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    Testing the plant pneumatic method to estimate xylem embolism resistance in stems of temperate trees 

    Zhang, Ya; Lamarque, Laurent J.; Torres-Ruiz, José M.; Schuldt, Bernhard; Karimi, Zohreh; Li, Shan; Qin, De-Wen; Bittencourt, Paulo; Burlett, Régis; Cao, Kun-Fang; et al.
    Delzon, SylvainOliveira, RafaelPereira, LucianoJansen, Steven
    Tree Physiology 2018; 38(7) p.1016-1025
    Methods to estimate xylem embolism resistance generally rely on hydraulic measurements, which can be far from straightforward. Recently, a pneumatic method based on air flow measurements of terminal branch ends was proposed to construct vulnerability curves by linking the amount of air extracted from a branch with the degree of embolism. We applied this novel technique for 10 temperate tree species, including six diffuse, two ring-porous and two gymnosperm species, and compared the pneumatic curves with hydraulic ones obtained from either the flow-centrifuge or the hydraulic-bench dehydration method. We found that the pneumatic method provides a good estimate of the degree of xylem embolism for all angiosperm species. The xylem pressure at 50% and 88% loss of hydraulic conductivity (i.e., Ψ50 and Ψ88) based on the methods applied showed a strongly significant correlation for all eight angiosperms. However, the pneumatic method showed significantly reduced Ψ50 values for the two conifers. Our findings suggest that the pneumatic method could provide a fast and accurate approach for angiosperms due to its convenience and feasibility, at least within the range of embolism resistances covered by our samples.
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    Velvet domain protein VosA represses the zinc cluster transcription factor SclB regulatory network for Aspergillus nidulans asexual development, oxidative stress response and secondary metabolism. 

    Thieme, Karl G.; Gerke, Jennifer; Sasse, Christoph; Valerius, Oliver; Thieme, Sabine; Karimi, Razieh; Heinrich, Antje K.; Finkernagel, Florian; Smith, Kristina; Bode, Helge B.; et al.
    Freitag, MichaelRam, Arthur F. J.Braus, Gerhard H.
    PLOS Genetics 2018; 14(7): Art. e1007511
    The NF-κB-like velvet domain protein VosA (viability of spores) binds to more than 1,500 promoter sequences in the filamentous fungus Aspergillus nidulans. VosA inhibits premature induction of the developmental activator gene brlA, which promotes asexual spore formation in response to environmental cues as light. VosA represses a novel genetic network controlled by the sclB gene. SclB function is antagonistic to VosA, because it induces the expression of early activator genes of asexual differentiation as flbC and flbD as well as brlA. The SclB controlled network promotes asexual development and spore viability, but is independent of the fungal light control. SclB interactions with the RcoA transcriptional repressor subunit suggest additional inhibitory functions on transcription. SclB links asexual spore formation to the synthesis of secondary metabolites including emericellamides, austinol as well as dehydroaustinol and activates the oxidative stress response of the fungus. The fungal VosA-SclB regulatory system of transcription includes a VosA control of the sclB promoter, common and opposite VosA and SclB control functions of fungal development and several additional regulatory genes. The relationship between VosA and SclB illustrates the presence of a convoluted surveillance apparatus of transcriptional control, which is required for accurate fungal development and the linkage to the appropriate secondary metabolism.
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    Crystal Structure of the Human tRNA Guanine Transglycosylase Catalytic Subunit QTRT1 

    Johannsson, Sven; Neumann, Piotr; Ficner, Ralf
    Biomolecules 2018; 8(3): Art. 81
    RNA modifications have been implicated in diverse and important roles in all kingdoms of life with over 100 of them present on tRNAs. A prominent modification at the wobble base of four tRNAs is the 7-deaza-guanine derivative queuine which substitutes the guanine at position 34. This exchange is catalyzed by members of the enzyme class of tRNA guanine transglycosylases (TGTs). These enzymes incorporate guanine substituents into tRNAAsp, tRNAAsn tRNAHis, and tRNATyr in all kingdoms of life. In contrast to the homodimeric bacterial TGT, the active eukaryotic TGT is a heterodimer in solution, comprised of a catalytic QTRT1 subunit and a noncatalytic QTRT2 subunit. Bacterial TGT enzymes, that incorporate a queuine precursor, have been identified or proposed as virulence factors for infections by pathogens in humans and therefore are valuable targets for drug design. To date no structure of a eukaryotic catalytic subunit is reported, and differences to its bacterial counterpart have to be deducted from sequence analysis and models. Here we report the first crystal structure of a eukaryotic QTRT1 subunit and compare it to known structures of the bacterial TGT and murine QTRT2. Furthermore, we were able to determine the crystal structure of QTRT1 in complex with the queuine substrate.
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    Comparative Genomics and Description of Putative Virulence Factors of Melissococcus plutonius, the Causative Agent of European Foulbrood Disease in Honey Bees 

