Juliet W. Welch

Population genomics and local adaptation in wild isolates of a model microbial eukaryote

Elucidating the connection between genotype, phenotype, and adaptation in wild populations is fundamental to the study of evolutionary biology, yet it remains an elusive goal, particularly for microscopic taxa, which comprise the majority of life. Even for microbes that can be reliably found in the wild, defining the boundaries of their populations and discovering ecologically relevant phenotypes has proved extremely difficult. Here, we have circumvented these issues in the microbial eukaryote Neurospora crassa by using a “reverse-ecology” population genomic approach that is free of a priori assumptions about candidate adaptive alleles. We performed Illumina whole-transcriptome sequencing of 48 individuals to identify single nucleotide polymorphisms. From these data, we discovered two cryptic and recently diverged populations, one in the tropical Caribbean basin and the other endemic to subtropical Louisiana. We conducted high-resolution scans for chromosomal regions of extreme divergence between these populations and found two such genomic “islands.” Through growth-rate assays, we found that the subtropical Louisiana population has a higher fitness at low temperature (10 °C) and that several of the genes within these distinct regions have functions related to the response to cold temperature. These results suggest the divergence islands may be the result of local adaptation to the 9 °C difference in average yearly minimum temperature between these two populations. Remarkably, another of the genes identified using this unbiased, whole-genome approach is the well-known circadian oscillator frequency, suggesting that the 2.4°–10.6° difference in latitude between the populations may be another important environmental parameter.

Gene amplification in yeast: CUP1 copy number regulates copper resistance

The CUP1 locus in yeast confers resistance to copper toxicity. We determined the molecular basis for copper resistance in three yeast strains, with differing degrees of resistance. Increased resistance to copper is associated with overproduction of a low molecular weight copper-binding protein, copper-chelatin. Increased chelatin synthesis results from amplification of the CUP1r gene and increased synthesis of the copper inducible mRNA. The copper resistance level of a given strain correlates directly with the gene copy number.Strains containing one copy and ten tandemly iterated copies of the CUP1 gene were studied. From the latter, a haploid strain with enhanced resistance was isolated following several selection cycles at elevated copper concentrations. This strain was disomic for chromosome VIII, the chromosome containing the CUP1 locus. The disomic chromosomes exhibit differential CUP1 gene amplification: 11 and 14 tandemly organized repeat units are found in the respective chromosome VIII homologues. We propose that the molecular mechanisms of gene amplification involve unequal sister chromatid exchange and intrachromosomal gene conversion, as well as disomy.

Genome Wide Association Identifies Novel Loci Involved in Fungal Communication

Understanding how genomes encode complex cellular and organismal behaviors has become the outstanding challenge of modern genetics. Unlike classical screening methods, analysis of genetic variation that occurs naturally in wild populations can enable rapid, genome-scale mapping of genotype to phenotype with a medium-throughput experimental design. Here we describe the results of the first genome-wide association study (GWAS) used to identify novel loci underlying trait variation in a microbial eukaryote, harnessing wild isolates of the filamentous fungus Neurospora crassa. We genotyped each of a population of wild Louisiana strains at 1 million genetic loci genome-wide, and we used these genotypes to map genetic determinants of microbial communication. In N. crassa, germinated asexual spores (germlings) sense the presence of other germlings, grow toward them in a coordinated fashion, and fuse. We evaluated germlings of each strain for their ability to chemically sense, chemotropically seek, and undergo cell fusion, and we subjected these trait measurements to GWAS. This analysis identified one gene, NCU04379 (cse-1, encoding a homolog of a neuronal calcium sensor), at which inheritance was strongly associated with the efficiency of germling communication. Deletion of cse-1 significantly impaired germling communication and fusion, and two genes encoding predicted interaction partners of CSE1 were also required for the communication trait. Additionally, mining our association results for signaling and secretion genes with a potential role in germling communication, we validated six more previously unknown molecular players, including a secreted protease and two other genes whose deletion conferred a novel phenotype of increased communication and multi-germling fusion. Our results establish protein secretion as a linchpin of germling communication in N. crassa and shed light on the regulation of communication molecules in this fungus. Our study demonstrates the power of population-genetic analyses for the rapid identification of genes contributing to complex traits in microbial species.

Evolution and diversity of a fungal self/nonself recognition locus.

