Pooled Segregant Sequencing Reveals Genetic Determinants of Yeast Pseudohyphal Growth

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.

假菌丝生长（pseudohyphal growth）响应是一种剧烈的形态转变过程，同时被认为是一种觅食机制：酵母细胞在此过程中会形成具有侵袭性且可在表面扩散的多细胞丝状体。假菌丝生长已被广泛研究，作为保守信号通路的模型系统，用于研究病原真菌中的应激响应、细胞形态发生以及真菌毒力调控机制。假菌丝生长的遗传调控基础极为复杂，丝状体形成过程至少需要500个基因参与；因此，假菌丝生长属于复杂性状，而连锁分析是解析复杂表型遗传基础的经典手段。本研究通过将两株酿酒酵母（Saccharomyces cerevisiae）丝状菌株（Σ1278b与SK1）分别与一株源自S288C的非丝状菌株进行杂交，开展连锁分析。随后，我们对减数分裂子代的丝状体形成能力进行检测，并通过全基因组测序对混合分离体的等位基因连锁关系进行定位。本次分析在一组基因中鉴定出了连锁信号，其中包括负调控因子SFL1：我们发现在具有侵袭性的SK1背景中，该基因携带一个提前终止密码子。极性基因PEA2的S288C等位基因编码亮氨酸409（而非甲硫氨酸），该等位基因与非侵袭表型相关联。在Σ1278b菌株中，pea2-M409L突变会导致侵袭性丝状体形成与延伸能力下降，Kss1p丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）通路报告基因的活性降低，单极出芽现象减少，且极性体蛋白Spa2p的结合能力减弱。SK1与S288C菌株在线粒体内膜蛋白Mdm32p的182和262位氨基酸存在变异，该变异会影响侵袭性生长以及线粒体网络结构。综上，本研究鉴定出了假菌丝生长的新决定因子，同时揭示了在特定基因组中，蛋白质复合物与细胞器结构的共同进化如何调控复杂表型的形成。