Submergence confers immunity mediated by the WRKY22 transcription factor in Arabidopsis

Abstract

Transcriptional control plays an important role in regulating submergence responses in plants. Although numerous genes are highly induced during hypoxia, their individual roles in hypoxic responses are still poorly understood. Here, we found that expression of genes that encode members of the WRKY transcription factor family was rapidly and strongly induced upon submergence in Arabidopsis thaliana, and this induction correlated with induction of a large portion of innate immunity marker genes. Furthermore, prior submergence treatment conferred higher resistance to the bacterial pathogen Pseudomonas syringae in Arabidopsis. Among the WRKY genes tested, WRKY22 had the highest level of induction during the early stages of submergence. Compared with the wild type, WRKY22 T-DNA insertion mutants wrky22-1 and wrky22-2 had lower disease resistance and lower induction of innate immunity markers, such as FLG22-INDUCED RECEPTOR-LIKE KINASE1 (FRK1) and WRKY53, after submergence. Furthermore, transcriptomic analyses of wrky22-2 and chromatin immunoprecipitation identified several potential targets of WRKY22, which included genes encoding a TIR domain-containing protein, a plant peptide hormone, and many OLIGO PEPTIDE TRANSPORTER genes, all of which may lead to induction of innate immunity. In conclusion, we propose that submergence triggers innate immunity in Arabidopsis via WRKY22, a response that may protect against a higher probability of pathogen infection either during or after flooding.

Figure 1.

Induction of Innate Immunity Markers and WRKY Genes in Response to Submergence. (A) Expression of WRKY genes is induced by submergence. Gene expression was determined by microarray analysis and validated by qRT-PCR in 9-d-old wild-type Arabidopsis (Columbia) seedlings from at least four independent biological replicates. (B) to (D) Innate immunity marker genes were responsive to submergence (C), but PR genes (B) and stress-regulated genes (D) were not (see Supplemental Table 1 online for gene information). The color scale indicates treatment-to-control ratio of expression in log₂ or in fold induction.

Figure 2.

Expression of Innate Immunity Marker Genes after Submergence. Five-week-old Columbia plants were submerged for 12 h and allowed to recover for up to 9 h, with samples collected at the indicated time points. Transcript levels were detected by qRT-PCR using specific primers. TUBULIN mRNA was used as an internal control. The data represent means ± sd from four to seven independent biological replicates.

Figure 3.

Plant Immunity Is Triggered by Submergence. (A) Disease symptoms assessed 4 d postinoculation (dpi) with P. syringae in submerged and control Columbia plants. Bar = 2 cm. (B) The levels of resistance were defined using a damage index based on the necrotic and chlorotic area of leaves (black, 100% leaf area; dark gray, equal to or >50% leaf area; light gray, <50% leaf area; white, no damage observed). The data represent means ± sd from four independent repeats. Statistical differences between submerged and control were determined using Student’s t test for the sum of 100% and ≥50% indexes. **P < 0.01. (C) Bacterial population at 0 and 4 d postinoculation in submerged and control Columbia plants. The data represent means ± sd from five independent repeats. Statistical differences between submerged and control plants were determined by Student’s t test. *P < 0.05. cfu, colony-forming units.

Figure 4.

WRKY22 Mediates Submergence-Triggered Immunity. (A) Transcript levels of WRKY22 and ADH1 were detected by qRT-PCR. Nine-day-old Columbia seedlings were in light, dark, or hypoxia (0.5% oxygen gas balanced with nitrogen in dark) for up to 9 h and were collected at specific time points (0, 1, 3, 6, and 9 h). TUBULIN mRNA was used as an internal control. The data represent means ± sd from seven independent biological replicates and were subjected to an one-way analysis of variance and Tukey’s honestly significant difference tests (P < 0.05). Data from the same time point with different lowercase letters were significantly different from each other. (B) Disease symptoms assessed 4 d postinoculation in submerged Columbia, wrky22-1, and wrky22-2 plants. Bar = 2 cm. (C) Levels of resistance in submerged Columbia, wrky22-1, and wrky22-2 plants. The levels of resistance were defined using a damage index based on the necrotic and chlorotic area of leaves, as described in Figure 3. The data represent means ± sd from eight independent repeats. Pairwise statistical differences between Columbia and WRKY22 mutants were determined by Student’s t test for the sum of 100% and ≥50% indexes. **P < 0.01. (D) Bacterial populations at 0 and 4 d postinoculation in submerged Columbia, wrky22-1, and wrky22-2 plants. The data represent means ± sd from five to six independent repeats. Statistical differences between Columbia and WRKY22 mutants were identified using Student’s t test. *P < 0.05.

Figure 5.

Innate Immunity Markers FRK1 and WRKY53 Are Transcriptionally Regulated by WRKY22. Transcript levels were detected by qRT-PCR using specific primers. TUBULIN mRNA was used as an internal control. The data represent means ± sd from four to eight independent biological replicates. *P < 0.05 and **P < 0.01 in Student’s t test.

Figure 6.

Several PRR-Related and MAMP-Induced Genes Are WRKY22 Targets under Submergence. Transcript levels of PRR-related (A) and MAMP-induced (B) genes were detected by qRT-PCR using specific primers. TUBULIN mRNA was used as an internal control. The data represent means ± sd from four to eight independent biological replicates. *P < 0.05 and **P < 0.01 in Student’s t test.

Figure 7.

Integrated Networks of WRKY22 Downstream Targets upon Submergence. Networks were constructed with a Web-based analysis tool STRING (Szklarczyk et al., 2011). Gene lists of WRKY22 downstream targets are integrated from qRT-PCR results (Figures 5 and 6; see Supplemental Figure 7 online), microarray analysis (asterisk-labeled genes in Supplemental Table 2 online), and ChIP-qPCR results (see Supplemental Table 3 online). These three types of experimental evidence are represented by three different line colors. The STRING database assembles information about co-occurrence, coexpression, databases, and text mining, which are also marked with different line colors. Nodes of genes were clustered to six groups by KMEANS in STRING and marked by a different color code.

Figure 8.

Models of WRKY22-Mediated Submergence-Triggered Responses. (A) Summary of WRKY22-mediated and -independent pathways under submergence. (B) Model of submergence-triggered immunity. (C) Putative molecular mechanism of submergence-triggered immunity. Solid lines indicate validated events. Dotted lines and question marks indicate predicted events.