Ke Chen

François Dorlhac De Borne

Emilie Julio

Julie Obszynski

Patrick Pale

Léon Otten
email: leon.otten@ibmp-cnrs.unistra.fr

Sequencing and functional analysis of cT-DNAs in Nicotiana

Keywords: Agrobacterium, N. tomentosiformis, cT-DNA, natural transformation, horizontal gene transfer, 6b, leaf polarity, plast genes N. tabacum; Lv: Linaria vulgaris. Plast protein Opine synthesis, natural transformant, T-DNA, Nicotiana tabacum

The bacterium Agrobacterium tumefaciens is well-known for its utilisation in plant genetic engineering where it serves as a gene vector. This bacterium and the related species Agrobacterium rhizogenes are phytopathogens that induce tumors and hairy roots respectively on susceptible plants like grapevine or fruit trees. Their phytopathogenicity is due to horizontal transfer of bacterial genes to the plant host, from a plasmid called the Ti (tumor-inducing) or Ri (root-inducing) plasmid. The subject of my Thesis concerns two particular aspects of this bacterium.

1. Their capacity to stably transform several plant species in nature, thereby yielding naturally transformed plants, especially in the genus Nicotiana. We have shown by deep sequencing of the Nicotiana tomentosiformis genome and by analysis of other recently published Nicotiana sequences the presence of five different Agrobacterium-derived sequences (cT-DNAs), totalling 65 kb, some of which carry intact genes. We have shown that two of them (TB-mas2' from N. tabacum and TE-6b from N. otophora) have biological activity. A detailed comparative study has allowed us to better understand the evolution of these cT-DNAs (Chen et al., 2014). The mas2' gene is well-known, it codes for the synthesis of desoxyfructosyl-glutamine (DFG) in tumors or roots induced by Agrobacterium. Recent work in our group has shown that the TB-mas2' gene is highly expressed in some N. tabacum cultivars and leads to the accumulation of detectable amounts of DFG. This work is presented as a manuscript to be submitted.

2.

. The TE-6b gene from N. otophora was placed under strong and constitutive promoter control and introduced into tobacco, where it was found to cause new types of morphological change, different from those observed for T-6b. The latter results are preliminary and will be presented as a complement to the work on T-6b. They indicate that the introduction of the TE-6b gene in the N. otophora ancestor could have caused a change in growth pattern, and might have favored the appearance of a new species.

Séquençage et analyse fonctionelle d'AND-cT dans Nicotiana Résumé:

La bactérie Agrobacterium tumefaciens est bien connue pour son utilisation en génie génétique végétale où elle sert comme vecteur de gènes. A l'origine, cette bactérie ainsi que l'espèce voisine Agrobacterium rhizogenes sont des bactéries phytopathogènes qui induisent respectivement des tumeurs et des racines anormales sur des plantes sensibles telles que la vigne ou des arbres fruitiers. L'action pathogène résulte d'un transfert horizontal de gènes de la bactérie vers l'hôte végétal, à partir d'un plasmide, le pTi (plasmide inducteur de tumeurs) ou pRi (plasmide inducteur de racines). Mon travail de thèse concerne deux aspects particuliers de cette bactérie.

1. Sa capacité à transformer durablement des espèces végétales dans la nature, donnant ainsi naissance à des plantes naturellement transformées, notamment dans le genre Nicotiana. Nous avons pu montrer par séquençage à haut débit du génome de N. tomentosiformis et par l'analyse d'autres séquences complètes de Nicotianées publiées récemment l'existence inattendue de 5 séquences venant d'Agrobacterium (cT-DNAs) avec une taille total de 65 kb, dont certaines portent des gènes intacts. Nous avons montré que deux de ces gènes (TB-mas2' de N. tabacum et TE-6b de N. otophora) ont une activité biologique. Une étude comparative approfondie a permis de mieux comprendre l'évolution de ces cT-DNAs (Chen et al., 2014). Le gène mas2' est bien connu, il code pour une enzyme qui catalyse la synthèse du désoxyfructosyl-glutamine (DFG) dans des tumeurs ou racines induites par Agrobacterium. Des résultats récents dans notre groupe portant sur le gène TB-mas2' montrent que ce gène est exprimé de façon très active dans plusieurs cultivars de N. tabacum, et y donne naissance à l'apparition de quantités mesurables de DFG. Ce travail est présenté sous forme d'un manuscrit à soumettre.

2. Une deuxième partie de la Thèse concerne les propriétés du gène T-6b, qui fait partie de l'ADN transféré par A. vitis souche Tm4 et provoque une croissance anormale caractérisée par l'apparition d'énations, sans que l'on connaisse son mode d'action. Le gène 6b fait partie de la famille des gènes plast (pour plasticité phénotypique), avec des effets différents et souvent remarquables sur la croissance des plantes. Le gène T-6b a été mis sous contrôle d'un promoteur inductible par le dexaméthasone, et des plantes de tabac transformées par cette construction ont été étudiées en détail, à différents moments après son induction. Un grand nombre de changements a été décrit incluant des analyses anatomiques montrant des modifications encore jamais décrites chez les plantes, comme par exemple l'apparition de méristèmes foliaires ectopiques à la base de trichomes, ou l'apparition de systèmes vasculaires ectopiques parallèles au système vasculaire normal avec un développement régulier menant à des structures complexes ordonnées [START_REF] Chen | Morphological analysis of the 6b oncogene-induced enation syndrome[END_REF]. Le gène TE-6b de N. otophora a été mis sous contrôle d'un promoteur fort constitutif et introduit dans des plantes de tabac, où il provoque des changements de croissance différents de ce qui a été observé pour le gène T-6b. Ces derniers résultats préliminaires sont présentés en complément des observations sur le gène T-6b. Ils indiquent que le transfert horizontal du gène TE-6b vers l'ancêtre de N. otophora aurait pu contribuer à une modification de la croissance et ainsi à la création d'une nouvelle espèce.

