The ITS1-5.8S-ITS2 sequence region in the Musaceae: structure, diversity and use in molecular phylogeny

Genes coding for 45S ribosomal RNA are organized in tandem arrays of up to several thousand copies and contain 18S, 5.8S and 26S rRNA units separated by internal transcribed spacers ITS1 and ITS2. While the rRNA units are evolutionary conserved, ITS show high level of interspecific divergence and have been used frequently in genetic diversity and phylogenetic studies. In this work we report on the structure and diversity of the ITS region in 87 representatives of the family Musaceae. We provide the first detailed information on ITS sequence diversity in the genus Musa and describe the presence of more than one type of ITS sequence within individual species. Both Sanger sequencing of amplified ITS regions and whole genome 454 sequencing lead to similar phylogenetic inferences. We show that it is necessary to identify putative pseudogenic ITS sequences, which may have negative effect on phylogenetic reconstruction at lower taxonomic levels. Phylogenetic reconstruction based on ITS sequence showed that the genus Musa is divided into two distinct clades--Callimusa and Australimusa and Eumusa and Rhodochlamys. Most of the intraspecific banana hybrids analyzed contain conserved parental ITS sequences, indicating incomplete concerted evolution of rDNA loci. Independent evolution of parental rDNA in hybrids enables determination of genomic constitution of hybrids using ITS. The observation of only one type of ITS sequence in some of the presumed interspecific hybrid clones warrants further study to confirm their hybrid origin and to unravel processes leading to evolution of their genomes.     

Lipid-induced filamentous growth in Ustilago maydis

The phytopathogenic fungus Ustilago maydis is obligately dependent on infection of maize to complete the sexual phase of its life cycle. Mating interactions between haploid, budding cells establish an infectious filamentous cell type that invades the host, induces large tumours and eventually forms large masses of black spores. The ability to switch from budding to filamentous growth is therefore critical for infection and completion of the life cycle, although the signals that influence the transition have not been identified from the host or the environment. We have found that growth in the presence of lipids promotes a filamentous phenotype that resembles the infectious cell type found in planta. In addition, the ability of the fungus to respond to lipids is dependent on both the cAMP signalling pathway and a Ras/MAPK pathway; these pathways are known to regulate mating, filamentous growth and pathogenesis in U. maydis. Overall, these results lead us to hypothesize that lipids may represent one of the signals that promote and maintain the filamentous growth of the fungus in the host environment.     

Metabolic discrimination of Catharanthus roseus leaves infected by phytoplasma using 1H-NMR spectroscopy and multivariate data analysis

A comprehensive metabolomic profiling of Catharanthus roseus L. G. Don infected by 10 types of phytoplasmas was carried out using one-dimensional and two-dimensional NMR spectroscopy followed by principal component analysis (PCA), an unsupervised clustering method requiring no knowledge of the data set and used to reduce the dimensionality of multivariate data while preserving most of the variance within it. With a combination of these techniques, we were able to identify those metabolites that were present in different levels in phytoplasma-infected C. roseus leaves than in healthy ones. The infection by phytoplasma in C. roseus leaves causes an increase of metabolites related to the biosynthetic pathways of phenylpropanoids or terpenoid indole alkaloids: chlorogenic acid, loganic acid, secologanin, and vindoline. Furthermore, higher abundance of Glc, Glu, polyphenols, succinic acid, and Suc were detected in the phytoplasma-infected leaves. The PCA of the (1)H-NMR signals of healthy and phytoplasma-infected C. roseus leaves shows that these metabolites are major discriminating factors to characterize the phytoplasma-infected C. roseus leaves from healthy ones. Based on the NMR and PCA analysis, it might be suggested that the biosynthetic pathway of terpenoid indole alkaloids, together with that of phenylpropanoids, is stimulated by the infection of phytoplasma.     

Fitness,risk taking,and spatial behavior covary with boldness in experimental vole populations

Individuals of a population may vary along a pace‐of‐life syndrome from highly fecund, short‐lived, bold, dispersive “fast” types at one end of the spectrum to less fecund, long‐lived, shy, plastic “slow” types at the other end. Risk‐taking behavior might mediate the underlying life history trade‐off, but empirical evidence supporting this hypothesis is still ambiguous. Using experimentally created populations of common voles (Microtus arvalis)—a species with distinct seasonal life history trajectories—we aimed to test whether individual differences in boldness behavior covary with risk taking, space use, and fitness. We quantified risk taking, space use (via automated tracking), survival, and reproductive success (via genetic parentage analysis) in 8 to 14 experimental, mixed‐sex populations of 113 common voles of known boldness type in large grassland enclosures over a significant part of their adult life span and two reproductive events. Populations were assorted to contain extreme boldness types (bold or shy) of both sexes. Bolder individuals took more risks than shyer ones, which did not affect survival. Bolder males but not females produced more offspring than shy conspecifics. Daily home range and core area sizes, based on 95% and 50% Kernel density estimates (20 ± 10 per individual, n = 54 individuals), were highly repeatable over time. Individual space use unfolded differently for sex‐boldness type combinations over the course of the experiment. While day ranges decreased for shy females, they increased for bold females and all males. Space use trajectories may, hence, indicate differences in coping styles when confronted with a novel social and physical environment. Thus, interindividual differences in boldness predict risk taking under near‐natural conditions and have consequences for fitness in males, which have a higher reproductive potential than females. Given extreme inter‐ and intra‐annual fluctuations in population density in the study species and its short life span, density‐dependent fluctuating selection operating differently on the sexes might maintain (co)variation in boldness, risk taking, and pace‐of‐life.     

Top‐down and bottom‐up control of periphyton by benthivorous fish and light supply in two streams

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GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness¹. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST)^2,3 and a C-terminal 10xHis tag⁴, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression⁵. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme⁶. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl₂ and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest.     

Community structure of soil oribatida (acari) two years after windthrow in the high tatra mountains

In November 2004 a catastrophic windstorm destroyed a large part of the spruce forest in the Tatra National Park (Slovakia). The majority of the windthrown area was cleared; only a small part was left uncleared, thereby allowing regeneration by natural succession. The aim of the present study was to assess the impact of the different forestry practices on soil Oribatida. Three different stands were selected for the study, where sampling took place in June and October 2006: control forest stands (REF), windthrown stands left for natural development (NEX) and clear-cut windthrown stands (EXT). The mean abundance of Oribatida recorded in REF and NEX stands was significantly higher than in EXT stands. Kruskal-Wallis test of mean abundance of adults as well as juveniles confirmed significant influence of treatment and date. The highest abundance of adults was found in control forest stands (REF). Post hoc multiple comparison proved significantly lower abundance of adults in clear-cut stands (EXT) compared with REF. The mean abundance of adults and juveniles was several times higher in stands left for natural development (NEX) than in EXT stands. The highest species richness was observed in REF, followed by NEX and EXT stands. Ordination method showed differences in species composition between studied treatments. Furthermore, a much lower abundance of Hermannia gibba, a dweller of leaf litter and upper soil layers, was recorded in cleared stands compared to the other stands. Indeed, windthrown stands had an obvious lower species richness than control stands. The ordination method used demonstrated a significant influence of both treatment and sampling date on the abundance and species richness of Oribatida. The present study showed that clear-cutting of wind-damaged spruce forest markedly decreases the abundance of soil Oribatida compared with windthrown forest stands left to natural succession.