A genomic approach to identify regulatory nodes in the transcriptional network of systemic acquired resistance in plants

Abstract

Many biological processes are controlled by intricate networks of transcriptional regulators. With the development of microarray technology, transcriptional changes can be examined at the whole-genome level. However, such analysis often lacks information on the hierarchical relationship between components of a given system. Systemic acquired resistance (SAR) is an inducible plant defense response involving a cascade of transcriptional events induced by salicylic acid through the transcription cofactor NPR1. To identify additional regulatory nodes in the SAR network, we performed microarray analysis on Arabidopsis plants expressing the NPR1-GR (glucocorticoid receptor) fusion protein. Since nuclear translocation of NPR1-GR requires dexamethasone, we were able to control NPR1-dependent transcription and identify direct transcriptional targets of NPR1. We show that NPR1 directly upregulates the expression of eight WRKY transcription factor genes. This large family of 74 transcription factors has been implicated in various defense responses, but no specific WRKY factor has been placed in the SAR network. Identification of NPR1-regulated WRKY factors allowed us to perform in-depth genetic analysis on a small number of WRKY factors and test well-defined phenotypes of single and double mutants associated with NPR1. Among these WRKY factors we found both positive and negative regulators of SAR. This genomics-directed approach unambiguously positioned five WRKY factors in the complex transcriptional regulatory network of SAR. Our work not only discovered new transcription regulatory components in the signaling network of SAR but also demonstrated that functional studies of large gene families have to take into consideration sequence similarity as well as the expression patterns of the candidates.

Figure 1. Differential Regulation of SAR by NPR1-Induced WRKY Factors

(A) A schematic representation of the strategy to identify NPR1 direct targets. SA treatment activates components of the SAR pathway upstream or independent of NPR1. Subsequent application of dexamethasone (DEX) triggers nuclear translocation of the NPR1-GR fusion protein to activate existing TGA transcription factors. Direct target genes of NPR1 are transcribed but not translated in the presence of the inhibitor cycloheximide (CHX) to prevent transcription of indirect target genes. ER, endoplasmic reticulum–resident proteins; TF, transcription factors; PR, pathogenesis-related proteins. (B) Mode of action for the NPR1-target WRKY factors. In WT, SA accumulation triggers nuclear localization of NPR1, which directly induces several WRKY genes. When SA levels are low, WRKY58 functions to prevent (blocked arrow) spurious activation of SAR (dotted lines). When SA levels are high, signaling through positive WRKY factors overcomes the negative effect of WRKY58 to activate (arrow) downstream gene transcription (solid lines). In addition, WRKY70 and WRKY54 prevent excessive SA accumulation (blocked arrow).

Figure 2. Induction of WRKY Genes by SAR Inducers and NPR1

Plotted here are log₂-transformed microarray data normalized by the GeneSpring package, showing the expression levels of six WRKY genes 0, 8, and 24 h after BTH treatment in WT (NPR1 +) and npr1 mutant (NPR1 −). The expression levels of WRKY59 and WRKY66 were too low to be detected under these conditions. Error bars represent standard deviations (SDs).

Figure 3. Resistance Defects of wrky18

(A) Plants were chemically induced with 60 μM BTH 24 h before inoculation with a high dose of Psm ES4326 (OD₆₀₀ = 0.001). As a control, uninduced plants were inoculated at the same time. Bacterial growth was scored 3 dpi. Each datapoint represents the average colony-forming units (cfu) from 16 leaf disks plotted on a log scale, with error bars indicating 95% confidence intervals. This experiment was repeated more than five times with similar results. (B) Plants were first inoculated with either P. syringae pv. phaseolicola avrB or 10 mM MgCl₂ on two lower leaves. Later (3 d), three upper leaves were inoculated with Psm ES4326 (OD₆₀₀ = 0.001). Leaf disks from the second inoculation were collected 3 dpi to measure bacterial growth. This experiment was carried out twice with similar results. (C and D) To examine wrky18 for an EDS phenotype, plants were inoculated with a low dose of Psm ES4326 (OD₆₀₀ = 0.0001). Bacterial growth was measured in 3 dpi (C), and disease symptoms were recorded in (D) 3 dpi. These experiments were performed more than five times with similar results.

Figure 4. Genes Affected by BTH, npr1, and wrky18 0, 8, and 24 h after Induction

Using ANOVA, the expression of 6,525 genes was found to be altered in WT following BTH treatment (p < 0.05) (after multiple testing correction using the method proposed by Benjamini and Hochberg to assess false discovery rate [22]). After applying a 2-fold change cutoff to these genes, the list was reduced to 2,280 genes, among which 1,147 were induced and 1,133 were repressed. From this list, a two-way ANOVA was applied between WT and npr1 data sets and between WT and wrky18 data sets to identify NPR1-dependent and WRKY18-dependent genes, respectively. (A) The Venn diagram shows that almost all BTH-responsive genes were NPR1-dependent (2,248/2,280; 99%) whereas the expression of 451 BTH-responsive genes (∼19.8%) was altered in the wrky18 mutant. (B) The expression levels of 2,280 BTH-dependent genes normalized by GeneSpring were plotted on log scale on the y-axis and in time order on the x-axis. Genes induced and repressed in WT are colored red and green, respectively. The profile of these genes in the npr1 mutant is also depicted. (C) The expression levels of 451 WRKY-dependent genes in WT and in wrky18 mutant were normalized by GeneSpring and plotted on log scale on the y-axis and in time order on the x-axis. Genes induced and repressed in WT are colored red and green, respectively. The majority of them showed either diminished induction (204 genes) or diminished repression (152 genes) in wrky18, in contrast to the robust response in WT and the almost complete lack of response in npr1.

Figure 5. Resistance Defects of wrky58

(A) Loss of WRKY58 confers resistance when plants were weakly induced with 30 mM BTH. (B) To examine the function of WRKY58, the wrky58 mutation was introduced into wrky18, and the effect was observed in an EDS test 3 dpi. w18 w58 represents the wrky18 wrky58 double mutant. Both (A) and (B) were performed twice with similar results.

Figure 6. Defects in WRKY70 and WRKY54 Result in SA Overaccumulation

(A) Plants were dipped in either a 10 mM MgCl₂ solution or a suspension of Psm ES4326 avrRpt2 to trigger SA production. Free SA was extracted and measured from three samples for each datapoint 3 dpi. Error bars represent SDs. This experiment was repeated twice with similar results. (B) The SA biosynthesis gene ICS1 is upregulated in the wrky54 wrky70 (w54 w70) double mutant. Relative transcript levels were determined by RT-qPCR after normalization to ubiquitin. Error bars represent SD from three PCR runs. (C) Lack of resistance in w54 w70, measured by bacterial growth 3 dpi with a high dose of Psm ES4326. (D and E) The wrky53 wrky70 (w53 w70) double mutant displays an EDS phenotype. Bacterial growth was measured in 3 dpi (D) and disease symptoms were recorded in 3 dpi (E). Both (C) and (D) were done three times each with similar results.