In silico reversal of repeat-induced point mutation (RIP) identifies the origins of repeat families and uncovers obscured duplicated genes

Background: Repeat-induced point mutation (RIP) is a fungal genome defence mechanism guarding against transposon invasion. RIP mutates the sequence of repeated DNA and over time renders the affected regions unrecognisable by similarity search tools such as BLAST. Results: DeRIP is a new software tool developed to predict the original sequence of a RIP-mutated region prior to the occurrence of RIP. In this study, we apply deRIP to the genome of the wheat pathogen Stagonospora nodorum SN15 and predict the origin of several previously uncharacterised classes of repetitive DNA. Conclusions: Five new classes of transposon repeats and four classes of endogenous gene repeats were identified after deRIP. The deRIP process is a new tool for fungal genomics that facilitates the identification and understanding of the role and origin of fungal repetitive DNA. DeRIP is open-source and is available as part of the RIPCAL suite at http://www.sourceforge.net/projects/ripcal

Two alternative recessive quantitative trait loci influence resistance to spring black stem and leaf spot in Medicago truncatula

Background Knowledge of the genetic basis of plant resistance to necrotrophic pathogens is incomplete and has been characterised in relatively few pathosystems. In this study, the cytology and genetics of resistance to spring black stem and leaf spot caused by Phoma medicaginis, an economically important necrotrophic pathogen of Medicago spp., was examined in the model legume M. truncatula. Results Macroscopically, the resistant response of accession SA27063 was characterised by small, hypersensitive-like spots following inoculation while the susceptible interaction with accessions A17 and SA3054 showed necrotic lesions and spreading chlorosis. No unique cytological differences were observed during early infection (<48 h) between the resistant and susceptible genotypes, except pathogen growth was restricted to one or a few host cells in SA27063. In both interactions reactive oxygen intermediates and phenolic compounds were produced, and cell death occurred. Two F2 populations segregating for resistance to spring black stem and leaf spot were established between SA27063 and the two susceptible accessions, A17 and SA3054. The cross between SA27063 and A17 represented a wider cross than between SA27063 and SA3054, as evidenced by higher genetic polymorphism, reduced fertility and aberrant phenotypes of F2 progeny. In the SA27063 × A17 F2 population a highly significant quantitative trait locus (QTL, LOD = 7.37; P < 0.00001) named resistance to the necrotroph P homa m edicaginis one (rnpm1) genetically mapped to the top arm of linkage group 4 (LG4). rnpm1 explained 33.6% of the phenotypic variance in the population's response to infection depicted on a 1–5 scale and was tightly linked to marker AW256637. A second highly significant QTL (LOD = 6.77; P < 0.00001), rnpm2, was located on the lower arm of LG8 in the SA27063 × SA3054 map. rnpm2 explained 29.6% of the phenotypic variance and was fine mapped to a 0.8 cM interval between markers h2_16a6a and h2_21h11d. rnpm1 is tightly linked to a cluster of Toll/Interleukin1 receptor-nucleotide binding site-leucine-rich repeat (TIR-NBS-LRR) genes and disease resistance protein-like genes, while no resistance gene analogues (RGAs) are apparent in the genomic sequence of the reference accession A17 at the rnpm2 locus. Conclusion The induction of defence responses and cell death in the susceptible interaction following infection by P. medicaginis suggested this pathogen is not negatively affected by these responses and may promote them. A QTL for resistance was revealed in each of two populations derived from crosses between a resistant accession and two different susceptible accessions. Both loci are recessive in nature, and the simplest explanation for the existence of two separate QTLs is the occurrence of host genotype-specific susceptibility loci that may interact with undetermined P. medicaginis virulence factors

Proteomic identification of extracellular proteins regulated by the Gna1 Gα subunit in Stagonospora nodorum

