The rapid production of reactive oxygen species (ROS burst) in plant defense activation provides second messenger signaling molecules and direct antimicrobials that are crucial for successful defense against pathogens and maintaining microbiota homeostasis. These ROS bursts require proper transcriptional and post-transcriptional regulation of NADPH oxidases (NOX, also known as RESPIRATORY BURST OXIDASE HOMOLOG or RBOH) that produce superoxide radicals in the apoplast (Kadota et al., 2015). Despite a growing body of knowledge about the regulation of NADPH oxidases in model plants, there remain gaps: (1) the mechanism of NOX repression is not well understood and (2) the regulation and function of NADPH oxidases remain largely unknown in non-model crop species, such as common bread wheat (Triticum aestivum), an allohexaploid cereal that is challenging to study with genetics.
In this issue, Ning Wang and colleagues (Wang et al., 2022) narrow these gaps by identifying a WRKY transcription factor (TF) that transcriptionally represses TaNOX10 in wheat. They first screened WRKY TFs in purple false brome (Brachypodium distachyon), a genetically more tractable model for cereals, and identified BdWRKY67 as a repressor of BdRBOHD and pathogen resistance. These findings allowed the authors to filter through the 171 wheat WRKY genes (Chen et al., 2019) for the BdWRKY67 homolog, TaWRKY19. TaWRKY19 has three homoeologs (genes from each of the three wheat subgenomes)—TaWRKY19-2A, TaWRKY19-2B, and TaWRKY19-2D—all of which are induced upon infection by the wheat pathogen Puccinia striiformis f. sp. tritici (Pst), a causal agent of wheat stripe rust disease. These homoeologs are partially redundant, but their simultaneous silencing by RNA interference (RNAi) resulted in plants that become more resistant to Pst with enhanced expression of TaNOX10, the wheat ortholog of BdRBOHD, consistent with the findings in B. distachyon.
Most impressively, using CRISPR-mediated genome editing, the authors further generated a stable knockout (ko) line in wheat lacking all three TaWRKY19 homoeologs. This triple tawrky19-ko mutant showed increased ROS accumulation upon Pst infection and enhanced resistance against the rust pathogen, corroborating the results from the RNAi lines (see Figure). These results supported the notion that TaWRKY19 is a potential genetic engineering target for protecting wheat from rust disease.
Figure.
A knockout mutant of TaWRKY19 in wheat shows increased ROS (H2O2) production (middle panel) and enhanced resistance against the rust pathogen P. striiformis f. sp. tritici. Adapted from Wang et al. (2022), Figure 8, C–E.
After finding the negative role of TaWRKY19 in ROS production and pathogen resistance, the authors tested for a mechanistic link. In vitro biochemical assays revealed that TaWRKY19 binds to a W-box motif, a canonical WRKY binding site, in the TaNOX10 promoter, and that this binding is required for the transcriptional repression of TaNOX10. Ectopic expression of TaNOX10 from a TaNOX10 promoter lacking the W-box deleted promoter, no longer subject to WRKY regulation, abolished the lower ROS production and pathogen susceptibility caused by constitutive expression of TaWRKY19. Taken together, TaWRKY19 suppresses ROS production by directly repressing TaNOX10 and this transcriptional repression causes susceptibility to the pathogen.
This study revealed a novel function of a WRKY TF in suppressing ROS generation in wheat. Yet, the biological role of this mechanism is still cryptic. In Nicotiana benthamiana, multiple WRKY TFs have been shown to induce ROS production by transactivating NbRBOHB, an ortholog of BdRBOHD and TaNOX10 (Adachi et al., 2015). It would be interesting to study whether and how WRKY TFs with contrasting functions on NADPH oxidases collaborate to control immunity in wheat and other plant species. ROS-suppressing WRKY TFs, such as TaWRKY19 and BdWRKY67, may play a role in fine-tuning ROS production, as overaccumulation of ROS can be detrimental to plant health. Other interesting questions are: do virulent pathogens exploit ROS-suppressing WRKY TFs to dampen host immune responses, and if so, how common is this type of exploitation? A deeper understanding of the WRKY-ROS axis in plant–microbe interactions will pave the way toward optimizing crop engineering in the future.
References
- Adachi H, Nakano T, Miyagawa N, Ishihama N, Yoshioka M, Katou Y, Yaeno T, Shirasu K, Yoshioka H (2015) WRKY transcription factors phosphorylated by MAPK regulate a plant immune NADPH oxidase in Nicotiana benthamiana. Plant Cell 27: 2645–2663 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chen X, Li C, Wang H, Guo Z (2019) WRKY transcription factors: Evolution, binding, and action. Phytopathol Res 1: 13 [Google Scholar]
- Kadota Y, Shirasu K, Zipfel C (2015) Regulation of the NADPH oxidase RBOHD during plant immunity. Plant Cell Physiol 56: 1472–1480 [DOI] [PubMed] [Google Scholar]
- Wang N, Fan X, He M, Hu Z, Tang C, Lin D, Gan P, Wang J, Huang X, Kang Z, et al. (2022) Transcriptional repression of TaNOX10 by TaWRKY19 compromises ROS generation and enhances wheat susceptibility to stripe rust. Plant Cell 34: 1784--1803 [DOI] [PMC free article] [PubMed] [Google Scholar]