Quantitative live-cell imaging of auxin and cytokinin signalling in developing feeding sites of <i>Heterodera schachtii</i>

Cyst nematodes are sedentary endoparasites that establish unique feeding structures (syncytia) inside their host roots. Crosstalk between auxin and cytokinin signaling is thought to play a crucial role in syncytia formation. However, the relative differences in space and time between auxin and cytokinin signaling during infection are largely unknown. Here, we used an auxin‒cytokinin double-reporter line to study the spatio-temporal dynamics of auxin and cytokinin signaling during the initiation and expansion of syncytia in <i>Arabidopsis thaliana</i> roots induced by the beet cyst nematode <i>Heterodera schachtii</i>. Continuous live-cell imaging of single juveniles showed that upon infection, cytokinin signaling increases rapidly, precedes auxin signaling, and occurs in nearly all cell types, also outside the syncytial area. Auxin signaling is restricted to the syncytial area, starts after a lag phase, and is induced after the first contours of the syncytial area have become visible. Within the syncytial area, the signaling domains overlap, and the elevated auxin response in nuclei is associated with a high cytokinin response. Quantitative analyses showed that cytokinin signaling is relatively high in cyst nematode infection sites when compared to signaling maxima in root tips, while auxin signaling is relatively weak when compared to signaling maxima in root tips.