Strains used in this study.

<div><p>The eukaryotic serine/threonine protein phosphatase PP2A is a heterotrimeric enzyme composed of a scaffold A subunit, a regulatory B subunit, and a catalytic C subunit. Of the four known B subunits, the B”‘ subunit (known as striatin) interacts with the multi-protein striatin-interacting phosphatase and kinase (STRIPAK) complex. Orthologs of STRIPAK components were identified in <i>Cryptococcus neoformans</i>, namely PP2AA/Tpd3, PP2AC/Pph22, PP2AB/Far8, STRIP/Far11, SLMAP/Far9, and Mob3. Structural modeling, protein domain analysis, and detected protein-protein interactions suggest <i>C</i>. <i>neoformans</i> STRIPAK is assembled similarly to the human and fungal orthologs. Here, STRIPAK components Pph22, Far8, and Mob3 were functionally characterized. Whole-genome sequencing revealed that mutations in STRIPAK complex subunits lead to increased segmental and chromosomal aneuploidy, suggesting STRIPAK functions in maintaining genome stability. We demonstrate that <i>PPH22</i> is a haploinsufficient gene: heterozygous <i>PPH22/pph22</i>Δ mutant diploid strains exhibit defects in hyphal growth and sporulation and have a significant fitness disadvantage when grown in competition against a wild-type diploid. Deletion mutants <i>pph22</i>Δ, <i>far8</i>Δ, and <i>mob3</i>Δ exhibit defects in mating and sexual differentiation, including impaired hyphae, basidia, and basidiospore production. Loss of either <i>PPH22</i> or <i>FAR8</i> in a haploid background leads to growth defects at 30°C, severely reduced growth at elevated temperature, abnormal cell morphology, and impaired virulence. Additionally, <i>pph22</i>Δ strains frequently accumulate suppressor mutations that result in overexpression of another putative PP2A catalytic subunit, <i>PPG1</i>. The <i>pph22</i>Δ and <i>far8</i>Δ mutants are also unable to grow in the presence of the calcineurin inhibitors cyclosporine A or FK506, and thus these mutations are synthetically lethal with loss of calcineurin activity. Conversely, <i>mob3</i>Δ mutants display increased thermotolerance, capsule production, and melanization, and are hypervirulent in a murine infection model. Taken together, these findings reveal that the <i>C</i>. <i>neoformans</i> STRIPAK complex plays an important role in genome stability, vegetative growth, sexual development, and virulence in this prominent human fungal pathogen.</p></div>