PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-16 (16)
 

Clipboard (0)
None

Select a Filter Below

Journals
Year of Publication
Document Types
1.  The transcriptome landscape of early maize meiosis 
BMC Plant Biology  2014;14:118.
Background
A major step in the higher plant life cycle is the decision to leave the mitotic cell cycle and begin the progression through the meiotic cell cycle that leads to the formation of gametes. The molecular mechanisms that regulate this transition and early meiosis remain largely unknown. To gain insight into gene expression features during the initiation of meiotic recombination, we profiled early prophase I meiocytes from maize (Zea mays) using capillary collection to isolate meiocytes, followed by RNA-seq.
Results
We detected ~2,000 genes as preferentially expressed during early meiotic prophase, most of them uncharacterized. Functional analysis uncovered the importance of several cellular processes in early meiosis. Processes significantly enriched in isolated meiocytes included proteolysis, protein targeting, chromatin modification and the regulation of redox homeostasis. The most significantly up-regulated processes in meiocytes were processes involved in carbohydrate metabolism. Consistent with this, many mitochondrial genes were up-regulated in meiocytes, including nuclear- and mitochondrial-encoded genes. The data were validated with real-time PCR and in situ hybridization and also used to generate a candidate maize homologue list of known meiotic genes from Arabidopsis.
Conclusions
Taken together, we present a high-resolution analysis of the transcriptome landscape in early meiosis of an important crop plant, providing support for choosing genes for detailed characterization of recombination initiation and regulation of early meiosis. Our data also reveal an important connection between meiotic processes and altered/increased energy production.
doi:10.1186/1471-2229-14-118
PMCID: PMC4032173  PMID: 24885405
Maize; Meiosis; Meiocytes; Mitochondria; RNA-seq; Transcriptome
2.  Passage through the mammalian gut triggers a phenotypic switch that promotes Candida albicans commensalism 
Nature genetics  2013;45(9):1088-1091.
Among ~5,000,000 fungal species,1 Candida albicans is exceptional in its lifelong association with humans, either within the gastrointestinal microbiome or as an invasive pathogen.2 Opportunistic infections are generally ascribed to defective host immunity 3 but may require specific microbial programs. Here, we report that exposure of C. albicans to the mammalian gut triggers a developmental switch, driven by the Wor1 transcription factor, to a commensal cell type. Wor1 expression was previously observed only in rare genetic backgrounds,4–6 where it controls a white-opaque switch for mating.4–7 We show that passage of wild-type cells through the murine gastrointestinal tract triggers WOR1 expression and a novel phenotypic switch. The resulting GUT (Gastrointestinally-IndUced Transition) cells differ morphologically and functionally from previously defined cell types, including opaque, and express a transcriptome that is optimized for the digestive tract. The white-GUT switch illuminates how a microorganism utilizes distinct genetic programs to transition between commensalism and invasive pathogenesis.
doi:10.1038/ng.2710
PMCID: PMC3758371  PMID: 23892606
3.  Sequencing-based large-scale genomics approaches with small numbers of isolated maize meiocytes 
High-throughput sequencing has become the large-scale approach of choice to study global gene expression and the distribution of specific chromatin marks and features. However, the limited availability of large amounts of purified cells made it very challenging to apply sequencing-based techniques in plant meiosis research in the past. In this paper, we describe a method to isolate meiocytes from maize anthers and detailed protocols to successfully perform RNA-seq, smRNA-seq, H3K4me3-ChIP-seq, and DNA bisulfite conversion sequencing with 5000–30,000 isolated maize male meiotic cells. These methods can be adjusted for other flowering plant species as well.
doi:10.3389/fpls.2014.00057
PMCID: PMC3933774  PMID: 24611068
meiocytes; meiosis; Illumina sequencing; RNA-seq; ChIP-seq; DNA methylation; small RNA; maize
4.  The meiotic transcriptome architecture of plants 
Although a number of genes that play key roles during the meiotic process have been characterized in great detail, the whole process of meiosis is still not completely unraveled. To gain insight into the bigger picture, large-scale approaches like RNA-seq and microarray can help to elucidate the transcriptome landscape during plant meiosis, discover co-regulated genes, enriched processes, and highly expressed known and unknown genes which might be important for meiosis. These high-throughput studies are gaining more and more popularity, but their beginnings in plant systems reach back as far as the 1960's. Frequently, whole anthers or post-meiotic pollen were investigated, while less data is available on isolated cells during meiosis, and only few studies addressed the transcriptome of female meiosis. For this review, we compiled meiotic transcriptome studies covering different plant species, and summarized and compared their key findings. Besides pointing to consistent as well as unique discoveries, we finally draw conclusions what can be learned from these studies so far and what should be addressed next.
