Spatially segregated SNARE protein interactions in living fungal cells

The machinery for trafficking proteins through the secretory pathway is well conserved in eukaryotes, from fungi to mammals. We describe the isolation of the snc1, sso1, and sso2 genes encoding exocytic SNARE proteins from the filamentous fungus Trichoderma reesei. The localization and interactions of the T. reesei SNARE proteins were studied with advanced fluorescence imaging methods. The SSOI and SNCI proteins co-localized in sterol-independent clusters on the plasma membrane in subapical but not apical hyphal regions. The vesicle SNARE SNCI also localized to the apical vesicle cluster within the Spitzenk?rper of the growing hyphal tips. Using fluorescence lifetime imaging microscopy and Foerster resonance energy transfer analysis, we quantified the interactions between these proteins with high spatial resolution in living cells. Our data showed that the site of ternary SNARE complex formation between SNCI and SSOI or SSOII, respectively, is spatially segregated. SNARE complex formation could be detected between SNCI and SSOI in subapical hyphal compartments along the plasma membrane, but surprisingly, not in growing hyphal tips, previously thought to be the main site of exocytosis. In contrast, SNCI.SSOII complexes were found exclusively in growing apical hyphal compartments. These findings demonstrate spatially distinct sites of plasma membrane SNARE complex formation in fungi and the existence of multiple exocytic SNAREs, which are functionally and spatially segregated. This is the first demonstration of spatially regulated SNARE interactions within the same membrane.     

Biogeography of the smooth snake (Coronella austriaca): origin and conservation of the northernmost population

Understanding historical range expansions and population demography can be crucial for the conservation and management of endangered species. In doing so, valuable information can be obtained regarding, for example, the identification of isolated populations, associations to particular habitats and distribution range shifts. As poikilotherms, snakes are vulnerable to environmental changes that can greatly shape their distribution ranges. Here we used mitochondrial data to elucidate the origin of the smooth snake population in Åland island, which is the northernmost location where the species is found. In Åland, we used mitochondrial and microsatellite data to fine‐map its spatial genetic structure, infer its demographic dynamics and determine its effective population size. We found three independent lineages, which expanded north from Iberian, the Balkans and Caucasus regions. The central lineage originating in the Balkans was the only one that reached Scandinavia. The Åland population belongs to this lineage and potentially colonized the island from the west via Sweden. This population appeared to be critically small and fragmented into two genetically isolated subpopulations. We discuss our results in light of previous findings regarding colonization routes in Europe and Scandinavia. Moreover, we discuss the origin and current genetic status of the Åland population relative to other co‐occurring snakes and suggest conservation measures based on our findings. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 114 , 426–435.     

Interactions and biocontrol of pathogenic Streptomyces strains co-occurring in potato scab lesions

Aims:  To test interactions between pathogenic strains of Streptomyces turgidiscabies , S. scabies and S. aureofaciens . To study biological control of S. turgidiscabies and S. scabies using the nonpathogenic Streptomyces strain (346) isolated from a scab lesion and a commercially available biocontrol agent ( S. griseoviridis strain K61; 'Mycostop').
Methods and Results:  Pathogenic strains of S. turgidiscabies and S. aureofaciens inhibited growth of S. scabies in vitro , whereas strain 346 and S. griseoviridis inhibited the pathogenic strains and were subsequently tested for control of scab in the greenhouse and field. Strains 346 and K61 suppressed development of common scab disease caused by S. turgidiscabies in the greenhouse. Strain 346 reduced incidence of S. turgidiscabies in scab lesions on potato tubers in the field.
Conclusions:  Streptomyces turgidiscabies shows antagonism against S. scabies that occurs in the same scab lesions and shares the ecological niche in the field. Biocontrol of S. turgidiscabies is possible with nonpathogenic Streptomyces strains but interactions may be complicated.
Significance and Impact of the Study:  Streptomyces turgidiscabies may have potential to displace S. scabies under the Scandinavian potato growing conditions. Biological control of the severe potato scab pathogen, S. turgidiscabies , is demonstrated for the first time. The results can be applied to enhance control of common scab.     