The fungus Stagonospora nodorum is the causal agent of stagonospora nodorum blotch (syn.leaf and glume blotch) disease of wheat. The Gna1-encoded Ga protein is an important signaltransduction component in the fungus, which is required for full pathogenicity, sporulationand extracellular depolymerase production. In this study, we sought to gaina better understanding of defects associated with the gna1 mutant by using twodimensionalgel electrophoresis to analyse the extracellular proteome for differences tothe wildtype. Mass spectrometry analysis of altered abundant protein spots and peptidematching to the Stagonospora nodorum genome database have led to the identification ofgenes implicated in cell wall degradation, proteolysis, RNA hydrolysis and aromatic compoundmetabolism. In addition, quantitative RT-PCR has demonstrated that some of theencoding genes showed differential expression throughout host infection. Implicationsof these proteins and their corresponding genes in fungal virulence are discussed.The fungus Stagonospora nodorum is the causal agent of stagonospora nodorum blotch (syn. leaf and glume blotch) disease of wheat. The Gna1-encoded Gα protein is an important signal transduction component in the fungus, which is required for full pathogenicity, sporulation and extracellular depolymerase production. In this study, we sought to gain a better understanding of defects associated with the gna1 mutant by using two-dimensional gel electrophoresis to analyse the extracellular proteome for differences to the wildtype. Mass spectrometry analysis of altered abundant protein spots and peptide matching to the Stagonospora nodorum genome database have led to the identification of genes implicated in cell wall degradation, proteolysis, RNA hydrolysis and aromatic compound metabolism. In addition, quantitative RT-PCR has demonstrated that some of the encoding genes showed differential expression throughout host infection. Implications of these proteins and their corresponding genes in fungal virulence are discussed

Heterologous Expression of the Pyrenophora tritici-repentis Effector Proteins ToxA and ToxB, and the Prevalence of Effector Sensitivity in Australian Cereal Crops

Here, we evaluate the expression of the proteinaceous effectors ToxA and ToxB, produced by the necrotrophic fungal pathogen Pyrenophora tritici-repentis, which confer tan spot disease susceptibility on wheat. These necrotrophic effectors were expressed in two heterologous systems: Escherichia coli and Pichia pastoris. The E. coli SHuffle system was demonstrated to be superior to P. pastoris in generating high-levels of recombinant proteins that were soluble and stable. In addition, protein extracts from P. pastoris induced non-specific chlorosis on wheat, postulated to be caused by co-purified glucanases secreted by the host. Up to 79.6 μg/ml of ToxB was obtained using the SHuffle system in the absence of the native signal peptide, whilst the ToxA yield was considerably lower at 3.2 μg/ml. Results indicated that a histidine tag at the ToxA C-terminus interfered with effector functionality. Heterologously expressed ToxA and ToxB were tested on a panel of Australian cereals, including 122 varieties of bread wheat, 16 durum, 20 triticale and 5 barley varieties, as well as common plant model species including tobacco and Arabidopsis thaliana. A varying degree of effector sensitivities was observed, with a higher ToxB sensitivity and prevalence in the durum and triticale varieties. ToxB-induced chlorosis was also detected on barley. The heterologous expression of effectors that are easily scalable, will facilitate effector-assisted selection of varieties in wheat breeding programs as well as the investigation of P. tritici-repentis effectors in host and non-host interactions

Costs of sea dikes – regressions and uncertainty estimates

Failure to consider the costs of adaptation strategies can be seen by decision makers as a barrier to implementing coastal protection measures. In order to validate adaptation strategies to sea-level rise in the form of coastal protection, a consistent and repeatable assessment of the costs is necessary. This paper significantly extends current knowledge on cost estimates by developing – and implementing using real coastal dike data – probabilistic functions of dike costs. Data from Canada and the Netherlands are analysed and related to published studies from the US, UK, and Vietnam in order to provide a reproducible estimate of typical sea dike costs and their uncertainty. We plot the costs divided by dike length as a function of height and test four different regression models. Our analysis shows that a linear function without intercept is sufficient to model the costs, i.e. fixed costs and higher-order contributions such as that due to the volume of core fill material are less significant. We also characterise the spread around the regression models which represents an uncertainty stemming from factors beyond dike length and height. Drawing an analogy with project cost overruns, we employ log-normal distributions and calculate that the range between 3x and x∕3 contains 95 % of the data, where x represents the corresponding regression value. We compare our estimates with previously published unit costs for other countries. We note that the unit costs depend not only on the country and land use (urban/non-urban) of the sites where the dikes are being constructed but also on characteristics included in the costs, e.g. property acquisition, utility relocation, and project management. This paper gives decision makers an order of magnitude on the protection costs, which can help to remove potential barriers to developing adaptation strategies. Although the focus of this research is sea dikes, our approach is applicable and transferable to other adaptation measures