doi:10.3389/fpls.2014.00220
PMCID: PMC4046320  PMID: 24926296
meiosis; transcriptome; meiocytes; anthers; RNA-seq; microarray
5.  Post-Transcriptional Regulation of the Sef1 Transcription Factor Controls the Virulence of Candida albicans in Its Mammalian Host 
PLoS Pathogens  2012;8(11):e1002956.
The yeast Candida albicans transitions between distinct lifestyles as a normal component of the human gastrointestinal microbiome and the most common agent of disseminated fungal disease. We previously identified Sef1 as a novel Cys6Zn2 DNA binding protein that plays an essential role in C. albicans virulence by activating the transcription of iron uptake genes in iron-poor environments such as the host bloodstream and internal organs. Conversely, in the iron-replete gastrointestinal tract, persistence as a commensal requires the transcriptional repressor Sfu1, which represses SEF1 and genes for iron uptake. Here, we describe an unexpected, transcription-independent role for Sfu1 in the direct inhibition of Sef1 function through protein complex formation and localization in the cytoplasm, where Sef1 is destabilized. Under iron-limiting conditions, Sef1 forms an alternative complex with the putative kinase, Ssn3, resulting in its phosphorylation, nuclear localization, and transcriptional activity. Analysis of sfu1 and ssn3 mutants in a mammalian model of disseminated candidiasis indicates that these post-transcriptional regulatory mechanisms serve as a means for precise titration of C. albicans virulence.
Author Summary
Candida albicans is a fungus that resides on the skin and in the gastrointestinal tract of humans and other mammals. However, this commensal organism is also capable of proliferating and causing disease in people who have received antibiotics, who are immunocompromised, or who have suffered injury to epithelial layers. We previously identified a novel transcription factor called Sef1 that promotes C. albicans virulence by activating the expression of iron uptake genes in iron-poor environments, such as the host bloodstream. However, in iron-replete environments such as the gastrointestinal niche, the SEF1 gene is repressed by a second transcription factor called Sfu1. Here, we report our discovery of a series of post-transcriptional regulatory events that determine the intracellular localization, stability, and activity of Sef1 protein. Mutants that disrupt these post-transcriptional events alter C. albicans virulence in a mammalian model of disseminated infection. The existence of multiple levels of regulation speaks to the importance of Sef1 in C. albicans virulence and suggests that close titration of Sef1 activity is important for adaptation to distinct microenvironments within the mammalian host.
doi:10.1371/journal.ppat.1002956
PMCID: PMC3486892  PMID: 23133381
6.  An iron homeostasis regulatory circuit with reciprocal roles in Candida albicans commensalism and pathogenesis 
Cell host & microbe  2011;10(2):118-135.
The mammalian gastrointestinal tract and bloodstream are highly disparate biological niches that differ in concentrations of nutrients such as iron. However, some commensal-pathogenic microorganisms, such as the yeast Candida albicans, thrive in both environments. We report the evolution of a transcription circuit in C. albicans that controls iron uptake and determines its fitness in both niches. Our analysis of DNA-binding proteins that regulate iron uptake by this organism suggests the evolutionary intercalation of a transcriptional activator called Sef1 between two broadly conserved iron-responsive transcriptional repressors, Sfu1 and Hap43. Sef1 activates iron uptake genes and promotes virulence in a mouse model of bloodstream infection, whereas Sfu1 represses iron uptake genes and is dispensable for virulence but promotes gastrointestinal commensalism. Thus, C. albicans can alternate between genetic programs conferring resistance to iron depletion in the bloodstream versus iron toxicity in the gut, and this may represent a fundamental attribute of gastrointestinal commensal-pathogens.
doi:10.1016/j.chom.2011.07.005
PMCID: PMC3165008  PMID: 21843869
7.  Characterization of a set of novel meiotically-active promoters in Arabidopsis 
BMC Plant Biology  2012;12:104.
