Thesis (Ph. D.)--University of Wisconsin--Madison, 1949. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 110-114).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1952. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves [58]-63).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1958. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 64-73).

Previous studies have detected the presence of the capsid protein (CP) and CP RNA of PVX in chloroplasts of transgenic plants expressing the PVX CP and 8kD genes from nuclear transgenes. CP RNA was also found in the chloroplasts of transgenic plants with mutations that eliminated either the CP or the 8kD protein. To further investigate the potential “RNA Tractor” activity of the PVX RNA, various constructs containing limited regions of the PVX 8kD and CP genes were produced and used to transform tobacco plants. RT-PCR analyses of chloroplastic RNA ascertained the presence of the RNA transcript within chloroplasts when as little as 125bp of the PVX sequence was used to transform plants. From this, it was concluded that the PVX proteins did not contribute to the movement of the viral RNA and that a region within the 125bp PVX sequence is acting as a chloroplastic “RNA Tractor”.

Previous studies have detected the presence of the capsid protein (CP) and CP RNA of PVX in chloroplasts of transgenic plants expressing the PVX CP and 8kD genes from nuclear transgenes. CP RNA was also found in the chloroplasts of transgenic plants with mutations that eliminated either the CP or the 8kD protein. To further investigate the potential “RNA Tractor” activity of the PVX RNA, various constructs containing limited regions of the PVX 8kD and CP genes were produced and used to transform tobacco plants. RT-PCR analyses of chloroplastic RNA ascertained the presence of the RNA transcript within chloroplasts when as little as 125bp of the PVX sequence was used to transform plants. From this, it was concluded that the PVX proteins did not contribute to the movement of the viral RNA and that a region within the 125bp PVX sequence is acting as a chloroplastic “RNA Tractor”.

Les Rémorines (REM) du groupe 1 sont des protéines spécifiques du monde végétale. Malgré leur caractère hydrophile elles sont localisées à la membrane plasmique. La phosphorylation des REM serait potentiellement impliquée dans la signalisation précoce et la défense des végétaux contre les pathogènes. Benschop et al. (2007) détecte AtREM1.3 (Arabidopsis thaliana, groupe 1b) phosphorylée en réponse au traitement par l'éliciteur générale flg22, tandis que Widjaja et al. (2008) a suggéré que la phosphorylation de AtREM1.2 est potentiellement impliquée dans la signalisation précoce à l'infection par Pseudomonas syringae. La fonction précise de la phosphorylation des protéines REM du groupe 1 reste inconnue. Des travaux antérieurs dans le laboratoire ont montré que le mouvement du virus X de la pomme de terre (PVX) est inversement corrélée à l'accumulation de StREM1.3 (Solanum tuberosum) et que StREM1.3 peut interagir physiquement avec la protéine de mouvement TRIPLE GENE BLOC Protein 1 (TGBp1) du PVX (Raffaele et al., 2009). Dans ce travail, nous avons étudié les mécanismes qui sous-tendent les interactions REM-TGBp1 et nous avons essayé de caractériser biochimiquement la kinase qui phosphoryle REM. Les conséquences physiologiques de l'interaction TGBp1 / StREM1.3 et de la phosphorylation de REM en terme de propagation des virus, d’inactivation génique post-transcriptionnelle, de régulation de l’ouverture des plasmodesmes, et d’activation de kinase ont également été étudiés. / The group 1 Remorin (REM) proteins are plant-specific oligomeric proteins that have been reported to localize to the plasma membrane despite their overall hydrophilic nature. There is evidence that the REM protein phosphorylation is potentially implicated in the early signaling and defense. Benschop et al. (2007) detected the AtREM1.3 (Arabidopsis thaliana group 1b of REM protein family) to be phosphorylated in response to treatment with the general elicitor flg22, while the Widjaja et al. (2008) suggested that the phosphorylation of AtREM1.2 is potentially implicated in early signaling upon infection with Pseudomonas syringae. The precise exact function of the group 1 REM protein phosphorylation remains unknown. Previous work in the laboratory showed that Potato virus X (PVX) movement is inversely correlated to potato StREM1.3 accumulation and that StREM1.3 can physically interacts with the movement protein TRIPLE GENE BLOCK PROTEIN 1 (TGBp1) from PVX (Raffaele et al., 2009). In this work, we studied the mechanism underlying the REM-TGBp1 interactions and we tried to characterise biochemically the kinase that phosphorylate REM. The physiological consequences of TGBp1/ StREM1.3 interaction and REM phosphorylation in terms of virus spreading, post-transcriptional gene silencing, plasmodesmata gating, kinase activation were also investigated.

