Abstract
The pseudohyphal growth response is a dramatic morphological transition and presumed foraging mechanism wherein yeast cells form invasive and surface-spread multicellular filaments. Pseudohyphal growth has been studied extensively as a model of conserved signaling pathways controlling stress responses, cell morphogenesis, and fungal virulence in pathogenic fungi. The genetic contribution to pseudohyphal growth is extensive, with at least 500 genes required for filamentation; as such, pseudohyphal growth is a complex trait, and linkage analysis is a classical means to dissect the genetic basis of a complex phenotype. Here, we implemented linkage analysis by crossing each of two filamentous strains of Saccharomyces cerevisiae (Σ1278b and SK1) with an S288C-derived non-filamentous strain. We then assayed meiotic progeny for filamentation and mapped allelic linkage in pooled segregants by whole-genome sequencing. This analysis identified linkage in a cohort of genes, including the negative regulator SFL1, which we find contains a premature stop codon in the invasive SK1 background. The S288C allele of the polarity gene PEA2, encoding Leu409 rather than Met, is linked with non-invasion. In Σ1278b, the pea2-M409L mutation results in decreased invasive filamentation and elongation, diminished activity of a Kss1p MAPK pathway reporter, decreased unipolar budding, and diminished binding of the polarisome protein Spa2p. Variation between SK1 and S288C in the mitochondrial inner membrane protein Mdm32p at residues 182 and 262 impacts invasive growth and mitochondrial network structure. Collectively, this work identifies new determinants of pseudohyphal growth, while highlighting the coevolution of protein complexes and organelle structures within a given genome in specifying complex phenotypes.
Highlights
The budding yeast Saccharomyces cerevisiae undergoes a pronounced growth transition in response to nitrogen limitation or glucose limitation, forming multicellular pseudohyphal filaments that can spread outward from a colony and/or invade the surface of a solid growth substrate [1,2]
We selected as parents the non-filamentous S288C-derived strain BY4741 and the filamentation-competent strains S1278b and SK1 [34,35]
The linkage analysis presented here identifies a broad gene set contributing to pseudohyphal growth (Fig. 6)
Summary
The budding yeast Saccharomyces cerevisiae undergoes a pronounced growth transition in response to nitrogen limitation or glucose limitation, forming multicellular pseudohyphal filaments that can spread outward from a colony and/or invade the surface of a solid growth substrate [1,2]. The molecular basis of yeast pseudohyphal growth has been studied extensively as a model of conserved signaling pathways controlling cell morphogenesis and polarity. Classic studies of pseudohyphal growth in S. cerevisiae have resulted most prominently in the identification of core pseudohyphal growth signaling modules encompassing the Kss1p mitogen-activated protein kinase (MAPK) cascade, the cAMP-dependent protein kinase A (PKA) pathway, and the AMP-activated protein kinase ortholog Snf1p [14,15,16,17,18,19,20]. Kss1p phosphorylates the Ste12p transcription factor, resulting in dissociation of the negative regulatory Dig1p and Dig2p interactors and binding of a Ste12pTec1p heterodimer to target promoters, such as the FLO11 promoter [21,22,23]. In response to glucose limitation, FLO11 transcription is regulated by Snf1p; the Snf1p-Gal83p isoform promotes cell adhesion during invasive filamentation by antagonizing Nrg1p- and Nrg2pmediated repression of FLO11 [19,27]
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