    Djukic, Marvin; Erler, Silvio; Leimbach, Andreas; Grossar, Daniela; Charrière, Jean-Daniel; Gauthier, Laurent; Hartken, Denise; Dietrich, Sascha; Nacke, Heiko; Daniel, Rolf; et al.
    Poehlein, Anja
    Genes 2018; 9(8): Art. 419
    In Europe, approximately 84% of cultivated crop species depend on insect pollinators, mainly bees. Apis mellifera (the Western honey bee) is the most important commercial pollinator worldwide. The Gram-positive bacterium Melissococcus plutonius is the causative agent of European foulbrood (EFB), a global honey bee brood disease. In order to detect putative virulence factors, we sequenced and analyzed the genomes of 14 M. plutonius strains, including two reference isolates. The isolates do not show a high diversity in genome size or number of predicted protein-encoding genes, ranging from 2.021 to 2.101 Mbp and 1589 to 1686, respectively. Comparative genomics detected genes that might play a role in EFB pathogenesis and ultimately in the death of the honey bee larvae. These include bacteriocins, bacteria cell surface- and host cell adhesion-associated proteins, an enterococcal polysaccharide antigen, an epsilon toxin, proteolytic enzymes, and capsule-associated proteins. In vivo expression of three putative virulence factors (endo-alpha-N-acetylgalactosaminidase, enhancin and epsilon toxin) was verified using naturally infected larvae. With our strain collection, we show for the first time that genomic differences exist between non-virulent and virulent typical strains, as well as a highly virulent atypical strain, that may contribute to the virulence of M. plutonius. Finally, we also detected a high number of conserved pseudogenes (75 to 156) per genome, which indicates genomic reduction during evolutionary host adaptation.
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    Establishment of Apomixis in Diploid F2 Hybrids and Inheritance of Apospory From F1 to F2 Hybrids of the Ranunculus auricomus Complex 

    Barke, Birthe H.; Daubert, Mareike; Hörandl, Elvira
    Frontiers in Plant Science 2018; 9: Art. 1111
    Hybridization and polyploidization play important roles in plant evolution but it is still not fully clarified how these evolutionary forces contribute to the establishment of apomicts. Apomixis, the asexual reproduction via seed formation, comprises several essential alterations in development compared to the sexual pathway. Furthermore, most natural apomicts were found to be polyploids and/or hybrids. The Ranunculus auricomus complex comprises diploid sexual and polyploid apomictic species and represents an excellent model system to gain knowledge on origin and evolution of apomixis in natural plant populations. In this study, the second generation of synthetically produced homoploid (2x) and heteroploid (3x) hybrids derived from sexual R. auricomus species was analyzed for aposporous initial cell formation by DIC microscopy. Complete manifestation of apomixis was determined by measuring single mature seeds by flow cytometric seed screen. Microscopic analysis of the female gametophyte formation indicated spontaneous occurrence of aposporous initial cells and several developmental irregularities. The frequency of apospory was found to depend on dosage effects since a significant increase in apospory was observed, when both F1 parents, rather than just one, were aposporous. Other than in the F1 generation, diploid Ranunculus F2 hybrids formed BIII seeds and fully apomictic seeds. The results indicate that hybridization rather than polyploidization seems to be the functional activator of apomictic reproduction in the synthetic Ranunculus hybrids. In turn, at least two hybrid generations are required to establish apomictic seed formation.
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    Gender Differences in the Recognition of Vocal Emotions 

