RNAi‐mediated endogene silencing in strawberry fruit: detection of primary and secondary siRNAs by deep sequencing

RNA interference (RNAi) has been exploited as a reverse genetic tool for functional genomics in the nonmodel species strawberry (Fragaria × ananassa) since 2006. Here, we analysed for the first time different but overlapping nucleotide sections (>200 nt) of two endogenous genes, FaCHS (chalcone synthase) and FaOMT (O‐methyltransferase), as inducer sequences and a transitive vector system to compare their gene silencing efficiencies. In total, ten vectors were assembled each containing the nucleotide sequence of one fragment in sense and corresponding antisense orientation separated by an intron (inverted hairpin construct, ihp). All sequence fragments along the full lengths of both target genes resulted in a significant down‐regulation of the respective gene expression and related metabolite levels. Quantitative PCR data and successful application of a transitive vector system coinciding with a phenotypic change suggested propagation of the silencing signal. The spreading of the signal in strawberry fruit in the 3′ direction was shown for the first time by the detection of secondary small interfering RNAs (siRNAs) outside of the primary targets by deep sequencing. Down‐regulation of endogenes by the transitive method was less effective than silencing by ihp constructs probably because the numbers of primary siRNAs exceeded the quantity of secondary siRNAs by three orders of magnitude. Besides, we observed consistent hotspots of primary and secondary siRNA formation along the target sequence which fall within a distance of less than 200 nt. Thus, ihp vectors seem to be superior over the transitive vector system for functional genomics in strawberry fruit.     

Molecular Variability and Evolution of the Pectate Lyase (<Emphasis Type="Italic">pel</Emphasis>-2) Parasitism Gene in Cyst Nematodes Parasitizing Different <Emphasis Type="Italic">Solanaceous</Emphasis> Plants

While pectate lyases are major parasitism factors in plant-parasitic nematodes, there is little information on the variability of these genes within species and their utility as pathotype or host range molecular markers. We have analysed polymorphisms of pectate lyase 2 (pel-2) gene, which degrades the unesterified polygalacturonate (pectate) of the host cell-wall, in the genus Globodera. Molecular variability of the pel-2 gene and the predicted protein was evaluated in populations of G. rostochiensis, G. pallida, G. “mexicana” and G. tabacum. Seventy eight pel-2 sequences were obtained and aligned. Point mutations were observed at 373 positions, 57% of these affect the coding part of the gene and produce 129 aa replacements. The observed polymorphism does not correlate either to the pathotypes proposed in potato cyst nematodes (PCN) or the subspecies described in tobacco cyst nematodes. The trees reveal a topology different from the admitted species topology as G. rostochiensis and G. pallida sequences are more similar to each other than to G. tabacum. Species-specific sites, potentially applicable for identification, and sites distinguishing PCN from tobacco cyst nematodes, were identified. As both G. rostochiensis and G. pallida display the same host range, but distinct from G. tabacum, which cannot parasitize potato plants, it is tempting to speculate that pel-2 genes polymorphism may be implicated in this adaptation, a view supported by the fact that no active pectate lyase 2 was found in G. “mexicana”, a close relative of G. pallida that is unable to develop on cultivated potato varieties.     

Enhanced antioxidant formula based on a selenium-supplemented carotenoid-producing yeast biomass

Carotenoid-producing yeast species such as Rhodotorula glutinis and Sporobolomyces roseus efficiently accumulated selenium from the growth medium. It was observed that incorporation of selenium into yeast cells during the growth inhibited production of beta-carotenoid and other carotenoid precursors (torularhodin and torulene). The yeasts with high content of the carotenoid pigments and selenium may be used for the preparation of a new type of antioxidant formula that could be directly applied for various human and animal diets. We have demonstrated that such a formula can only be produced by separate processes of the cultivation of red yeasts and a subsequent sorption of selenium into the cells.     

Ligand binding induces a conformational change in ifnar1 that is propagated to its membrane-proximal domain

The type I interferon (IFN) receptor plays a key role in innate immunity against viral and bacterial infections. Here, we show by intramolecular Förster resonance energy transfer spectroscopy that ligand binding induces substantial conformational changes in the ectodomain of ifnar1 (ifnar1-EC). Binding of IFNα2 and IFNβ induce very similar conformations of ifnar1, which were confirmed by single-particle electron microscopy analysis of the ternary complexes formed by IFNα2 or IFNβ with the two receptor subunits ifnar1-EC and ifnar2-EC. Photo-induced electron-transfer-based fluorescence quenching and single-molecule fluorescence lifetime measurements revealed that the ligand-induced conformational change in the membrane-distal domains of ifnar1-EC is propagated to its membrane-proximal domain, which is not involved in ligand recognition but is essential for signal activation. Temperature-dependent ligand binding studies as well as stopped-flow fluorescence experiments corroborated a multistep conformational change in ifnar1 upon ligand binding. Our results thus suggest that the relatively intricate architecture of the type I IFN receptor complex is designed to propagate the ligand binding event to and possibly even across the membrane by conformational changes.     

Effects of common atopy-associated amino acid substitutions in the IL-4 receptor alpha chain on IL-4 induced phenotypes

The human IL-4 receptor alpha chain gene (IL4R) is highly polymorphic and controversial reports have been published with respect to the association of different single nucleotide polymorphisms (SNPs) with atopy markers. Here we analyzed the functional and associational relevance of common IL4R coding SNPs. Transfection of B cell lines expressing the IL-4R variant V75+R576 did not result in enhanced IL-4 induced CD23 expression compared to cell lines expressing the wild type IL-4R alpha chain. Transfection of the IL-4R variant P503 into a murine T cell line did not influence IL-4 induced T-cell proliferation compared to wild type constructs. Analysis of six IL4R coding SNPs (I75V, E400A, C431R, S436L, S503P, Q576R) and common haplotypes (frequency 0.05%) in blood donors (n=300) did not indicate a significant association with elevated serum IgE level. Moreover, the most informative IL4R coding SNPs (I75V, C431R, Q576R) and related two- and three-point haplotypes (frequency 0.05%) were analyzed in a second, extended group of blood donors (n=689). Again, no significant association with elevated serum IgE was detectable. We conclude that common coding SNPs in the IL4R gene are unlikely to contribute significantly to increased IgE levels and variations outside the coding region may influence atopy susceptibility.     

Multiscale Investigation of Sodium-Ion Battery Anodes: Analytical Techniques and Applications

The anode/electrolyte interface behavior, and by extension, the overall cell performance of sodium-ion batteries is determined by a complex interaction of processes that occur at all components of the electrochemical cell across a wide range of size- and timescales. Single-scale studies may provide incomplete insights, as they cannot capture the full picture of this complex and intertwined behavior. Broad, multiscale studies are essential to elucidate these processes. Within this perspectives article, several analytical and theoretical techniques are introduced, and described how they can be combined to provide a more complete and comprehensive understanding of sodium-ion battery (SIB) performance throughout its lifetime, with a special focus on the interfaces of hard carbon anodes. These methods target various length- and time scales, ranging from micro to nano, from cell level to atomistic structures, and account for a broad spectrum of physical and (electro)chemical characteristics. Specifically, how mass spectrometric, microscopic, spectroscopic, electrochemical, thermodynamic, and physical methods can be employed to obtain the various types of information required to understand battery behavior will be explored. Ways are then discussed how these methods can be coupled together in order to elucidate the multiscale phenomena at the anode interface and develop a holistic understanding of their relationship to overall sodium-ion battery function.