One of the most powerful techniques for attributing functions to genes in uni- and multicellular organisms is comprehensive analysis of mutant traits. In this study, systematic and quantitative analyses of mutant traits are achieved in the budding yeast Saccharomyces cerevisiae by investigating morphological phenotypes. Analysis of fluorescent microscopic images of triple-stained cells makes it possible to treat morphological variations as quantitative traits. Deletion of nearly half of the yeast genes not essential for growth affects these morphological traits. Similar morphological phenotypes are caused by deletions of functionally related genes, enabling a functional assignment of a locus to a specific cellular pathway. The high-dimensional phenotypic analysis of defined yeast mutant strains provides another step toward attributing gene function to all of the genes in the yeast genome.

The unicellular green alga Haematococcus pluvialis accumulates large amounts of the red ketocarotenoid astaxanthin to protect against environmental stresses. Haematococcus cells that accumulate astaxanthin in the central part (green-red cyst cells) respond rapidly to intense light by distributing astaxanthin diffusively to the peripheral part of the cell within 10 min after irradiation. This response is reversible: when astaxanthin-diffused cells were placed in the dark, astaxanthin was redistributed to the center of the cell. Although Haematococcus possesses several pigments other that astaxanthin, the subcellular distribution and content of each pigment remain unknown. Here, we analyzed the subcellular dynamics and localization of major pigments such as astaxanthin, β-carotene, lutein, and chlorophylls under light irradiation using time-lapse and label-free hyperspectral imaging analysis. Fluorescence microscopy and freeze-fracture transmission electron microscopy showed that, preceding/following exposure to light, astaxanthin colocalized with lipid droplets, which moved from the center to the periphery through pathways in a chloroplast. This study revealed that photoresponse dynamics differed between astaxanthin and other pigments (chlorophylls, lutein, and β-carotene), and that only astaxanthin freely migrates from the center to the periphery of the cell through a large, spherical, cytoplasm-encapsulating chloroplast as a lipid droplet. We consider this to be the Haematococcus light-protection mechanism.

A rise in resistance to current antifungals necessitates strategies to identify alternative sources of effective fungicides. We report the discovery of poacic acid, a potent antifungal compound found in lignocellulosic hydrolysates of grasses. Chemical genomics using Saccharomyces cerevisiae showed that loss of cell wall synthesis and maintenance genes conferred increased sensitivity to poacic acid. Morphological analysis revealed that cells treated with poacic acid behaved similarly to cells treated with other cell wall-targeting drugs and mutants with deletions in genes involved in processes related to cell wall biogenesis. Poacic acid causes rapid cell lysis and is synergistic with caspofungin and fluconazole. The cellular target was identified; poacic acid localized to the cell wall and inhibited β-1,3-glucan synthesis in vivo and in vitro, apparently by directly binding β-1,3-glucan. Through its activity on the glucan layer, poacic acid inhibits growth of the fungi Sclerotinia sclerotiorum and Alternaria solani as well as the oomycete Phytophthora sojae. A single application of poacic acid to leaves infected with the broad-range fungal pathogen S. sclerotiorum substantially reduced lesion development. The discovery of poacic acid as a natural antifungal agent targeting β-1,3-glucan highlights the potential side use of products generated in the processing of renewable biomass toward biofuels as a source of valuable bioactive compounds and further clarifies the nature and mechanism of fermentation inhibitors found in lignocellulosic hydrolysates. Significance The search for new antifungal compounds is struggling to keep pace with emerging fungicide resistance. Through chemoprospecting of an untapped reservoir of inhibitory compounds, lignocellulosic hydrolysates, we have identified a previously undescribed antifungal agent, poacic acid. Using both chemical genomics and morphological analysis together for the first time, to our knowledge, we identified the cellular target of poacic acid as β-1,3-glucan. Through its action on the glucan layer of fungal cell walls, poacic acid is a natural antifungal agent against economically significant fungi and oomycete plant pathogens. This work highlights the chemical diversity within lignocellulosic hydrolysates as a source of potentially valuable chemicals.

