Jasmonic acid (JA), ethylene (ET) and salicylic acid (SA) are the three major phytohormones coordinating a plant's defense response to pathogenic attack. While JA and ET are assumed to primarily control the defense against necrotrophic pathogens, SA-induced defense responses target mainly biotrophic microbes, and can include drastic measures such as programmed cell death as part of the plant's hypersensitive response (HR). Fusarium oxysporum is a hemibiotrophic fungal pathogen of several plant species, including many important food crops, and the model plant species Arabidopsis thaliana. Colonization of the plant's root vascular tissue by the fungus eventually results in wilting and plant death. A general role for JA, ET and SA in combating infection and colonization of the plant by F. oxysporum has been demonstrated, but their distinct roles and modes of action have so far not been described. Here, using high resolution microscopy with fluorescent marker lines of A. thaliana roots infected with F. oxysporum we show that SA acts spatially separate from JA/ET, in a distinct set of root cells immediately neighboring the fungal colonization site. JA and ET, however, act together in a different, but also clearly defined set of cells, slightly removed from the colonization site. SA induces HR in the cells directly in contact with already colonized tissue, that presumably can no longer be rescued from infection, stopping the spread of colonization. In comparison, JA and ET act in a group of cells slightly removed from the HR site, initiating a defense response to actively resist the invader. These results show how the three phytohormones act together, but spatially and functionally separate from each other, to fight this hemibiotrophic pathogen. Such a high-resolution analysis to resolve the plant's immune response to pathogenic infection on an individual cell level and in intact tissue has so far been lacking.