Repetitive DNA Reeling by the Cascade-Cas3 Complex in Nucleotide Unwinding Steps
Loeff, Luuk ; Brouns, Stan J.J. ; Joo, Chirlmin - \ 2018
Molecular Cell 70 (2018)3. - ISSN 1097-2765 - p. 385 - 394.
adaptive - Cas3 - cascade - CRISPR - FRET - helicase - immunity - interference - single-molecule
CRISPR-Cas provides RNA-guided adaptive immunity against invading genetic elements. Interference in type I systems relies on the RNA-guided Cascade complex for target DNA recognition and the Cas3 helicase/nuclease protein for target degradation. Even though the biochemistry of CRISPR interference has been largely covered, the biophysics of DNA unwinding and coupling of the helicase and nuclease domains of Cas3 remains elusive. Here, we employed single-molecule Förster resonance energy transfer (FRET) to probe the helicase activity with high spatiotemporal resolution. We show that Cas3 remains tightly associated with the target-bound Cascade complex while reeling the DNA using a spring-loaded mechanism. This spring-loaded reeling occurs in distinct bursts of 3 bp, which underlie three successive 1-nt unwinding events. Reeling is highly repetitive, allowing Cas3 to repeatedly present its inefficient nuclease domain with single-strand DNA (ssDNA) substrate. Our study reveals that the discontinuous helicase properties of Cas3 and its tight interaction with Cascade ensure controlled degradation of target DNA only. Loeff et al. report on a single-molecule fluorescence analysis of the E. coli CRISPR-Cas3 protein. The Cas3 protein uses a spring-loaded unwinding mechanism, reeling the target DNA 3 bp at a time. Facilitated by slipping, Cas3 repeatedly presents its intrinsically inefficient nuclease domain with DNA substrate, which may contribute to promoting a robust immune response.
Data from: Balancing food and density-dependence in the spatial distribution of an interference-prone forager
Dokter, Adriaan M. ; Loon, E.E. ; Rappoldt, Cornelis ; Oosterbeek, Kees ; Baptist, M.J. ; Bouten, Willem ; Ens, Bruno J. - \ 2017
Wageningen University & Research
resource selection - density-dependence - eurasian oystercatcher - interference - intertidal mudflat - ideal free distribution - benthic survey - Haematopus ostralegus - Cerastoderma edule - Ensis directus
Foraging distributions are thought to be density-dependent, because animals not only select for a high availability and quality of resources, but also avoid conspecific interference. Since these processes are confounded, their relative importance in shaping foraging distributions remains poorly understood. Here we aimed to rank the contribution of density-dependent and density-independent effects on the spatio-temporal foraging patterns of eurasian oystercatchers. In our intertidal study area, tides caused continuous variation in oystercatcher density, providing an opportunity to disentangle conspecific interference and density-independent interactions with the food landscape. Spatial distributions were quantified using high-resolution individual tracking of foraging activity and location. In a model environment that included a realistic reconstruction of both the tides and the benthic food, we tested a family of behaviour-based optimality models against these tracking data. Density-independent interactions affected spatial distributions much more strongly than conspecific interference, even in an interference-prone species like oystercatchers. Spatial distributions were governed by avoidance of bill injury costs, selection for high interference-free intake rates and a decreasing availability of benthic bivalve prey after their exposure. These density-independent interactions outweighed interference competition in terms of effect size. We suggest that the bottleneck in our mechanistic understanding of foraging distributions may be primarily the role of density-independent prey attributes unrelated to intake rates, like damage costs in the case of oystercatchers foraging on perilous prey. At a landscape scale, above the finest inter-individual distances, effects of conspecific interaction on spatial distributions may have been overemphasised.