Functional genomics relies on two essential parameters: the sensitivity of phenotypic measures and the power to detect genomic perturbations that cause phenotypic variations. In model organisms, two types of perturbations are widely used. Artificial mutations can be introduced in virtually any gene and allow the systematic analysis of gene function via mutants fitness. Alternatively, natural genetic variations can be associated to particular phenotypes via genetic mapping. However, the access to genome manipulation and breeding provided by model organisms is sometimes counterbalanced by phenotyping limitations. Here we investigated the natural genetic diversity of Saccharomyces cerevisiae cellular morphology using a very sensitive high-throughput imaging platform. We quantified 501 morphological parameters in over 50,000 yeast cells from a cross between two wild-type divergent backgrounds. Extensive morphological differences were found between these backgrounds. The genetic architecture of the traits was complex, with evidence of both epistasis and transgressive segregation. We mapped quantitative trait loci (QTL) for 67 traits and discovered 364 correlations between traits segregation and inheritance of gene expression levels. We validated one QTL by the replacement of a single base in the genome. This study illustrates the natural diversity and complexity of cellular traits among natural yeast strains and provides an ideal framework for a genetical genomics dissection of multiple traits. Our results did not overlap with results previously obtained from systematic deletion strains, showing that both approaches are necessary for the functional exploration of genomes. A familiar face or a dog breed is easily recognized because morphology of individuals differs according to their genetic backgrounds. For single-cell organisms, morphology reduces to the shape and size of cellular features. Microbiologists noticed that the shape of S. cerevisiae cells (baker's yeast) differs from one strain to another, but these differences were usually described qualitatively. We used a high-throughput imaging platform to study the morphology of yeast cells when they divide. Cells were stained with three fluorescent dyes so that their periphery, their DNA, and their actin could be recognized, and their images were analysed by a specialized software program. Numerous morphological differences were found between two distant strains of S. cerevisiae. By crossing these two strains, we performed quantitative genetics: several loci controlling morphological variations were found on the genome, and correlations were made between gene expression and morphology changes. Using bioinformatics, we showed that the results obtained do not overlap with previous results obtained from yeast cells in which specific genes are deleted. The study, therefore, illustrates how mutagenesis and the use of natural genetic variations provide complementary knowledge.

Vanillin, generated by acid hydrolysis of lignocellulose, acts as a potent inhibitor of the growth of the yeast Saccharomyces cerevisiae. Here, we investigated the cellular processes affected by vanillin using high-content, image-based profiling. Among 4,718 non-essential yeast deletion mutants, the morphology of those defective in the large ribosomal subunit showed significant similarity to that of vanillin-treated cells. The defects in these mutants were clustered in three domains of the ribosome: the mRNA tunnel entrance, exit and backbone required for small subunit attachment. To confirm that vanillin inhibited ribosomal function, we assessed polysome and messenger ribonucleoprotein granule formation after treatment with vanillin. Analysis of polysome profiles showed disassembly of the polysomes in the presence of vanillin. Processing bodies and stress granules, which are composed of non-translating mRNAs and various proteins, were formed after treatment with vanillin. These results suggest that vanillin represses translation in yeast cells.

Droplet microfluidics has become a powerful tool in precision medicine, green biotechnology, and cell therapy for single-cell analysis and selection by virtue of its ability to effectively confine cells. However, there remains a fundamental trade-off between droplet volume and sorting throughput, limiting the advantages of droplet microfluidics to small droplets (<10 pl) that are incompatible with long-term maintenance and growth of most cells. We present a sequentially addressable dielectrophoretic array (SADA) sorter to overcome this problem. The SADA sorter uses an on-chip array of electrodes activated and deactivated in a sequence synchronized to the speed and position of a passing target droplet to deliver an accumulated dielectrophoretic force and gently pull it in the direction of sorting in a high-speed flow. We use it to demonstrate large-droplet sorting with ~20-fold higher throughputs than conventional techniques and apply it to long-term single-cell analysis of Saccharomyces cerevisiae based on their growth rate.