Egg wash treatments
For the treatment of different B. nigra accessions (Fig. 1), egg wash was prepared following a recently published protocol (Caarls et al., 2023). In short, Pieris brassicae eggs were collected on filter paper pinned underneath a B. oleracea leaf in a cage containing twenty mated females. Egg clutches laid on the paper were cut out and submersed in 1 mL 2-(N-morpholino) methanesulfonic acid (MES) buffer per 400 eggs, overnight (16h) without disturbance. The solution (egg wash) was pipetted into a new tube the next morning. For this experiment, two 10 μl drops of egg wash were applied to each of the youngest fully developed leaves. Drops of an equivalent amount of MES buffer were applied as negative control.
For the treatment of the genetic mapping populations and to compare egg-induced HR to pathogen-induced HR, a protocol resulting in a more concentrated egg wash was used (Bassetti et al., 2022). In brief,P. brassicae egg clutches were collected from Brussel sprout leaves within 24 h after oviposition. Eggs were carefully removed with a stainless-steel lab spatula without breaking them and placed in an Eppendorf tube together with demineralised water in a ratio of ~1000 eggs per 1 ml of water. After an overnight incubation at room temperature, the liquid phase was retained and stored at -20 °C. Then, two 5 μl drops of egg wash were applied to each of the youngest fully developed leaves. Drops of an equivalent amount of demineralised water were applied as negative control.