Cas3-Derived Target DNA Degradation Fragments Fuel Primed CRISPR Adaptation
Künne, Tim ; Kieper, Sebastian N. ; Bannenberg, Jasper W. ; Vogel, Anne ; Miellet, Willem R. ; Klein, Misha ; Depken, Martin ; Suarez-Diez, Maria ; Brouns, Stan J.J. - \ 2016
Molecular Cell 63 (2016)5. - ISSN 1097-2765 - p. 852 - 864.
adaptive immunity - Cas1 - Cas2 - Cas3 - Cascade - CRISPR-Cas - interference - phage resistance - priming - spacer acquisition
Prokaryotes use a mechanism called priming to update their CRISPR immunological memory to rapidly counter revisiting, mutated viruses, and plasmids. Here we have determined how new spacers are produced and selected for integration into the CRISPR array during priming. We show that Cas3 couples CRISPR interference to adaptation by producing DNA breakdown products that fuel the spacer integration process in a two-step, PAM-associated manner. The helicase-nuclease Cas3 pre-processes target DNA into fragments of about 30–100 nt enriched for thymine-stretches in their 3′ ends. The Cas1-2 complex further processes these fragments and integrates them sequence-specifically into CRISPR repeats by coupling of a 3′ cytosine of the fragment. Our results highlight that the selection of PAM-compliant spacers during priming is enhanced by the combined sequence specificities of Cas3 and the Cas1-2 complex, leading to an increased propensity of integrating functional CTT-containing spacers.
Flavivirus RNAi suppression: decoding non-coding RNA
Pijlman, G.P. - \ 2014
Current Opinion in Virology 7 (2014). - ISSN 1879-6257 - p. 55 - 60.
west-nile-virus - aedes-aegypti mosquitos - double-stranded-rna - arbovirus infection - antiviral immunity - albopictus cells - subgenomic rna - messenger-rna - nss protein - interference
Flaviviruses are important human pathogens that are transmitted by invertebrate vectors, mostly mosquitoes and ticks. During replication in their vector, flaviviruses are subject to a potent innate immune response known as antiviral RNA interference (RNAi). This defense mechanism is associated with the production of small interfering (si)RNA that lead to degradation of viral RNA. To what extent flaviviruses would benefit from counteracting antiviral RNAi is subject of debate. Here, the experimental evidence to suggest the existence of flavivirus RNAi suppressors is discussed. I will highlight the putative role of non-coding, subgenomic flavivirus RNA in suppression of RNAi in insect and mammalian cells. Novel insights from ongoing research will reveal how arthropod-borne viruses modulate innate immunity including antiviral RNAi.
Degenerate target sites mediate rapid primed CRISPR adaptation
Fineran, P.C. ; Gerritzen, M.J.H. ; Suarez Diez, M. ; Künne, T.A. ; Boekhorst, J. ; Hijum, S.A.F.T. van; Staals, R.H.J. ; Brouns, S.J.J. - \ 2014
Proceedings of the National Academy of Sciences of the United States of America 111 (2014)16. - ISSN 0027-8424 - p. E1629 - E1638.
adaptive immune-systems - escherichia-coli - cas systems - streptococcus-thermophilus - bacterial immunity - defense system - foreign dna - dual-rna - recognition - interference
Prokaryotes encode adaptive immune systems, called CRISPR-Cas (clustered regularly interspaced short palindromic repeats–CRISPR associated), to provide resistance against mobile invaders, such as viruses and plasmids. Host immunity is based on incorporation of invader DNA sequences in a memory locus (CRISPR), the formation of guide RNAs from this locus, and the degradation of cognate invader DNA (protospacer). Invaders can escape type I-E CRISPR-Cas immunity in Escherichia coli K12 by making point mutations in the seed region of the protospacer or its adjacent motif (PAM), but hosts quickly restore immunity by integrating new spacers in a positive-feedback process termed “priming.” Here, by using a randomized protospacer and PAM library and high-throughput plasmid loss assays, we provide a systematic analysis of the constraints of both direct interference and subsequent priming in E. coli. We have defined a high-resolution genetic map of direct interference by Cascade and Cas3, which includes five positions of the protospacer at 6-nt intervals that readily tolerate mutations. Importantly, we show that priming is an extremely robust process capable of using degenerate target regions, with up to 13 mutations throughout the PAM and protospacer region. Priming is influenced by the number of mismatches, their position, and is nucleotide dependent. Our findings imply that even outdated spacers containing many mismatches can induce a rapid primed CRISPR response against diversified or related invaders, giving microbes an advantage in the coevolutionary arms race with their invaders.
