Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Sun, 25 Nov 2018 17:26:26 Ustilago maydis phosphodiesterases play a role in the dimorphic switch and in pathogenicity Charu Agarwal, Kavita B. Aulakh, Kaly Edelen, Michael Cooper, R. Margaret Wallen, Seth Adams, David J. Schultz and Michael H. Perlin Correspondence Michael H. Perlin mhperl01@louisville.edu Received 13 June 2012 Accepted 28 February 2013 Department of Biology, Program on Disease Evolution, University of Louisville, Louisville, KY 40208, USA Components of the cAMP (cyclic AMP) signalling cascades are conserved from fungi to humans, and are particularly important for fungal dimorphism and pathogenicity. Previous work has described two phosphodiesterases, UmPde1 and UmPde2, in Ustilago maydis which show strong phosphodiesterase activity. We further characterized the biological function(s) of these phosphodiesterases in U. maydis. Specifically, we examined their possible role(s) in regulation of the cAMP-dependent protein kinase A (PKA) pathway and their roles in filamentous growth and pathogenicity. We found that UmPde1, which shares 35 % similarity with Cryptococcus neoformans Pde1, also displays functional homology with this enzyme. UmPde1 complements the capsule-formation defect of C. neoformans strains deleted for Pde1. In U. maydis, the cell morphology of the umpde1 deletion mutant resembled the multiple budding phenotypes seen with the ubc1 mutant, which lacks the regulatory subunit of PKA. Interestingly, on low-ammonium medium, umpde2 deletion strains showed a reduction in filamentation that was comparable to that of ubc1 deletion strains; however, umpde1 deletion strains showed normal filamentation on low- ammonium medium. Furthermore, both the ubc1 deletion strain in which the PKA pathway was constitutively active and the umpde1 deletion strains were significantly reduced in pathogenicity, while the umpde2 deletion strains showed a trend for reduced pathogenicity compared with wild- type strains. These data support a role for the phosphodiesterases UmPde1 and UmPde2 in regulating the U. maydis cAMP-dependent PKA pathway through modulation of cAMP levels, thus affecting dimorphic growth and pathogenicity. INTRODUCTION Signalling pathways are important for interactions between diverse organisms. These cascades mediate responses by communicating different environmental cues to the nuclei and thus function to change the transcriptome of the organism. The fact that several components of signalling cascades are conserved from fungi to humans, despite divergence over 800 million years ago (Lengeler et al., 2000), indicates a high degree of conservation in the signalling pathway components and reflects the fun- damental importance of signalling cascades. For fungal systems, mitogen activated protein kinase (MAPK) and cyclic AMP-dependent protein kinase A (cAMP-PKA) signalling cascades are known to regulate the dimorphic switch from budding to filamentous growth (Garcı´a-Pedrajas et al., 2008a). In several well-studied fungal examples, the highly conserved cAMP-PKA signal- ling pathway has been found to function in parallel with the MAPK pathway, and is important for pseudohyphal growth, virulence factor production, asexual and sexual spore production, mating, mycotoxin production, patho- genicity and many other processes (Hicks et al., 1997; Pan and Heitman, 1999; Hicks et al., 2005; Klosterman et al., 2007; Ramanujam & Naqvi, 2010). The effect of cyclic AMP (cAMP) in eukaryotes, including fungi, can be transmitted via action of the cAMP- dependent protein kinase A (PKA) signalling pathway. Consequently, levels of cAMP play an important role in regulation of PKA-mediated responses. In this regard, adenylate cyclase is responsible for the production of cAMP from ATP, while phosphodiesterases (PDEs) are responsible for hydrolysis of cAMP into 59-AMP. Thus, these two enzymes act in opposition to rapidly alter the level of cAMP pools and thus affect the duration of cAMP signalling (Conti & Beavo, 2007). Such regulation plays a role in a wide array of cellular and developmental processes. In yeasts, such as Saccharomyces cerevisiae and Schizosaccharomyces pombe, cAMP signalling regulates the Abbreviations: cAMP, cyclic AMP; cAMP-PKA, cyclic AMP-dependent protein kinase A; PDE, phosphodiesterase; p.i., post-infection; PKA, protein kinase A. Four supplementary figures and three supplementary tables are available with the online version of this paper. Microbiology (2013), 159, 857–868 DOI 10.1099/mic.0.061234-0 061234 G 2013 SGM Printed in Great Britain 857