Role of ABA and Callose in Resistance to P. irregulare.
(A) Endogenous ABA levels (ng/g fresh weight) in Arabidopsis wild-type (Col-0) and JA/ET/SA/ABA-related mutant plants (coi1-16, ein2-5, sid2-1, and aba2-12) after infection with P. irregulare for 0, 6, and 12 h. Seedlings were grown on Johnson's media plates for 7 d prior to infection. Values are means ± se of three independent replicate experiments.
(B) Susceptibility of Arabidopsis ABA-related mutants (biosynthesis, aba2-12 and aao3-2; insensitive, abi4-1) and a callose-deficient mutant (pmr4) to P. irregulare compared with the wild type (Col-0). Mean disease area (mm²) per infected leaf 24 h after infection. Asterisks indicate significant difference (P < 0.05) from Col-0 using the Student's t test.
(C) B. cinerea (open bars) and A. brassicicola (closed bars) infection of Arabidopsis ABA-related mutants (biosynthesis, aba2-12 and aao3-2; insensitive, abi4-1) compared with the wild type (Col-0). Mean disease area (mm²) per infected leaf either 4 (B. cinerea) or 10 (A. brassicicola) d after infection. Asterisks indicate significant difference (P < 0.05) from Col-0 using the Student's t test.
(D) to (G) Callose deposition in Arabidopsis following infection with P. irregulare.
(D) Localized callose deposition (apposition [app]) around site of pathogen contact. Plant cell wall (cw) and P. irregulare hyphae (hy) are also indicated. Bar = 5 μm.
(E) Normal callose deposition in wild-type (Col-0) Arabidopsis following P. irregulare infection (haustoria-like infection structure [hl]) encompassed the entire plant cell wall (cw). Bar = 30 μm.
(F) Callose deposition in the Arabidopsis ABA mutant aao3-2 following P. irregulare infection (haustoria-like infection structure [hl]) encompassed the entire plant cell wall (cw). Bar = 15 μm.
(G) Spotted callose deposition (sp) within the Arabidopsis callose mutant (pmr4) cell wall (cw) following infection with P. irregulare and production of haustoria-like infection structure (hl). Bar = 30 μm.

Meta-Analysis of ABA-Dependent Gene Data.
Cluster view of 38 aba2-12–dependent genes also induced/repressed by P. irregulare in wild-type (Col-0) plants, with available expression data from JA- and ABA-treated experiments within the GENEVESTIGATOR database (Zimmermann et al., 2004). Genes (rows) were clustered with the TIGR multi-experiment viewer using the Pearsons uncentered distance and complete linkage. Gene subclusters of interest that are discussed in the text are indicated by labeled vertical bars.

JA and JA-Precursor Levels in Wild-Type and ABA Mutant Plants.
Endogenous JA and 12-oxo-phytodienoic acid (OPDA) (ng/g fresh weight) levels in Arabidopsis wild-type (Col-0) and ABA mutant (aba2-12) plants following infection with P. irregulare for 0, 6, and 12 h. Seedlings were grown on Johnson's media plates for 7 d prior to infection. Values are means ± se of three independent replicate experiments.

Microscopy Analysis of Arabidopsis Infection by P. irregulare.
(A) Appresorial (ap) formation on leaf epidermis. Penetration pegs (pp) may also be observed (Trypan blue stain). Bar = 30 μm.
(B) Multidigitate haustoria-like structure (hl) formed upon invasion of the epidermal cell by runner hyphae (rh). The cell wall (cw) is also fluorescent due to callose deposition (Analine blue stain). Bar = 25 μm.
(C) Constricted hyphae (hy) penetration through the root cell wall (cw) (Trypan blue stain). Bar = 5 μm.
(D) Localized plant cell death upon direct infection of P. irregulare via runner hyphae (rh). Bar = 25 μm.
(E) Trailing necrosis (tn) of infected mesophyll cells. Bar = 25 μm.