Background
Homologous recombination, together with selection, laid the foundation for traditional plant breeding. The recombination process that takes place during meiotic cell division is crucial for the creation of novel variations of highly desired traits by breeders. Gaining control over this process is important for molecular breeding to achieve more precise, large-scale and quicker plant improvement. As conventional ubiquitous promoters are neither tissue-specific nor efficient in driving gene expression in meiocytes, promoters with high meiotic activities are potential candidates for manipulating the recombination process. So far, only a few meiotically-active promoters have been reported. Recently developed techniques to profile the transcriptome landscape of isolated meiocytes provided the means to discover promoters from genes that are actively expressed in meiosis.
Results
In a screen for meiotically-active promoters, we examined ten promoter sequences that are associated with novel meiotic candidate genes. Each promoter was tested by expressing a GFP reporter gene in Arabidopsis. Characterization of regulatory regions revealed that these meiotically-active promoters possessed conserved motifs and motif arrangement. Some of the promoters unite optimal properties which are invaluable for meiosis-directed studies such as delivering specific gene expression in early meiosis I and/or meiosis II. Furthermore, the examination of homologs of the corresponding genes within green plants points to a great potential of applying the information from Arabidopsis to other species, especially crop plants.
Conclusions
We identified ten novel meiotically-active promoters; which, along with their homologs, are prime candidates to specifically drive gene expression during meiosis in plants and can thus provide important tools for meiosis study and crop breeding.
doi:10.1186/1471-2229-12-104
PMCID: PMC3462685  PMID: 22776406
Meiosis; Homologous recombination; Promoter; GFP; cis-regulatory elements; Plant molecular breeding
8.  Identification and Characterization of Sclerotinia sclerotiorum NADPH Oxidases▿† 
Applied and Environmental Microbiology  2011;77(21):7721-7729.
Numerous studies have shown both the detrimental and beneficial effects of reactive oxygen species (ROS) in animals, plants, and fungi. These organisms utilize controlled generation of ROS for signaling, pathogenicity, and development. Here, we show that ROS are essential for the pathogenic development of Sclerotinia sclerotiorum, an economically important fungal pathogen with a broad host range. Based on the organism's completed genome sequence, we identified two S. sclerotiorum NADPH oxidases (SsNox1 and SsNox2), which presumably are involved in ROS generation. RNA interference (RNAi) was used to examine the function of SsNox1 and SsNox2. Silencing of SsNox1 expression indicated a central role for this enzyme in both virulence and pathogenic (sclerotial) development, while inactivation of the SsNox2 gene resulted in limited sclerotial development, but the organism remained fully pathogenic. ΔSsnox1 strains had reduced ROS levels, were unable to develop sclerotia, and unexpectedly correlated with significantly reduced oxalate production. These results are in accordance with previous observations indicating that fungal NADPH oxidases are required for pathogenic development and are consistent with the importance of ROS regulation in the successful pathogenesis of S. sclerotiorum.
doi:10.1128/AEM.05472-11
PMCID: PMC3209176  PMID: 21890677
9.  Meiosis-Specific Loading of the Centromere-Specific Histone CENH3 in Arabidopsis thaliana 
PLoS Genetics  2011;7(6):e1002121.