    Lausen, Adi; Schacht, Annekathrin
    Frontiers in Psychology 2018; 9: Art. 882
    The conflicting findings from the few studies conducted with regard to gender differences in the recognition of vocal expressions of emotion have left the exact nature of these differences unclear. Several investigators have argued that a comprehensive understanding of gender differences in vocal emotion recognition can only be achieved by replicating these studies while accounting for influential factors such as stimulus type, gender-balanced samples, number of encoders, decoders, and emotional categories. This study aimed to account for these factors by investigating whether emotion recognition from vocal expressions differs as a function of both listeners' and speakers' gender. A total of N = 290 participants were randomly and equally allocated to two groups. One group listened to words and pseudo-words, while the other group listened to sentences and affect bursts. Participants were asked to categorize the stimuli with respect to the expressed emotions in a fixed-choice response format. Overall, females were more accurate than males when decoding vocal emotions, however, when testing for specific emotions these differences were small in magnitude. Speakers' gender had a significant impact on how listeners' judged emotions from the voice. The group listening to words and pseudo-words had higher identification rates for emotions spoken by male than by female actors, whereas in the group listening to sentences and affect bursts the identification rates were higher when emotions were uttered by female than male actors. The mixed pattern for emotion-specific effects, however, indicates that, in the vocal channel, the reliability of emotion judgments is not systematically influenced by speakers' gender and the related stereotypes of emotional expressivity. Together, these results extend previous findings by showing effects of listeners' and speakers' gender on the recognition of vocal emotions. They stress the importance of distinguishing these factors to explain recognition ability in the processing of emotional prosody.
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    Heavy Metal-Induced Expression of PcaA Provides Cadmium Tolerance to Aspergillus fumigatus and Supports Its Virulence in the Galleria mellonella Model 

    Bakti, Fruzsina; Sasse, Christoph; Heinekamp, Thorsten; Pócsi, István; Braus, Gerhard H.
    Frontiers in Microbiology 2018; 9: Art. 744
    Most of the metal transporters in Aspergillus fumigatus are yet uncharacterized. Their role in fungal metabolism and virulence remains unclear. This paper describes the novel PIB-type cation ATPase PcaA, which links metal homeostasis and heavy metal tolerance in the opportunistic human pathogen A. fumigatus. The protein possesses conserved ATPase motif and shares 51% amino acid sequence identity with the Saccharomyces cerevisiae cadmium exporter Pca1p. A pcaA deletion, an overexpression and a gfp-pcaA complementation strain of A. fumigatus were constructed and their heavy metal susceptibilities were studied. The pcaA knock out strain showed drastically decreased cadmium tolerance, however, its growth was not affected by the exposure to high concentrations of copper, iron, zinc, or silver ions. Although the lack of PcaA had no effect on copper adaption, we demonstrated that not only cadmium but also copper ions are able to induce the transcription of pcaA in A. fumigatus wild type Af293. Similarly, cadmium and copper ions could induce the copper exporting ATPase crpA. These data imply a general response on the transcriptomic level to heavy metals in A. fumigatus through the induction of detoxification systems. Confocal microscopy of the gfp-pcaA complementation strain expressing functional GFP-PcaA supports the predicted membrane localization of PcaA. The GFP-PcaA fusion protein is located in the plasma membrane of A. fumigatus in the presence of cadmium ions. Virulence assays support a function of PcaA for virulence of A. fumigatus in the Galleria mellonella wax moth larvae model, which might be linked to the elimination of reactive oxygen species.
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    Changes in the microstructure of compact and trabecular bone tissues of mice subchronically exposed to alcohol 

    Martiniakova, Monika; Sarocka, Anna; Babosova, Ramona; Grosskopf, Birgit; Kapusta, Edyta; Goc, Zofia; Formicki, Grzegorz; Omelka, Radoslav
    2018; 25(1): Art. 8
    Background: Alcohol is one of the most commonly consumed neurotoxins by humans. Its negative effect on bone health is known for a long time. However, its impact on qualitative and quantitative 2D characteristics of the compact bone is still unclear. Therefore, the aim of this study was to investigate in detail the effects of subchronic alcohol exposure on compact and trabecular bone tissues microstructure of laboratory mice using 2D and 3D imaging methods. Ten clinically healthy 12 weeks-old mice (males) were randomly divided into two groups. Animals from experimental group (group E; n = 5) drank a solution composed of 15% ethanol and water (1.7 g 100% ethanol kg-1 b.w. per day) for 8 weeks, while those from control group (group C; n = 5) drank only water. Results: Subchronic exposure to alcohol leads to several changes in qualitative 2D characteristics of the compact bone such as the presence of primary vascular radial bone tissue in pars anterior of endosteal border and a higher number of resorption lacunae (five times more) in the middle part of substantia compacta. Morphometrical 2D evaluations of the compact bone showed significantly increased sizes of primary osteons' vascular canals (p < 0.05) in mice from the experimental group (E group). Sizes of Haversian canals and secondary osteons were not affected by alcohol consumption. In mice from the E group, significantly lower values for relative bone volume and bone mineral density of the compact bone were observed. In the trabecular bone, decreased values for bone volume, trabecular number, trabecular thickness and bone surface (p < 0.05) were documented. Conclusions: Alcohol decreased not only bone volume and density of the compact bone, but it also reduced trabecular bone volume and leads to trabecular thinning. It caused vasodilation of primary osteons' vascular canals and increased porosity in the compact bone.
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    A CRISPR-Cas9-Based Toolkit for Fast and Precise In Vivo Genetic Engineering of Bacillus subtilis Phages 