Identification, cloning and characterization of the tomato TCP transcription factor family
Parapunova, V.A. ; Busscher, M. ; Busscher-Lange, J. ; Lammers, M. ; Karlova, R.B. ; Bovy, A.G. ; Angenent, G.C. ; Maagd, R.A. de - \ 2014
BMC Plant Biology 14 (2014). - ISSN 1471-2229
mads-box gene - arabidopsis-thaliana - fruit-development - leaf development - high-throughput - plant-growth - dna-binding - expression - time - interference
Background: TCP proteins are plant-specific transcription factors, which are known to have a wide range of functions in different plant species such as in leaf development, flower symmetry, shoot branching, and senescence. Only a small number of TCP genes has been characterised from tomato (Solanum lycopersicum). Here we report several functional features of the members of the entire family present in the tomato genome. Results: We have identified 30 Solanum lycopersicum SlTCP genes, most of which have not been described before. Phylogenetic analysis clearly distinguishes two homology classes of the SlTCP transcription factor family - class I and class II. Class II differentiates in two subclasses, the CIN-TCP subclass and the CYC/TB1 subclass, involved in leaf development and axillary shoots formation, respectively. The expression patterns of all members were determined by quantitative PCR. Several SlTCP genes, like SlTCP12, SlTCP15 and SlTCP18 are preferentially expressed in the tomato fruit, suggesting a role during fruit development or ripening. These genes are regulated by RIN (RIPENING INHIBITOR), CNR (COLORLESS NON-RIPENING) and SlAP2a (APETALA2a) proteins, which are transcription factors with key roles in ripening. With a yeast one-hybrid assay we demonstrated that RIN binds the promoter fragments of SlTCP12, SlTCP15 and SlTCP18, and that CNR binds the SlTCP18 promoter. This data strongly suggests that these class I SlTCP proteins are involved in ripening. Furthermore, we demonstrate that SlTCPs bind the promoter fragments of members of their own family, indicating that they regulate each other. Additional yeast one-hybrid studies performed with Arabidopsis transcription factors revealed binding of the promoter fragments by proteins involved in the ethylene signal transduction pathway, contributing to the idea that these SlTCP genes are involved in the ripening process. Yeast two-hybrid data shows that SlTCP proteins can form homo and heterodimers, suggesting that they act together in order to form functional protein complexes and together regulate developmental processes in tomato.
A heritable antiviral RNAi response limits Orsay virus infection in Caenorhabditis elegans N2
Sterken, M.G. ; Snoek, L.B. ; Bosman, K.J. ; Daamen, J. ; Riksen, J.A.G. ; Bakker, J. ; Pijlman, G.P. ; Kammenga, J.E. - \ 2014
PLoS ONE 9 (2014)2. - ISSN 1932-6203
c. elegans - gene-expression - interference - requirements - inheritance - environment - diversity - pathways - genotype - immunity
Orsay virus (OrV) is the first virus known to be able to complete a full infection cycle in the model nematode species Caenorhabditis elegans. OrV is transmitted horizontally and its infection is limited by antiviral RNA interference (RNAi). However, we have no insight into the kinetics of OrV replication in C. elegans. We developed an assay that infects worms in liquid, allowing precise monitoring of the infection. The assay revealed a dual role for the RNAi response in limiting Orsay virus infection in C. elegans. Firstly, it limits the progression of the initial infection at the step of recognition of dsRNA. Secondly, it provides an inherited protection against infection in the offspring. This establishes the heritable RNAi response as anti-viral mechanism during OrV infections in C. elegans. Our results further illustrate that the inheritance of the anti-viral response is important in controlling the infection in the canonical wild type Bristol N2. The OrV replication kinetics were established throughout the worm life-cycle, setting a standard for further quantitative assays with the OrV-C. elegans infection model.
