Transgenic sequences are frequently lost in Phytophthora parasitica transformants without reversion of the transgene-induced silenced state

Little data exist on the mechanism and stability of transformation in Phytophthora parasitica, a major oomycete parasite of plants. Here, we studied the stability of drug-resistant protoplast transformants by analyzing single-zoospore derivatives. We show that the transgenic sequences are not stably integrated into the chromosomes, resulting in the loss of drug resistance in single-zoospore derivatives. However, in strains where the P. parasitica gene encoding the CBEL elicitor was silenced by transformation with sense or antisense constructs, silencing is not reversed when the transgenic sequences are lost. This suggests that instability of P. parasitica transformants is not an obstacle for loss-of-function studies in this organism.     

Diazepam binding inhibitor promotes progenitor proliferation in the postnatal SVZ by reducing GABA signaling

The subventricular zone (SVZ) of the lateral ventricles is the largest neurogenic niche of the postnatal brain. New SVZ-generated neurons migrate via the rostral migratory stream to the olfactory bulb (OB) where they functionally integrate into preexisting neuronal circuits. Nonsynaptic GABA signaling was previously shown to inhibit SVZ-derived neurogenesis. Here we identify the endogenous protein diazepam binding inhibitor (DBI) as a positive modulator of SVZ postnatal neurogenesis by regulating GABA activity in transit-amplifying cells. We performed DBI loss- and gain-of-function experiments in vivo at the peak of postnatal OB neuron generation in mice and demonstrate that DBI enhances proliferation by preventing SVZ progenitors to exit the cell cycle. Furthermore, we provide evidence that DBI exerts its effect on SVZ progenitors via its octadecaneuropeptide proteolytic product (ODN) by inhibiting GABA-induced currents. Together our data reveal a regulatory mechanism by which DBI counteracts the inhibitory effect of nonsynaptic GABA signaling on subventricular neuronal proliferation.     

Population structure analysis provides insights into the infection biology and invasion strategies of Kretzschmaria deusta in trees

Infection biology and invasion strategies of Kretzschmaria deusta were investigated through the analysis of patterns of colonization and population diversity in three case studies, comprising different host species. Molecular analysis and isolation assays performed on stem sections at different heights indicated a prevalent heart rot mode of expansion. Random amplified microsatellites – RAMs and somatic incompatibility assays on isolates allowed detection, in one case, of a genet occupying the entire decay column and, in the other cases, of several different genets in each individual tree. Hypotheses on modes of arrival and entrance of K. deusta in trees are discussed on the basis of the distribution of genets and population genetics analyses. Significant correlation (Spearman ρ = 1.0; p < 0.001) between the number of genets and the number of areas delimited by pseudosclerotial plates (PSPs) on the decayed portions of stem sections suggests PSPs are interaction zone lines between different individuals of K. deusta occupying adjacent decay columns.     

Two neighboring residues of loop A of the alpha1 subunit point towards the benzodiazepine binding site of GABAA receptors

Benzodiazepines are widely used drugs exerting sedative, anxiolytic, muscle relaxant, and anticonvulsant effects by acting through specific high affinity binding sites on some GABA(A) receptors. It is important to understand how these ligands are positioned in this binding site. We are especially interested here in the conformation of loop A of the alpha(1)beta(2)gamma(2) GABA(A) receptor containing a key residue for the interaction of benzodiazepines: alpha(1)H101. We describe a direct interaction of alpha(1)N102 with a diazepam- and an imidazobenzodiazepine-derivative. Our observations help to better understand the conformation of this region of the benzodiazepine pocket in GABA(A) receptor.     

The relative efficiency of homology-directed repair has distinct effects on proper anaphase chromosome separation

Homology-directed repair (HDR) is essential to limit mutagenesis, chromosomal instability (CIN) and tumorigenesis. We have characterized the consequences of HDR deficiency on anaphase, using markers for incomplete chromosome separation: DAPI-bridges and Ultra-fine bridges (UFBs). We show that multiple HDR factors (Rad51, Brca2 and Brca1) are critical for complete chromosome separation during anaphase, while another chromosome break repair pathway, non-homologous end joining, does not affect chromosome segregation. We then examined the consequences of mild versus severe HDR disruption, using two different dominant-negative alleles of the strand exchange factor, Rad51. We show that mild HDR disruption is viable, but causes incomplete chromosome separation, as detected by DAPI-bridges and UFBs, while severe HDR disruption additionally results in multipolar anaphases and loss of clonogenic survival. We suggest that mild HDR disruption favors the proliferation of cells that are prone to CIN due to defective chromosome separation during anaphase, whereas, severe HDR deficiency leads to multipolar divisions that are prohibitive for cell proliferation.     

