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 <i>Puccinia striiformis</i> f. sp. <i>tritici</i> (<i>Pst</i>) 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-<i>Pst</i> 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 <i>TaGR-RBP</i> compromises wheat resistance to <i>Pst</i>. Furthermore, CRISPR-Cas9-mediated knockout of <i>TaGR-RBP</i> resulted in reduced resistance to <i>Pst</i>, 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 <i>Nicotiana benthamiana</i> 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 <i>Pst</i>. 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.