Temporal global expression data reveal known and novel salicylate-impacted processes and regulators mediating powdery mildew growth and reproduction on Arabidopsis

Abstract

Salicylic acid (SA) is a critical mediator of plant innate immunity. It plays an important role in limiting the growth and reproduction of the virulent powdery mildew (PM) Golovinomyces orontii on Arabidopsis (Arabidopsis thaliana). To investigate this later phase of the PM interaction and the role played by SA, we performed replicated global expression profiling for wild-type and SA biosynthetic mutant isochorismate synthase1 (ics1) Arabidopsis from 0 to 7 d after infection. We found that ICS1-impacted genes constitute 3.8% of profiled genes, with known molecular markers of Arabidopsis defense ranked very highly by the multivariate empirical Bayes statistic (T(2) statistic). Functional analyses of T(2)-selected genes identified statistically significant PM-impacted processes, including photosynthesis, cell wall modification, and alkaloid metabolism, that are ICS1 independent. ICS1-impacted processes include redox, vacuolar transport/secretion, and signaling. Our data also support a role for ICS1 (SA) in iron and calcium homeostasis and identify components of SA cross talk with other phytohormones. Through our analysis, 39 novel PM-impacted transcriptional regulators were identified. Insertion mutants in one of these regulators, PUX2 (for plant ubiquitin regulatory X domain-containing protein 2), results in significantly reduced reproduction of the PM in a cell death-independent manner. Although little is known about PUX2, PUX1 acts as a negative regulator of Arabidopsis CDC48, an essential AAA-ATPase chaperone that mediates diverse cellular activities, including homotypic fusion of endoplasmic reticulum and Golgi membranes, endoplasmic reticulum-associated protein degradation, cell cycle progression, and apoptosis. Future work will elucidate the functional role of the novel regulator PUX2 in PM resistance.

Figure 1.

Venn diagrams showing genes with differential expression in wild-type Arabidopsis (PM) or in the ics1 mutant versus the wild type (PM:ICS1-impacted) over the time course of PM infection or in response to exogenous BTH (data from Wang et al., 2006). A, ATH1 genes. B, Transcription factors. PM and PM:ICS1-impacted genes were selected with log₁₀(T²) ≥ 2.5. BTH-responsive genes exhibited significantly altered expression in wild-type versus BTH-treated samples, selected using ANOVA with P < 0.05 and 2-fold change cutoff (Wang et al., 2006).

Figure 2.

Expression profile plots for clusters of genes selected based on differences in temporal expression for wild-type (WT) T²-selected data (A) or ics1 versus wild-type T²-selected data (B). For wild-type clusters, log₂ expression values for 0, 1, 3, 5, and 7 dpi (I) and 7-d UI are shown. For ICS1 clusters, log₂ expression values for 0, 0.25, 1, 3, 5, and 7 dpi for wild-type and ics1 are displayed. Data for biological replicates are shown sequentially.

Figure 3.

Patterns of ICS1-impacted transcriptional responses. Gene expression profiles for the wild type (black) and ics1 mutant (gray) over the time course of PM infection from 0 (UI) to 7 dpi. Expression profiles for four paired (wild-type and mutant) biological replicates are shown for members of each ICS1 cluster: cluster 1 (A–D), cluster 2 (E), cluster 3 (F), cluster 4 (G), cluster 5 (H), and cluster 6 (I).

Figure 4.

PUX2 expression in response to pathogens. A, In response to G. orontii. The wild type is shown in black, and ics1 is shown in gray. Crosses indicate UI 7-d samples. B, In response to P. syringae pv maculicola ES4326 (Psm) or mock treatment (5 mm MgSO₄). Data are from the NASCArray database (Craigon et al., 2004), experiment 414 (submitted by R. Mitra and J. Glazebrook).

Figure 5.

PUX2 T-DNA insertion mutants exhibit reduced PM reproduction. A, Macroscopic phenotype. Wild-type (Col-0), pux2-1 (SALK_148507), and pux2-2 (SALK_145175) UI whole plants were photographed at 6 weeks. G. orontii-infected leaves were rated and photographed at 11 dpi. Number of plants with disease rating is indicated as described by Reuber et al. (1998) as follows: 0, no visible symptoms of infection; 4, approximately 100% coverage of infected leaves. Bars = 1 cm. B, Microscopic phenotype. Leaves were harvested at 5 dpi and stained with trypan blue for visualization of fungus or cell death. c, Initial germinated conidia; cp, conidiophore. Bars = 100 μm. Independent experiments showed similar results.