not shown). Found at: doi:10.1371/journal.pgen.1000545.s002 (1.09 MB TIF) Figure S3 Morphology, cell death, PR-1, and R gene expression and SA/SAG levels in ssi2 eds1-2 fad7-1 and ssi2 eds1-2 fad7-1 fad8-1 plants. (A) Comparison of the morphological phenotypes displayed by 4-week-old soil-grown wt (SSI2), ssi2, ssi2 eds1, ssi2 fad7, ssi2 fad7 fad8, ssi2 eds1 fad7, and ssi2 eds1 fad7 fad8 plants. (B) Microscopy of trypan blue-stained leaves from indicated geno- types. (C) Expression of PR-1 indicated genotypes. Total RNA was PLoS Genetics | www.plosgenetics.org 15 July 2009 | Volume 5 | Issue 7 | e1000545