    Schilling, Tobias; Dietrich, Sascha; Hoppert, Michael; Hertel, Robert
    Viruses 2018; 10(5): Art. 241
    Phages are currently under discussion as a solution for the antibiotic crisis, as they may cure diseases caused by multi-drug-resistant pathogens. However, knowledge of phage biology and genetics is limited, which impedes risk assessment of therapeutic applications. In order to enable advances in phage genetic research, the aim of this work was to create a toolkit for simple and fast genetic engineering of phages recruiting Bacillus subtilis as host system. The model organism B. subtilis represents a non-pathogenic surrogate of its harmful relatives, such as Bacillus anthracis or Bacillus cereus. This toolkit comprises the application CutSPR, a bioinformatic tool for rapid primer design, and facilitates the cloning of specific CRISPR-Cas9-based mutagenesis plasmids. The employment of the prophage-free and super-competent B. subtilis TS01 strain enables an easy and fast introduction of specific constructs for in vivo phage mutagenesis. Clean gene deletions and a functional clean gene insertion into the genome of the model phage vB_BsuP-Goe1 served as proof of concept and demonstrate reliability and high efficiency. The here presented toolkit allows comprehensive investigation of the diverse phage genetic pool, a better understanding of phage biology, and safe phage applications.
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    Reconstructing geographical parthenogenesis: effects of niche differentiation and reproductive mode on Holocene range expansion of an alpine plant. 

    Kirchheimer, Bernhard; Wessely, Johannes; Gattringer, Andreas; Hülber, Karl; Moser, Dietmar; Schinkel, Christoph C. F.; Appelhans, Marc; Klatt, Simone; Caccianiga, Marco; Dellinger, Agnes; et al.
    Guisan, AntoineKuttner, MichaelLenoir, JonathanMaiorano, LuigiNieto-Lugilde, DiegoPlutzar, ChristophSvenning, Jens-ChristianWillner, WolfgangHörandl, ElviraDullinger, Stefan
    Ecology Letters 2018; 21(3) p.392-401
    Asexual taxa often have larger ranges than their sexual progenitors, particularly in areas affected by Pleistocene glaciations. The reasons given for this 'geographical parthenogenesis' are contentious, with expansion of the ecological niche or colonisation advantages of uniparental reproduction assumed most important in case of plants. Here, we parameterized a spread model for the alpine buttercup Ranunculus kuepferi and reconstructed the joint Holocene range expansion of its sexual and apomictic cytotype across the European Alps under different simulation settings. We found that, rather than niche broadening or a higher migration rate, a shift of the apomict's niche towards colder conditions per se was crucial as it facilitated overcoming of topographical barriers, a factor likely relevant for many alpine apomicts. More generally, our simulations suggest potentially strong interacting effects of niche differentiation and reproductive modes on range formation of related sexual and asexual taxa arising from their differential sensitivity to minority cytotype disadvantage.
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    A toolkit for Nannochloropsis oceanica CCMP1779 enables gene stacking and genetic engineering of the eicosapentaenoic acid pathway for enhanced long-chain polyunsaturated fatty acid production 

    Poliner, Eric; Pulman, Jane A.; Zienkiewicz, Krzysztof; Childs, Kevin; Benning, Christoph; Farré, Eva M.
    Plant Biotechnology Journal 2018; 16(1) p.298-309
    Nannochloropsis oceanica is an oleaginous microalga rich in ω3 long-chain polyunsaturated fatty acids (LC-PUFAs) content, in the form of eicosapentaenoic acid (EPA). We identified the enzymes involved in LC-PUFA biosynthesis in N. oceanica CCMP1779 and generated multigene expression vectors aiming at increasing LC-PUFA content in vivo. We isolated the cDNAs encoding four fatty acid desaturases (FAD) and determined their function by heterologous expression in S. cerevisiae. To increase the expression of multiple fatty acid desaturases in N. oceanica CCMP1779, we developed a genetic engineering toolkit that includes an endogenous bidirectional promoter and optimized peptide bond skipping 2A peptides. The toolkit also includes multiple epitopes for tagged fusion protein production and two antibiotic resistance genes. We applied this toolkit, towards building a gene stacking system for N. oceanica that consists of two vector series, pNOC-OX and pNOC-stacked. These tools for genetic engineering were employed to test the effects of the overproduction of one, two or three desaturase-encoding cDNAs in N. oceanica CCMP1779 and prove the feasibility of gene stacking in this genetically tractable oleaginous microalga. All FAD overexpressing lines had considerable increases in the proportion of LC-PUFAs, with the overexpression of Δ12 and Δ5 FAD encoding sequences leading to an increase in the final ω3 product, EPA.
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    Cyanophage-encoded lipid desaturases: oceanic distribution, diversity and function 

