Fig. 4.

LDs are important for spore germination and spore wall integrity. (A) Frequency of tetrad formation and spore germination in the different strains. Tetrad formation frequency=(number of asci with four spores/number of total zygotes)×100%. At least 600 tetrads were scored for each strain. After 3-5 days of sporulation, the rate of spore germination of each strain was determined by assessing spore colony formation through tetrad analysis (42 tetrads were dissected for each strain). Spores that failed to form colonies were further confirmed for germination using a dissecting microscope. Germination frequency=(number of germinated spores/number of total spores)×100%. A lys1⁺-integrating plasmid carrying dga1⁺ or plh1⁺ was used to restore the germination efficiency of the dga1Δplh1Δ mutant. (B) The dga1Δplh1Δ mutant possessed few LDs. The fluorescent dye BODIPY was used for LD labeling. The white dashed line outlines the sporulating cell Scale bar: 5 µm. (C) Examples of spore colony formation in the dga1Δplh1Δ mutant. (D) Representative morphological changes of spore germination over time in the wild-type or the dga1Δplh1Δ spore. Scale bar: 6 µm. (E) The germination defect of the dga1Δplh1Δ spores was time-dependent. The cells were subjected to sporulation on an ME plate for 2, 4, 8, or 16 days. At each time point, spore germination was assayed by tetrad dissection on the YES plate (three independent experiments per strain; 14 tetrads were dissected per experiment). Germination frequency=(number of germinated spores/number of total spores)×100%. The graph and the error bar represent mean and standard deviation, respectively. (F) The spore wall was improperly assembled in the dga1Δplh1Δ mutant. Isp3-GFP was used to visualize the outermost layer of the spore wall. Isp3-GFP fluorescence signals were evenly distributed on the surface of the wild-type ascospores, whereas Isp3-GFP exhibited aggregate formation and uneven decoration of the ascospores of the dga1Δplh1Δ mutant. Scale bar: 5 µm.