The ROSA26-iPSC Mouse: A Conditional,Inducible, and Exchangeable Resource for Studying Cellular (De)Differentiation

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Highlights? Conditional and inducible reprogrammable mouse model presented ? Allows removal of the Yamanaka factors after reprogramming ? Fully and partially reprogrammed endothelial cells generated ? Lineage-directed differentiation of iPSCs toward beating cardiomyocytes     

Quasispecies Analysis of JC Virus DNA Present in Urine of Healthy Subjects

JC virus is a human polyomavirus that infects the majority of people without apparent symptoms in healthy subjects and it is the causative agent of progressive multifocal leucoencephalopathy (PML), a disorder following lytic infection of oligodendrocytes that mainly manifests itself under immunosuppressive conditions. A hallmark for JC virus isolated from PML-brain is the presence of rearrangements in the non-coding control region (NCCR) interspersed between the early and late genes on the viral genome. Such rearrangements are believed to originate from the archetype JC virus which is shed in urine by healthy subjects and PML patients. We applied next generation sequencing to explore the non-coding control region variability in urine of healthy subjects in search for JC virus quasispecies and rearrangements reminiscent of PML. For 61 viral shedders (out of a total of 254 healthy subjects) non-coding control region DNA and VP1 (major capsid protein) coding sequences were initially obtained by Sanger sequencing. Deletions between 1 and 28 nucleotides long appeared in ∼24.5% of the NCCR sequences while insertions were only detected in ∼3.3% of the samples. 454 pyrosequencing was applied on a subset of 54 urine samples demonstrating the existence of JC virus quasispecies in four subjects (∼7.4%). Hence, our results indicate that JC virus DNA in urine is not always restricted to one unique virus variant, but can be a mixture of naturally occurring variants (quasispecies) reflecting the susceptibility of the non-coding control region for genomic rearrangements in healthy individuals. Our findings pave the way to explore the presence of viral quasispecies and the altered viral tropism that might go along with it as a potential risk factor for opportunistic secondary infections such as PML.     

Chloroplasts assemble the major subunit FaeG of Escherichia coli F4 (K88) fimbriae to strand-swapped dimers

F4 fimbriae encoded by the fae operon are the major colonization factors associated with porcine neonatal and postweaning diarrhoea caused by enterotoxigenic Escherichia coli (ETEC). Via the chaperone/usher pathway, the F4 fimbriae are assembled as long polymers of the major subunit FaeG, which also possesses the adhesive properties of the fimbriae. Intrinsically, the incomplete fold of fimbrial subunits renders them unstable and susceptible to aggregation and/or proteolytic degradation in the absence of a specific periplasmic chaperone. In order to test the possibility of producing FaeG in plants, FaeG expression was studied in transgenic tobacco plants. FaeG was directed to different subcellular compartments by specific targeting signals. Targeting of FaeG to the chloroplast results in much higher yields than FaeG targeting to the endoplasmic reticulum or the apoplast. Two chloroplast-targeted FaeG variants were purified from tobacco plants and crystallized. The crystal structures show that chloroplasts circumvent the absence of the fimbrial assembly machinery by assembling FaeG into strand-swapped dimers. Furthermore, the structures reveal how FaeG combines the structural requirements of a major fimbrial subunit with its adhesive role by grafting an additional domain on its Ig-like core.     

Priming of Wheat with the Green Leaf Volatile Z-3-Hexenyl Acetate Enhances Defense against Fusarium graminearum But Boosts Deoxynivalenol Production

