AbstractFusarium oxysporum, a global soil-borne pathogen, causes severe disease in various cultivated plants. The mechanism underlying infection and resistance remains largely elusive.Vernicia fordii, known as the tung tree, suffers from disease caused byF. oxysporumf. sp.fordiis(Fof-1), while its sister speciesV. montanadisplays high resistance toFof-1. To investigate the process of infection and resistance ability, we demonstrated thatFof-1 can penetrate the epidermis of root hairs and then centripetally invade the cortex and phloem in both species. Furthermore,Fof-1 spread upwards through the root xylem in susceptibleV. fordiitrees, whereas it failed to infect the root xylem in resistantV. montanatrees. We found that D6 PROTEIN KINASE LIKE 2 (VmD6PKL2) was specifically expressed in the lateral root xylem and was induced afterFof-1 infection in resistant trees. Transgenic analysis inArabidopsisand tomato revealed thatVmD6PKL2significantly enhanced resistance in both species, whereas thed6pkl2mutant displayed reduced resistance againstFof-1. Additionally, VmD6PKL2 was identified to interact directly with synaptotagmin (VmSYT3), which is specifically expressed in the root xylem and mediates the negative regulation responding toFof-1. Our data suggested thatVmD6PKL2could act as a resistance gene againstFof-1 through suppression ofVmSYT3-mediated negative regulation in the lateral root xylem of the resistant species. These findings provide novel insight intoFusariumwilt resistance in plants.