Molecular tools for isolate and community studies of Pyrenomycete fungi

The Pyrenomycetes, defined physiologically by the formation of a flask-shaped fruiting body present in the sexual form, are a monophyletic group of fungi that consist of a wide diversity of populations including human and plant pathogens. Based on sequence analysis of 18S ribosomal DNA (rDNA), rDNA regions conserved among the Pyrenomycetes but divergent among other organisms were identified and used to develop selective PCR primers and a highly specific primer set. The primers presented here were used to amplify large portions of the 18S rDNA as well as the entire internal transcribed spacer (ITS) region (ITS 1, 5.8S rDNA, and ITS 2). In addition to database searches, the specificity of the primers was verified by PCR amplification of DNA extracted from pure culture isolates and by sequence analysis of fungal rDNA PCR-amplified from environmental samples. In addition, denaturing gradient gel electrophoresis (DGGE) analyses were performed on closely related Colletotrichum isolates serving as a model pathogenic genus of the Pyrenomycetes. Although both ITS and 18S rDNA DGGE analyses of Colletotrichum were consistent with a phylogeny established from sequence analysis of the ITS region, DGGE analysis of the ITS region was found to be more sensitive than DGGE analysis of the 18S rDNA. This study introduces molecular tools for the study of Pyrenomycete fungi by the development of two specific primers, demonstration of the enhanced sensitivity of ITS-DGGE for typing of closely related isolates and application of these tools to environmental samples.     

Failure of the Tomato Trans-Acting Short Interfering RNA Program to Regulate AUXIN RESPONSE FACTOR3 and ARF4 Underlies the Wiry Leaf Syndrome

Interfering with small RNA production is a common strategy of plant viruses. A unique class of small RNAs that require microRNA and short interfering (siRNA) biogenesis for their production is termed trans-acting short interfering RNAs (ta-siRNAs). Tomato (Solanum lycopersicum) wiry mutants represent a class of phenotype that mimics viral infection symptoms, including shoestring leaves that lack leaf blade expansion. Here, we show that four WIRY genes are involved in siRNA biogenesis, and in their corresponding mutants, levels of ta-siRNAs that regulate AUXIN RESPONSE FACTOR3 (ARF3) and ARF4 are reduced, while levels of their target ARFs are elevated. Reducing activity of both ARF3 and ARF4 can rescue the wiry leaf lamina, and increased activity of either can phenocopy wiry leaves. Thus, a failure to negatively regulate these ARFs underlies tomato shoestring leaves. Overexpression of these ARFs in Arabidopsis thaliana, tobacco (Nicotiana tabacum), and potato (Solanum tuberosum) failed to produce wiry leaves, suggesting that the dramatic response in tomato is exceptional. As negative regulation of orthologs of these ARFs by ta-siRNA is common to land plants, we propose that ta-siRNA levels serve as universal sensors for interference with small RNA biogenesis, and changes in their levels direct species-specific responses.     

Live reporting for hypoxia: Hypoxia sensor–modified mesenchymal stem cells as in vitro reporters

Natural oxygen gradients occur in tissues of biological organisms and also in the context of three-dimensional (3D) in vitro cultivation. Oxygen diffusion limitation and metabolic oxygen consumption by embedded cells produce areas of hypoxia in the tissue/matrix. However, reliable systems to detect oxygen gradients and cellular response to hypoxia in 3D cell culture systems are still missing. In this study, we developed a system for visualization of oxygen gradients in 3D using human adipose tissue–derived mesenchymal stem cells (hAD-MSCs) modified to stably express a fluorescent genetically engineered hypoxia sensor HRE-dUnaG. Modified cells retained their stem cell characteristics in terms of proliferation and differentiation capacity. The hypoxia-reporter cells were evaluated by fluorescence microscopy and flow cytometry under variable oxygen levels (2.5%, 5%, and 7.5% O₂). We demonstrated that reporter hAD-MSCs output is sensitive to different oxygen levels and displays fast decay kinetics after reoxygenation. Additionally, the reporter cells were encapsulated in bulk hydrogels with a variable cell number, to investigate the sensor response in model 3D cell culture applications. The use of hypoxia-reporting cells based on MSCs represents a valuable tool for approaching the genuine in vivo cellular microenvironment and will allow a better understanding of the regenerative potential of AD-MSCs.     

