Bacterial strain used

E.coli (OD600:100)

Setting up the primary and secondary cultures

Take out the bacterial glycerol stock from -80^oC
Dip a sterilized inoculating loop in the stock, streak the culture on a freshly prepared LB-Agar plate, and incubate the plate at 37^oC overnight.
Once there is visible growth (single colonies) the next day, take out one single colony, inoculate it in the required amount of liquid LB (Luria-Bertani) media (primary culture), and incubate it at 37^oC in a shaking incubator till the time the optical density at 600 nm (OD600) reaches between 0.4–0.6.
Once the primary culture reaches OD600 between 0.4–0.6, take out 100-200ml of the culture, re-suspend it in the required amount of liquid LB media, and incubate at 37^oC in a shaking incubator (secondary culture).

Note: For each set of experiments, the secondary culture was freshly prepared from the primary culture.

Infection experiments

For larval infections, once the secondary bacterial culture reached OD600 around 0.5, the cultures were concentrated by centrifugation.
The pellet formed post centrifugation was re-suspended in phosphate-buffered saline (1X PBS) to the appropriate OD600 value (mentioned above).
For all the analyses, synchronized third instar larval batches were used. The larvae were washed thrice with sterile ddH2O and pricked at the postero-lateral part using a fine insect pin (Austerlitz insect pins)/tungsten pin: Fine Scientific Tools) dipped in bacterial suspension.

For proper holding of the pin, it was mounted on a pin holder (Fine Scientific tool: Cat. No. 91606-07).

Note: The larvae should be pricked gently; the pin should penetrate the cuticle with minimal damage to internal tissues/organs. Before dissection, check out the melanization spots to ensure that the larvae have been infected. The spots that appear at the site of pricking/injury correspond to the activation of the melanization cascade.

Mock injections were done using 1X PBS dipped pins.
Once infected, larval batches were transferred to food plates and reared at 25^oC till dissection.
All observations were made 4 hours post-infection for the experiments related to this study (https://doi.org/10.7554/eLife.67158).