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1. Deletion of the Candida albicans TLO gene family using CRISPR-Cas9 mutagenesis allows characterisation of functional differences in α-, β- and γ- TLO gene function

   https://doi.org/10.1371/journal.pgen.1011082  

   Fletcher, Jessica; O’Connor-Moneley, James; Frawley, Dean; Flanagan, Peter R.; Alaalm, Leenah; Menendez-Manjon, Pilar; Estevez, Samuel Vega; Hendricks, Shane; Woodruff, Andrew L.; Buscaino, Alessia; et al (December 2023, PLOS Genetics)

   Forche, Anja (Ed.)

   TheCandida albicansgenome contains between ten and fifteen distinctTLOgenes that all encode a Med2 subunit of Mediator. In order to investigate the biological role of Med2/Tlo inC.albicanswe deleted all fourteenTLOgenes using CRISPR-Cas9 mutagenesis. ChIP-seq analysis showed that RNAP II localized to 55% fewer genes in thetloΔ mutant strain compared to the parent, while RNA-seq analysis showed that thetloΔ mutant exhibited differential expression of genes required for carbohydrate metabolism, stress responses, white-opaque switching and filamentous growth. Consequently, thetloΔ mutant grows poorly in glucose- and galactose-containing media, is unable to grow as true hyphae, is more sensitive to oxidative stress and is less virulent in the wax worm infection model. Reintegration of genes representative of the α-, β- and γ-TLOclades resulted in the complementation of the mutant phenotypes, but to different degrees.TLOα1could restore phenotypes and gene expression patterns similar to wild-type and was the strongest activator of glycolytic and Tye7-regulated gene expression. In contrast, the two γ-TLOgenes examined (i.e.,TLOγ5 and TLOγ11) had a far lower impact on complementing phenotypic and transcriptomic changes. Uniquely, expression ofTLOβ2in thetloΔmutant stimulated filamentous growth in YEPD medium and this phenotype was enhanced when Tloβ2 expression was increased to levels far in excess of Med3. In contrast, expression of reintegratedTLOgenes in atloΔ/med3Δdouble mutant background failed to restore any of the phenotypes tested, suggesting that complementation of these Tlo-regulated processes requires a functional Mediator tail module. Together, these data confirm the importance of Med2/Tlo in a wide range ofC.albicanscellular activities and demonstrate functional diversity within the gene family which may contribute to the success of this yeast as a coloniser and pathogen of humans. 

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2. Virulence phenotypes result from interactions between pathogen ploidy and genetic background

   https://doi.org/10.1002/ece3.6619  

   Feistel, Dorian J.; Elmostafa, Rema; Hickman, Meleah A. (August 2020, Ecology and Evolution)

   Abstract Studying fungal virulence is often challenging and frequently depends on many contexts, including host immune status and pathogen genetic background. However, the role of ploidy has often been overlooked when studying virulence in eukaryotic pathogens. Since fungal pathogens, including the human opportunistic pathogenCandida albicans, can display extensive ploidy variation, assessing how ploidy impacts virulence has important clinical relevance. As an opportunistic pathogen,C. albicanscauses nonlethal, superficial infections in healthy individuals, but life‐threatening bloodstream infections in individuals with compromised immune function. Here, we determined how both ploidy and genetic background ofC. albicansimpacts virulence phenotypes in healthy and immunocompromised nematode hosts by characterizing virulence phenotypes in four near‐isogenic diploid and tetraploid pairs of strains, which included both laboratory and clinical genetic backgrounds. We found thatC. albicansinfections decreased host survival and negatively impacted host reproduction, and we leveraged these two measures to survey both lethal and nonlethal virulence phenotypes across the multipleC. albicansstrains. In this study, we found that regardless of pathogen ploidy or genetic background, immunocompromised hosts were susceptible to fungal infection compared to healthy hosts. Furthermore, for each host context, we found a significant interaction betweenC. albicansgenetic background and ploidy on virulence phenotypes, but no global differences between diploid and tetraploid pathogens were observed. 

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3. Genetic variation in aneuploidy prevalence and tolerance across Saccharomyces cerevisiae lineages

   https://doi.org/10.1093/genetics/iyab015  

   Scopel, Eduardo F; Hose, James; Bensasson, Douda; Gasch, Audrey P (April 2021, Genetics)

   Hahn, M (Ed.)

   Abstract Individuals carrying an aberrant number of chromosomes can vary widely in their expression of aneuploidy phenotypes. A major unanswered question is the degree to which an individual’s genetic makeup influences its tolerance of karyotypic imbalance. Here we investigated within-species variation in aneuploidy prevalence and tolerance, using Saccharomyces cerevisiae as a model for eukaryotic biology. We analyzed genotypic and phenotypic variation recently published for over 1,000 S. cerevisiae strains spanning dozens of genetically defined clades and ecological associations. Our results show that the prevalence of chromosome gain and loss varies by clade and can be better explained by differences in genetic background than ecology. The relationships between lineages with high aneuploidy frequencies suggest that increased aneuploidy prevalence emerged multiple times in S. cerevisiae evolution. Separate from aneuploidy prevalence, analyzing growth phenotypes revealed that some genetic backgrounds—such as the European Wine lineage—show fitness costs in aneuploids compared to euploids, whereas other clades with high aneuploidy frequencies show little evidence of major deleterious effects. Our analysis confirms that chromosome gain can produce phenotypic benefits, which could influence evolutionary trajectories. These results have important implications for understanding genetic variation in aneuploidy prevalence in health, disease, and evolution. 