Costs of sea dikes – regressions and uncertainty estimates

Failure to consider the costs of adaptation strategies can be seen by decision makers as a barrier to implementing coastal protection measures. In order to validate adaptation strategies to sea-level rise in the form of coastal protection, a consistent and repeatable assessment of the costs is necessary. This paper significantly extends current knowledge on cost estimates by developing – and implementing using real coastal dike data – probabilistic functions of dike costs. Data from Canada and the Netherlands are analysed and related to published studies from the US, UK, and Vietnam in order to provide a reproducible estimate of typical sea dike costs and their uncertainty. We plot the costs divided by dike length as a function of height and test four different regression models. Our analysis shows that a linear function without intercept is sufficient to model the costs, i.e. fixed costs and higher-order contributions such as that due to the volume of core fill material are less significant. We also characterise the spread around the regression models which represents an uncertainty stemming from factors beyond dike length and height. Drawing an analogy with project cost overruns, we employ log-normal distributions and calculate that the range between 3x and x∕3 contains 95 % of the data, where x represents the corresponding regression value. We compare our estimates with previously published unit costs for other countries. We note that the unit costs depend not only on the country and land use (urban/non-urban) of the sites where the dikes are being constructed but also on characteristics included in the costs, e.g. property acquisition, utility relocation, and project management. This paper gives decision makers an order of magnitude on the protection costs, which can help to remove potential barriers to developing adaptation strategies. Although the focus of this research is sea dikes, our approach is applicable and transferable to other adaptation measures

Estimated financial returns from two white pine plantations, Station Bulletin, no.496

The Bulletin is a publication of the New Hampshire Agricultural Experiment Station, College of Life Sciences and Agriculture, University of New Hampshire, Durham, New Hampshire

The impact of temperature on insecticide toxicity against the malaria vectors Anopheles arabiensis and Anopheles funestus

BACKGROUND: It is anticipated that malaria elimination efforts in Africa will be hampered by increasing resistance to the limited arsenal of insecticides approved for use in public health. However, insecticide susceptibility status of vector populations evaluated under standard insectary test conditions can give a false picture of the threat, as the thermal environment in which the insect and insecticide interact plays a significant role in insecticide toxicity. METHODS: The effect of temperature on the expression of the standard WHO insecticide resistance phenotype was examined using Anopheles arabiensis and Anopheles funestus strains: a susceptible strain and the derived resistant strain, selected in the laboratory for resistance to DDT or pyrethroids. The susceptibility of mosquitoes to the pyrethroid deltamethrin or the carbamate bendiocarb was assessed at 18, 25 or 30 degrees C. The ability of the pyrethroid synergist piperonyl-butoxide (PBO) to restore pyrethroid susceptibility was also assessed at these temperatures. RESULTS: Temperature impacted the toxicity of deltamethrin and bendiocarb. Although the resistant An. funestus strain was uniformly resistant to deltamethrin across temperatures, increasing temperature increased the resistance of the susceptible An. arabiensis strain. Against susceptible An. funestus and resistant An. arabiensis females, deltamethrin exposure at temperatures both lower and higher than standard insectary conditions increased mortality. PBO exposure completely restored deltamethrin susceptibility at all temperatures. Bendiocarb displayed a consistently positive temperature coefficient against both susceptible and resistant An. funestus strains, with survival increasing as temperature increased. CONCLUSIONS: Environmental temperature has a marked effect on the efficacy of insecticides used in public health against important African malaria vectors. Caution must be exercised when drawing conclusions about a chemical's efficacy from laboratory assays performed at only one temperature, as phenotypic resistance can vary significantly even over a temperature range that could be experienced by mosquitoes in the field during a single day. Similarly, it might be inappropriate to assume equal efficacy of a control tool over a geographic area where local conditions vary drastically. Additional studies into the effects of temperature on the efficacy of insecticide-based interventions under field conditions are warranted

Nanoindentation at elevated temperatures

Relating the creep response observed with high temperature instrumented indentation experiments to macroscopic uniaxial creep response is of great practical value. In this review, we present an overview of various methods currently being used to measure creep at small scales with instrumented indentation, with a focus on geometrically self-similar indenters, and their relative merits and demerits from an experimental perspective. A comparison of the various methods to use those instrumented indentation results to predict the uniaxial power law creep response of a wide range of materials (stress exponent of 1 to 8), will be presented to assess their validity. The interplay of size dependent hardness effects, strain rate effects and temperature effects will also be discussed. The extension of rapid testing and mapping techniques to high temperatures will also be demonstrated. Figure 1 shows a map of hardness vs position in a carbide containing steel at 300 degrees C. These techniques are extended to stress exponent and pre-exponential maps determined at high temperatures. Please click Additional Files below to see the full abstract