Evaluating fundamental life-history traits for Tobacco etch potyvirus

Los virus de ARN son probablemente algunos de los parásitos más extendidos que se pueden encontrar en todas las formas de vida. Estos agentes infecciosos parecen particularmente propensos a causar enfermedades emergentes en plantas, seres humanos y otros animales. Sus habilidades para escapar al sistema inmune, evadir estrategias antivirales o para infectar a nuevas especies son una consecuencia directa de su enorme capacidad para evolucionar rápidamente. Comprender los procesos básicos del cambio evolutivo puede ser unos de los principales pasos necesarios en el diseño de nuevas estrategias de intervención. Desde una perspectiva evolutiva, una de las principales características de los virus de ARN es su capacidad para mutar. El conocimiento de las tasas de mutación y el espectro molecular de mutaciones espontáneas son importantes para entender la evolución de la composición genética de las poblaciones virales. Estudios anteriores han demostrado que la tasa de mutaciones espontáneas de los virus de ARN varía 14 ampliamente entre 0.01 y 2 mutaciones por genoma y generación, ocupando los virus de plantas la parte inferior de esta escala de valores. En este estudio, se propuso analizar el espectro mutacional y la tasa de mutación del virus del grabado del tabaco (Tobacco etch virus, TEV), como modelo para los virus de ARN de cadena positiva. Nuestro experimento minimiza la acción purificadora de la selección en el espectro mutacional, dando así una imagen exacta de qué tipo de mutación ha producido la replicasa viral. Hemos calculado la tasa de mutación espontánea de este virus, hallándose en el intervalo de valores entre 10-6 - 10-5 mutaciones por sitio y generación. Nuestras estimaciones se encuentran en el mismo rango de valores que los anteriormente descritos para otros virus ARN de plantas. La recombinación de un virus de ARN es un parámetro evolutivo que contribuye significativamente a la diversidad y a la evolución de los virus. La tasa de recombinación depende de dos parámetros: de la frecuencia de intercambio genético entre genomas virales dentro de una célula infectada del huésped y de la frecuencia de las células doblemente-infectadas. A pesar 15   de la importancia del conocimiento de dichos factores, actualmente solo se ha estimado experimentalmente la tasa de recombinación del retrovirus virus del mosaico de la coliflor (Cauliflower mosaic virus, CaMV) (Froissart et al., 2005), mediante una cuantificación in vivo y directa de la tasa de recombinación de dicho virus durante la infección de un huésped. Dicha tasa se estimó en 4×10-5 eventos de recombinación por nucleótido y por ciclo de replicación. En los potyvirus, observaciones in vivo han demostrado que aislados del mismo virus se segregan espacialmente durante infecciones mixtas. Esta segregación debería reducir al mínimo la posibilidad de dos genotipos de infectar las mismas células y así limitar eventos de recombinación durante la replicación del ARN viral. Para conciliar estas observaciones, hemos evaluado la tasa de recombinación y la multiplicidad de infección (MOI) del virus TEV, in vivo. La tasa de recombinación se estimó en 1.03×10-5 eventos de recombinación por nucleótido y generación. Este valor se encuentra en el mismo orden de magnitud que la tasa de mutación del TEV, lo que sugiere que la recombinación tiene una importancia 16   comparable a la mutación puntual en la creación de variabilidad. La multiplicidad de infección celular se define como el número de genomas virales que logran infectar de manera eficaz una célula. Dos estudios recientes han mostrado estimaciones in vivo de la MOI para el virus del mosaico del tabaco (Tobacco mosaic virus, TMV) y del CaMV, gracias a métodos sofisticados que miden la distribución de dos genotipos virales en las células del huésped. Aquí presentamos un análisis detallado de la dinámica temporal y espacial de la MOI celular durante la colonización de una planta por el TEV. Observamos una baja frecuencia tanto del número de células infectadas por el virus (media ± SD: 0.100 ± 0.073), como de las infectadas por dos genotipos del TEV (media ± SD: 0.012 ± 0.023). Se usó un nuevo método basado en un modelo de selección para determinar la MOI. Los valores de MOI predichos fueron bajos, oscilando entre 1.0 (hoja tres, 3 días después de la inoculación (dpi)) a 1.6 (hoja cuatro, 7 dpi). Por último, la alta diversidad genética de las poblaciones virales da lugar a una nube de variantes, todas 17   vinculadas a través de mutación, que interactúan y contribuyen colectivamente, conocido también por el término de cuasiespecies. Las poblaciones virales pueden adaptarse rápidamente a sus entornos dinámicos, y son notablemente inestables en determinadas condiciones como hemos