    Roitman, Sheila; Hornung, Ellen; Flores-Uribe, José; Sharon, Itai; Feussner, Ivo; Béjà, Oded
    The ISME Journal 2018; 12(2) p.343-355
    Cyanobacteria are among the most abundant photosynthetic organisms in the oceans; viruses infecting cyanobacteria (cyanophages) can alter cyanobacterial populations, and therefore affect the local food web and global biochemical cycles. These phages carry auxiliary metabolic genes (AMGs), which rewire various metabolic pathways in the infected host cell, resulting in increased phage fitness. Coping with stress resulting from photodamage appears to be a central necessity of cyanophages, yet the overall mechanism is poorly understood. Here we report a novel, widespread cyanophage AMG, encoding a fatty acid desaturase (FAD), found in two genotypes with distinct geographical distribution. FADs are capable of modulating the fluidity of the host's membrane, a fundamental stress response in living cells. We show that both viral FAD (vFAD) families are Δ9 lipid desaturases, catalyzing the desaturation at carbon 9 in C16 fatty acid chains. In addition, we present a comprehensive fatty acid profiling for marine cyanobacteria, which suggests a unique desaturation pathway of medium- to long-chain fatty acids no longer than C16, in accordance with the vFAD activity. Our findings suggest that cyanophages are capable of fiddling with the infected host's membranes, possibly leading to increased photoprotection and potentially enhancing viral-encoded photosynthetic proteins, resulting in a new viral metabolic network.
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    Dendrochronologically dated pine stumps document phase-wise bog expansion at a northwest German site between ca. 6700 and ca. 3400 BC 

    Achterberg, Inke Elisabeth Maike; Eckstein, Jan; Birkholz, Bernhard; Bauerochse, Andreas; Leuschner, Hanns Hubert
    Climate of the Past 2018; 14(1) p.85-100
    The investigated northwest German mire site at "Totes Moor" is densely covered with subfossil pine stumps (Pinus sylvestris L.) from the fen–bog transition. This facilitates the spatio-temporal reconstruction of mire development, which is based on 212 in situ tree stumps in the case study presented here. Six dendrochronologically dated site chronologies together cover 2345 years between 6703 and 3403 BC. The gaps in between are 6 to 550 years long. Additionally, a floating chronology of 309 years, containing 30 trees, was radiocarbon-dated to the beginning of the 7th millennium cal BC. Peat-stratigraphical survey was carried out additionally, and elevations a.s.l. were determined at several locations. Tree dying-off phases, which indicate water level rise at the site, mostly in context of the local fen–bog transition, are evident for ca. 6600–6450, ca. 6350–5750, ca. 5300–4900, ca. 4700–4550, ca. 3900–3850, ca. 3700–3600, ca. 3500–3450 and ca. 3400 BC. The spatial distribution of the dated in situ trees illustrates the phase-wise expansion of raised bog over fen peat at the site. The documented bog expansion pulses likely correspond to climatic wet sifts.
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    Long-Term Outcomes of a Multimodal Day-Clinic Treatment for Chronic Pain under the Conditions of Routine Care 

    Preis, Mira A.; Vögtle, Elisabeth; Dreyer, Nele; Seel, Stefanie; Wagner, Ruth; Hanshans, Klaus; Reyersbach, Renate; Pieh, Christoph; Mühlberger, Andreas; Probst, Thomas
    Pain Research and Management 2018; 2018 p.1-7
    Chronic pain has high prevalence rates and is one of the top causes of years lived with disability. The aim of the present study was to evaluate the long-term effects of a multimodal day-clinic treatment for chronic pain. The sample included 183 chronic pain patients (114 females and 69 males; 53.3 ± 9.8 years) who participated in a four-week multimodal day-clinic treatment for chronic pain. The patients’ average current pain intensity (NRS), sensory and affective pain (Pain Perception Scale), and depression and anxiety (HADS) were assessed at pre- and posttreatment, as well as at three follow-ups (one month, six months, and twelve months after completion of the treatment). Multilevel models for discontinuous change were performed to evaluate the change of the outcome variables. Improvements from pretreatment to posttreatment and from pretreatment to all follow-ups emerged for pain intensity (NRS; 0.54 ≤ d ≤ 0.74), affective pain (Pain Perception Scale; 0.24 ≤ d ≤ 0.47), depression (HADS; 0.38 ≤ d ≤ 0.53), and anxiety (HADS; 0.26 ≤ d ≤ 0.43) (all ). Sensory pain as assessed with the Pain Perception Scale did not show any significant change. Patients suffering from chronic pain benefited from the multimodal pain treatment up to twelve months after completion of the treatment.
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    High-level accumulation of oleyl oleate in plant seed oil by abundant supply of oleic acid substrates to efficient wax ester synthesis enzymes. 

