Mécanismes moléculaires à l'origine de la pathogenicité de phytovirus de betterave sucrière transmis par un vecteur tellurique

Le virus des nervures jaunes et nécrotiques de la betterave (Beet necrotic yellow vein virus, BNYVV) est l agent infectieux responsable de la rhizomanie de la betterave sucrière, une maladie caractérisée par une prolifération anarchique du chevelu racinaire. Le Beet soil-borne mosaic virus (BSBMV) appartient également au genre Benyvirus mais n est retrouvé qu en Amérique du Nord. Ce virus, identifié pour la première fois au Texas, est morphologiquement et génétiquement semblable au BNYVV mais sérologiquement éloigné. Compte tenu des différences moléculaires existant, le BSBMV et BNYVV correspondent à deux espèces virales distinctes. Mon projet de thèse a consisté à étudier les interactions moléculaires entre le BNYVV et le BSBMV et rechercher les mécanismes impliqués dans la pathogénicité de ces deux virus. Des clones complets cDNA infectieux du BNYVV étaient disponibles, tout comme ceux de BSBMV. Compte tenu de l aspect versatile de l obtention de transcrits infectieux de ces différents clones, j ai entrepris de produire des clones cDNA de chacun des ARN viraux sous contrôle d un promoteur constitutive végétal pour initier l infection par agroinfiltration. Les plantes hôtes Chenopodium quinoa et Nicotiana benthamiana ont été inoculées par des transcrits et agroinfiltrées pour initier l infection virale et étudier l interaction entre les ARN génomiques 1 et 2 des deux virus et étudier les propriétés de constructions chimères. En parallèle à ce travail, j ai réalisé la caractérisation du suppresseur de RNA silencing du BSBMV en le comparant à celui du BNYVV.The genus Benyvirus includes the most important and widespread sugar beet viruses transmitted through the soil by the plasmodiophorid Polymyxa betae. In particular Beet necrotic yellow vein virus (BNYVV), the leading infectious agent that affects sugar beet, causes an abnormal rootlet proliferation known as rhizomania. Beet soil-borne mosaic virus (BSBMV) is widely distributed in the United States and, up to date has not been reported in others countries. My PhD project aims to investigate molecular interactions between BNYVV and BSBMV and the mechanisms involved in the pathogenesis of these viruses.BNYVV full-length infectious cDNA clones were available as well as full-length cDNA clones of BSBMV RNA-1, -2, -3 and -4. Handling of these cDNA clones in order to produce in vitro infectious transcripts need sensitive and expensive steps, so Ideveloped agroclones of BNYVV and BSBMV RNAs, as well as viral replicons allowing the expression of different proteins.Chenopodium quinoa and Nicotiana benthamiana plants have been infected with in vitro transcripts and agroclones to investigate the interaction between BNYVV and BSBMV RNA-1 and -2 and the behavior of artificial viral chimeras. Simultaneously I characterized BSBMV p14 and demonstrated that it is a suppressor of posttranscriptional gene silencing sharing common features with BNYVV p14.STRASBOURG-Bib.electronique 063 (674829902) / SudocSudocFranceF

Étude des interactions Benyvirus-Polymyxa betae par génétique inverse (Rôle de la protéine codée par l'ARN4 dans la transmission)

Les virus Beet soil-borne mosaic virus (BSBMV) et Beet necrotic yellow vein virus (BNYVV, agent de la Rhizomanie) font partie du genre Benyvirus. Des clones d ADNc infectieux de BSBMV ont été obtenus et ont permis d entreprendre l étude des interactions moléculaires entre plante et Benyvirus sur les bases des différences biologiques des deux virus. Les transcrits infectieux de BSBMV peuvent substituer ceux du BNYVV et ainsi produire des recombinants chimériques.La transmission par Polymyxa betae est liée à la présence de l ARN4 et un ARN4 de 1730 nts de BSBMV, non encore décrit, a été caractérisé. Nous avons démontré que cet ARN4 pouvait remplacer celui du BNYVV lors de la transmission et que seule l expression de la protéine p32 suffit à la transmission du virus. La mutagenèse de la protéine p32 étiquetée a été réalisée et a permis de rechercher les domaines essentiels à ses fonctions.Beet soil-borne mosaic virus (BSBMV) and Beet necrotic yellow vein virus (BNYVV, responsible for Rhizomania disease) belong to the Benyvirus genus. BSBMV full-length cDNA clones were produced to investigate molecular interactions between plant and benyviruses exploiting BSBMV/BNYVV biological and molecular divergences. BSBMV Full-length infectious cDNA clones can substitute BNYVV RNA-1 or -2 to produce chimeric viral progenies. Virus transmission by Polymyxa betae is directly linked to the viral RNA4 and a 1,730 nts long BSBMV RNA4, not previously described, has been molecularly characterized. We demonstrated that BSBMV RNA4 could substitute BNYVV RNA4 for an efficient transmission. Replicon mediated expression of the BSBMV p32 protein complemented RNA4 defective strain demonstrating for the first time that the p32 protein but not full-length RNA4 is essential for Benyvirus transmission. Mutagenesis or Flag or GFP-tagged p32 has been to investigate the domains of BSBMV p32 protein essential for its properties.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