Centromere behavior is specialized in meiosis I, so that sister chromatids of homologous chromosomes are pulled toward the same side of the spindle (through kinetochore mono-orientation) and chromosome number is reduced. Factors required for mono-orientation have been identified in yeast. However, comparatively little is known about how meiotic centromere behavior is specialized in animals and plants that typically have large tandem repeat centromeres. Kinetochores are nucleated by the centromere-specific histone CENH3. Unlike conventional histone H3s, CENH3 is rapidly evolving, particularly in its N-terminal tail domain. Here we describe chimeric variants of CENH3 with alterations in the N-terminal tail that are specifically defective in meiosis. Arabidopsis thaliana cenh3 mutants expressing a GFP-tagged chimeric protein containing the H3 N-terminal tail and the CENH3 C-terminus (termed GFP-tailswap) are sterile because of random meiotic chromosome segregation. These defects result from the specific depletion of GFP-tailswap protein from meiotic kinetochores, which contrasts with its normal localization in mitotic cells. Loss of the GFP-tailswap CENH3 variant in meiosis affects recruitment of the essential kinetochore protein MIS12. Our findings suggest that CENH3 loading dynamics might be regulated differently in mitosis and meiosis. As further support for our hypothesis, we show that GFP-tailswap protein is recruited back to centromeres in a subset of pollen grains in GFP-tailswap once they resume haploid mitosis. Meiotic recruitment of the GFP-tailswap CENH3 variant is not restored by removal of the meiosis-specific cohesin subunit REC8. Our results reveal the existence of a specialized loading pathway for CENH3 during meiosis that is likely to involve the hypervariable N-terminal tail. Meiosis-specific CENH3 dynamics may play a role in modulating meiotic centromere behavior.
Author Summary
There are two types of cell division in eukaryotes. Mitosis produces cells with identical copies of the genome, while meiosis produces gametes with half the number of chromosomes found in the parent cell. Faithful genome inheritance is controlled by centromeres, chromosomal structures that allow duplicated chromosomes to be pulled apart correctly during cell division. Centromeres are differentially configured during meiosis (relative to mitosis) so chromosome number can be reduced by half. Centromeres are built upon a specialized DNA packing protein, CENH3. Here we describe altered forms of CENH3 that are loaded correctly during mitosis but are severely depleted from centromeres in meiotic cells. As CENH3 is essential for chromosome inheritance, plants expressing these versions of the protein are sterile because they produce very few viable gametes. Differential loading of CENH3 during meiosis may play a role in modulating chromosome inheritance to form haploid gametes.
doi:10.1371/journal.pgen.1002121
PMCID: PMC3111537  PMID: 21695238
10.  Meiosis-specific gene discovery in plants: RNA-Seq applied to isolated Arabidopsis male meiocytes 
BMC Plant Biology  2010;10:280.
Background
Meiosis is a critical process in the reproduction and life cycle of flowering plants in which homologous chromosomes pair, synapse, recombine and segregate. Understanding meiosis will not only advance our knowledge of the mechanisms of genetic recombination, but also has substantial applications in crop improvement. Despite the tremendous progress in the past decade in other model organisms (e.g., Saccharomyces cerevisiae and Drosophila melanogaster), the global identification of meiotic genes in flowering plants has remained a challenge due to the lack of efficient methods to collect pure meiocytes for analyzing the temporal and spatial gene expression patterns during meiosis, and for the sensitive identification and quantitation of novel genes.
Results
A high-throughput approach to identify meiosis-specific genes by combining isolated meiocytes, RNA-Seq, bioinformatic and statistical analysis pipelines was developed. By analyzing the studied genes that have a meiosis function, a pipeline for identifying meiosis-specific genes has been defined. More than 1,000 genes that are specifically or preferentially expressed in meiocytes have been identified as candidate meiosis-specific genes. A group of 55 genes that have mitochondrial genome origins and a significant number of transposable element (TE) genes (1,036) were also found to have up-regulated expression levels in meiocytes.
Conclusion
These findings advance our understanding of meiotic genes, gene expression and regulation, especially the transcript profiles of MGI genes and TE genes, and provide a framework for functional analysis of genes in meiosis.
doi:10.1186/1471-2229-10-280
PMCID: PMC3018465  PMID: 21167045
11.  Systematic genetic analysis of virulence in the human fungal pathogen Cryptococcus neoformans 
Cell  2008;135(1):174-188.