Influence of “protective” symbionts throughout the different steps of an aphid–parasitoid interaction

Microbial associates are widespread in insects, some conferring a protection to their hosts against natural enemies like parasitoids. These protective symbionts may affect the infection success of the parasitoid by modifying behavioral defenses of their hosts, the development success of the parasitoid by conferring a resistance against it or by altering life-history traits of the emerging parasitoids. Here, we assessed the effects of different protective bacterial symbionts on the entire sequence of the host-parasitoid interaction (i.e., from parasitoid attack to offspring emergence) between the pea aphid, Acyrthosiphon pisum, and its main parasitoid, Aphidius ervi and their impacts on the life-history traits of the emerging parasitoids. To test whether symbiont-mediated phenotypes were general or specific to particular aphid–symbiont associations, we considered several aphid lineages, each harboring a different strain of either Hamiltonella defensa or Regiella insecticola, two protective symbionts commonly found in aphids. We found that symbiont species and strains had a weak effect on the ability of aphids to defend themselves against the parasitic wasps during the attack and a strong effect on aphid resistance against parasitoid development. While parasitism resistance was mainly determined by symbionts, their effects on host defensive behaviors varied largely from one aphid–symbiont association to another. Also, the symbiotic status of the aphid individuals had no impact on the attack rate of the parasitic wasps, the parasitoid emergence rate from parasitized aphids nor the life-history traits of the emerging parasitoids. Overall, no correlations between symbiont effects on the different stages of the host–parasitoid interaction was observed, suggesting no trade-offs or positive associations between symbiont-mediated phenotypes. Our study highlights the need to consider various sequences of the host-parasitoid interaction to better assess the outcomes of protective symbioses and understand the ecological and evolutionary dynamics of insect–symbiont associations.     

BNIP3 promotes HIF‐1α‐driven melanoma growth by curbing intracellular iron homeostasis

BNIP3 is a mitophagy receptor with context‐dependent roles in cancer, but whether and how it modulates melanoma growth in vivo remains unknown. Here, we found that elevated BNIP3 levels correlated with poorer melanoma patient’s survival and depletion of BNIP3 in B16‐F10 melanoma cells compromised tumor growth in vivo. BNIP3 depletion halted mitophagy and enforced a PHD2‐mediated downregulation of HIF‐1α and its glycolytic program both in vitro and in vivo. Mechanistically, we found that BNIP3‐deprived melanoma cells displayed increased intracellular iron levels caused by heightened NCOA4‐mediated ferritinophagy, which fostered PHD2‐mediated HIF‐1α destabilization. These effects were not phenocopied by ATG5 or NIX silencing. Restoring HIF‐1α levels in BNIP3‐depleted melanoma cells rescued their metabolic phenotype and tumor growth in vivo, but did not affect NCOA4 turnover, underscoring that these BNIP3 effects are not secondary to HIF‐1α. These results unravel an unexpected role of BNIP3 as upstream regulator of the pro‐tumorigenic HIF‐1α glycolytic program in melanoma cells.     

Optimization of sampling procedures for DNA‐based diagnosis of wood decay fungi in standing trees

Aims: To develop fast and reliable sampling procedures for DNA‐based diagnosis of wood decay fungi in standing trees. Methods and Results: A total of 250 trees were tested for the presence of a suite of wood decay fungi by collecting wood frass obtained by drilling each tree once with a 4‐mm‐diameter, 43‐cm‐long bit. We identified at least one of 11 target wood decay fungi in 56 trees through multiplex PCR assays. The presence of target wood decay taxa was further investigated in these 56 trees, by analysing independently wood from each of six drillings. Results were then compared with those obtained using sampling schemes differing in terms of number and position of drillings. Samples of 1–4 drillings were either analysed separately, and the results were combined, or pooled together before analysis was performed. In comparison with taxa identified by the analysis of six drillings, diagnostic efficiency ranged from 56·6% for the scheme based on a single drill to 96·8% for the scheme based on four drillings analysed separately. Both schemes significantly differ (P < 0·05) from those based on two and three drillings, whose efficiency was 72·6% and 83·9%, respectively. Diagnostic efficiency of pooled samples was comparable to that of samples analysed separately. Conclusions: Highest diagnostic efficiency was obtained by analysing wood from four drillings. It is advisable to pool samples deriving from different drillings to reduce laboratory costs. Significance and Impact of the Study: Fast and reliable sampling procedures make DNA‐based diagnosis more suitable for tree inspection procedures.