Macromolecular relationships of liver nuclei and amino acid composition of total histones during bovine ontogeny

The weight of the liver and the macromolecular composition of the liver and its nuclei during bovine ontogeny were compared. The findings suggest that bovine liver growth proceeds in three phases. The macromolecular composition of the liver nuclei was found to remain fairly constant throughout bovine ontogeny.     

The conserved aromatic residue W122 is a determinant of potyviral coat protein stability,replication, and cell-to-cell movement in plants

Coat proteins (CPs) play critical roles in potyvirus cell-to-cell movement. However, the underlying mechanism controlling them remains unclear. Here, we show that substitutions of alanine, glutamic acid, or lysine for the conserved residue tryptophan at position 122 (W¹²²) in tobacco vein banding mosaic virus (TVBMV) CP abolished virus cell-to-cell movement in Nicotiana benthamiana plants. In agroinfiltrated N. benthamiana leaf patches, both the CP and RNA accumulation levels of three W¹²² mutant viruses were significantly reduced compared with those of wild-type TVBMV, and CP accumulated to a low level similar to that of a replication-deficient mutant. The results of polyprotein transient expression experiments indicated that CP instability was responsible for the significantly low CP accumulation levels of the three W¹²² mutant viruses. The substitution of W¹²² did not affect CP plasmodesmata localization or virus particle formation; however, the substitution significantly reduced the number of virus particles. The wild-type TVBMV CP could complement the reduced replication and abolished cell-to-cell movement of the mutant viruses. When the codon for W¹²² was mutated to that for a different aromatic residue, phenylalanine or tyrosine, the resultant mutant viruses moved systemically and accumulated up to 80% of the wild-type TVBMV level. Similar results were obtained for the corresponding amino acids of W¹²² in the watermelon mosaic virus and potato virus Y CPs. Therefore, we conclude that the aromatic ring in W¹²² in the core domain of the potyviral CP is critical for cell-to-cell movement through the effects on CP stability and viral replication.     

The Influence of Infective Dose on the Virulence of a Generalist Pathogen in Rainbow Trout (Oncorhynchus mykiss) and Zebra Fish (Danio rerio)

Pathogen density and genetic diversity fluctuate in the outside-host environment during and between epidemics, affecting disease emergence and the severity and probability of infections. Although the importance of these factors for pathogen virulence and infection probability has been acknowledged, their interactive effects are not well understood. We studied how an infective dose in an environmentally transmitted opportunistic fish pathogen, Flavobacterium columnare, affects its virulence both in rainbow trout, which are frequently infected at fish farms, and in zebra fish, a host that is not naturally infected by F. columnare. We used previously isolated strains of confirmed high and low virulence in a single infection and in a co-infection. Infection success (measured as host morbidity) correlated positively with dose when the hosts were exposed to the high-virulence strain, but no response for the dose increase was found when the hosts were exposed to the low-virulence strain. Interestingly, the co-infection resulted in poorer infection success than the single infection with the high-virulence strain. The rainbow trout were more susceptible to the infection than the zebra fish but, in both species, the effects of the doses and the strains were qualitatively similar. We suggest that as an increase in dose can lead to increased host morbidity, both the interstrain interactions and differences in infectivity in different hosts may influence the severity and consequently the evolution of disease. Our results also confirm that the zebra fish is a good laboratory model to study F. columnare infection.     

Suppression of RNAi by dsRNA-Degrading RNaseIII Enzymes of Viruses in Animals and Plants