SUMMARY
The fungus Cryptococcus neoformans is a leading cause of mortality and morbidity among HIV-infected individuals. We utilized the completed genome sequence and optimized methods for homologous DNA replacement using high-velocity particle bombardment to engineer 1,201 gene knockout mutants. We screened this resource in vivo for proliferation in murine lung tissue and in vitro for three well-recognized virulence attributes — polysaccharide capsule formation, melanization, and growth at body temperature. We identified dozens of previously uncharacterized genes that affect these known attributes as well as 40 infectivity mutants without obvious defects in these traits. The latter mutants affect predicted regulatory factors, secreted proteins, and immune-related factors, and represent powerful tools for elucidating novel virulence mechanisms. In particular, we describe a GATA family transcription factor that inhibits phagocytosis by murine macrophages independently of the capsule, indicating a previously unknown mechanism of innate immune modulation.
doi:10.1016/j.cell.2008.07.046
PMCID: PMC2628477  PMID: 18854164
12.  Tomato QM-Like Protein Protects Saccharomyces cerevisiae Cells against Oxidative Stress by Regulating Intracellular Proline Levels 
Exogenous proline can protect cells of Saccharomyces cerevisiae from oxidative stress. We altered intracellular proline levels by overexpressing the proline dehydrogenase gene (PUT1) of S. cerevisiae. Put1p performs the first enzymatic step of proline degradation in S. cerevisiae. Overexpression of Put1p results in low proline levels and hypersensitivity to oxidants, such as hydrogen peroxide and paraquat. A put1-disrupted yeast mutant deficient in Put1p activity has increased protection from oxidative stress and increased proline levels. Following a conditional life/death screen in yeast, we identified a tomato (Lycopersicon esculentum) gene encoding a QM-like protein (tQM) and found that stable expression of tQM in the Put1p-overexpressing strain conferred protection against oxidative damage from H2O2, paraquat, and heat. This protection was correlated with reactive oxygen species (ROS) reduction and increased proline accumulation. A yeast two-hybrid system assay was used to show that tQM physically interacts with Put1p in yeast, suggesting that tQM is directly involved in modulating proline levels. tQM also can rescue yeast from the lethality mediated by the mammalian proapoptotic protein Bax, through the inhibition of ROS generation. Our results suggest that tQM is a component of various stress response pathways and may function in proline-mediated stress tolerance in plants.
doi:10.1128/AEM.02428-05
PMCID: PMC1489650  PMID: 16751508
13.  Cdc42 Is Required for Proper Growth and Development in the Fungal Pathogen Colletotrichum trifolii 
Eukaryotic Cell  2006;5(1):155-166.
Cdc42 is a highly conserved small GTP-binding protein that is involved in regulating morphogenesis in eukaryotes. In this study, we isolated and characterized a highly conserved Cdc42 gene from Colletotrichum trifolii (CtCdc42), a fungal pathogen of alfalfa. CtCdc42 is, at least in part, functionally equivalent to Saccharomyces cerevisiae Cdc42p, since it restores the temperature-sensitive phenotype of a yeast Cdc42p mutant. Inhibition of CtCdc42 by expression of an antisense CtCdc42 or a dominant negative form of CtCdc42 (DN Cdc42) resulted in appressorium differentiation under noninductive conditions, suggesting that CtCdc42 negatively regulates pathogenic development in this fungus. We also examined the possible linkage between CtCdc42 and Ras signaling. Expression of a dominant active Cdc42 (DA Cdc42) in C. trifolii leads to aberrant hyphal growth under nutrient-limiting conditions. This phenotype was similar to that of our previously reported dominant active Ras (DA Ras) mutant. Also consistent with our observations of the DA Ras mutant, high levels of reactive oxygen species (ROS) were observed in the DA Cdc42 mutant, and proline restored the wild-type phenotype. Moreover, overexpression of DN Cdc42 resulted in a significant decrease in spore germination, virtually no hyphal branching, and earlier sporulation, again similar to what we observed in a dominant negative Ras (DN Ras) mutant strain. Interestingly, coexpression of DA Cdc42 with DN Ras resulted in germination rates close to wild-type levels, while coexpression of DN Cdc42 with the DA Ras mutant restored the wild-type phenotype. These data suggest that CtCdc42 is positioned as a downstream effector of CtRas to regulate spore germination and pathogenic development.
doi:10.1128/EC.5.1.155-166.2006
PMCID: PMC1360247  PMID: 16400178
14.  The Arabidopsis thaliana PARTING DANCERS Gene Encoding a Novel Protein Is Required for Normal Meiotic Homologous RecombinationD⃞ 
Molecular Biology of the Cell  2006;17(3):1331-1343.