demostrado en este trabajo durante la evolución del TEV en el caso de redundancia genética y funcional. Después de varios pasos a tiempos largos de infección en plantas transgénicas que expresan la replicasa viral NIb, se observaron grandes deleciones y múltiples partículas defectuosas. Este resultado demuestra la gran plasticidad del genoma de los virus ARN y su capacidad para eliminar cualquier carga genética innecesaria.RNA viruses are probably some of the most pervasive parasites that can be found in all life forms. These infectious agents seem particularly prone to causing emerging diseases in plants, humans and other animals. Their abilities to escape the immune system, evade antiviral strategies or to jump to new species are a direct consequence of their enormous capacity to evolve quickly. Understanding basic processes of evolutionary change may be a necessary primary step in designing new intervention strategies. One of the key characteristics of RNA viruses, especially from an evolutionary perspective, is their capacity for mutation. Knowing mutation rates and the molecular spectrum of spontaneous mutations is important to understanding how the genetic composition of viral populations evolves. Previous studies have shown that the rate of spontaneous mutations for RNA viruses widely varies between 0.01 and 2 mutations per genome and generation, with plant RNA viruses always occupying the lower side of this range. Here we analyse the spontaneous mutational spectrum 20 and the mutation rate of Tobacco etch potyvirus (TEV), a model system of positive sense RNA viruses. Our experimental set up minimizes the action of purifying selection on the mutational spectrum, thus giving a picture of what types of mutations are produced by the viral replicase. We have estimated that the spontaneous mutation rate for this virus was in the range 10-6 - 10-5 mutations per site and generation. Our estimates are in the same biological ballpark as previous values reported for plant RNA viruses. Recombination is a virus characteristic that also contributes significantly to the diversity and evolution of viruses. The recombination rate depends on two parameters: the frequency of genetic exchange between viral genomes within an infected host cell and the frequency of co-infected cells. Despite this importance, only one direct quantification of the in vivo recombination rate for an RNA virus during host infection has been reported: the in vivo recombination rate for Cauliflower mosaic virus (CaMV) (Froissart et al., 2005) was reported to be 4×10-5 events per nucleotide site and per replication cycle. In plant potyviruses, in vivo observations have 21   shown that strains of the same virus segregate spatially during mixed infections. This segregation shall minimize the chances of two genotypes to co-infect the same cells and henceforth, precludes template switching and recombination events during genomic RNA replication. To reconcile these confronting observations, we have evaluated the in vivo TEV recombination rate and multiplicity of infection (MOI). TEV recombination rate was estimated to 1.03×10−5 recombination events per nucleotide site and generation. This value is in the same order of magnitude than TEV mutation rate, suggesting that recombination should be at least as important as point mutation in creating variability. The multiplicity of cellular infection is defined as the number of viral genome infecting effectively a cell. Two recent studies have reported in vivo MOI estimates for Tobacco mosaic virus (TMV) and CaMV, using sophisticated approaches to measure the distribution of two virus genotypes over host cells. Here, we present a detailed analysis of spatial and temporal dynamics of the cellular MOI during colonization of a host plant by TEV. We observe a low frequency of virus-infected 22   cells (mean ± SD: 0.100 ± 0.073), and cells infected by both virus variants (mean ± SD: 0.012 ± 0.023). A new, model-selection- based method was used to determine the MOI, and the predicted MOIs values were low, ranging from 1.0 (leaf three, 3 days post inoculation (dpi)) to 1.6 (leaf four, 7 dpi). Finally, high genetic diversity in viral population gives rise to a cloud of variants; linked through mutation, interacting and contributing collectively; also called quasi-species. Viral populations can rapidly adapt to dynamic environments, remaining remarkably unstable under certain conditions as observed during evolution of TEV in case of genetic and functional redundancy. Large deletions and multiple defective particles were observed after various passages of long time TEV infection in transgenics plants expressing the viral replicase NIb. This result demonstrates the great genome plasticity of RNA virus and their capacity to eliminate any useless genetic load