Background Self/nonself discrimination is an essential feature for pathogen recognition and graft rejection and is a ubiquitous phenomenon in many organisms. Filamentous fungi, such as Neurospora crassa, provide a model for analyses of population genetics/evolution of self/nonself recognition loci due to their haploid nature, small genomes and excellent genetic/genomic resources. In N. crassa, nonself discrimination during vegetative growth is determined by 11 heterokaryon incompatibility (het) loci. Cell fusion between strains that differ in allelic specificity at any of these het loci triggers a rapid programmed cell death response. Methodology/Principal Findings In this study, we evaluated the evolution, population genetics and selective mechanisms operating at a nonself recognition complex consisting of two closely linked loci, het-c (NCU03493) and pin-c (NCU03494). The genomic position of pin-c next to het-c is unique to Neurospora/Sordaria species, and originated by gene duplication after divergence from other species within the Sordariaceae. The het-c pin-c alleles in N. crassa are in severe linkage disequilibrium and consist of three haplotypes, het-c1/pin-c1, het-c2/pin-c2 and het-c3/pin-c3, which are equally frequent in population samples and exhibit trans-species polymorphisms. The absence of recombinant haplotypes is correlated with divergence of the het-c/pin-c intergenic sequence. Tests for positive and balancing selection at het-c and pin-c support the conclusion that both of these loci are under non-neutral balancing selection; other regions of both genes appear to be under positive selection. Our data show that the het-c2/pin-c2 haplotype emerged by a recombination event between the het-c1/pin-c1 and het-c3/pin-c3 approximately 3–12 million years ago. Conclusions/Significance These results support models by which loci that confer nonself discrimination form by the association of polymorphic genes with genes containing HET domains. Distinct allele classes can emerge by recombination and positive selection and are subsequently maintained by balancing selection and divergence of intergenic sequence resulting in recombination blocks between haplotypes.

Unequal crossing-over and gene conversion at the amplifiedCUP1 locus of yeast

SummaryMeiotic recombination was analyzed between two twelve-copy arrays of a gene amplification at theCUP1 locus ofSaccharomyces cerevisiae. Utilizing Southern analysis to identify spores with non-parental repeat arrays, we find that approximately 11% of a sample with 202 unselected tetrads possess at least one nonparental spore array. Both reciprocal and non-reciprocal changes are observed. The data suggest a model in which frequent mispairing among identical copies of the 2.0 kb repeat unit leads to the formation of unpaired loops containing integral numbers of repeat units. In this model, conversions involving the loops lead to non-reciprocal changes in arrays: about half are associated with reciprocal exchange, and net increases in repeat unit numbers occur about as frequently as net decreases. Thus, the known properties of gene conversion can account for all the segregations we observe.

Multicopy CUP1 plasmids enhance cadmium and copper resistance levels in yeast

SummaryA 3.3 kb fragment of yeast genomic DNA was isolated by screening a genomic library constructed in the high copy number 2 micron plasmid YEp351 vector for clones capable of enhancing the degree of resistance of Saccharomyces cerevisiae strain MW3070-8B to cadmium. The insert contained two complete copies of the CUP1 gene open reading frame (183 bp), including the upstream promoter sequences (450 bp) with two conserved metal responsive cis-acting elements. Northern analysis showed that addition of cadmium (0.02 μM) or copper (50 μM) to overnight liquid cultures of yeast induced expression of CUP1 transcripts from both chromosomal and plasmid-borne gene copies. The cloned 3.3 kb DNA in a high copy number plasmid restored copper resistance to the sensitive strain LS70-313Δ, deleted for the CUP1 gene (cup1Δ), but failed to restore cadmium resistance. Thus, CUP1 gene expression in yeast appears to be influenced differently by cadmium and copper ions. Resistance to heavy metal poisoning resulted from enhanced gene product levels attributable to amplification of the CUP1 gene as well as to increased transcriptions. Two distinct gene product levels mediate cadmium and copper resistance; a higher gene product level was required to confer cadmium resistance.