    Yu, Dan; Hornung, Ellen; Iven, Tim; Feussner, Ivo
    Biotechnology for Biofuels 2018; 11 p.1-14: Art. 53
    Background: Biotechnology enables the production of high-valued industrial feedstocks from plant seed oil. The plant-derived wax esters with long-chain monounsaturated acyl moieties, like oleyl oleate, have favorite properties for lubrication. For biosynthesis of wax esters using acyl-CoA substrates, expressions of a fatty acyl reductase (FAR) and a wax synthase (WS) in seeds are sufficient. Results: For optimization of the enzymatic activity and subcellular localization of wax ester synthesis enzymes, two fusion proteins were created, which showed wax ester-forming activities in Saccharomyces cerevisiae. To promote the formation of oleyl oleate in seed oil, WSs from Acinetobactor baylyi (AbWSD1) and Marinobacter aquaeolei (MaWS2), as well as the two created fusion proteins were tested in Arabidopsis to evaluate their abilities and substrate preference for wax ester production. The tested seven enzyme combinations resulted in different yields and compositions of wax esters. Expression of a FAR of Marinobacter aquaeolei (MaFAR) with AbWSD1 or MaWS2 led to a high incorporation of C18 substrates in wax esters. The MaFAR/TMMmAWAT2-AbWSD1 combination resulted in the incorporation of more C18:1 alcohol and C18:0 acyl moieties into wax esters compared with MaFAR/AbWSD1. The fusion protein of a WS from Simmondsia chinensis (ScWS) with MaFAR exhibited higher specificity toward C20:1 substrates in preference to C18:1 substrates. Expression of MaFAR/AbWSD1 in the Arabidopsis fad2 fae1 double mutant resulted in the accumulation of oleyl oleate (18:1/18:1) in up to 62 mol% of total wax esters in seed oil, which was much higher than the 15 mol% reached by MaFAR/AbWSD1 in Arabidopsis Col-0 background. In order to increase the level of oleyl oleate in seed oil of Camelina, lines expressing MaFAR/ScWS were crossed with a transgenic high oleate line. The resulting plants accumulated up to >40 mg g seed-1 of wax esters, containing 27-34 mol% oleyl oleate. Conclusions: The overall yields and the compositions of wax esters can be strongly affected by the availability of acyl-CoA substrates and to a lesser extent, by the characteristics of wax ester synthesis enzymes. For synthesis of oleyl oleate in plant seed oil, appropriate wax ester synthesis enzymes with high catalytic efficiency and desired substrate specificity should be expressed in plant cells; meanwhile, high levels of oleic acid-derived substrates need to be supplied to these enzymes by modifying the fatty acid profile of developing seeds.
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    Design and Manual to Construct Rainout-Shelters for Climate Change Experiments in Agroecosystems 

    Kundel, Dominika; Meyer, Svenja; Birkhofer, Herbert; Fliessbach, Andreas; Mäder, Paul; Scheu, Stefan; van Kleunen, Mark; Birkhofer, Klaus
    Frontiers in Environmental Science 2018; 6: Art. 14
    Climate change models predict reduced summer precipitations for most European countries, including more frequent and extreme summer droughts. Rainout-shelters which intercept part of the natural precipitation provide an effective tool to investigate effects of different precipitation levels on biodiversity and ecosystem functioning. In this study, we evaluate and describe in detail a fixed-location rainout-shelter (2.5 × 2.5 m) with partial interception of natural rainfall. We provide a complete parts list, a construction manual and detailed CAD drawings allowing to rebuild and use these shelters for rainfall manipulation studies. In addition, we describe a rainout-shelter control treatment giving the possibility to quantify and account for potential shelter artifacts. To test the rainout-shelters, we established the following three treatments each in eight winter wheat plots of the agricultural long-term farming system comparison trial DOK in Therwil (Switzerland): (1) A rainout-shelter with 65% interception of rainfall, (2) a rainout-shelter control without interception of rainfall, and (3) an ambient control. The rainout-shelter effectively excluded 64.9% of the ambient rainfall, which is very close to the a priori calculated exclusion of 65.1%. In comparison to the ambient control plots, gravimetric soil moisture decreased under the rainout-shelter by a maximum of 11.1 percentage points. Air temperature under the rainout-shelter differed little from the ambient control (−0.55◦C in 1.2 m height and +0.19◦C in 0.1 m height), whereas soil temperatures were slightly higher in periods of high ambient temperature (+1.02◦C), but remained basically unaffected in periods of low ambient temperature (+0.14◦C). A maximum edge effect of 0.75 m defined a sampling area of 1 × 1 m under the rainout-shelter. The rainout-shelters presented here, proved to sustain under heavy weather and they were well-suited to be used in agricultural fields where management operations require the removal of the rainout-shelters for management operations. Overall, the results confirmed the good performance of the presented rainout-shelters regarding rainout-shelter artifacts, predictable rain exclusion, and feasibility for experimental studies in agricultural fields.
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    The 'forma specialis' issue in Fusarium: A case study in Fusarium solani f. sp. pisi 

