Experimental validation of top ranked candidate genes.

Co-cultures of D. discoideum (D.d.) and bacteria were generated by serial dilutions of axenically grown D. discoideum amoebae with a large excess of K. pneumoniae bacteria such that the number of amoebae plated in each spot was between 10 and 10⁴ as indicated above each column. The relevant genotypes of the amoebae strains are indicated on the left of each row. The co-cultures were plated on SM agar plates and incubated in a humid chamber at 22°C. Images were taken at 2 and 3 days after plating to show the progression of amoebae growth in time. The larger white opaque spots are lawns of the K. pneumoniae bacteria. Growth of the D. discoideum amoebae results in the formation of plaques within the opaque spots in cases of low amoebae cell density or clearing of much or all of the opaque spots in cases of high amoebae cell density. Upon complete clearing of the bacteria, the amoebae starve and begin to develop, producing white protruding multicellular structures within the lawn (e.g. AX4, 10⁴ cells, day 3), which have no significance in this assay. Growth of the Collage-predicted knockout strains was compared to the wild type (AX4, top row) and to the most severe mutant available (tirA–, bottom row). Each experiment was performed in duplicate. Representative images of three independent experiments are shown.