Ustilaginoidea virens secreted effector UvSec117 hijacks OsWRKY31‐OsAOC module to suppress jasmonic acid‐mediated immunity in rice

Rice false smut (RFS) caused by Ustilaginoidea virens is one of the most important disease in rice (Oryza sativa)-growing regions worldwide. RFS not only causes rice yield losses but also potentially threatens human and animal health by producing cyclopeptide mycotoxins (Sun et al., 2020). Introducing genetically encoded resistance is an environmentally friendly, economical approach to controlling plant diseases (Yu et al., 2023). However, at present, the varieties and gene resources of resistance to RFS are still extremely scarce, and it is difficult to identify major resistance genes against RFS. Uncovering the functions of the U. virens effectors and molecular mechanism of the rice, U. virens interaction can help to identify molecular probes for discovering disease resistance-related genes (Wang and Kawano, 2022). In previous studies, we identified UvSec117 as a key virulence effector in U. virens, and found rice transcription factor OsWRKY31 in a screen for proteins that interact with UvSec117 (Chen et al., 2022). WRKY transcription factors have many regulatory roles in development and response to biotic/abiotic stresses in plants (Wang et al., 2023). However, little is known about the regulatory functions of WRKY genes in the plant resistance to grain-infecting pathogens. In this work, we confirmed interactions between UvSec117 and OsWRKY31 in a directed yeast two-hybrid assay (Figure 1a; Data S1). In a co-immunoprecipitation (Co-IP) assay by rice protoplasts transiently co-expressing OsWRKY31-Flag and UvSec117-GFP constructs, UvSec117 was immunoprecipitated by OsWRKY31 (Figure 1b). In a pull-down assay using recombinant OsWRKY31-GST and UvSec117-His purified from Escherichia coli, OsWRKY31-GST was pulled down by His beads coated with UvSec117-His (Figure 1c). We also validated the interaction between UvSec117 and OsWRKY31 by a luciferase complementation imaging (LCI) assay in N. benthamiana leaves (Figure 1d). When we transiently co-expressed UvSec117-cYFP and OsWRKY31-nYFP constructs in rice protoplasts and performed a bimolecular fluorescence complementation (BiFC) assay, we detected YFP (yellow fluorescent protein) fluorescence in the nucleus (Figure 1e). Collectively, these results suggest that UvSec117 interacts with OsWRKY31 in vivo and in vitro. To explore the role of OsWRKY31 in resistance against RFS fungus or other rice pathogens, we generated OsWRKY31 knockout mutant plants (wrky31) (Figure S1a) and OsWRKY31-overexpressing transgenic rice lines (OsWRKY31-OE) (Figure S1b). The agronomic traits of wrky31 and OsWRKY31-OE plants were similar to those of wild-type Nipponbare (NPB) (Figure S1c,d). Following inoculation with different rice pathogens, OsWRKY31-OE plants were less susceptible and wrky31 plants were more susceptible to the RFS, bacterial blight, rice blast and sheath blight than NPB plants (Figure 1f–i), indicating that OsWRKY31 positively regulates the resistance of rice to multiple diseases. To identify global targets of the transcription factor OsWRKY31, we performed chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) using OsWRKY31-OE plants with an anti-Flag antibody. In total, we identified 4626 peaks (1054 target genes, Data S2). A significant majority (> 60%) of these peaks are located within genic regions, with the modifications being highly enriched at the promoters of protein-coding genes (Figure 1j). MEME (Multiple EM for Motif Elicitation) analysis revealed that most OsWRKY31-bound DNA motifs contained the sequence TTGTACTT, GGGCCCAC or CCCCTTTT (Figure 1k). Gene ontology (GO) analysis revealed that the target genes were enriched for induced systemic resistance and salicylic acid (SA)/jasmonic acid (JA)-mediated signalling pathways (Figure 1l). RT-qPCR showed that the key JA biosynthesis gene OsAOC (ALLENE OXIDE CYCLASE) is significantly downregulated in wrky31-1 plants, and ChIP-qPCR confirmed that OsWRKY31 binds to the OsAOC promoter (Figure 1m). Knockout of OsAOC in rice enhances its susceptibility to RFS (Figure 1n). OsWRKY31 expression in N. benthamiana significantly enhanced firefly luciferase (LUC) activity derived from the OsAOCpro-LUC reporter. Co-infiltration of UvSec117 with OsAOCpro-LUC inhibited OsWRKY31-induced LUC activity, whereas co-infiltration of GFP, did not (Figure 1o). Yeast one-hybrid results showed that OsWRKY31 can bind the promoter of OsAOC (Figure 1p). In an electrophoretic mobility shift assay (EMSA) assay, OsWRKY31-His specifically bound to the OsAOC promoter; addition of unlabelled competitive probe decreased this binding. Preincubation with UvSec117 reduced the DNA-binding activity of OsWRKY31 (Figure 1q), indicating that UvSec117 directly inhibits the DNA-binding activity of OsWRKY31. Moreover, the contents of JA were significantly lower in wrky31-1 than in NPB rice spikelets; in HE-1 (Heterologous expression of UvSec117 transgenic plants) relative to EV (empty vector transgenic plants) rice spikelets (Figure 1r). These results indicate that OsWRKY31 regulating the JA-mediated defence was suppressed by UvSec117. In this study, we found that the transcription factor OsWRKY31 functions as a key positive regulator to broad-spectrum disease resistance. Here, we provide a comprehensive genome-wide binding map of OsWRKY31 and its regulatory network, and further describe a previously unknown regulatory role where OsWRKY31 mediates the JA-mediated signalling pathway to regulate plant immunity. Collectively, this study unveils a pivotal virulence strategy employed by U. virens, the secretory effector UvSec117 inhibits OsWRKY31 binding to target gene promoters like OsAOC, thereby suppressing JA-mediated defence (Figure 1s). Moreover, this investigation highlights the critical role of OsWRKY31 as a crucial component in orchestrating multi-pathogen resistance, further underscoring its significance in plant defence mechanisms. The OsWRKY31-OE lines generated in this study may provide valuable germplasm resources for rice disease resistance breeding, which has important theoretical and practical value. This study was funded by the National Natural Science Foundation of China (32100465, 32302302 and 32172372) and the 'Pioneer' and 'Leading Goose' R&D Program of Zhejiang (2023C02018). The authors declare no conflict of interest. Y.D. J.T. and G.Y. performed most of the experiments. Q.X. performed the data analyses. L.Z., H.L., H.W., Z.W., Y.F., J.H., J.C. and X-L.C. provided technical support. X-Y.C. and L.Z. wrote and revised the manuscript. All authors have read and approved the final manuscript. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Data S1 Materials and methods. Data S2 ChIP-seq data statistics of the OsWRKY31. Figure S1 OsWRKY31 transgenic rice plants were achieved without adverse effects on plant growth or yield. (a) Mutations identified within sgRNA target sites of OsWRKY31 in rice generated by CRISPR/Cas9-mediated genome editing. (b) RT-qPCR of OsWRKY31 expression in NPB and OsWRKY31-OE transgenic rice plants. (c, d) Morphology and agronomic traits of mature wild-type NPB, OsWRKY31-OE and wrky31 plants grown in the field. Data are means ± SD (n = 3 unless otherwise indicated). The P-values were determined by Tukey's multiple comparison tests compared to NPB. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.