P. irregulare Infection of Arabidopsis Defense Mutants.
(A) Infection of JA/ET/SA-related mutants. Mean disease area (MDA) per infected leaf (mm²) 24 h after inoculation with a mycelial plug. Asterisks indicate significant difference (P < 0.05) from Col-0 using the Student's t test.
(B) Endogenous JA and SA levels (ng/g fresh weight [FW]) in Arabidopsis wild-type (Col-0) and JA/ET/SA-related mutant plants (coi1-16, ein2-5, and sid2-1) after infection with P. irregulare for 0, 6, and 12 h (h after infection [HAI]). Seedlings were grown on Johnson's media plates for 7 d prior to infection. Values are means ± se of three independent replicate experiments.
(C) Infection of nonhormonal mutants. Asterisks indicate significant difference (P < 0.05) from Col-0 (or Landsberg in the case of Ler) using the Student's t test.

GO Categorization of Arabidopsis Genes Differentially Expressed after Inoculation with P. irregulare using FatiGo.
(A) 129 JA/ET/SA-dependent genes categorized at biological process level 5.
(B) 296 JA/ET/SA-independent genes categorized at biological process level 3.
(C) 149 JA/ET/SA-independent genes, previously identified as having a cellular physiological or metabolic function (first two categories in [B]), categorized at biological process level 9.
Percentages relate to total number of genes with an ontology at each level. Only categories >5% of total number of genes shown.

Expression of JA/ET/SA-Dependent Genes.
Hierarchical cluster analysis of 129 genes differentially expressed in wild-type (Col-0) Arabidopsis and at least one of the hormone-deficient mutants (sid2-1, ein2-5, and coi1-1) compared with wild-type plants following P. irregulare infection (P). Genes (rows) and experiments (columns) were clustered with The Institute for Genomic Research (TIGR) multi-experiment viewer using Euclidean distance and complete linkage. Gene subclusters of interest that are discussed in the text are indicated by labeled vertical bars to the right of the image.

Meta-Analysis of JA/ET/SA-Dependent Gene Data.
Cluster of JA/ET/SA-dependent genes with available expression data from hormonal and pathogen treatments within the GENEVESTIGATOR database (expression data available for only 107 of the 129 genes; Zimmermann et al., 2004). Genes (rows) and experiments (columns) were clustered with the TIGR multi-experiment viewer using Euclidean distance and complete linkage. Gene subclusters of interest that are discussed in the text are indicated by labeled vertical bars. Overrepresented cis-elements within each gene cluster were identified by TAIR motif analysis, Botany Beowulf Cluster Promomer, and Gibbs motif sampler. Statistical significance (P value from binomial distribution) is shown in parenthesis where possible.

Meta-Analysis of JA/ET/SA-Independent Gene Data.
Tree view of Arabidopsis–P. irregulare infection (P) experiments with GENEVESTIGATOR (stress response) experiments when cluster analysis included only JA/ET/SA-independent genes. Thus, the four within-genotype comparisons [Col-0(P)-Col-0, ein2(P)-ein2, coi1(P)-coi1, and sid2(P)-sid2] show very similar gene expression patterns and cluster together. Data were clustered with the TIGR multi-experiment viewer using Euclidean distance and complete linkage.

Meta-Analysis of JA/ET/SA-Independent Gene Data.
Cluster view of 296 JA/ET/SA-independent genes (selected with a false discovery rate <1% and log ratio >1.5) with available expression data from the most closely related experiments within the GENEVESTIGATOR database (Zimmermann et al., 2004). Genes (rows) and experiments (columns) were clustered with the TIGR multi-experiment viewer using the Pearsons uncentered distance and complete linkage. Gene subclusters of interest that are discussed in the text are indicated by labeled vertical bars. Overrepresented (black lettering) and underrepresented (green lettering) cis-elements within each gene cluster were identified by TAIR motif analysis, Botany Beowulf Cluster Promomer, and Gibbs motif sampler. WBS, WRKY binding site; ARE, anthocyanin regulatory element. Statistical significance (z-score significance [WRKY binding site and ARE] and P value from binomial distribution [all others]) is shown in parenthesis.