Large-Scale Analysis of Yeast Filamentous Growth by Systematic Gene Disruption and Overexpression

Abstract

Under certain conditions of nutrient stress, the budding yeast Saccharomyces cerevisiae initiates a striking developmental transition to a filamentous form of growth, resembling developmental transitions required for virulence in closely related pathogenic fungi. In yeast, filamentous growth involves known mitogen-activated protein kinase and protein kinase A signaling modules, but the full scope of this extensive filamentous response has not been delineated. Accordingly, we have undertaken the first systematic gene disruption and overexpression analysis of yeast filamentous growth. Standard laboratory strains of yeast are nonfilamentous; thus, we constructed a unique set of reagents in the filamentous ⌺1278b strain, encompassing 3627 integrated transposon insertion alleles and 2043 overexpression constructs. Collectively, we analyzed 4528 yeast genes with these reagents and identified 487 genes conferring mutant filamentous phenotypes upon transposon insertion and/or gene overexpression. Using a fluorescent protein reporter integrated at the MUC1 locus, we further assayed each filamentous growth mutant for aberrant protein levels of the key flocculence factor Muc1p. Our results indicate a variety of genes and pathways affecting filamentous growth. In total, this filamentous growth gene set represents a wealth of yeast biology, highlighting 84 genes of uncharacterized function and an underappreciated role for the mitochondrial retrograde signaling pathway as an inhibitor of filamentous growth. INTRODUCTIONIn its most familiar growth form, the baker's yeast Saccharomyces cerevisiae divides mitotically by budding, forming two independent and separate daughter cells from a single mother cell. In response to specific environmental cues, however, some strains of S. cerevisiae are capable of forming multicellular filaments-chains of cells that remain physically connected after cytokinesis (Gimeno et al., 1992;Kron, 1997;Madhani and Fink, 1998). In yeast, this form of filamentous growth is thought to constitute a foraging mechanism initiated under conditions of limited nutrient availability (Gimeno et al., 1992;Liu et al., 1993;Cullen and Sprague, 2000). Similar filamentous growth transitions are evident in many fungal species; in particular, many fungal pathogens transition between unicellular and filamentous growth forms, and, in fact, this transition is required for virulence in most of these organisms (Alspaugh et al., 1997;Lo et al., 1997b). For example, in the opportunistic human pathogen Candida albicans, environmental cues of temperature, pH, and serum source have been found to trigger a distinct morphogenetic program resulting in the transition from a cellular yeast form to a filamentous growth form (Liu et al., 1994; Singh et al., 1997). Furthermore, a mutant strain of C. albicans impaired in its ability to undergo filamentous growth is avirulent in a mouse model of disseminated candidiasis (Lo et al., 1997a). Thus, filamentous growth is relevant to our understanding of fungal pathogenesis, and the budding ye...

Systematic measurement of transcription factor-DNA interactions by targeted mass spectrometry identifies candidate gene regulatory proteins

“…Mot3 was a particularly attractive candidate for functional studies based on this analysis combined with previous studies that revealed Mot3 to be a repressor of both filamentation and invasive growth (20,33). Consistent with our prediction that Mot3 functions as a repressor of FLO11 expression, we found that overexpression of Mot3 represses FLO11 expression when cells are grown on low-nitrogen, SLAD medium ( Fig.…”

“…Mot3 is an attenuator of signaling responses (32) that negatively regulates filamentous and invasive growth (20,33). Our SRM evidence indicates that Mot3 binds segments 6 and 7 of the FLO11 promoter (Table 1 and Fig.…”

A Profile of Differentially Abundant Proteins at the Yeast Cell Periphery during Pseudohyphal Growth

“…The genotype of the haploid Y825 strain is MATa ura3-52 leu2⌬0; the genotype of the diploid Y825/6 strain is ura3-52/ura3-52 leu2⌬0/leu2⌬0 (30,31). Haploid deletion mutants were constructed in strain Y825 by homologous recombination using the PCR-based strategy of Baudin et al (47).…”

“…Filamentous growth signaling networks target a broad set of proteins, including a subset of proteins at the yeast cell periphery (31)(32)(33)(34)(35). In particular, proteins at the plasma membrane and cell wall contribute to the pseudohyphal growth response by mediating cell-cell and cell-substrate adhesion, nutrient * This work was supported, in whole or in part, by National Institutes of Health transport, and receptor-linked signaling events (1, 36 -38).…”

Budding off: bringing functional genomics toCandida albicans

“…Overexpression of candidate genes can therefore be used to complement studies of gene disruption [150]. A number of regulatable promoters have been developed to regulate gene expression, including the MET3 promoter [151], the MAL2 promoter [152] and tetracyclineregulated promoters (Tet-OFF and Tet-ON) [153,154].…”