De nos jours, les agents pathogènes des plantes, engendrent encore des pertes agricoles importantes. A terme, la solution la plus adaptée semble être la création de variétés de plantes cultivées présentant une résistance génétique à large spectre et durable. Créer de telles plantes nécessite la compréhension des mécanismes de résistance mis en jeu dans des pathosystèmes modèles, tels que celui impliquant le gène de résistance Rx et le virus X de la pomme de terre. Dans ce système, la protéine Rx intervient dans la perception d’un facteur d'avirulence issu du virus: sa protéine de capside (Cp). Cependant, les mécanismes moléculaires déclenchant la résistance chez la plante exprimant Rx demeurent obscurs. Cette étude s’est focalisée sur l’étape de reconnaissance de l’éliciteur viral par Rx. Une première approche a permis la mise en évidence d’un éliciteur minimal de 90 acides amine. L’étude d’un fragment légèrement plus grand par des méthodes de biochimie structurale, tend à exclure un modèle selon lequel, la différence de structuration tertiaire entre Cp élicitrice et Cp non élicitrice déterminerait la reconnaissance par Rx. Une seconde approche, de biologie moléculaire, a mis en évidence deux protéines hôte interagissant avec ce petit fragment éliciteur. L'étude s’est focalisée sur un gène codant un facteur de transcription, nbERF5, et a révélé que cette protéine interagit aussi bien avec les Cp de souches de Potexvirus avirulentes que virulentes dans la résistance liée à Rx, mais également avec la protéine de résistance Rx et l’intéracteur direct: RanGAP2. La poursuite de la caractérisation de cet ERF, permettra de déterminer son importance dans la résistance liée à Rx. / Nowadays, phytopathogenic agents are still causing significant agricultural losses. The most suitable option appears to be the creation of crop species carrying a genetic durable and broad spectrum resistance. In order to create such varieties, we need to understand the mechanisms underlying resistance, involved in model Pathosystems, Such as the one composed of the resistance gene Rx and the potato virus X. In that system, the host gene encodes a protein assimilated to a receptor implicated in the perception of an avirulence factor produced by the virus: its capside protein (Cp). Nevertheless, the molecular mechanisms triggering the resistance remain largely unknown. This study has been focused on the elicitor recognition mediated by Rx. A first approach led to the identification of a minimal elicitor containing 90 amino acids has. The structural characterization of a slightly larger protein fragment using biochemical methods suggested that the difference in the tertiary structuration of both elicitor and non-elicitor Cp would not be the determinant of Rx mediated recognition. Second, a molecular approach led to the discovery of two host proteins interacting with the small elicitor fragment. The work was focused on a transcription factor, nbERF5 and showed that this protein interacts similarly with elicitor or non-elicitor Cps of Rx mediated resistance. Interestingly, this gene product is able to directly interact with the Rx protein, but also with the direct interactor of Rx: RanGAP2, protein required for the Rx mediated resistance efficiency. Further characterization of this ethylen response factor will help us to understand its role in Rx mediated resistance.

Production of vaccines and pharmaceutical proteins in plants is a promising nascent technology with a great potential to provide high-quality, safe and non-expensive production and delivery platform. In this work we studied the experimental vaccine against human papillomavirus based on modified plant pathogen - Potato virus X (PVX). The experimental vaccine is based on PVX virus particles decorated with genetically fused HPV-E7 oncoprotein. These chimeric virus particles should be able to activate strong and specific cellular immune response. However the modification of the PVX coat protein with such relatively large fused protein might influence its ability to form particles. In this work we have characterized some properties of such chimeric virus particles like solubility or ability infect host plant. (In Czech)

The latest model of tertiary structure of capsid protein of potato virus X (PVX CP) was used as a template to design new insertion sites suitable for the preparation of PVX-based antigen presentation system. Based on this model, seven insertion sites (A-G) located in putative surface loops were tested. As an antigen inserted into these sites was used 17 amino acids long epitope derived from human papillomavirus type 16 E7 oncoprotein (E7 epitope) fused with either 6xHis tag or StrepII tag in both possible orientations (6xHis-E7 and E7-6xHis, StrepII-E7 and E7-StrepII). Prior to plant expression, modified PVX CPs were expressed in Escherichia coli MC1061. The results showed that only PVX CP carrying StrepII-E7 or E7-StrepII in the insertion site A formed virus particles. The results from transient expression experiments with modified PVX CPs in Nicotiana benthamiana showed that only the insertion site A (located between 24th and 25th amino acid in the PVX CP) could tolerate all tested inserts. Importantly, viral particles were detected only in the presence of StrepII tag and their stability was affected by the insert orientation (StrepII-E7 vs. E7-StrepII) as only the viral particles presenting E7-StrepII could be purified. Besides the preparation of PVX-based antigen presentation system, an...

A movement protein Potato virus X (PVX) chimera virus (PVX.GFP(CH3)) bearing the grapevine virus Grapevine rupestris stem pitting-associated virus (GRSPaV) triple gene block proteins (TGB) (denoted P1, P2 and P3) instead of the PVX TGB was delivered into N. benthamiana and other related species by agro-inoculation. This movement protein PVX chimera virus was found to be unable to support the local and systemic movement of PVX in cis. Local and systemic movement of this PVX chimera virus was restored in trans by the dianthovirus Red clover necrotic mosaic virus (RCNMV) movement protein and by a PVX TGB rescue virus that replaced the GRSPaV TGB with the PVX TGB (PVX.GFP(Rescue)). However, a PVX TGB hybrid chimera virus (PVX.GFP(HY2)) containing PVX P1 and the GRSPaV TGB had limited cell-to-cell, but not systemic, movement.