Structure-based cleavage mechanism of Thermus thermophilus Argonaute DNA guide strand-mediated DNA target cleavage
Sheng, G. ; Zhao, H. ; Wang, J. ; Rao, Y. ; Tian, W. ; Swarts, D.C. ; Oost, J. van der; Patel, D.J. ; Wang, Y. - \ 2014
Proceedings of the National Academy of Sciences of the United States of America 111 (2014)2. - ISSN 0027-8424 - p. 652 - 657.
crystal-structure - rna recognition - silencing complex - substrate-specificity - slicer activity - piwi protein - paz domain - human risc - endonuclease - interference
We report on crystal structures of ternary Thermus thermophilus Argonaute (TtAgo) complexes with 5'-phosphorylated guide DNA and a series of DNA targets. These ternary complex structures of cleavage-incompatible, cleavage-compatible, and postcleavage states solved at improved resolution up to 2.2 Å have provided molecular insights into the orchestrated positioning of catalytic residues, a pair of Mg2+ cations, and the putative water nucleophile positioned for in-line attack on the cleavable phosphate for TtAgo-mediated target cleavage by a RNase H-type mechanism. In addition, these ternary complex structures have provided insights into protein and DNA conformational changes that facilitate transition between cleavage-incompatible and cleavage-compatible states, including the role of a Glu finger in generating a cleavage-competent catalytic Asp-Glu-Asp-Asp tetrad. Following cleavage, the seed segment forms a stable duplex with the complementary segment of the target strand
Structure and Activity of the RNA-Targeting Type III-B CRISPR-Cas Complex of Thermus thermophilus
Staals, R.H.J. ; Agari, Y. ; Maki-Yonekura, S. ; Zhu, Y. ; Taylor, D.W. ; Duijn, E. van; Barendregt, A. ; Vlot, M. ; Koehorst, J.J. ; Sakamoto, K. ; Masuda, A. ; Dohmae, N. ; Schaap, P.J. ; Doudna, J.A. ; Heck, A. ; Yonekura, K. ; Oost, J. van der; Shinkai, A. - \ 2013
Molecular Cell 52 (2013)1. - ISSN 1097-2765 - p. 135 - 145.
of-flight instrument - mass-spectrometry - escherichia-coli - silencing complex - antiviral defense - immune-system - protein - interference - transcription - recognition
The CRISPR-Cas system is a prokaryotic host defense system against genetic elements. The Type III-B CRISPR-Cas system of the bacterium Thermus thermophilus, the TtCmr complex, is composed of six different protein subunits (Cmr1-6) and one crRNA with a stoichiometry of Cmr112131445361:crRNA1. The TtCmr complex copurifies with crRNA species of 40 and 46 nt, originating from a distinct subset of CRISPR loci and spacers. The TtCmr complex cleaves the target RNA at multiple sites with 6 nt intervals via a 5' ruler mechanism. Electron microscopy revealed that the structure of TtCmr resembles a "sea worm" and is composed of a Cmr2-3 heterodimer "tail," a helical backbone of Cmr4 subunits capped by Cmr5 subunits, and a curled "head" containing Cmr1 and Cmr6. Despite having a backbone of only four Cmr4 subunits and being both longer and narrower, the overall architecture of TtCmr resembles that of Type I Cascade complexes
A high density recombination map of the pig reveals a correlation between sex-specific recombination and GC content
Tortereau, F.J.D. ; Servin, B. ; Frantz, L.A.F. ; Megens, H.J.W.C. ; Milan, D. ; Rohrer, G. ; Schook, L.B. ; Groenen, M.A.M. - \ 2012
BMC Genomics 13 (2012). - ISSN 1471-2164
biased gene conversion - multipoint linkage analysis - porcine genome - hot-spots - sus-scrofa - evolution - rates - humans - mice - interference
Background: The availability of a high-density SNP genotyping chip and a reference genome sequence of the pig (Sus scrofa) enabled the construction of a high-density linkage map. A high-density linkage map is an essential tool for further fine-mapping of quantitative trait loci (QTL) for a variety of traits in the pig and for a better understanding of mechanisms underlying genome evolution. Results: Four different pig pedigrees were genotyped using the Illumina PorcineSNP60 BeadChip. Recombination maps for the autosomes were computed for each individual pedigree using a common set of markers. The resulting genetic maps comprised 38,599 SNPs, including 928 SNPs not positioned on a chromosome in the current assembly of the pig genome (build 10.2). The total genetic length varied according to the pedigree, from 1797 to 2149 cM. Female maps were longer than male maps, with a notable exception for SSC1 where male maps are characterized by a higher recombination rate than females in the region between 91-250 Mb. The recombination rates varied among chromosomes and along individual chromosomes, regions with high recombination rates tending to cluster close to the chromosome ends, irrespective of the position of the centromere. Correlations between main sequence features and recombination rates were investigated and significant correlations were obtained for all the studied motifs. Regions characterized by high recombination rates were enriched for specific GC-rich sequence motifs as compared to low recombinant regions. These correlations were higher in females than in males, and females were found to be more recombinant than males at regions where the GC content was greater than 0.4. Conclusions: The analysis of the recombination rate along the pig genome highlighted that the regions exhibiting higher levels of recombination tend to cluster around the ends of the chromosomes irrespective of the location of the centromere. Major sex-differences in recombination were observed: females had a higher recombination rate within GC-rich regions and exhibited a stronger correlation between recombination rates and specific sequence features.