Nanomechanical testing study of the elementary deformation mechanisms in the Ti2AlN and Cr2AlC MAX phases

Abstract: Deformation mechanisms in MAX phases are still not well understood. The complex mechanical behavior of these materials, including mechanical hysteresis, arises both from their crystallography, with a nanolayered structure alternating nitride or carbide layers with metal atoms layers, and from their macroscopic polycrystalline structure, composed of platelets-like grains. In order to distinguish from these two contributions, we focused our study at the sub-micrometer scale, in order to probe the mechanical response of individual grains. Please click Additional Files below to see the full abstract

Deformation twinning in Cr2AlC MAX phase single crystals: A nanomechanical testing study

In a recent study [1], we observed and characterized for the first time deformation twinning in the Ti2AlN MAX phase deformed at high temperature (800°C) by Berkovich nanoindentation. Since plastic deformation in these nanolayered materials was believed to be governed only by basal plane dislocations involved in kink band mechanisms, this result has shed a new light on the mechanical behavior of MAX phases. In order to go further in the understanding of twinning deformation mechanisms in MAX phases, we performed a study in Cr2AlC single crystal, deformed at room temperature by spherical nanoindentation and by micropillar compression tests, in such an orientation that the basal plane was edge on, to inhibit basal dislocations and to promote twinning. Please click Download on the upper right corner to see the full abstract

Portevin‐Le Chatelier effect in AlMg3% studied using elevated temperature nanoindentation

The Portevin-Le Chatelier (PLC) is a plastic instability observed in different alloys, and particularly in aluminum alloys, which is characterized by a serrated flow during plastic deformation. The PLC effect originates from the competition between gliding of mobile dislocations and pinning of these dislocations by diffusing solute atoms. This dynamic strain hardening leads to a negative strain rate sensitivity which is often used to characterize or quantify the PLC effect. The PLC effect has been widely investigated in the case of stress-strain curves obtained in macroscopic uni-axial tests. However, in the case of the aluminum matrix composites Al/AlCuFe, it has been observed that copper atoms diffuse during the material synthesis form the reinforcement particles to the aluminum matrix. The aluminum matrix thus presents a heterogeneous concentration of copper atoms leading to local PLC effect. Nanoindentation test is the best way to characterize locally this mechanical effect. However, strain rate is not a convenient parameter for nanoindentation tests since the complex strain field below the indent, as well as the increase of the contact area during the test, makes difficult the definition a single strain value. Another way to investigate a local PLC effect would be thus the perform nanoindentation tests at different temperatures rather than different strain rates. This poster will present experimental results from elevated temperature nanoindentation studies on an AlMg3% alloy, used as a model material for easy comparison with uniaxial tests, in the temperature range from 25-300°C. The experiments were performed in displacement controlled mode in a recently developed vacuum high temperature nanoindenter based on active surface referencing and non-contact tip and sample heating. In this configuration, the PLC effect appears as successive load drops on the loading curves. The temperatures of the tip and the sample surface were calibrated and matched in order to minimize thermal drift. With increasing temperature, the magnitude of load drop decreased whereas its occurrence frequency increased. The load drop magnitude and its occurrence frequency were statistically analyzed for different temperatures of testing. The results will be discussed in terms of an expanding plastic volume beneath the indenter interacting with the solute atoms in the complex stress field of the indenter

Mechanical hysteresis of the MAX phase Ti2AlN: A nano-mechanical testing study

MAX phases are nano-lamellar ternary carbides and nitrides, with a hexagonal crystallographic structure. These materials combine several properties of metals and ceramics, which give them a high potential for technological applications. Their mechanical properties are characterized by a high stiffness and a relatively low yield strength More surprisingly, deformation tests on MAX phases reveal a mechanical hysteresis. At a macroscopic scale, in polycrystalline samples, several studies have shown that this behavior could be related to load transfers from grain to grain. However, a mechanical hysteresis is also observed in single crystals. In this work, the mechanical hysteresis and the plasticity of the MAX phase Ti2AlN has been studied at small scale by using nanoindentation tests with a spherical tip and micro-pillar compression tests. In both cases, cyclic loadings have been applied in single grains, for different crystallographic orientations, previously determined by EBSD. These cyclic loadings, with partial unloadings (cf. figure 1), have revealed a same behavior in nanoindentation tests and in micro-pillars compression test. In both cases, the unloading curves show an elastic behavior followed by a plastic recovery at low load. Furthermore, this mechanical hysteresis is related to the crystallographic orientation since the energy dissipated during the cycles is shown to be minimum when the basal plane is perpendicular or parallel to the indentation (or compression) axis. Please click Additional Files below to see the full abstract