Priming refers to a mechanism whereby plants are sensitized to respond faster and/or more strongly to future pathogen attack. Here, we demonstrate that preexposure to the green leaf volatile Z-3-hexenyl acetate (Z-3-HAC) primed wheat (Triticum aestivum) for enhanced defense against subsequent infection with the hemibiotrophic fungus Fusarium graminearum. Bioassays showed that, after priming with Z-3-HAC, wheat ears accumulated up to 40% fewer necrotic spikelets. Furthermore, leaves of seedlings showed significantly smaller necrotic lesions compared with nonprimed plants, coinciding with strongly reduced fungal growth in planta. Additionally, we found that F. graminearum produced more deoxynivalenol, a mycotoxin, in the primed treatment. Expression analysis of salicylic acid (SA) and jasmonic acid (JA) biosynthesis genes and exogenous methyl salicylate and methyl jasmonate applications showed that plant defense against F. graminearum is sequentially regulated by SA and JA during the early and later stages of infection, respectively. Interestingly, analysis of the effect of Z-3-HAC pretreatment on SA- and JA-responsive gene expression in hormone-treated and pathogen-inoculated seedlings revealed that Z-3-HAC boosts JA-dependent defenses during the necrotrophic infection stage of F. graminearum but suppresses SA-regulated defense during its biotrophic phase. Together, these findings highlight the importance of temporally separated hormone changes in molding plant health and disease and support a scenario whereby the green leaf volatile Z-3-HAC protects wheat against Fusarium head blight by priming for enhanced JA-dependent defenses during the necrotrophic stages of infection.Biogenic volatile organic compounds (BVOCs) are known regulators of communication of sedentary plants with their direct environment (Dudareva et al., 2006). Besides attracting pollinators (Pichersky and Gershenzon, 2002), repelling insect herbivores (Birkett et al., 2010), and exerting direct antimicrobial properties (Friedman et al., 2002), BVOCs can act as an alarm signal to warn neighboring plants of an imminent herbivorous or pathogen attack (Heil and Ton, 2008) or serve as an intraplant signal for the induction of resistance (Karban et al., 2006). Engelberth et al. (2004) found that maize (Zea mays) seedlings emitted the green leaf volatiles (GLVs) Z-3-hexenal, Z-3-hexenol (Z-3-HOL), and Z-3-hexenyl acetate (Z-3-HAC) after they had been infested with caterpillars of Spodoptera exigua. Neighboring uninfested seedlings that had been exposed to these GLVs subsequently showed a considerable higher production of the plant defense hormone jasmonic acid (JA) after treatment with caterpillar regurgitant. This form of induced resistance is called priming. Plants in a primed state display faster and/or stronger activation of defense pathways when challenged by microbial pathogens, herbivorous insects, or abiotic stresses (Conrath, 2009). Exposure to these priming signals does not entail a direct activation of costly defense mechanisms but rather a stronger up-regulation of defense pathways when the plant is actually under attack (van Hulten et al., 2006). Besides resulting in a stronger induction of the JA pathway, priming also has been shown to enhance defense associated with the salicylic acid (SA) pathway, which plays a critical role in plant defense against biotrophic pathogens (Conrath et al., 2006; Jung et al., 2009).The lion’s share of attention on the use of GLVs in induced resistance has been directed to plant-insect interactions. However, the literature regarding priming by GLVs in plant-pathogen interactions remains scarce (Heil, 2014). Few studies have been performed investigating the effect of priming by GLVs on plant-fungus interactions (Scala et al., 2013a, and refs. therein). For example, hexanoic acid, a molecule with a similar structure to GLVs, has been shown to act as a priming agent in tomato (Solanum lycopersicum) plants against an infection by the necrotrophic fungus Botrytis cinerea, leading to a reduced accumulation of reactive oxygen species in primed plants (Vicedo et al., 2009; Kravchuk et al., 2011; Finiti et al., 2014). Since the GLVs E-2-hexenal (E-2-HAL), Z-3-HOL, E-2-hexenol, and Z-3-HAC also have been reported to be emitted by perennial ryegrass (Lolium perenne) after infection with Fusarium poae (Pańka et al., 2013) and by wheat (Triticum aestivum) seedlings after infection with Fusarium graminearum (Piesik et al., 2011), one may speculate that GLVs not only serve as a priming agent against the impending threat of herbivorous insects but rather constitute a general warning and priming mechanism against insects, bacteria, and fungi alike.Fusarium head blight (FHB) is an important disease in cereals caused by a complex of Fusarium spp., of which the hemibiotroph F. graminearum is one of the most prevalent (Parry et al., 1995; Goswami and Kistler, 2004; Audenaert et al., 2009). Besides yield losses of up to 40%, FHB also confers quality losses because of the production of mycotoxins such as deoxynivalenol (DON; Parry et al., 1995; Bottalico and Perrone, 2002; Vanheule et al., 2014).The hemiobiotrophic nature of F. graminearum entails that its lifestyle is characterized by a biotrophic phase followed by a necrotrophic phase. During the biotrophic phase, spores will germinate and hyphae will grow extracellularly and intercellularly. To counteract fungal colonization during the biotrophic phase, the host plant will accumulate hydrogen peroxide (H₂O₂) to induce programmed cell death. However, H₂O₂ acts as a signal for F. graminearum to produce DON, which in turn creates a positive feedback loop leading to increased H₂O₂ and DON production, clearing the way for F. graminearum to further colonize the host plant (Desmond et al., 2008). Plant defense against the biotrophic and necrotrophic phases generally has been linked to SA- and JA-related pathways, respectively (Glazebrook, 2005). This was also found in the study by Ding et al. (2011). They reported higher endogenous SA concentrations during the first hours of infection, followed by a rise in JA concentrations later on. However, plant defense against pathogens is regulated by a whole array of plant hormones, between which an intricate cross talk exists (Pieterse et al., 2012). One of the best-studied antagonistic signaling pathways is between SA and JA (Thaler et al., 2002; Pieterse et al., 2012). Research investigating the hormonal modulation of plant immunity has been done primarily in dicots. The negative relationship between SA and JA also seems to be conserved in rice (Oryza sativa), another monocot (De Vleesschauwer et al., 2013). Because of the presence of this possible antagonistic signaling and the hemibiotrophic lifestyle of F. graminearum, it is important to look more closely to the effect of priming on these two defense pathways in wheat.Here, we show that preexposure of wheat to the GLV Z-3-HAC primes wheat plants for an enhanced defense against a future infection with F. graminearum. Furthermore, our results indicate that pretreatment with Z-3-HAC leads to a stronger activation of JA-related defense while exerting suppressive effects on SA-responsive gene expression. Lastly, we found evidence that enhanced plant defense led to increased DON production by F. graminearum.     