Cascade-mediated binding and bending of negatively supercoiled DNA
Westra, E.R. ; Nilges, B. ; Erp, P.B. ; Oost, J. van der; Dame, R.T. ; Brouns, S.J.J. - \ 2012
RNA Biology 9 (2012)9. - ISSN 1547-6286 - p. 1134 - 1138.
crispr-cas systems - immune-system - structural basis - rna - bacteria - archaea - defense - interference - recognition - mechanism
Prokaryotes possess various defense mechanisms against invading DNA. Adaptive defense by CRISPR/Cas relies on incorporation of invader DNA sequences in the host genome. In Escherichia coli, processed transcripts of these incorporated sequences (crRNAs) guide Cascade-mediated invader DNA recognition. ( 1) (-) ( 4) Cascade is a multisubunit ribonucleoprotein complex, consisting of one crRNA and five proteins: Cse1, Cse2, Cas7, Cas5 and Cas6e. ( 1) (, ) ( 2) Cascade-mediated DNA recognition requires a conserved sequence adjacent to the target (protospacer adjacent motif, PAM) and a negatively supercoiled DNA topology. ( 3) (, ) ( 4) While Cse1 carries out PAM recognition, ( 5) the Cascade structure suggests that Cse2 may interact with target DNA in the PAM-distal end of the protospacer. ( 6) Using Electrophoretic Mobility Shift Assays, we here describe the function of the Cse1 and Cse2 subunits in the context of protospacer recognition on negatively supercoiled DNA. While Cse1 is required for nonspecific DNA binding, Cse2 appears to be important for specific binding, presumably by mediating stabilizing interactions with the displaced strand, the R-loop, or both. Furthermore, we performed Scanning Force Microscopy using linearized DNA molecules, which facilitates accurate and reliable measurements of Cascade-mediated bending. This analysis reveals that Cascade binding induces flexibility in the DNA target, most likely due to single stranded DNA regions flanking the R-loop
Can epigenetic control explain pronounced within plant heterogeneity of meiosis in a translocation trisome of Secale L.?
Sybenga, J. - \ 2012
Genome 55 (2012)1. - ISSN 0831-2796 - p. 257 - 264.
recombination frequencies - meiotic recombination - interference - rye
Meiotic metaphase I configuration frequencies were determined in different tillers of genetically related plants of rye (Secale cereale L.) heterozygous for reciprocal translocation T248W (between chromosome arms 1RS and 6RS) and with an additional (telocentric) arm 1RS. Seventeen different configurations could be recognized, grouped into three categories. Very different configuration frequencies were found not only between sister plants from the same parents but also between tillers of the same plant grown under identical conditions (climate chambers at 15 °C and 20 °C). The heterogeneity reflects variation in chromosome pairing and crossing over, and is variable and unpredictable. Anthers within florets were homogeneous. Between tiller heterogeneity is insufficient to explain differences between sister plants. It is ascribed to random somatic variation in the conditions of the chromatin which, at meiosis, govern chromosome pairing. During sexual differentiation, these conditions are fixed and subsequent cell lineages have the same pairing and crossing over characteristics. As homology search is an activity of DNA, this control of pairing and crossing over, consistent over long cell lineages, may be considered to be epigenetic even when no realistic suggestions concerning its character can be given
Trisomy greatly enhances interstitial crossing over in a translocation heterozygote of Secale
Sybenga, J. ; Verhaar, H.M. ; Botje, D.G.A. - \ 2012
Genome 55 (2012)1. - ISSN 0831-2796 - p. 15 - 25.
