Adenylate cyclase A amplification and functional diversification during Polyspondylium pallidum development

BACKGROUND: In Dictyostelium discoideum (Ddis), adenylate cyclase A (ACA) critically generates the cAMP oscillations that coordinate aggregation and morphogenesis. Unlike group 4 species like Ddis, other groups do not use extracellular cAMP to aggregate. However, deletion of cAMP receptors (cARs) or extracellular phosphodiesterase (PdsA) in Polyspondylium pallidum (Ppal, group 2) blocks fruiting body formation, suggesting that cAMP oscillations ancestrally control post-aggregative morphogenesis. In group 2, the acaA gene underwent several duplications. We deleted the three Ppal aca genes to identify roles for either gene and tested whether Ppal shows transient cAMP-induced cAMP accumulation, which underpins oscillatory cAMP signalling. RESULTS: In contrast to Ddis, pre-aggregative Ppal cells did not produce a pulse of cAMP upon stimulation with the cAR agonist 2′H-cAMP, but acquired this ability after aggregation. Deletion of Ppal aca1, aca2 and aca3 yielded different phenotypes. aca1ˉ cells showed relatively thin stalks, aca2ˉ showed delayed secondary sorogen formation and aca3ˉ formed less aggregation centers. The aca1ˉaca2ˉ and aca1ˉaca3ˉ mutants combined individual defects, while aca2ˉaca3ˉ and aca1ˉaca3ˉaca2ˉ additionally showed > 24 h delay in aggregation, with only few aggregates with fragmenting streams being formed. The fragments developed into small fruiting bodies with stalk and spore cells. Aggregation was restored in aca2ˉaca3ˉ and aca1ˉaca3ˉaca2ˉ by 2.5 mM 8Br-cAMP, a membrane-permeant activator of cAMP-dependent protein kinase (PKA). Like Ddis, Ppal sorogens also express the adenylate cyclases ACR and ACG. We found that prior to aggregation, Ddis acaˉ/ACG cells produced a pulse of cAMP upon stimulation with 2′H-cAMP, indicating that cAMP oscillations may not be dependent on ACA alone. CONCLUSIONS: The three Ppal replicates of acaA perform different roles in stalk morphogenesis, secondary branch formation and aggregation, but act together to enable development by activating PKA. While even an aca1ˉaca3ˉaca2ˉ mutant still forms (some) fruiting bodies, suggesting little need for ACA-induced cAMP oscillations in this process, we found that ACG also mediated transient cAMP-induced cAMP accumulation. It, therefore, remains likely that post-aggregative Ppal morphogenesis is organized by cAMP oscillations, favouring a previously proposed model, where cAR-regulated cAMP hydrolysis rather than its synthesis dominates oscillatory behaviour. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13227-022-00203-7.