Room temperature deformation in the Fe7_7Mo6_6 μ\mu-Phase

The role of TCP phases in deformation of superalloys and steels is still not fully resolved. In particular, the intrinsic deformation mechanisms of these phases are largely unknown including the active slip systems in most of these complex crystal structures. Here, we present a first detailed investigation of the mechanical properties of the Fe7Mo6 {\mu}-phase at room temperature using microcompression and nanoindentation with statistical EBSD-assisted slip trace analysis and TEM imaging. Slip occurs predominantly on the basal and prismatic planes, resulting also in decohesion on prismatic planes with high defect density. The correlation of the deformation structures and measured hardness reveals pronounced hardening where interaction of slip planes occurs and prevalent deformation at pre-existing defects.Comment: Accepted manuscript in International Journal of Plasticit

Estimation of the in vivo recombination rate for a plant RNA virus

[EN] Phylogenomic evidence suggested that recombination is an important evolutionary force for potyviruses, one of the larger families of plant RNA viruses. However, mixed-genotype potyvirus infections are marked by low levels of cellular coinfection, precluding template switching and recombination events between virus genotypes during genomic RNA replication. To reconcile these conflicting observations, we evaluated the in vivo recombination rate (r(g)) of Tobacco etch virus (TEV; genus Potyvirus, family Potyviridae) by coinfecting plants with pairs of genotypes marked with engineered restriction sites as neutral markers. The recombination rate was then estimated using two different approaches: (i) a classical approach that assumed recombination between marked genotypes can occur in the whole virus population, rendering an estimate of r(g)=7.762x10(-8) recombination events per nucleotide site per generation, and (ii) an alternative method that assumed recombination between marked genotypes can occur only in coinfected cells, rendering a much higher estimate of r(g)=3.427x10(-8) recombination events per nucleotide site per generation. This last estimate is similar to the TEV mutation rate, suggesting that recombination should be at least as important as point mutation in creating variability. Finally, we compared our mutation and recombination rate estimates to those reported for animal RNA viruses. Our analysis suggested that high recombination rates may be an unavoidable consequence of selection for fast replication at the cost of low fidelity.We thank Francisca de la Iglesia and Angels Prosper for excellent technical assistance, Jose A. Dare's for methodological advice, Jose M. Cuevas for critical reading of the manuscript, and other lab members for helpful discussions. This work was supported by the Spanish Secretaria de Estado de Investigacion, Desarrollo e Innovacion (grants BFU2009-06993 and BFU2012-30805). N. T. was supported,by a pre-doctoral fellowship from the former Spanish Ministerio de Ciencia e Innovacion.Tromas, N.; Zwart, MP.; Poulain, M.; Elena Fito, SF. (2014). Estimation of the in vivo recombination rate for a plant RNA virus. Journal of General Virology. 95:724-732. https://doi.org/10.1099/vir.0.060822-0S7247329

Toward the understanding of the brittle to ductile transition at low size in silicon: Experiments and simulations

