22 research outputs found

    Specific detection and quantification of virulent/avirulent Phytophthora infestans isolates using a real-time PCR assay that targets polymorphisms of the Avr3a gene.

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    Equipe 6International audienceMolecular tools that allow intraspecific quantification and discrimination of pathogen isolates are useful to assess fitness of competitors during mixed infections. However, methods that were developed for quantifying Phytophthora infestans are only specific at the species level. Here, we reported a TaqMan-based real-time PCR assay allowing, according to the specificity of the used probes, an accurate quantification of different proportions of two genetically distinct clones of P. infestans in mixed fractions. Indeed, in addition to a primer specific to P. infestans, two primers and two TaqMan(®) probes that target single-nucleotide polymorphisms located in the Avr3a/avr3a virulence gene sequence were designed. The reliability of the method was tested on serially diluted fractions containing plasmid DNA with either the Avr3a or the avr3a sequences at concentrations ranging from 10(2) to 10(8) copies per μl. Based on its specificity, sensitivity and repeatability, the proposed assay allowed a quantification of the targeted DNA sequence in fractions with a Avr3a/avr3a ratio in the range 1/99 to 99/1. The reliability of the test was also checked for counting zoospores. Applications for future research in P. infestans/host quantitative interactions were also discussed

    The novel gene Ny-1 on potato chromosome IX confers hypersensitive resistance to Potato virus Y and is an alternative to Ry genes in potato breeding for PVY resistance

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    Hypersensitive resistance (HR) is an efficient defense strategy in plants that restricts pathogen growth and can be activated during host as well as non-host interactions. HR involves programmed cell death and manifests itself in tissue collapse at the site of pathogen attack. A novel hypersensitivity gene, Ny-1, for resistance to Potato virus Y (PVY) was revealed in potato cultivar Rywal. This is the first gene that confers HR in potato plants both to common and necrotic strains of PVY. The locus Ny-1 mapped on the short arm of potato chromosome IX, where various resistance genes are clustered in Solanaceous genomes. Expression of HR was temperature-dependent in cv. Rywal. Strains PVYO and PVYN, including subgroups PVYNW and PVYNTN, were effectively localized when plants were grown at 20°C. At 28°C, plants were systemically infected but no symptoms were observed. In field trials, PVY was restricted to the inoculated leaves and PVY-free tubers were produced. Therefore, the gene Ny-1 can be useful for potato breeding as an alternative donor of PVY resistance, because it is efficacious in practice-like resistance conferred by Ry genes

    Phylogeography and Molecular Evolution of Potato virus Y

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    Potato virus Y (PVY) is an important plant pathogen, whose host range includes economically important crops such as potato, tobacco, tomato, and pepper. PVY presents three main strains (PVYO, PVYN and PVYC) and several recombinant forms. PVY has a worldwide distribution, yet the mechanisms that promote and maintain its population structure and genetic diversity are still unclear. In this study, we used a pool of 77 complete PVY genomes from isolates collected worldwide. After removing the effect of recombination in our data set, we used Bayesian techniques to study the influence of geography and host species in both PVY population structure and dynamics. We have also performed selection and covariation analyses to identify evolutionarily relevant amino acid residues. Our results show that both geographic and host-driven adaptations explain PVY diversification. Furthermore, purifying selection is the main force driving PVY evolution, although some indications of positive selection accounted for the diversification of the different strains. Interestingly, the analysis of P3N-PIPO, a recently described gene in potyviruses, seems to show a variable length among the isolates analyzed, and this variability is explained, in part, by host-driven adaptation

    Fitness costs associated with unnecessary virulence factors and life history traits: evolutionary insights from the potato late blight pathogen Phytophthora infestans.

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    BACKGROUND: In gene-for-gene models of plant-pathogen interactions, the existence of fitness costs associated with unnecessary virulence factors still represents an issue, both in evolutionary biology and agricultural sciences. Measuring such costs experimentally has proven difficult, especially in pathogens not readily amenable to genetic transformation, since the creation of isogenic lines differing only by the presence or absence of avirulence genes cannot be achieved in many organisms. Here, we circumvented this difficulty by comparing fitness traits in groups of Phytophthora infestans isolates sharing the same multilocus fingerprint, but differing by their virulence/avirulence spectrum. RESULTS: Fitness was assessed from calculations derived from the basic reproduction number, combining several life history traits (latent period, spore density and lesion growth rate) evaluated on leaflets of the potato cultivar Bintje, which is free of resistance genes. A statistically significant fitness cost was found in isolates virulent to the R10 resistance gene. That cost was due to a lower spore production in virulent isolates; however, the latent period was shorter in virulent isolates. Similar trends, although not statistically significant, were observed for the other genes tested. CONCLUSION: The data likely reflect the adaptive response of the pathogen to the cost associated with virulence. They suggest strong trade-offs between life history traits related to pathogenicity and adaptive biology of pathogens

    Microsatellite markers reveal two admixed genetic groups and an ongoing displacement within the French population of the invasive plant pathogen Phytophthora infestans

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    Potato late blight is an example of a re-emerging disease of plants. Phytophthora infestans was first introduced into Europe during the 19th century, where it caused the Irish potato famine. During the 20th century several additional introduction events have been suspected, especially in the mid-70s due to the import of large quantities of potato needed after the shortage caused by drought in 1976. Here, we investigate the genetic population structure of Phytophthora infestans, at the first stages of a recent invasion process in France. A total of 220 isolates was collected from 20 commercial fields of the potato susceptible cultivar Bintje, during two consecutive years (2004 and 2005). Clustering analyses based on eight recently developed microsatellite markers reveal that French P. infestans populations are made of two differentiated genetic clusters of isolates (FST = 0.19). This result suggests multiple introductions of P. infestans into France, either through the introduction of a composite population of isolates or through the successive introduction of isolates having differentiated genetic backgrounds. Both clusters identified have a strong clonal structure and are similar regarding genetic diversity and mating type composition. The maintenance of differentiation between the two genetic clusters should result from the low or non-existent contribution of sexual reproduction in French P. infestans populations

    Surface plasmon resonance for monitoring the interaction of Potato virus Y with monoclonal antibodies

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    BGPI : équipe 6International audienceSurface plasmon resonance (SPR)-based biosensors have been widely utilized for measuring interactions of a variety of molecules. Fewer examples include higher biological entities such as bacteria and viruses, and even fewer deal with plant viruses. Here, we describe the optimization of an SPR sensor chip for evaluation of the interaction of the economically relevant filamentous Potato virus Y(PVY) with monoclonal antibodies. Different virus isolates were efficiently and stably bound to a previously immobilized polyclonal antibody surface, which remained stable over subsequent injection regeneration steps. The ability of the biosensor to detect and quantify PVY particles was compared with ELISA and RT-qPCR. Stably captured virus surfaces were successfully used to explore kinetic parameters of the interaction of a panel of monoclonal antibodies with two PVY isolates representing the main viral serotypes N and O. In addition, the optimized biosensor proved to be suitable for evaluating whether two given monoclonal antibodies compete for the same epitope within the viral particle surface. The strategy proposed in this work can help to improve existing serologic diagnostic tools that target PVY and will allow investigation of the inherent serological variability of the virus and exploration for new interactions of PVY particles with other proteins. (C) 2013 Elsevier Inc. All rights reserved
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