Integrating large‐scale geographic patterns in flight morphology,flight characteristics and sexual selection in a range‐expanding damselfly

While geographic trait variation along environmental clines is widespread, associated patterns in sexual selection remain largely unexplored. Geographic patterns in sexual selection may be expected if 1) phenotypes vary geographically and sexual selection is dependent on the local phenotypes in the population, and if 2) sexual selection is influenced by geographically structured environmental conditions. We quantified geographic variation in flight‐related traits and flight performance in mated and unmated males and tested for geographic variation in sexual selection on these traits in the poleward range‐expanding damselfly Coenagrion scitulum across a set of eleven core and edge populations ordered along thermal gradients in the larval and in the adult stage. We found little support for trait differentiation between core and edge populations, instead we found considerable geographic trait variation along the larval and adult thermal gradients. As expected under time constraints, body mass decreased with shorter larval growth seasons. Lower temperatures during the adult flight period were associated with a higher body mass, a higher flight speed and a higher fat content; these traits likely evolved to buffer flight ability at suboptimal temperatures and to optimize starvation resistance. Across the large geographic scale, we found a consistent higher flight duration in mated males. Instead, sexual selection for higher fat content was stronger in populations with lower adult flight temperatures and sexual selection for lower body mass acted only in edge populations. Our results indicate sexual selection on flight performance to be consistent over a large geographic scale and this despite the clear geographic patterns in sexual selection on the underlying morphological traits. Our results highlight that to fully understand the fitness implications of geographically changing trait patterns, researchers should consider the entire phenotype–performance–fitness axis and incorporate effects of geographically structured life‐stage specific environmental conditions on this axis.