Post‐Translational Modifications of TOE3 Regulate Antiviral Defense in Tobacco

CK2αL phosphorylates TOE3 and enhances its stability, while FBXL1 promotes the ubiquitination‐degradation of TOE3 via the 26S proteasome. Compared to CK2αL‐phosphorylated TOE3, nonphosphorylated TOE3 shows a much higher affinity for FBXL1 and is more susceptible to ubiquitination‐degradation, thus facilitating viral infection. The regulatory mechanisms provide insights into the roles of TOE3's PTMs in tobacco antiviral defense. Abstract Post‐translational modifications (PTMs) affect the function of transcription factors and regulate plant immune responses. APETALA2 (AP2) transcription factor TARGET OF EAT3 (TOE3) plays a pivotal role in plant antiviral immunity. However, little is known about the impact of PTMs on TOE3 function. Here, that casein kinase II α subunit‐like protein (CK2αL) is identified to interact with TOE3, phosphorylating it at serine 58 (S58) and threonine 128 (T128). Overexpression of CK2αL enhances the stability of TOE3 to upregulate the expression of defense‐related genes, thereby improving tobacco resistance to tobacco mosaic virus (TMV). Additionally, the F‐box protein FBXL1 interacts with TOE3 and promotes the ubiquitination‐degradation of TOE3 through the 26S proteasome, and overexpression of FBXL1 facilitates TMV infection in tobacco. Importantly, CK2αL‐phosphorylated TOE3 exhibits a lower binding affinity to FBXL1 compared to the nonphosphorylated TOE3, thereby protecting TOE3 from FBXL1‐mediated degradation by the 26S proteasome. The stable TOE3 activates the expression of defense‐related genes to enhance the resistance of plants to viral infection. Taken together, the findings demonstrate a mechanism by which phosphorylation and ubiquitination cooperatively regulate the stability of TOE3 to fine‐tune antiviral immunity in tobacco. CK2αL phosphorylates TOE3 and enhances its stability, while FBXL1 promotes the ubiquitination-degradation of TOE3 via the 26S proteasome. Compared to CK2αL-phosphorylated TOE3, nonphosphorylated TOE3 shows a much higher affinity for FBXL1 and is more susceptible to ubiquitination-degradation, thus facilitating viral infection. The regulatory mechanisms provide insights into the roles of TOE3's PTMs in tobacco antiviral defense. Abstract Post-translational modifications (PTMs) affect the function of transcription factors and regulate plant immune responses. APETALA2 (AP2) transcription factor TARGET OF EAT3 (TOE3) plays a pivotal role in plant antiviral immunity. However, little is known about the impact of PTMs on TOE3 function. Here, that casein kinase II α subunit-like protein (CK2αL) is identified to interact with TOE3, phosphorylating it at serine 58 (S58) and threonine 128 (T128). Overexpression of CK2αL enhances the stability of TOE3 to upregulate the expression of defense-related genes, thereby improving tobacco resistance to tobacco mosaic virus (TMV). Additionally, the F-box protein FBXL1 interacts with TOE3 and promotes the ubiquitination-degradation of TOE3 through the 26S proteasome, and overexpression of FBXL1 facilitates TMV infection in tobacco. Importantly, CK2αL-phosphorylated TOE3 exhibits a lower binding affinity to FBXL1 compared to the nonphosphorylated TOE3, thereby protecting TOE3 from FBXL1-mediated degradation by the 26S proteasome. The stable TOE3 activates the expression of defense-related genes to enhance the resistance of plants to viral infection. Taken together, the findings demonstrate a mechanism by which phosphorylation and ubiquitination cooperatively regulate the stability of TOE3 to fine-tune antiviral immunity in tobacco. Advanced Science, Volume 12, Issue 42, November 13, 2025.