A Septin from the Filamentous Fungus A. nidulans Induces Atypical Pseudohyphae in the Budding Yeast S. cerevisiae

PLOS ONE, Mar 2010

Background Septins, novel cytoskeletal proteins, form rings at the bases of emerging round buds in yeasts and at the bases of emerging elongated hyphal initials in filamentous fungi. Methodology/Principal Findings When introduced into the yeast Saccharomyces cerevisiae, the septin AspC from the filamentous fungus Aspergillus nidulans induced highly elongated atypical pseudohyphae and spore-producing structures similar to those of hyphal fungi. AspC induced atypical pseudohyphae when S. cerevisiae pseudohyphal or haploid invasive genes were deleted, but not when the CDC10 septin gene was deleted. AspC also induced atypical pseudohyphae when S. cerevisiae genes encoding Cdc12-interacting proteins Bem4, Cla4, Gic1 and Gic2 were deleted, but not when BNI1, a Cdc12-interacting formin gene, was deleted. AspC localized to bud and pseudohypha necks, while its S. cerevisiae ortholog, Cdc12, localized only to bud necks. Conclusions/Significance Our results suggest that AspC competes with Cdc12 for incorporation into the yeast septin scaffold and once there alters cell shape by altering interactions with the formin Bni1. That introduction of the A. nidulans septin AspC into S. cerevisiae induces a shift from formation of buds to formation of atypical pseudohyphae suggests that septins play an important role in the morphological plasticity of fungi.

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A Septin from the Filamentous Fungus A. nidulans Induces Atypical Pseudohyphae in the Budding Yeast S. cerevisiae

Momany M (2010) A Septin from the Filamentous Fungus A. nidulans Induces Atypical Pseudohyphae in the Budding Yeast S. cerevisiae. PLoS ONE 5(3): e9858. doi:10.1371/journal.pone.0009858 A Septin from the Filamentous Fungus A. nidulans Induces Atypical Pseudohyphae in the Budding Yeast S. cerevisiae Rebecca Lindsey 0 Youngsil Ha 0 Michelle Momany 0 Robert Alan Arkowitz, CNRS UMR6543, Universite de Nice, Sophia Antipolis, France 0 Department of Plant Biology, University of Georgia , Athens, Georgia , United States of America Background: Septins, novel cytoskeletal proteins, form rings at the bases of emerging round buds in yeasts and at the bases of emerging elongated hyphal initials in filamentous fungi. Methodology/Principal Findings: When introduced into the yeast Saccharomyces cerevisiae, the septin AspC from the filamentous fungus Aspergillus nidulans induced highly elongated atypical pseudohyphae and spore-producing structures similar to those of hyphal fungi. AspC induced atypical pseudohyphae when S. cerevisiae pseudohyphal or haploid invasive genes were deleted, but not when the CDC10 septin gene was deleted. AspC also induced atypical pseudohyphae when S. cerevisiae genes encoding Cdc12-interacting proteins Bem4, Cla4, Gic1 and Gic2 were deleted, but not when BNI1, a Cdc12interacting formin gene, was deleted. AspC localized to bud and pseudohypha necks, while its S. cerevisiae ortholog, Cdc12, localized only to bud necks. Conclusions/Significance: Our results suggest that AspC competes with Cdc12 for incorporation into the yeast septin scaffold and once there alters cell shape by altering interactions with the formin Bni1. That introduction of the A. nidulans septin AspC into S. cerevisiae induces a shift from formation of buds to formation of atypical pseudohyphae suggests that septins play an important role in the morphological plasticity of fungi. - Funding: This work was supported by NSF grant MCB0211787 to MM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Septins were first discovered in S. cerevisiae where they form a scaffold that organizes the bud site and are a component of the morphogenesis checkpoint that coordinates budding with mitosis [1,2,3]. Five of the seven S. cerevisiae septins (Cdc3, Cdc10, Cdc11, Cdc12 and Shs1) co-localize as a ring or collar to the neck region where the daughter bud emerges from the mother cell. At least 35 other proteins, including cell wall biosynthetic enzymes and cell cycle regulators, localize to the bud neck region in a septindependent manner [4,5]. Mutation of any of the core septins, CDC3, CDC10, CDC11, or CDC12, prevents formation of the septin ring, resulting in elongated buds and mitotic delay [2]. During sexual reproduction, the Spr3 and Spr28 septins localize with Cdc3, Cdc11 and Cdc12 at the leading edge of the prospore membrane as it encapsulates the nucleus [6,7,8]. Cdc12 occupies the central position of the core heteropolymeric septin complex in S. cerevisiae, binding to both Cdc11 and Cdc3 [9]. In the filamentous fungus Aspergillus nidulans four of the five septins (AspA, AspB, AspC and AspD) are orthologs of the S. cerevisiae core septins and localize as a ring or collar to the region where the hypha emerges from the conidium [10,11,12]. AspA is orthologous to S. cerevisiae Cdc11. AspB is orthologous to Cdc3. AspC is orthologous to Cdc12 and AspD is orthologous to Cdc10. Loss of AspC prevents the proper localization of AspA and visa versa consistent with participation in a heteropolymeric septin complex like that in S. cerevisiae [13]. Materials and Methods Strains and growth conditions Yeast strains and plasmids used in this study are listed in Tables 1, 2 and 3. Yeast strains were incubated in YPD (rich medium), SC (synthetic complete medium with amino acids omitted as necessary for plasmid selection), sporulation medium or SLAD (sigma strains) [14] at 30uC unless otherwise stated. Plasmids were constructed using standard methods [14,15]. A. nidulans AspC was amplified from cDNA using the Advantage High Fidelity 2 PCR kit (Clontech Laboratories, Inc, Mountain View, CA), cloned into PCR 2.1 TOPO (Invitrogen Corp., Carlsbad, CA) and sequenced. For expression in S. cerevisiae, aspC was cloned behind the inducible GAL1 promoter or the BY4741with pRL20 (PGAL1-aspC) and pJT2044 (CDC12-GFP) Sc295 with pRL16 (PGAL1-aspC, URA3) Sc295 with pRS316GU (empty vector) BY4741 with pRS316GU (empty vector) BY4741 with pRL16 (PGAL1-aspC, URA3) BY4741 with pRL19 (PADH1-aspC1, URA3) Sc295 with pRL16 (PGAL1-aspC, URA3) BY4741 with pHY33 (PGAL1-GFP-aspC) CDC12/cdc12D with pYH33 (PGAL1-GFP-aspC) CDC12/cdc12D with pYH35 (PADH1-GFP-aspC) a his3D1 leu2D0 met15D0 ura3D0 a his3D1 leu2D0 lys2D0 ura3D0 a ura3-52 his7 ade2 trp1-289 cdc3-1ts a ura3-52 his7 tyr1 ade2 lys2 cdc11-1ts a ura3-52 trp1-28 (...truncated)


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Rebecca Lindsey, Youngsil Ha, Michelle Momany. A Septin from the Filamentous Fungus A. nidulans Induces Atypical Pseudohyphae in the Budding Yeast S. cerevisiae, PLOS ONE, 2010, Volume 5, Issue 3, DOI: 10.1371/journal.pone.0009858