cereale l - rye - recombination - interference
Chromosomal rearrangements, including reciprocal translocations, may prevent recombinational transfer of genes from a donor genotype to a recipient, especially when the gene is located in an interstitial segment. The effect of trisomy of chromosome arm 1RS on recombination was studied in translocation heterozygote T248W of rye (Secale cereale), involving chromosome arms 1RS and 6RS. (Pro)metaphase I configuration frequencies were analyzed. Crossing over, estimated as chiasma parameters, in five genetically different euploid heterozygotes was compared with those of 10 different single arm trisomics. The addition of 1RS greatly altered the crossing over pattern around the translocation break point, with a special increase in the interstitial segment of 6RS and adjoining regions, normally hardly accessible to recombination. Furthermore, there was considerable variation between plants of closely related genotypes. Heterogeneity widens the distribution of crossing overs, including segments normally not accessible to recombination, but decreases average recombination in other segments. The extra chromosome and abnormal segregants are eliminated by using the trisomic as the pollen parent
Whether ideal free or not, predatory mites distribute so as to maximize reproduction
Hammen, T. van der; Montserrat, M. ; Sabelis, M.W. ; Roos, A.M. ; Janssen, A. - \ 2012
Oecologia 169 (2012)1. - ISSN 0029-8549 - p. 95 - 104.
free distribution models - unequal competitors - egg predation - interference - density - prey - phytoseiidae - oviposition - tests
Ideal free distribution (IFD) models predict that animals distribute themselves such that no individual can increase its fitness by moving to another patch. Many empirical tests assume that the interference among animals is independent of density and do not quantify the effects of density on fitness traits. Using two species of predatory mites, we measured oviposition as a function of conspecific density. Subsequently, we used these functions to calculate expected distributions on two connected patches. We performed an experimental test of the distributions of mites on two such connected patches, among which one had a food accessibility rate that was twice as high as on the other. For one of the two species, Iphiseius degenerans, the distribution matched the expected distribution. The distribution also coincided with the ratio of food accessibility. The other species, Neoseiulus cucumeris, distributed itself differently than expected. However, the oviposition rates of both species did not differ significantly from the expected oviposition rates based on experiments on single patches. This suggests that the oviposition rate of N. cucumeris was not negatively affected by the observed distribution, despite the fact that N. cucumeris did not match the predicted distributions. Thus, the distribution of one mite species, I. degenerans, was in agreement with IFD theory, whereas for the other mite species, N. cucumeris, unknown factors may have influenced the distribution of the mites. We conclude that density-dependent fitness traits provide essential information for explaining animal distributions
A fitness assay for comparing RNAi effects across multiple C. elegans genotypes
Elvin, M. ; Snoek, L.B. ; Frejno, M. ; Klemstein, U. ; Kammenga, J.E. ; Poulin, G. - \ 2011
BMC Genomics 12 (2011). - ISSN 1471-2164 - 14 p.
life-history traits - caenorhabditis-elegans - gene - interference - rde-1
Background - RNAi technology by feeding of E. coli containing dsRNA in C. elegans has significantly contributed to further our understanding of many different fields, including genetics, molecular biology, developmental biology and functional genomics. Most of this research has been carried out in a single genotype or genetic background. However, RNAi effects in one genotype do not reveal the allelic effects that segregate in natural populations and contribute to phenotypic variation. Results - Here we present a method that allows for rapidly comparing RNAi effects among diverse genotypes at an improved high throughput rate. It is based on assessing the fitness of a population of worms by measuring the rate at which E. coli is consumed. Critically, we demonstrate the analytical power of this method by QTL mapping the loss of RNAi sensitivity (in the germline) in a recombinant inbred population derived from a cross between Bristol and a natural isolate from Hawaii. Hawaii has lost RNAi sensitivity in the germline. We found that polymorphisms in ppw-1 contribute to this loss of RNAi sensitivity, but that other loci are also likely to be important. Conclusions - In summary, we have established a fast method that improves the throughput of RNAi in liquid, that generates quantitative data, that is easy to implement in most laboratories, and importantly that enables QTL mapping using RNAi.
