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.