First report of Barley yellow mosaic virus in Barley in Spain

The presence of Barley mild mosaic virus (BaMMV) and the weakly serological detection of Barley yellow mosaic virus (BaYMV) were reported in Spain (1); both viruses are members of the genus Bymovirus (family _Potyviridae_). Random and symptomatic surveys were conducted during February and March of 2003 in barley fields in north eastern Spain to determine the occurrence of BaMMV and BaYMV. Leaves from 316 samples collected in 15 fields were analyzed using enzyme-linked immunosorbent assay (ELISA) with commercial antisera specific for BaYMV and BaMMV (Loewe Biochemica, Munich) as well as antisera against both viruses (provided by T Klumen). Positive ELISA samples were further analyzed using reverse transcription-polymerase chain reaction (RT-PCR) with specific primers that amplify 445 bp of BaMMV (1) and 433 bp of BaYMV (2). Complete agreement was observed between the ELISA and RT-PCR results. Mixed infections of BaYMV and BaMMV were detected in 10 samples, BaYMV in 5 samples and BaMMV in 3 samples. Samples positive for both viruses that exhibited clear mosaic symptoms were collected in 2 fields. RT-PCR products from 5 BaYMV-infected samples were cloned and sequenced and showed 96 to 98 per cent identity to BaYMV isolates previously reported from Europe (Genbank Accession Nos. AJ1515479-85 and X95695-7) and 92 to 95 per cent identity with isolates reported from Asia (GenBank Accession Nos. AB023585-96, AJ132268, AJ224619-22, AJ224624-28, AF536944-46, AF536948-58, D01091, D00544, and Z24677). Sequence identity of Spanish isolates was 96 to 99 per cent. To our knowledge, this is the 1st report of BaYMV infecting barley in Spain and illustrates the association of both Bymoviruses infecting barley

Preliminary work for full -lenght CDNA clones construction of Italian Soil-borne cereal mosaic virus isolate

A mosaic disease of winter wheat was first described in the USA in 1919 and later was world wide associated to the Furovirus Soil-borne wheat mosaic virus (SBWMV), transmitted by the soil-borne plasmodiophorid Polymyxa graminis. Subsequent sequence analyises divided American, European and Chinese isolates into different species within the Furovirus genus: wheat mosaic disease is generally caused by the species SBWMV in the United States, Brazil and Canada, Soil-borne cereal mosaic virus (SBCMV ) in Europe and Chinese wheat mosaic virus (CWMV) in Asia. In Italy, SBCMV was first reported in 1960 on cultivars of common wheat (Triticum aestivum) grown in the Po valley, and subsequently also on durum wheat (Triticum durum) in the central and southern Italian regions. SBCMV has been shown to cause grain yield reductions of about 50-70% on the most susceptible varieties in Italy. The use of resistant varieties, due to the persistent nature of the virus within the soil, is the only practical and economical viable mean of control. The SBWMV, SBCMV and CWMV genomes consist of two plus-sense strands of RNA. RNA1 (about 7 kb) contains three open reading frames which encode for the RNA polymerase, the methyltransferase-helicase and the cell-to-cell movement proteins, The RNA2 (about 3.7 kb) encodes for the coat protein, for protein involved on lasmodiophorid transmission of the virus, pathogenicity determinant, suppressor of RNA silencing and for 25 kDa protein with unknown function. An Italian isolate of SBCMV has been derived from infected wheat plants collected in a field near Bologna in 2007. Instability of a viral cDNA insert in a bacterial plasmid vector has been previously reported with a number of plus-sense RNA viruses in the process of construction of full-length cDNA clones. Several approaches have been examined as a solution for this problem with the aim to produce cDNA clones of the complete SBCMV genome. Strategies based on the use of several plasmid vectors with different markers and high- or low-copy numbers, cDNA insertion in both orientations in the plasmid, insertion of multiple introns into a full-length cDNA, use of E. coli and Bacillus subtilis strains with different phenotypes have been employed in attempting the construction of the full-length SBCMV RNA-1 cDNA clone. Two clones, containing the 5’-terminal 3.9-kb region and the 3’-terminal 3.1-kb region, have been produced and a full-length form can be reconstituted from them. Plasmid containing SBCMV RNA-2 cDNA has been obtained cloning full-length PCR product. Biological activity of the clones has been tested by rub-inoculation on Chenopodium quinoa and T. durum plants of the in vitro transcripts produced from plasmids