Complex Haploinsufficiency-Based Genetic Analysis of the NDR/Lats Kinase Cbk1 Provides Insight into Its Multiple Functions in Candida albicans

Although the analysis of genetic interactions and networks is a powerful approach to understanding biology, it has not been applied widely to the pathogenic yeast Candida albicans . Here, we describe the use of both screening and directed genetic interaction studies based on complex haploinsufficiency to probe the function of the R egulation of A ce2 and M orphogenesis (RAM) pathway in C. albicans . A library of 5200 Tn7 -mutagenized derivatives of a parental strain heterozygous at CBK1 , the key kinase in the RAM pathway, was screened for alterations in serum-induced filamentation. Following confirmation of phenotypes and identification of insertion sites by sequencing, a set of 36 unique double heterozygous strains showing complex haploinsufficiency was obtained. In addition to a large set of genes regulated by the RAM transcription factor Ace2, genes related to cell wall biosynthesis, cell cycle, polarity, oxidative stress, and nitrogen utilization were identified. Follow-up analysis led to the first demonstration that the RAM pathway is required for oxidative stress tolerance in a manner related to the two-component-regulated kinase Chk1 and revealed a potential direct connection between the RAM pathway and the essential Mps1 spindle pole-related kinase. In addition, genetic interactions with CDC42 -related genes MSB1 , a putative scaffold protein, and RGD3 , a putative Rho GTPase-activating protein (GAP) were identified. We also provide evidence that Rgd3 is a GAP for Cdc42 and show that its localization and phosphorylation are dependent on Cbk1.