Predation risk, stoichiometric plasticity and ecosystem elemental cycling

It is widely held that herbivore growth and production is limited by dietary nitrogen (N) that in turn constrains ecosystem elemental cycling. Yet, emerging evidence suggests that this conception of limitation may be incomplete, because chronic predation risk heightens herbivore metabolic rate and shifts demand from N-rich proteins to soluble carbohydrate-carbon (C). Because soluble C can be limiting, predation risk may cause ecosystem elemental cycling rates and stoichiometric balance to depend on herbivore physiological plasticity. We report on a stoichiometrically explicit ecosystem model that investigates this problem. The model tracks N, and soluble and recalcitrant C through ecosystem compartments. We evaluate how soluble plant C influences C and N stocks and flows in the presence and absence of predation risk. Without risk, herbivores are limited by N and respire excess C so that plant-soluble C has small effects only on elemental stocks and flows. With predation risk, herbivores are limited by soluble C and release excess N, so plant-soluble C critically influences ecosystem elemental stocks flows. Our results emphasize that expressing ecosystem stoichiometric balance using customary C : N ratios that do not distinguish between soluble and recalcitrant C may not adequately describe limitations on elemental cycling.     

Modulation of DNA conformations through the formation of alternative high-order HU-DNA complexes

HU is an abundant, highly conserved protein associated with the bacterial chromosome. It belongs to a small class of proteins that includes the eukaryotic proteins TBP, SRY, HMG-I and LEF-I, which bind to DNA non-specifically at the minor groove. HU plays important roles as an accessory architectural factor in a variety of bacterial cellular processes such as DNA compaction, replication, transposition, recombination and gene regulation. In an attempt to unravel the role this protein plays in shaping nucleoid structure, we have carried out fluorescence resonance energy transfer measurements of HU-DNA oligonucleotide complexes, both at the ensemble and single-pair levels. Our results provide direct experimental evidence for concerted DNA bending by HU, and the abrogation of this effect at HU to DNA ratios above about one HU dimer per 10-12 bp. These findings support a model in which a number of HU molecules form an ordered helical scaffold with DNA lying in the periphery. The abrogation of these nucleosome-like structures for high HU to DNA ratios suggests a unique role for HU in the dynamic modulation of bacterial nucleoid structure.     

Contrasting patterns of Y chromosome variation in Ashkenazi Jewish and host non-Jewish European populations

The molecular basis of more than 25 genetic diseases has been described in Ashkenazi Jewish populations. Most of these diseases are characterized by one or two major founder mutations that are present in the Ashkenazi population at elevated frequencies. One explanation for this preponderance of recessive diseases is accentuated genetic drift resulting from a series of dispersals to and within Europe, endogamy, and/or recent rapid population growth. However, a clear picture of the manner in which neutral genetic variation has been affected by such a demographic history has not yet emerged. We have examined a set of 32 binary markers (single nucleotide polymorphisms; SNPs) and 10 microsatellites on the non-recombining portion of the Y chromosome (NRY) to investigate the ways in which patterns of variation differ between Ashkenazi Jewish and their non-Jewish host populations in Europe. This set of SNPs defines a total of 20 NRY haplogroups in these populations, at least four of which are likely to have been part of the ancestral Ashkenazi gene pool in the Near East, and at least three of which may have introgressed to some degree into Ashkenazi populations after their dispersal to Europe. It is striking that whereas Ashkenazi populations are genetically more diverse at both the SNP and STR level compared with their European non-Jewish counterparts, they have greatly reduced within-haplogroup STR variability, especially in those founder haplogroups that migrated from the Near East. This contrasting pattern of diversity in Ashkenazi populations is evidence for a reduction in male effective population size, possibly resulting from a series of founder events and high rates of endogamy within Europe. This reduced effective population size may explain the high incidence of founder disease mutations despite overall high levels of NRY diversity.Electronic Supplementary Material Supplementary material is available in the online version of this article at D.M. Behar and D. Garrigan contributed equally to this workElectronic database information: URLs for the data in this article are as follows:ARLEQUIN,