Geminiviral replication enhancer hijacks a negative regulator of cytokinin signaling to facilitate viral infection.

Geminiviruses are widespread across the globe and cause devastating diseases in food and medicinal crops. Their C3 proteins have long been known to enhance viral replication; however, the underlying molecular mechanisms remain poorly understood. In this study, we show that transgenic plants overexpressing the C3 protein of tobacco curly shoot geminivirus (TbCSV) exhibit hypersensitive responses to cytokinin (CK) treatment, which largely restores the attenuated viral replication observed in the TbCSV C3 mutant (TbCSV). We identified NbTAF12b, a negative regulator of CK signaling in Nicotiana benthamiana, as a C3-interacting protein that attenuates TbCSV replication. TbCSV C3 not only inhibits the transcription of NbTAF12b but also competes with NbRR1 and NbKMD17 for binding to the NbTAF12b protein. This competition disrupts the formation of the NbTAF12b-NbKMD-NbRR heterotrimer and promotes NbRR1 accumulation, thereby enhancing CK signaling and ultimately facilitating TbCSV replication. C3 proteins from other distantly related geminiviruses exhibit similar 3D structures and also target NbTAF12b in vivo, suggesting that this mechanism is conserved among geminiviruses. These findings shed new light on the molecular mechanism by which TbCSV C3 facilitates viral replication through the modulation of CK signaling and provide potential molecular targets for engineering virus-resistant plants.