Structures of the RNA-guided surveillance complex from a bacterial immune system
Wiedenheft, B. ; Jore, M.M. ; Brouns, S.J.J. ; Oost, J. van der - \ 2011
Nature 477 (2011). - ISSN 0028-0836 - p. 486 - 489.
crispr rna - electron-microscopy - target recognition - seed sequence - prokaryotes - interference - repeats - dna - endoribonuclease - maturation
Bacteria and archaea acquire resistance to viruses and plasmids by integrating short fragments of foreign DNA into clustered regularly interspaced short palindromic repeats (CRISPRs). These repetitive loci maintain a genetic record of all prior encounters with foreign transgressors1, 2, 3, 4, 5, 6. CRISPRs are transcribed and the long primary transcript is processed into a library of short CRISPR-derived RNAs (crRNAs) that contain a unique sequence complementary to a foreign nucleic-acid challenger7, 8, 9, 10, 11, 12. In Escherichia coli, crRNAs are incorporated into a multisubunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defence), which is required for protection against bacteriophages13, 14. Here we use cryo-electron microscopy to determine the subnanometre structures of Cascade before and after binding to a target sequence. These structures reveal a sea-horse-shaped architecture in which the crRNA is displayed along a helical arrangement of protein subunits that protect the crRNA from degradation while maintaining its availability for base pairing. Cascade engages invading nucleic acids through high-affinity base-pairing interactions near the 5' end of the crRNA. Base pairing extends along the crRNA, resulting in a series of short helical segments that trigger a concerted conformational change. This conformational rearrangement may serve as a signal that recruits a trans-acting nuclease (Cas3) for destruction of invading nucleic-acid sequences
Rapid Establishment of a Regular Distribution of Adult Tropical Drosophila Parasitoids in a Multi-Patch Environment by Patch Defence Behaviour.
Jong, P.W. de; Hemerik, L. ; Gort, G. ; Alphen, J.J.M. van - \ 2011
PLoS ONE 6 (2011)7. - ISSN 1932-6203 - 7 p.
interspecific host discrimination - asobara-tabida nees - time allocation - adaptive superparasitism - solitary parasitoids - insect parasitoids - wasps - interference - strategies - efficiency
Females of the larval parasitoid of Drosophila, Asobara citri, from sub-Saharan Africa, defend patches with hosts by fighting and chasing conspecific females upon encounter. Females of the closely related, palearctic species Asobara tabida do not defend patches and often search simultaneously in the same patch. The effect of patch defence by A. citri females on their distribution in a multi-patch environment was investigated, and their distributions were compared with those of A. tabida. For both species 20 females were released from two release-points in replicate experiments. Females of A. citri quickly reached a regular distribution across 16 patches, with a small variance/mean ratio per patch. Conversely, A. tabida females initially showed a clumped distribution, and after gradual dispersion, a more Poisson-like distribution across patches resulted (variance/mean ratio was closer to 1 and higher than for A. citri). The dispersion of A. tabida was most probably an effect of exploitation: these parasitoids increasingly made shorter visits to already exploited patches. We briefly discuss hypotheses on the adaptive significance of patch defence behaviour or its absence in the light of differences in the natural history of both parasitoid species, notably the spatial distribution of their hosts.
Alternative Stable States Driven by Density-Dependent Toxicity
Heide, T. van der; Nes, E.H. van; Katwijk, M.M. van; Scheffer, M. ; Hendriks, A.J. ; Smolders, A.J.P. - \ 2010
Ecosystems 13 (2010)6. - ISSN 1432-9840 - p. 841 - 850.
