VvDLK negatively regulates cold tolerance by interacting with Vv14-3-3A in grapevine

Sudden cold snaps due to blurred seasonality are a rising challenge to agriculture in times of climate change. Swift activation of cold signaling is crucial for resilience. Dual-localization kinesins convey the cold signal from the plasma membrane to the nucleus, where this protein modulates cold-dependent gene expression. Using the grapevine homologue as paradigm, we investigated the functional context of VvDLK. We combined live cell and immunofluorescence imaging with pharmacological interference, physiological analysis, and protein-interaction assays upon stable expression in grapevine cells and tomato, or CRISPR-Cas–mediated inactivation of the tomato homologue. Overexpression of VvDLK was associated with earlier and more pronounced cold-induced elimination of microtubules in both tomato plants as heterologous and grapevine cells as homologous hosts, followed by reduced cold tolerance, evident from cellular, physiological, and molecular readouts. Conversely, inactivation of the tomato VvDLK homologue SlDLK improved cold tolerance. VvDLK interacted specifically with Vv14-3-3A, and a 14-3-3 inhibitor attenuated cold-induced microtubule loss. ... mehrOverexpression of Vv14-3-3A also increased cold susceptibility. In addition, VvDLK and Vv14-3-3A both interacted with VvCBF4, while VvDLK reduced VvCBF4 protein accumulation and altered its intranuclear distribution under cold stress. Together, these findings suggest that VvDLK is associated with cold-induced microtubule elimination and attenuates cold tolerance through a signaling module involving Vv14-3-3A and VvCBF4.