Identification and characterization of LFD1, a novel protein involved in membrane merger during cell fusion in Neurospora crassa

Despite its essential role in development, molecular mechanisms of membrane merger during cell–cell fusion in most eukaryotic organisms remain elusive. In the filamentous fungus Neurospora crassa, cell fusion occurs during asexual spore germination, where genetically identical germlings show chemotropic interactions and cell–cell fusion. Fusion of germlings and hyphae is required for the formation of the interconnected mycelial network characteristic of filamentous fungi. Previously, a multipass membrane protein, PRM1, was characterized and acts at the step of bilayer fusion in N. crassa. Here we describe the identification and characterization of lfd‐1, encoding a single pass transmembrane protein, which is also involved in membrane merger. lfd‐1 was identified by a targeted analysis of a transcriptional profile of a transcription factor mutant (Δpp‐1) defective in germling fusion. The Δlfd‐1 mutant shows a similar, but less severe, membrane merger defect as a ΔPrm1 mutant. By genetic analyses, we show that LFD1 and PRM1 act independently, but share a redundant function. The cell fusion frequency of both Δlfd‐1 and ΔPrm1 mutants was sensitive to extracellular calcium concentration and was associated with an increase in cell lysis, which was suppressed by a calcium‐dependent mechanism involving a homologue to synaptotagmin.

Mutagenesis of yeast MW104-1B strain has identified the uncharacterized PMS6 DNA mismatch repair gene locus and additional alleles of existing PMS1, PMS2 and MSH2 genes

The haploid yeast Saccharomyces cerevisiae MW104-1B strain was disomic for chromosome III (n + 1) and carried DNA mismatches at three different heteroallelic loci; leu2 (leu2-1/leu2-27), thr4 (thr4-1/thr4-16) and his4 (his4-4/his4-519) (Williamson, 1984). We mutagenized the MW104-1B strain and identified seven mutant isolates that display elevated mitotic/meiotic prototrophs due to mismatch repair failures at heteroallelic loci. Three mutants (pms1, pms2 and pms3) isolated earlier from MW104-1B were shown to correct in vitro constructed plasmids with defined DNA mismatches (G/T, A/C, G/G, etc.) poorly (Kramer et al., 1989a). Complementation tests were performed by crossing all seven new mutant isolates to pms1 and pms2 mutants and assaying for mutant phenotype in the diploids. Four mutant isolates failed to complement the two known pms alleles (pms1-1 and pms2-1). Two other mutant isolates complemented the pms1-1 and pms2-1 alleles, but failed to complement each other and were named as the pms5-1 allele of an uncharacterized gene (PMS5). One other mutant isolate complemented the pms1-1, pms2-1 and pms5-1 alleles and was named as the pms6-1 allele of another uncharacterized gene (PMS6). Subsequently, the pms5-1 mutant allele was shown to be complemented by a plasmid borne yeast MSH2 gene, implying that it is an allele of MSH2 (PMS5). The human homologs (hMSH2 and hMLH1) of two yeast DNA mismatch repair genes (MSH2 and MLH1) have been cloned recently and shown to be responsible for hereditary nonpolypnosis colon cancer (HNPCC) (Fishel et al., 1993; Leach et al., 1993; Bronner et al., 1994; Papadopoulos et al., 1994).

Gene conversions within the Cup1r region from heterologous crosses in Saccharomyces cerevisiae

SummaryMeiotic recombination among unselected tetrads was analyzed genetically and physically in a heterologous cross where one parent carried six copies of a 1.6 kb CUP1 repeat while the other parent carried seven copies of a 1.1 kb repeat. In the heterologous cross, 140 unselected, complete tetrads were subjected to Southern analysis and 20% exhibited meiotic copy number alterations at the CUP1 locus. Most events, more than 75%, involved only a single spore of a tetrad, and were largely intrachromosomal or sister chromatid events. However, some conversions and associated cross-overs between homologs were also observed. We propose that the high level of heterologies interferes with homologous exchanges and leads to an increase in intrachromosomal events.

Chapter 4 Synchronous Mating in Yeasts

Publisher Summary This chapter focuses on synchronous mating in yeasts. The development of methods for hybridizing yeasts has concerned investigators in fungal biology and fermentation technology for several decades. Matings among haploid yeast cells generate zygotes that typically produce diploid buds by mitosis. Although the chapter focuses on heterothallic mating systems at the haploid-diploid level, the methods presented can be applied to homothallic strains or other ploidy levels. The chapter presents methods for producing synchronously mating populations of Saccharomyces cerevisiae . The experimental approaches of different researchers in this organism and in Hansenula wingei are briefly reviewed. A detailed comparison of all procedures as regards methodological approaches and zygotic yields is also included in the chapter. Modifications are suggested that might circumvent variability arising from differences in sex factor production, agglutinability, or response to factors. Suggestions are also presented in the chapter for new approaches to achieve synchronized yeast mating and the application of such systems to the analysis of mating itself or as a tool for analyzing other developmental sequences.