    Šišić, Adnan; Baćanović-Šišić, Jelena; Al-Hatmi, Abdullah M. S.; Karlovsky, Petr; Ahmed, Sarah A.; Maier, Wolfgang; de Hoog, G. Sybren; Finckh, Maria R.
    Scientific Reports 2018; 8(1) p.1252-1252
    The Fusarium solani species complex (FSSC) has been studied intensively but its association with legumes, particularly under European agro-climatic conditions, is still poorly understood. In the present study, we investigated phylogenetic relationships and aggressiveness of 79 isolates of the FSSC collected from pea, subterranean clover, white clover and winter vetch grown under diverse agro-climatic and soil conditions within Temperate and Mediterranean Europe. The isolates were characterized by sequencing tef1 and rpb2 loci and by greenhouse aggressiveness assays. The majority of the isolates belonged to two lineages: the F. pisi comb. nov. lineage (formerly F. solani f. sp. pisi) mainly accommodating German and Swiss isolates, and the Fusisporium (Fusarium) solani lineage accommodating mainly Italian isolates. Based on the results of aggressiveness tests on pea, most of the isolates were classified as weakly to moderately aggressive. In addition, using one model strain, 62 accessions of 10 legume genera were evaluated for their potential to host F. pisi, the species known mainly as a pathogen of pea. A total of 58 accessions were colonized, with 25 of these being asymptomatic hosts. These results suggest a broad host range for F. pisi and challenge the forma specialis naming system in Fusarium.
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    Allene oxide synthase, allene oxide cyclase and jasmonic acid levels in Lotus japonicus nodules. 

    Zdyb, Anna; Salgado, Marco G.; Demchenko, Kirill N.; Brenner, Wolfram G.; Płaszczyca, Małgorzata; Stumpe, Michael; Herrfurth, Cornelia; Feussner, Ivo; Pawlowski, Katharina
    PLOS ONE 2018; 13(1): Art. e0190884
    Jasmonic acid (JA), its derivatives and its precursor cis-12-oxo phytodienoic acid (OPDA) form a group of phytohormones, the jasmonates, representing signal molecules involved in plant stress responses, in the defense against pathogens as well as in development. Elevated levels of JA have been shown to play a role in arbuscular mycorrhiza and in the induction of nitrogen-fixing root nodules. In this study, the gene families of two committed enzymes of the JA biosynthetic pathway, allene oxide synthase (AOS) and allene oxide cyclase (AOC), were characterized in the determinate nodule-forming model legume Lotus japonicus JA levels were to be analysed in the course of nodulation. Since in all L. japonicus organs examined, JA levels increased upon mechanical disturbance and wounding, an aeroponic culture system was established to allow for a quick harvest, followed by the analysis of JA levels in whole root and shoot systems. Nodulated plants were compared with non-nodulated plants grown on nitrate or ammonium as N source, respectively, over a five week-period. JA levels turned out to be more or less stable independently of the growth conditions. However, L. japonicus nodules formed on aeroponically grown plants often showed patches of cells with reduced bacteroid density, presumably a stress symptom. Immunolocalization using a heterologous antibody showed that the vascular systems of these nodules also seemed to contain less AOC protein than those of nodules of plants grown in perlite/vermiculite. Hence, aeroponically grown L. japonicus plants are likely to be habituated to stress which could have affected JA levels.
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    Transient Activation of Apomixis in Sexual Neotriploids May Retain Genomically Altered States and Enhance Polyploid Establishment 