While bulk silicon is brittle at temperatures below 600-700K, the compression of nanopillars has shown that a decrease of the diameter below few hundreds of nanometers could change the silicon behavior from brittle to ductile [1,2]. This size effect cannot be explained by the initial defect density like in metals, because pristine silicon nano-objects do not contain residual defects. In these conditions the cracks and/or the dislocations nucleation should take origin at the surface. The identification of the parameters governing the brittle to ductile transition in size and the understanding of the mechanisms are the key points to further develop the MEMS and NEMS technology or to prevent the failure of microelectronic components based on the silicon strained technology. Nowadays the respective improvements in simulations and experiments allow to investigate the mechanical properties of objects of similar sizes, close to hundreds of nanometers. We have then used both approaches - experiments and simulations – to understand the mechanisms at the origin of cracks and dislocations nucleation in such nanopillars. Experimentally,nanopillars with diameters of 100 nm and heights of 300 nm are obtained by lithography. They are deformed in compression by a flat punch nano-indentor under controlled-displacement mode at room temperature, and analyzed by scanning electron microscopy and high resolution transmission electron microscopy. In simulation, nanopillars up to 44 nm in diameter and height are investigated under compression and tension in controlled-displacement too, with a temperature ranging from 1 to 600K. The atomic interactions in silicon are modeled by two different semi-empirical potentials, Stillinger Weber and a Modified Embedded-Atom-Method (MEAM), both fitted to better reproduce the ductile and brittle properties of bulk silicon. Under compressive load (Fig. 1), both approaches reveal a ductile behavior with similar stress-strain curves, and large shear bands of amorphous silicon along the slip plane. In addition the simulations enlighten the formation of stacking fault plane in the anti-twining shear stress direction at the onset of plasticity, not yet confirmed by experiments (work in progress). The simulations under tensile load (Fig. 2) show the nucleation of perfect dislocations from the surface that can lead to cavity opening when they interact [3]. We observe first that the height of the nanopillars must be higher than 20 nm to allow the cavity opening, and second that the brittle to ductile transition is controlled by the diameter of the nanopillars, as observed experimentally in compression. The deformation of pillars with large diameters operates by cavity expansion leading to the brittle fracture, while pillars with smaller diameters are deformed by dislocations gliding leading to ductile fracture. Finally, the simulations in temperature seem to corroborate the fact that the size of the brittle to ductile transition could increase with temperature, as presumed experimentally [2]

Ecological selection of siderophore-producing microbial taxa in response to heavy metal contamination

This is the author accepted manuscript. The final version is available from Wiley via the DOI in this record.Some microbial public goods can provide both individual and community-wide benefits, and are open to exploitation by non-producing species. One such example is the production of metal-detoxifying siderophores. Here, we investigate whether conflicting selection pressures on siderophore production by heavy metals - a detoxifying effect of siderophores, and exploitation of this detoxifying effect - result in a net increase or decrease. We show that the proportion of siderophore-producing taxa increases along a natural heavy metal gradient. A causal link between metal contamination and siderophore production was subsequently demonstrated in a microcosm experiment in compost, in which we observed changes in community composition towards taxa that produce relatively more siderophores following copper contamination. We confirmed the selective benefit of siderophores by showing that taxa producing large amounts of siderophore suffered less growth inhibition in toxic copper. Our results suggest that ecological selection will favour siderophore-mediated decontamination, with important consequences for potential remediation strategies.This work was funded by the AXA Research Fund and BBSRC (BB/K003240) and NERC (NE/P001130) research councils to AB. SOB was funded by a “Bridging the Gaps” award and PhD scholarship from the University of Exeter. NT was funded by the Horizon 2020 Framework Programme under the Marie Sklodowska-Curie grant agreement (656647). AML was supported by Marie Curie International Incoming Fellowships within the EU Seventh Framework Programme. AB acknowledges support from the Royal Society

Nanoindentation cartography in Al/Al-Cu-Fe composites: Correlation between chemical heterogeneities and mechanical properties

During the last two decades, nanoindentation testing has become a commonly used technique for measuring surface mechanical properties such as hardness or elastic modulus. With devices equipped with a motorized X-Y table, it is now possible to perform large regular nanoindentation arrays in order to make an accurate statistics of the mechanical properties. This method is particularly interesting to study heterogeneous materials. The statistical analysis, associated to mathematical deconvolution methods allows identifying the properties of each individual phase. Furthermore, hardness or elastic modulus maps can be then established and compared to other local properties such as microstructure, crystallographic orientation or chemical composition. The nanoindentation cartography method has been used to study the mechanical properties of a metal matrix composite (Aluminum matrix with ω-Al-Cu-Fe reinforcement particles, synthesized by sparking plasma sintering) (cf. figure 1). Emphasize has been placed on the Aluminum matrix properties, where the detailed analysis of the individual nanoindentation curves shows serrated behavior characteristic of Portevin-Le Chatelier effect associated to dislocation pinning by solute atoms. The comparison between chemical (SEM – EDXS analysis) and hardness maps as well as the quantitative analysis of the deformation curves gives evidence of a strong correlation between the chemical heterogeneities and mechanical properties of the Aluminum matrix