Recent studies of meiotic recombination in the budding yeast and the model plant Arabidopsis thaliana indicate that meiotic crossovers (COs) occur through two genetic pathways: the interference-sensitive pathway and the interference-insensitive pathway. However, few genes have been identified in either pathway. Here, we describe the identification of the PARTING DANCERS (PTD) gene, as a gene with an elevated expression level in meiocytes. Analysis of two independently generated transferred DNA insertional lines in PTD showed that the mutants had reduced fertility. Further cytological analysis of male meiosis in the ptd mutants revealed defects in meiosis, including reduced formation of chiasmata, the cytological appearance of COs. The residual chiasmata in the mutants were distributed randomly, indicating that the ptd mutants are defective for CO formation in the interference-sensitive pathway. In addition, transmission electron microscopic analysis of the mutants detected no obvious abnormality of synaptonemal complexes and apparently normal late recombination nodules at the pachytene stage, suggesting that the mutant's defects in bivalent formation were postsynaptic. Comparison to other genes with limited sequence similarity raises the possibility that PTD may present a previously unknown function conserved in divergent eukaryotic organisms.
doi:10.1091/mbc.E05-09-0902
PMCID: PMC1382321  PMID: 16394097
15.  A CDC42 Homologue in Claviceps purpurea Is Involved in Vegetative Differentiation and Is Essential for Pathogenicity 
Eukaryotic Cell  2005;4(7):1228-1238.
Claviceps purpurea, a biotrophic pathogen of cereals, has developed a unique pathogenic strategy including an extended period of unbranched directed growth in the host's style and ovarian tissue to tap the vascular system. Since the small GTPase Cdc42 has been shown to be involved in cytoskeleton organization and polarity in other fungi, we investigated the role of Cdc42 in the development and pathogenicity of C. purpurea. Expression of heterologous dominant-active (DA) and dominant-negative (DN) alleles of Colletotrichum trifolii in a wild strain of C. purpurea had significant impact on vegetative differentiation: whereas DA Ctcdc42 resulted in loss of conidiation and in aberrant cell shape, expression of DN Ctcdc42 stimulated branching and conidiation. Deletion of the endogenous Cpcdc42 gene was not lethal but led to a phenotype comparable to that of DN Ctcdc42 transformants. ΔCpcdc42 mutants were nonpathogenic; i.e., they induced no disease symptoms. Cytological analysis (light microscopy and electron microscopy) revealed that the mutants can penetrate and invade the stylar tissue. However, invasive growth was arrested in an early stage, presumably induced by plant defense reactions (necrosis or increased production of reactive oxygen species), which were never observed in wild-type infection. The data show a significant impact of Cpcdc42 on vegetative differentiation and pathogenicity in C. purpurea.
doi:10.1128/EC.4.7.1228-1238.2005
PMCID: PMC1168960  PMID: 16002649
16.  Colletotrichum trifolii TB3 Kinase, a COT1 Homolog, Is Light Inducible and Becomes Localized in the Nucleus during Hyphal Elongation 
Eukaryotic Cell  2002;1(4):626-633.
Colletotrichum trifolii is a fungal pathogen responsible for anthracnose disease of alfalfa. Previously, a serine/threonine protein kinase gene from this fungus (TB3), which is a functional homolog of the Neurospora crassa COT1 kinase, has been isolated in our laboratory and appears to be associated with hyphal elongation and branching. In this report we show that light treatment rapidly induces TB3 expression and hyphal branching frequency. Western analysis showed TB3 localization in both the cytoplasm and nucleus, but not in membranes. Moreover, indirect immunofluorescence indicated that TB3 levels were most abundant in the nucleus. To further evaluate the subcellular distribution of TB3, a TB3::GFP fusion construct was inserted into C. trifolii. Results indicated that the cellular location of TB3 changed during fungal growth and development. Consistent with previous observations, TB3 was localized in both the cytoplasm and the nucleus but was preferentially localized in the nucleus during extended hyphal growth. The amino terminus of TB3 contains two relatively long polyglutamine repeats. Yeast-based assays showed that these polyglutamine tracts can activate transcription. These results suggest that TB3 may be positioned in a signaling cascade regulating proper hyphal growth and development by functioning as a transcription factor.
doi:10.1128/EC.1.4.626-633.2002
PMCID: PMC117997  PMID: 12456010

Results 1-16 (16)