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4. Host-Induced Genome Instability Rapidly Generates Phenotypic Variation across Candida albicans Strains and Ploidy States

   https://doi.org/10.1128/mSphere.00433-20  

   Smith, Amanda C.; Hickman, Meleah A. (June 2020, mSphere)

   Mitchell, Aaron P. (Ed.)

   ABSTRACT Candida albicans is an opportunistic fungal pathogen of humans that is typically diploid yet has a highly labile genome tolerant of large-scale perturbations including chromosomal aneuploidy and loss-of-heterozygosity events. The ability to rapidly generate genetic variation is crucial for C. albicans to adapt to changing or stressful environments, like those encountered in the host. Genetic variation occurs via stress-induced mutagenesis or can be generated through its parasexual cycle, in which tetraploids arise via diploid mating or stress-induced mitotic defects and undergo nonmeiotic ploidy reduction. However, it remains largely unknown how genetic background contributes to C. albicans genome instability in vitro or in the host environment. Here, we tested how genetic background, ploidy, and the host environment impacts C. albicans genome stability. We found that host association induced both loss-of-heterozygosity events and genome size changes, regardless of genetic background or ploidy. However, the magnitude and types of genome changes varied across C. albicans strain background and ploidy state. We then assessed if host-induced genomic changes resulted in fitness consequences on growth rate and nonlethal virulence phenotypes and found that many host-derived isolates significantly changed relative to their parental strain. Interestingly, diploid host-associated C. albicans predominantly decreased host reproductive fitness, whereas tetraploid host-associated C. albicans increased host reproductive fitness. Together, these results are important for understanding how host-induced genomic changes in C. albicans alter its relationship with the host. IMPORTANCE Candida albicans is an opportunistic fungal pathogen of humans. The ability to generate genetic variation is essential for adaptation and is a strategy that C. albicans and other fungal pathogens use to change their genome size. Stressful environments, including the host, induce C. albicans genome instability. Here, we investigated how C. albicans genetic background and ploidy state impact genome instability, both in vitro and in a host environment. We show that the host environment induces genome instability, but the magnitude depends on C. albicans genetic background. Furthermore, we show that tetraploid C. albicans is highly unstable in host environments and rapidly reduces in genome size. These reductions in genome size often resulted in reduced virulence. In contrast, diploid C. albicans displayed modest host-induced genome size changes, yet these frequently resulted in increased virulence. Such studies are essential for understanding how opportunistic pathogens respond and potentially adapt to the host environment. 

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5. Molecular mechanisms underlying the evolution of a color polyphenism by genetic accommodation in the tobacco hornworm, Manduca sexta

   https://doi.org/10.1073/pnas.2425004122  

   Suzuki, Yuichiro; Amaya, Stephanie; Gonzalez, Paula; Becerril, Daniela; Aquit, Surisadai; Davis, Maya; Hoesel, Madeline; Chou, Elizabeth; Khong, Hesper; Zaia, Kathryn; et al (March 2025, Proceedings of the National Academy of Sciences)

   How organisms evolve under extreme environmental changes is a critical question in the face of global climate change. Genetic accommodation is an evolutionary process by which natural selection acts on novel phenotypes generated through repeated encounters with extreme environments. In this study, polyphenic and monophenic strains of theblackmutant tobacco hornworm,Manduca sexta, were evolved via genetic accommodation of heat stress-induced phenotypes, and the molecular differences between the two strains were explored. Transcriptomic analyses showed that epigenetic and hormonal differences underlie the differences between the two strains and their distinct responses to temperature. DNA methylation had diverged between the two strains potentially mediating genetic assimilation. Juvenile hormone (JH) signaling in the polyphenic strain was temperature sensitive, whereas in the monophenic strain, JH signaling remained low at all temperatures. Although 20-hydroxyecdysone titers were elevated under heat shock conditions in both strains, the strains did not differ in the titers. Tyrosine hydroxylase was also found to differ between the two strains at different temperatures, and its expression could be modulated by topical application of a JH analog. Finally, heat shock of unselectedblackmutants demonstrated that the expression of the JH-response gene,Krüppel-homolog 1(Kr-h1), increased within the first 30 min of heat shock, suggesting that JH levels respond readily to thermal stress. Our study highlights the critical role that hormones and epigenetics play during genetic accommodation and potentially in the evolution of populations in the face of climate change. 

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