SIGS vs HIGS: a study on the efficacy of two dsRNA delivery strategies to silence Fusarium FgCYP51 genes in infected host and non-host plants

CYP3RNA, a double-stranded (ds)RNA designed to concomitantly target the two sterol 14α-demethylase genes FgCYP51A and FgCYP51B and the fungal virulence factor FgCYP51C, inhibits the growth of the ascomycete fungus Fusarium graminearum (Fg) in vitro and in planta. Here we compare two different methods (setups) of dsRNA delivery, viz. transgene expression (host-induced gene silencing, HIGS) and spray application (spray-induced gene silencing, SIGS), to assess the activity of CYP3RNA and novel dsRNA species designed to target one or two FgCYP51 genes. Using Arabidopsis and barley, we found that dsRNA designed to target two FgCYP51 genes inhibited fungal growth more efficiently than dsRNA targeting a single gene, although both dsRNA species reduced fungal infection. Either dsRNA delivery method reduced fungal growth stronger than anticipated from previous mutational knock-out (KO) strategies, where single gene KO had no significant effect on fungal viability. Consistent with the strong inhibitory effects of the dsRNAs on fungal development in both setups, we detected to a large extent dsRNA-mediated co-silencing of respective non-target FgCYP51 genes. Together, our data further support the valuation that dsRNA applications have an interesting potential for pesticide target validation and gene function studies, apart from their potential for crop protection.     

Temperature-dependent competition hierarchy: a mechanism stabilizing the phenological strategy in the scorpionfly Panorpa communis L.

Previous studies of the phenologies and the different microclimatic patterns of the distribution of the scorpionflies Panorpa communis L. and Panorpa vulgaris Imhoff and Labram 1836 showed different phenological strategies. In P. communis , a species foraging at shadowy and cool places only, a majority of 90% of the individuals are univoltine; however, approximately 10% of the offspring of the first annual generation are bivoltine. This proportion remained unchanged in the Freiburg population over 8 years. Differently, all individuals of P. vulgaris foraging equally frequent at sunny and warm as at shadowy and cool places are bivoltine. The proximate cause of bivoltinism in both species is a heritable variation of different ‘day length thresholds’ triggering diapause‐free development if natural day length exceeds these thresholds. As selection favours maximal temporal exploitation of food availability it remains obscure why in P. communis the number of diapause‐free developing individuals does not increase continuously from year to year although this phenotype reproduces twice a year. Therefore, in the present paper, we focus on the following main questions. Does the competitive inferiority of P. communis in the presence of P. vulgaris at the temperature regime of the late summer function as a mechanism maintaining the majority of individuals of P. communis univoltine, by dramatically reducing the fitness of the bivoltine ones? As a long‐term evolutionary change in the frequency of bivoltine individuals in P. communis solely depends on the lifetime reproductive success of the females, we here consider the influence of interspecific competition and temperature conditions on the reproductive success of the females of P. communis only. Five lines of evidence suggest that the mechanism of maintaining univoltinism in P. communis is primarily because of differences in the ability of each species to exploit dead arthropod resources: (1) these species show complete diet overlap; (2) dead arthropods are limiting resources for both species of scorpionflies as indicated by positive demographic effects with increased food availability; (3) in competition with P. vulgaris at high temperatures, P. communis is competitively inferior in the ability to detect and exploit dead arthropods; (4) this reduced resource acquisition of female P. communis translates into significant reductions in the survivorship, body condition, fecundity and lifetime reproductive success; (5) exploitation competition does account for these negative demographic effects on second‐generation females of P. communis more than interference competition.     

