Ulwcd 0

Dictyostelium discoideum cells were washed with SB and deposited on SB agar (1%) plates at a cell density 5 × 10⁵ cells/cm². Subsequently, plates were incubated in a dark, moist chamber at 22°C. The morphogenetic stages were imaged under the stereomicroscope (Olympus ULWCD 0.30) at time points mentioned (Supplementary Figure 4). Dictyostelium discoideum development on SB agar was monitored in three independent experiments.

Slug pattern analyses were performed as described in Parkinson et al. (2009 (link)). For prestalk reporter (ecmAO-RFP) examination, D. discoideum amoebae carrying the ecmAO-RFP plasmid were mixed with D. discoideum amoebae at 20:80 ratio and seeded on a SB agar (1%) plate at a cell density 5 × 10⁵ cells/cm². For prespore reporter (pSA-RFP) examination, D. discoideum amoebae (20%) were mixed with D. discoideum amoebae marked with pSA-RFP (80%) and deposited on SB agar plates. The plates were incubated in a dark, moist chamber at 22°C until the migrating slugs were formed and were imaged under the stereomicroscope (Olympus ULWCD 0.30) (Supplementary Figure 4). Two independent experiments were performed in duplicates and at least ten slugs were imaged per plate.

Dictyostelium discoideum cells were harvested from Petri dishes, 2.5 × 10⁷ cells were sedimented, and the pellets were resuspended in 50 μl SB. This high density cell suspension was deposited on SB agar (1%) plates and incubated in a dark, moist chamber with an unidirectional light source until migrating slugs were formed (~18 h). Slug infection experiments were performed following Chen et al. (2007 (link)) with some modifications. The slugs were injured with a sterile needle (23G × 1^1/4, B|BRAUN Injekt F) and a dense bacterial suspension, which was prepared from an overnight culture, was layered on the injured slugs. K. pneumoniae expressing GFP reporter (KpGFP) and E. coli expressing DsRed reporter were used for infecting the injured slugs (see Supplementary Table 1). The slugs infected with Ec DsRed were imaged under the stereo microscope (Olympus ULWCD 0.30) 8 h post infection and those slugs infected with KpGFP were imaged 20 h and 24 h post infection. KpGFP was grown with ampicillin (1 mg/ml) for 12 h at 37°C. Ec DsRed was grown with isopropyl β-D-1-thiogalactopyranoside (IPTG, 100 mM) and kanamycin (150 μg/ml). Three independent experiments were performed and at least ten slugs were imaged per experiment for each strain [Ax2, aplD⁻, and aplD⁻(+)].

Dictyostelium discoideum cells were allowed to form slugs on SB agar (1%) plates containing ethidium bromide (EtBr, 3 μg/ml) as described in Chen et al. (2007 (link)). After 3 h, Sentinel cells present in the migrating slugs were visualized under the stereomicroscope (Olympus ULWCD 0.30). Three independent experiments were performed and at least ten slugs were imaged per experiment for each strain (Ax2 and aplD⁻).