TaGR-RBP, a Glycine-rich RNA-binding Protein in Wheat, Activates Rust Resistance Through ROS Burst

Glycine-rich RNA-binding proteins (GR-RBPs) play pivotal roles in regulating plant responses to abiotic and biotic stresses. However, the involvement of GR-RBPs in wheat immune responses to Puccinia striiformis f. sp. tritici (Pst) remains poorly understood. Here, we report the identification of 58 GR-RBP genes in wheat and provide a functional characterization of TaGR-RBP, which exhibits significant upregulation during wheat-Pst interactions. Subcellular localization analysis demonstrated that TaGR-RBP localizes in both the cytoplasm and the nucleus. Virus-induced gene silencing (VIGS) using Barley Stripe Mosaic Virus (BSMV) revealed that transient silencing of TaGR-RBP compromises wheat resistance to Pst. Furthermore, CRISPR-Cas9-mediated knockout of TaGR-RBP resulted in reduced resistance to Pst, as evidenced by diminished hydrogen peroxide (H₂O₂) accumulation and enhanced fungal proliferation, confirming its positive regulatory role in wheat immunity. Transient expression of TaGR-RBP in Nicotiana benthamiana induced Reactive Oxygen Species (ROS) accumulation. AlphaFold3 predictions and an RNA electrophoretic mobility shift assay (REMSA) validated the RNA-binding activity of TaGR-RBP. Overall, these findings imply the functional significance of GR-RBPs in wheat resistance against Pst. Deciphering the function of TaGR-RBP in plant immunity provides a theoretical basis for engineering disease-resistant wheat and guides the development of sustainable strategies for disease control.