Two Rac paralogs regulate polarized growth in the human fungal pathogen Cryptococcus neoformans q Elizabeth Ripley Ballou a,b , Kyla Selvig a,b , Jessica L. Narloch a , Connie B. Nichols a , J. Andrew Alspaugh a,b,⇑ a Department of Medicine, Duke University School of Medicine, Durham, NC 27710, USA b Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC 27710, USA article info Article history: Received 28 March 2013 Accepted 20 May 2013 Available online xxxx Keywords: Cryptococcus neoformans Rac GTPase Paralogs Polarization Hyphal growth ROS abstract A genome wide analysis of the human fungal pathogen Cryptococcus neoformans var. grubii has revealed a number of duplications of highly conserved genes involved in morphogenesis. Previously, we reported that duplicate Cdc42 paralogs provide C. neoformans with niche-specific responses to environmental stresses: Cdc42 is required for thermotolerance, while Cdc420 supports the formation of titan cells. The related Rho-GTPase Rac1 has been shown in C. neoformans var. neoformans to play a major role in fil- amentation and to share Cdc42/Cdc420 binding partners. Here we report the characterization of a second Rac paralog in C. neoformans, Rac2, and describe its overlapping function with the previously described CnRac, Rac1. Further, we demonstrate that the Rac paralogs play a primary role in polarized growth via the organization of reactive oxygen species and play only a minor role in the organization of actin. Finally, we provide preliminary evidence that pharmacological inhibitors of Rac activity and actin stabil- ity have synergistic activity. Ó 2013 The Authors. Published by Elsevier Inc. All rights reserved. 1. Introduction Gene and genome duplications offer potential benefits to micro- organisms by facilitating adaptation to a variety of environments. In addition to providing protection against deleterious mutations, duplicated genes provide genetic plasticity through neofunctional- ization, subfunctionalization, and gene conservation (Force et al., 1999; Hahn, 2009; Ohno, 1970). In the case of Saccharomyces cere- visiae, an ancient whole genome duplication may have allowed this fungal lineage to acquire the process of anaerobic fermentation (Wolfe and Shields, 1997). Experimental models of gene evolution in Salmonella enterica demonstrate similar examples of gene ampli- fication and divergence in the development of metabolic pathways (Näsvall et al., 2012). Beyond simple gene duplication events, genetic redundancy can also be provided by related gene families. For example, the related Rho-like GTPases Cdc42 and Rac serve distinct primary functions in divergent fungal species such as Penicillium marneffei, Ustilago maydis, and Aspergillus nidulans (Bassilana and Arkowitz, 2006; Boyce et al., 2001, 2003; Harris, 2011; Mahlert et al., 2006; Virag et al., 2007). However, in each of these species, deletion of both of these genes is synthetically lethal, demonstrating their potential for functional overlap (Boyce et al., 2005; Mahlert et al., 2006; Virag et al., 2007). In Cryptococcus neoformans, several duplicated gene pairs con- trol cellular processes required for pathogenesis and develop- ment. These gene duplication events appear to have arisen in the absence of a whole genome duplication event (Loftus et al., 2005). However, these duplications span multiple pathways and cellular processes (Ballou et al., 2010; Missal et al., 2005; Pukkila-Worley et al., 2005; Salas et al., 1996; Wang et al., 2002). For example, the cyclophillin-encoding genes CPA1 and CPA2, major and minor paralogs respectively, play shared and distinct roles in cell growth, mating, and virulence, in addition to their conserved roles in the detoxification of cyclosporine A (Wang et al., 2001). Similarly, duplicated Ras proteins are in- volved in mating, polarity, and thermotolerance (Alspaugh et al., 2000; Waugh et al., 2002). RAS1 appears to provide the majority of the inducible Ras activity required for survival within the infected host. However, the simultaneous deletion of both the RAS1 and the RAS2 genes results in a dramatic cell growth defect under otherwise permissive incubation conditions. In addition to functional redundancy, duplicated genes in C. neoformans have undergone subfunctionalization. The highly con- served Rho-GTPase encoded by CnCDC42 is required for growth at 37 °C and for virulence (Ballou et al., 2010). A paralogous gene, 1087-1845/$ - see front matter Ó 2013 The Authors. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.fgb.2013.05.006 q This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike License, which permits non- commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. ⇑ Corresponding author. Address: 1543 Hospital South, Box 3355 DUMC, Durham, NC 27710, USA. E-mail address: andrew.alspaugh@duke.edu (J.A. Alspaugh). Fungal Genetics and Biology xxx (2013) xxx–xxx Contents lists available at SciVerse ScienceDirect Fungal Genetics and Biology journal homepage: www.elsevier.com/locate/yfgbi Please cite this article in press as: Ballou, E.R., et al. Two Rac paralogs regulate polarized growth in the human fungal pathogen Cryptococcus neoformans. Fungal Genet. Biol. (2013), http://dx.doi.org/10.1016/j.fgb.2013.05.006