Xanthomonas albilineans is able to move outside of the sugarcane xylem despite its reduced genome and the absence of a Hrp type III secretion system.

Xanthomonas albilineans, the causal agent of leaf scald disease of sugarcane, is a pathogen that experienced genome reduction during its speciation. Additionally, this xanthomonad is notably missing the Hrp type III secretion system and the xanthan gene cluster that are commonly found in pathogenic Xanthomonas species. X. albilineans was up to now considered as limited to the xylem of sugarcane. However, recently published studies suggested that X. albilineans was able to invade tissues other than the xylem of sugarcane leaves but the occurrence of X. albilineans outside the xylem has not been clearly proven. In this study, we used confocal microscopy and transmission electron microscopy to investigate the localization of this pathogen in diseased leaves and stalks of sugarcane. Three sugarcane cultivars with different levels of resistance to leaf scald were inoculated with the green fluorescent protein labelled X. albilineans strains XaFL07-1 (from Florida) and GPE PC73 (from Guadeloupe). Sections of sugarcane leaves and stalks were examined 8-60 days after inoculation in order to localize X. albilineans in the different plant tissues. Confocal microscopy observation of symptomatic leaves confirmed the presence of the pathogen in the protoxylem and the metaxylem, however, X. albilineans was also observed in the phloem, the parenchyma and the bulliform cells of the leaves. Similarly, the protoxylem and the metaxylem of infected sugarcane stalks were invaded by X. albilineans. Surprisingly, the pathogen was also observed in apparently intact storage cells of the stalk and in the intercellular spaces between these cells. Several of these observations made by confocal microscopy have been confirmed by transmission electron microscopy. X. albilineans can therefore no longer be considered as a xylem-limited pathogen. To our knowledge, this is the first description of a plant pathogenic bacterium invading apparently intact non-vascular plant tissue and multiplying in parenchyma cells. The mechanisms and virulence factors used by X. albilineans to enter and invade different tissues of sugarcane remain to be identified. (Résumé d'auteur

Patterns of sequence polymorphism in the fleshless berry locus in cultivated and wild Vitis vinifera accessions

<p>Abstract</p> <p>Background</p> <p>Unlike in tomato, little is known about the genetic and molecular control of fleshy fruit development of perennial fruit trees like grapevine (<it>Vitis vinifera </it>L.). Here we present the study of the sequence polymorphism in a 1 Mb grapevine genome region at the top of chromosome 18 carrying the <it>fleshless berry </it>mutation (<it>flb</it>) in order, first to identify SNP markers closely linked to the gene and second to search for possible signatures of domestication.</p> <p>Results</p> <p>In total, 62 regions (17 SSR, 3 SNP, 1 CAPS and 41 re-sequenced gene fragments) were scanned for polymorphism along a 3.4 Mb interval (85,127-3,506,060 bp) at the top of the chromosome 18, in both <it>V. vinifera cv</it>. Chardonnay and a genotype carrying the <it>flb </it>mutation, <it>V. vinifera cv</it>. Ugni Blanc mutant. A nearly complete homozygosity in Ugni Blanc (wild and mutant forms) and an expected high level of heterozygosity in Chardonnay were revealed. Experiments using qPCR and BAC FISH confirmed the observed homozygosity. Under the assumption that <it>flb </it>could be one of the genes involved into the domestication syndrome of grapevine, we sequenced 69 gene fragments, spread over the <it>flb </it>region, representing 48,874 bp in a highly diverse set of cultivated and wild <it>V. vinifera </it>genotypes, to identify possible signatures of domestication in the cultivated <it>V. vinifera </it>compartment. We identified eight gene fragments presenting a significant deviation from neutrality of the Tajima's D parameter in the cultivated pool. One of these also showed higher nucleotide diversity in the wild compartments than in the cultivated compartments. In addition, SNPs significantly associated to berry weight variation were identified in the <it>flb </it>region.</p> <p>Conclusions</p> <p>We observed the occurrence of a large homozygous region in a non-repetitive region of the grapevine otherwise highly-heterozygous genome and propose a hypothesis for its formation. We demonstrated the feasibility to apply BAC FISH on the very small grapevine chromosomes and provided a specific probe for the identification of chromosome 18 on a cytogenetic map. We evidenced genes showing putative signatures of selection and SNPs significantly associated with berry weight variation in the <it>flb </it>region. In addition, we provided to the community 554 SNPs at the top of chromosome 18 for the development of a genotyping chip for future fine mapping of the <it>flb </it>gene in a F2 population when available.</p

Boire et déboires du bac-fish sur petits chromosomes de plantes

National audienceAfin de développer des compétences et une expertise dans le domaine de la cytogénétique moléculaire végétale présentant des problématiques spécifiques, le département de Génétique et d’Amélioration des Plantes a mis en place une plate-forme (PF) s’appuyant sur les deux laboratoires de cytogénétique traditionnelle travaillant sur les espèces Brassica et Blé. L’objectif étant de mutualiser les moyens techniques, les investissements et les compétences. La plate-forme située au sein de l’UMR APBV-Le Rheu doit répondre aux besoins des équipes de recherche de l'unité mais également à l'ensemble des équipes du Département. Cette capacité d’ouverture à hauteur de 50% vers les unités extérieures nous permettant ainsi d’ouvrir le champ des thématiques de la PF et permettre le développement des nouveaux outils qui seront ensuite transférés vers d’autres espèces. La plate-forme a ainsi développé un ensemble d’outils de cytogénomique permettant de répondre aux projets scientifiques d’études des génomes chez les plantes supérieures : • Caractérisation cytogénétique du matériel végétal impliquant des hybrides interspécifiques ou des espèces polyploïdes permettant l’identification des chromosomes de chaque espèce ou introgressions, • Evolution structurale et stabilisation des génomes chez des espèces polyploïdes, • Cartes cytogénétiques faisant la liaison entre la cartographie génétique et physique, • Cartographie physique fine de BAC sur chromosomes en méiose (stade pachytène) Actuellement dans le cadre de trois projets multi-espèces, nous mettons l’accent sur l‘optimisation de la technique du « BAC-FISH multi-espèces » devant permettre [1] l’assignation de clone BAC à un chromosome particulier dans le cadre du projet de séquençage du génome de la Tomate, [2] la caractérisation des modifications structurales lors de la stabilisation des colzas synthétiques et de la nature des appariements homologues ou homéologues à la méiose, [3] l’étude de microsyntenie dans deux régions impliquée dans la résistance aux maladies (mildiou et oïdium) chez la vigne. Tomate/Brassica/Vigne, 3 espèces à « petits chromosomes » présentant des réponses différentes : succès et problématiques du BAC-FISH sur petits chromosomes de plantes