    Hojsgaard, Diego
    Frontiers in Plant Science 2018; 9: Art. 230
    Polyploid genomes evolve and follow a series of dynamic transfigurations along with adaptation and speciation. The initial formation of a new polyploid individual within a diploid population usually involves a triploid bridge, a two-step mechanism of cell fusions between ubiquitous (reduced) and rare (unreduced) gametes. The primary fusion event creates an intermediate triploid individual with unbalanced genome sets, a situation of genomic-shock characterized by gene expression dysregulation, high dosage sensitivity, disturbed cell divisions, and physiological and reproductive attributes drastically altered. This near-sterile neotriploid must produce (even) eupolyploids through secondary fusion events to restore genome steadiness, meiotic balance, and fertility required for the demographic establishment of a nascent lineage. Natural conditions locate several difficulties to polyploid establishment, including the production of highly unbalanced and rarely unreduced (euploid) gametes, frequency-dependent disadvantages (minority cytotype exclusion), severe fitness loss, and ecological competition with diploid parents. Persistence and adaptation of neopolyploids depend upon genetic and phenotypic novelty coupled to joint selective forces that preserve shock-induced genomic changes (subgenome homeolog partitioning) and drive meiotic (reproductive) stabilization and ecological diversification. Thus, polyploid establishment through the triploid bridge is a feasible but not ubiquitous process that requires a number of low-probability events and singular circumstances. Yet, frequencies of polyploids suggest that polyploid establishment is a pervasive process. To explain this disparity, and supported in experimental evidence, I propose that situations like hybridization and ploidy-state transitions associated to genomic shock and substantial developmental alterations can transiently activate apomixis as a mechanism to halt genomic instability and cancel factors restraining neopolyploid’s sexual fertility, particularly in triploids. Apomixis –as a temporal alternative to sex– skip meiosis and syngamy, and thus can freeze genomic attributes, avoid unbalanced chromosomal segregation and increase the formation of unreduced euploid gametes, elude frequency-dependent reproductive disadvantages by parthenogenetic development of the embryo and permissive development of endosperm during seed formation, and increase the effective population size of the neopolyploid lineage favoring the formation rate of eupolyploids compared to aneuploids. The subsequent action of genome resilience mechanisms that alleviate transcriptomic shock and selection upon gene interactions might restore a stable meiosis and sexual fertility within few generations, as observed in synthetic polyploids. Alternatively, provided that resilience mechanisms fail, the neopolyploid might retain apomixis and hold genomically and transcriptionally altered states for many generations.
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    Comparative genome and phenotypic analysis of three Clostridioides difficile strains isolated from a single patient provide insight into multiple infection of C. difficile. 

    Groß, Uwe; Brzuszkiewicz, Elzbieta; Gunka, Katrin; Starke, Jessica; Riedel, Thomas; Bunk, Boyke; Spröer, Cathrin; Wetzel, Daniela; Poehlein, Anja; Chibani, Cynthia; et al.
    Bohne, WolfgangOvermann, JörgZimmermann, OrtrudDaniel, RolfLiesegang, Heiko
    BMC Genomics 2018; 19(1) p.1-14
    BACKGROUND: Clostridioides difficile infections (CDI) have emerged over the past decade causing symptoms that range from mild, antibiotic-associated diarrhea (AAD) to life-threatening toxic megacolon. In this study, we describe a multiple and isochronal (mixed) CDI caused by the isolates DSM 27638, DSM 27639 and DSM 27640 that already initially showed different morphotypes on solid media. RESULTS: The three isolates belonging to the ribotypes (RT) 012 (DSM 27639) and 027 (DSM 27638 and DSM 27640) were phenotypically characterized and high quality closed genome sequences were generated. The genomes were compared with seven reference strains including three strains of the RT 027, two of the RT 017, and one of the RT 078 as well as a multi-resistant RT 012 strain. The analysis of horizontal gene transfer events revealed gene acquisition incidents that sort the strains within the time line of the spread of their RTs within Germany. We could show as well that horizontal gene transfer between the members of different RTs occurred within this multiple infection. In addition, acquisition and exchange of virulence-related features including antibiotic resistance genes were observed. Analysis of the two genomes assigned to RT 027 revealed three single nucleotide polymorphisms (SNPs) and apparently a regional genome modification within the flagellar switch that regulates the fli operon. CONCLUSION: Our findings show that (i) evolutionary events based on horizontal gene transfer occur within an ongoing CDI and contribute to the adaptation of the species by the introduction of new genes into the genomes, (ii) within a multiple infection of a single patient the exchange of genetic material was responsible for a much higher genome variation than the observed SNPs.
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