Direct Interference on Competition in a Chemostat

Thispaperstudiestheglobaldynamicsofcompetitioninchemostatinwhichtwopopulationsofmicrooganismscompeteexploitativelyforasingle,essential,nonreproducing,growth-limitingsubstrateandthereisadirectinterferencebetweencompetitors.Inordertounderstandthedifferencesintheeffectsofintraspecificandinterspecificinterference,thebothcasesareconsideredrespectively.Keywords:##4Populationdynamicsecology;;chemostat;;competition;;interference;;interspecific;;intraspecific;;principalofcompetitiveexclusion     

Reproductive interference explains persistence of aggression between species

Interspecific territoriality occurs when individuals of different species fight over space, and may arise spontaneously when populations of closely related territorial species first come into contact. But defence of space is costly, and unless the benefits of excluding heterospecifics exceed the costs, natural selection should favour divergence in competitor recognition until the species no longer interact aggressively. Ordinarily males of different species do not compete for mates, but when males cannot distinguish females of sympatric species, females may effectively become a shared resource. We model how reproductive interference caused by undiscriminating males can prevent interspecific divergence, or even cause convergence, in traits used to recognize competitors. We then test the model in a genus of visually orienting insects and show that, as predicted by the model, differences between species pairs in the level of reproductive interference, which is causally related to species differences in female coloration, are strongly predictive of the current level of interspecific aggression. Interspecific reproductive interference is very common and we discuss how it may account for the persistence of interspecific aggression in many taxonomic groups.     

Estimation of proteins in the presence of polyethylenimine

Polyethylenimine (PEI) interferes strongly in all commonly employed protein estimation assays. Here, a strategy to remove this interference is developed. Polyethylenimine is selectively removed by adsorption from a mixture containing PEI and lysozyme using carboxymethyl-cellulose, a cation-exchange resin between an ionic strength of 0.35–1 M (pH 6.0), conditions under which lysozyme remains in solution and can be accurately estimated.     

RNAi-mediated knockdown of the Rhau helicase preferentially depletes proteins with a Guanine-quadruplex motif in the 5'-UTR of their mRNA

Guanine-quadruplex (G-quadruplex) structures in mRNAs have been shown to modulate gene expression. However, the overall biological relevance of this process is under debate, as cellular helicases unwind G-quadruplex structures. The helicase Rhau (encoded by the DHX36 gene) was reported to be the major source of RNA G-quadruplex resolving activity in lysates of human cells. In the current study, we depleted Rhau by RNAi-mediated silencing and analyzed the effect on proteins whose mRNAs harbor a G-quadruplex motif in their 5'-UTRs. A targeted investigation of the proto-oncogenes Bcl-2 and NRAS, which are well-known examples for the translational repression of G-quadruplex structures, did not reveal effects caused by Rhau silencing. We therefore carried out a global analysis of changes in protein levels by label-free quantification using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Following Rhau knockdown, of all the identified proteins, only 1.9% were significantly downregulated to at least 70%. According to a bioinformatic analysis with the QGRS mapper, 33% of the downregulated proteins were predicted to harbor a G-quadruplex motif in the 5'-UTR of their respective mRNAs, compared to only 11% in the complete dataset. This indicates that in an unexpectedly small set of genes, in which G-quadruplex motifs are unusually common in the 5'-UTR of their mRNAs, Rhau helicase is responsible for the regulation of their expression.     

BACE1 RNA interference improves spatial memory and attenuates Aβ burden in a streptozotocin‐induced tau hyperphosphorylated rat model

Both senile plaques and intracellular neurofibrillary tangles are important pathological characteristics in Alzheimer's disease. However, the relationship between Aβ deposition and tau hyperphosphorylation is unknown. In this study, the increased levels of full‐length amyloid precursor protein (APP), APP C‐terminal fragment (β‐CTF) and BACE1 were found in streptozotocin‐induced tau hyperphosphorylation models by quantitative polymerase chain reaction, Western blotting and immunohistochemistry methods. In the previous studies, few strategies focusing on inhibiting β‐secretase (BACE1) in a tau hyperphosphorylation model were utilized. Here, BACE1 RNAi was used to treat the streptozotocin‐induced tau hyperphosphorylation animal models. BACE1 RNAi treatment improved the behavioural ability of animal models and reduced the amount of Aβ1‐40 and Aβ1‐42, accompanied by decreasing the levels of BACE1 and β‐CTF. Our results demonstrated that neurological defects and neurotoxic fragments, including Aβ and β‐CTF, were eliminated by BACE1 RNAi in the tau hyperphosphorylated model, implying the efficiency and safety of BACE1RNAi treatment against Alzheimer's disease. Copyright © 2014 John Wiley & Sons, Ltd.