eelgrass zostera-marina - distinguishing resource competition - fresh-water wetlands - vegetation dieback - baltic sea - ecosystems - sulfide - phytotoxicity - interference - populations
Many populations are exposed to naturally occurring or synthetic toxicants. An increasing number of studies demonstrate that the toxicity of such compounds is not only dependent on the concentration or load, but also on the biomass or density of exposed organisms. At high biomass, organisms may be able to alleviate adverse effects of the toxicant by actively lowering ambient concentrations through either a joint detoxification mechanism or growth dilution. We show in a conceptual model that this mechanism may potentially lead to alternative stable states if the toxicant is lethal at low densities of organisms, whereas a high density is able to reduce the toxicant concentrations to sub-lethal levels. We show in an example that this effect may be relevant in real ecosystems. In an earlier published experimental laboratory study, we demonstrated that ammonia toxicity in eelgrass is highly dependent on the eelgrass shoot density. Here, we used the results of these experiments to construct a model describing the complex interactions between the temperate seagrass Zostera marina and potentially lethal ammonia. Analyses of the model show that alternative stable states are indeed present over wide ranges of key-parameter settings, suggesting that the mechanism might be important especially in sheltered, eutrophicated estuaries where mixing of the water layer is poor. We argue that the same mechanism could cause alternative stable states in other biological systems as well.
BNYVV-derived dsRNA confers resistance to rhizomania disease of sugar beet as evidenced by a novel transgenic hairy root approach
Pavli, R. ; Panopoulos, N.J. ; Goldbach, R.W. ; Skaracis, G.N. - \ 2010
Transgenic Research 19 (2010)5. - ISSN 0962-8819 - p. 915 - 922.
double-stranded-rna - yellow-vein-virus - beta-vulgaris l - gene-expression - mediated resistance - antisense rna - interference - plants - suppression - transformation
Agrobacterium rhizogenes-transformed sugar beet hairy roots, expressing dsRNA from the Beet necrotic yellow vein virus replicase gene, were used as a novel approach to assess the efficacy of three intron-hairpin constructs at conferring resistance to rhizomania disease. Genetically engineered roots were similar in morphology to wild type roots but were characterized by a profound abundancy, rapid growth rate and, in some cases, plagiotropic development. Upon challenge inoculation, seedlings showed a considerable delay in symptom development compared to untransformed or vector-transformed seedlings, expressing dsRNA from an unrelated source. The transgenic root system of almost all seedlings contained no or very low virus titer while the non-transformed aerial parts of the same plants were found infected, leading to the conclusion that the hairy roots studied were effectively protected against the virus. This readily applicable novel method forms a plausible approach to preliminarily evaluate transgenic rhizomania resistance before proceeding in transformation and whole plant regeneration of sugar beet, a tedious and time consuming process for such a recalcitrant crop species
Effects of inbreeding and genetic modification on Aedes aegypti larval competition and adult energy reserves
Koenraadt, C.J.M. ; Kormaksson, M. ; Harrington, L.C. - \ 2010
Parasites & Vectors 3 (2010). - ISSN 1756-3305 - 11 p.
yellow-fever mosquito - environment interactions - transgenic mosquitos - rapid-determination - borne diseases - fitness - diptera - culicidae - strain - interference
Background - Genetic modification of mosquitoes offers a promising strategy for the prevention and control of mosquito-borne diseases. For such a strategy to be effective, it is critically important that engineered strains are competitive enough to serve their intended function in population replacement or reduction of wild mosquitoes in nature. Thus far, fitness evaluations of genetically modified strains have not addressed the effects of competition among the aquatic stages and its consequences for adult fitness. We therefore tested the competitive success of combinations of wild, inbred and transgenic (created in the inbred background) immature stages of the dengue vector Aedes aegypti in the presence of optimal and sub-optimal larval diets. Results - The wild strain of Ae. aegypti demonstrated greater performance (based on a composite index of survival, development rate and size) than the inbred strain, which in turn demonstrated greater performance than the genetically modified strain. Moreover, increasing competition through lowering the amount of diet available per larva affected fitness disproportionately: transgenic larvae had a reduced index of performance (95-119%) compared to inbred (50-88%) and wild type larvae (38-54%). In terms of teneral energy reserves (glycogen, lipid and sugar), adult wild type mosquitoes had more reserves directly available for flight, dispersal and basic metabolic functions than transgenic and inbred mosquitoes. Conclusions - Our study provides a detailed assessment of inter- and intra-strain competition across aquatic stages of wild type, inbred, and transgenic mosquitoes and the impact of these conditions on adult energy reserves. Although it is not clear what competitive level is adequate for success of transgenic strains in nature, strong gene drive mechanisms are likely to be necessary in order to overcome competitive disadvantages in the larval stage that carryover to affect adult fitness