Route of a multipartite nanovirus within its aphid vector

BGPI : équipe 2BGPI : équipe 2Nanoviruses are multipartite ssDNA viruses transmitted by aphid vectors. Their mode of transmission is believed to be of the circulative non-propagative type [1, 2], but a recent analysis of the relative frequency of the distinct genome segments in aphids versus in plants indicated a more complex relationship [3]. Previous microscopy studies using immunofluorescence against the coat protein of Banana bunshy top virus (BBTV) confirmed that the virus is detected solely in gut and principal salivary gland cells of its aphid vector Pentalonia nigronervosa [4]. However, the subcellular localization and the cell compartment(s) with which the virus associates during transcytosis could not be determined [5]. Another unresolved question is whether, as demonstrated in monopartite viruses, nanoviruses are submitted to strong bottleneck during aphid transmission (see the abstract by Romain Gallet). In that case, when the virus particles traverse cellular barrier, different particles containing different segments may be separated, increasing the chance of loosing genomic information (loosing segments) and thus aborting transmission. We have revisited the cycle of a nanovirus, the Faba bean necrotic stunt virus, within the body of its aphid vector, Acyrthosiphon pisum, using a combination of FISH (Fluorescent In Situ Hybridization), immunolabelling, confocal and electron microscopy. FISH labeling of viral DNA indicates that the virus massively accumulates in all cells of the midgut but is virtually absent in all other regions of the gut. At the salivary glands, the virus is very much restricted not only to the principal salivary glands but also to a single cell type within these glands. No signal could ever be detected in any other organ. When using DNA-FISH probes specific to distinct segments, we could demonstrate that all segments are always colocalized within the midgut, and to a lesser extent also within the salivary gland cells. This observation contrasts with the situation in infected plant cells (see abstract from Anne Sicard), and indicate that the size of the viral population traversing cellular barriers within the aphid is often sufficiently large to prevent the loss of rare genome segments. Finally, we have indication that the genome segment N controls internalization of the virus within aphid gut cells. We currently investigate the subcellular compartment with which the virus associates, and the mode of action of the NSP protein (product of gene N

Localizing Genome Segments and Protein Products of a Multipartite Virus in Host Plant Cells

International audienceA founding paradigm in virology is that the spatial unit of the viral replication cycle is an individual cell. This concept applied to multipartite viruses-which have a genome composed of two or more nucleic acid segments, each individually encapsulated-implies that all segments constituting a viral genome need to coinfect the same host cell for replication to occur. Would this requirement be verified, it would constitute a major cost for extreme cases of multipartition such as the Faba bean necrotic stunt virus (FBNSV, nanovirus) whose genome is composed of eight complementary segments, each encoding a single gene (Grigoras et al., 2009). To address this question, we followed the distribution of the FBNSV genome segments by fluorescence in situ hybridization combined to immunolocalization of the replication-controlling viral protein within the cells of the host plant: Vicia Faba.A rapid and efficient protocol to localize viral transcripts in plant and insect hosts has been developed earlier (Ghanim et al., 2009). We here improve this method by using random-primed labeled probes and apply it to the detection and quantification of the individual segments composing the FBNSV genome. Moreover, we combine this technique with immunolocalization so that both viral segments and proteins can be visualized within the same samples

Breaking dogmas: the plant vascular pathogen Xanthomonas albilineans is able to invade non-vascular tissues despite its reduced genome

BGPI : équipe 3Xanthomonas albilineans, the causal agent of sugarcane leaf scald, is missing the Hrp type III secretion system that is used by many Gram-negative bacteria to colonize their host. Until now, this pathogen was considered as strictly limited to the xylem of sugarcane. We used confocal laser scanning microscopy, immunocytochemistry and transmission electron microscopy (TEM) to investigate the localization of X. albilineans in diseased sugarcane. Sugarcane plants were inoculated with strains of the pathogen labelled with a green fluorescent protein. Confocal microscopy observations of symptomatic leaves confirmed the presence of the pathogen in the protoxylem and metaxylem; however, X. albilineans was also observed in phloem, parenchyma and bulliform cells of the infected leaves. Similarly, vascular bundles of infected sugarcane stalks were invaded by X. albilineans. Surprisingly, the pathogen was also observed in apparently intact storage cells of the stalk and in intercellular spaces between these cells. Most of these observations made by confocal microscopy were confirmed by TEM. The pathogen exits the xylem following cell wall and middle lamellae degradation, thus creating openings to reach parenchyma cells. This is the first description of a plant pathogenic vascular bacterium invading apparently intact non-vascular plant tissues and multiplying in parenchyma cells