Certain RNA and DNA viruses that infect plants, insects, fish or poikilothermic animals encode Class 1 RNaseIII endoribonuclease-like proteins. dsRNA-specific endoribonuclease activity of the RNaseIII of rock bream iridovirus infecting fish and Sweet potato chlorotic stunt crinivirus (SPCSV) infecting plants has been shown. Suppression of the host antiviral RNA interference (RNAi) pathway has been documented with the RNaseIII of SPCSV and Heliothis virescens ascovirus infecting insects. Suppression of RNAi by the viral RNaseIIIs in non-host organisms of different kingdoms is not known. Here we expressed PPR3, the RNaseIII of Pike-perch iridovirus, in the non-hosts Nicotiana benthamiana (plant) and Caenorhabditis elegans (nematode) and found that it cleaves double-stranded small interfering RNA (ds-siRNA) molecules that are pivotal in the host RNA interference (RNAi) pathway and thereby suppresses RNAi in non-host tissues. In N. benthamiana, PPR3 enhanced accumulation of Tobacco rattle tobravirus RNA1 replicon lacking the 16K RNAi suppressor. Furthermore, PPR3 suppressed single-stranded RNA (ssRNA)—mediated RNAi and rescued replication of Flock House virus RNA1 replicon lacking the B2 RNAi suppressor in C. elegans. Suppression of RNAi was debilitated with the catalytically compromised mutant PPR3-Ala. However, the RNaseIII (CSR3) produced by SPCSV, which cleaves ds-siRNA and counteracts antiviral RNAi in plants, failed to suppress ssRNA-mediated RNAi in C. elegans. In leaves of N. benthamiana, PPR3 suppressed RNAi induced by ssRNA and dsRNA and reversed silencing; CSR3, however, suppressed only RNAi induced by ssRNA and was unable to reverse silencing. Neither PPR3 nor CSR3 suppressed antisense-mediated RNAi in Drosophila melanogaster. These results show that the RNaseIII enzymes of RNA and DNA viruses suppress RNAi, which requires catalytic activities of RNaseIII. In contrast to other viral silencing suppression proteins, the RNaseIII enzymes are homologous in unrelated RNA and DNA viruses and can be detected in viral genomes using gene modeling and protein structure prediction programs.     

Development of FRET-based high-throughput screening for viral RNase III inhibitors

The class 1 ribonuclease III (RNase III) encoded by Sweet potato chlorotic stunt virus (CSR3) suppresses RNA silencing in plant cells and thereby counters the host antiviral response by cleaving host small interfering RNAs, which are indispensable components of the plant RNA interference (RNAi) pathway. The synergy between sweet potato chlorotic stunt virus and sweet potato feathery mottle virus can reduce crop yields by 90%. Inhibitors of CSR3 might prove efficacious to counter this viral threat, yet no screen has been carried out to identify such inhibitors. Here, we report a novel high-throughput screening (HTS) assay based on fluorescence resonance energy transfer (FRET) for identifying inhibitors of CSR3. For monitoring CSR3 activity via HTS, we used a small interfering RNA substrate that was labelled with a FRET-compatible dye. The optimized HTS assay yielded 109 potential inhibitors of CSR3 out of 6,620 compounds tested from different small-molecule libraries. The three best inhibitor candidates were validated with a dose–response assay. In addition, a parallel screen of the selected candidates was carried out for a similar class 1 RNase III enzyme from Escherichia coli (EcR3), and this screen yielded a different set of inhibitors. Thus, our results show that the CSR3 and EcR3 enzymes were inhibited by distinct types of molecules, indicating that this HTS assay could be widely applied in drug discovery of class 1 RNase III enzymes.     

Disruption or aposematism? Significance of dorsal zigzag pattern of European vipers

European vipers (genus Vipera) are venomous and often have a distinctive dorsal zigzag pattern. The zigzag pattern of vipers has been suggested to be an example of disruptive colouration which reduces the detectability of a snake. However, recent studies suggest that the patterns have an aposematic function, although those experiments did not exclude the possibility of disruptive colouration. We used plasticine replicas of snakes to examine whether the zigzag pattern of European vipers provides protection from avian predator attacks via disruptive or aposematic function, or if the zigzag pattern might simultaneously serve both antipredatory functions. Experiments were conducted in the Coto Doñana National Park southern Spain. In the experiment, predation pressure caused by birds was compared between zigzag pattern (patterns were painted with and without disruptive effect i.e. breaking body outline or not), classical disruptive colouration (non-randomly placed patterns that breaks body outline) and control markings (replicas with length wise stripes and models without painted pattern) on natural and controlled backgrounds. We found that zigzag patterned snake replicas suffered less predation than striped ones regardless of the background, providing further evidence that the zigzag pattern of European vipers functions as a warning signal against predators. However, we did not find evidence that the zigzag pattern involves a disruptive effect.