Mots clefs: Agrobacterium, N. tomentosiformis, cT-DNA, naturella transformation, transfert horizontal du gène, 6b, polarité des feuilles, plast gène years help during my thesis in Strasbourg. He is so patient and precise to explain our topic, related topics, each experiment and its principle. We make efforts together to enhance my scope of knowledge and also vocabulary in plant molecular field. He also encouraged me to learn French in order to get used to a French lab. It is doubtless to say, if there is no help of him, it would be impossible for me to finish my PhD these. I really enjoy the time working with him.

I would like to thank our collaborators Dr. François Dorlhac de Borne and Emilie Julie from Imperial Tobacco of Bergerac. As a coauthor of my first paper, François offered very important data e.g. RNA expression levels of mas1' and mas2' from different tobacco cultivars. Emilie helped to send lots of tobacco cultivars seeds. It is important to mention that they offered me a chance to present my topic in an international tobacco conference in Paris. I got a valuable experience there.

I am very grateful to another collaborator Dr. Ernö Szegedi from Hungary even I have never met him. He helped to improve the detection method of deoxyfructosyl-glutamine by paper electrophoresis a lot. He works so effcient that as soon as we propose questions, he answers immediately and sends us samples or Agrobacteria we need. I would like to thank Dr. Patrick Pale and Julie Obszynski from Chemical Institute of Strasbourg University. Since they are experts in chemical field, they gave us quite a lot of advices and help on synthesis and detection of the amadoric compound deoxyfructosyl-glutamine. Only with their help, the mas2' gene study can have further progress.

I have to thank my colleagues Patrick Achard, Jean Michel, Lali Achard and Thomas Regnault.

Since we work in a same lab, they helped me a lot in many field not only scientific research but also language and culture. We together created a very good research environment.

Many thanks to all people in IBMP, specially greenhouse team and sequencing technician Malek Alioua. Thanks for their hard and prescise work. I am so happy to know all these people and to be friends with them. I like the experience in IBMP. It's a big treasure for my life. Agrobacteria suspension .................................................................................................. II.1.2.2 Tobacco leaf transformation ................................................................................................................. ................................................................................................................. II.1.4.2 Western blot ..........................................................................................................................................

II.1.2.1 Preparation of

I. Agrobacterium T-DNA transformation

I.1. Agrobacterium

The plant pathogen Agrobacterium was first discovered in 1897 by Fridiano Cavara in Napoli, Italy (Reviewed in [START_REF] Kado | Historical account on gaining insights on the mechanism of crown gall tumorigenesis induced by Agrobacterium tumefaciens[END_REF]. He isolated an unknown bacterium from grapevine crown gall which he named Bacterium tumefaciens, the name was later changed to Phytomonas tumefaciens and finally to Agrobacterium tumefaciens. Agrobacterium is a typical soil bacterium that belongs to the Rhizobiaceae family. Initially, Agrobacteria were classified into three types: A.

tumefaciens, A. rhizogenes and A. radiobacter according to their effect on plants: tumorigenic, rhizogenic and non-pathogenic, respectively [START_REF] Conn | Validity of the genus Alcaligenes[END_REF]. Later, a biovar (or biotype) classification was used [START_REF] Kerr | Biotypes of Agrobacterium radiobacter var. tumefaciens and their biological control[END_REF] based on chromosomal characteristics.

Biovar3 was subsequently named A. vitis [START_REF] Ophel | Agrobacterium vitis-new species for strains of Agrobacterium biovar 3 from grapevine[END_REF]. Apart from these Agrobacterium species, others have been described, like A. rubi [START_REF] Hildebrand | Cane gall of brambles caused by Phytomonas rubi n. sp[END_REF] and A.

larrymoorei (Bouzart and Jones, 2001). Most Agrobacteria are not host-specific. A. vitis is an interesting exception because it has only been found on grapevines [START_REF] Burr | Crown gall of grape: biology and disease management[END_REF].

A new way of classifying Agrobacteria is based on genome sequence analysis. However, care should be used to compare these sequences since some sequences are not stable. During

I.2. Agrobacterium Ti and Ri plasmids

Agrobacteria carry tumor-inducing (Ti) plasmids or root-inducing (Ri) plasmids with T-DNA (transferred-DNA) sequences which are transferred to the plant cell upon infection. However, most natural Agrobacterium isolates lack Ti/Ri plasmids and are non-virulent [START_REF] Burr | Crown gall of grape: biology and disease management[END_REF]. Expression of T-DNAs results in a proliferation of transformed and non-transformed cells, thus causing tumors (A. tumefaciens, figure 2B) or hairy roots (A. rhizogenes, figure 2C) [START_REF] Binns | The Agrobacterium oncogenes. The Rhizobiaceae: molecular biology of model plant-associated bacteria[END_REF]. The structures of the Ti and Ri plasmids are highly diverse and have been traditionally classified according to the type of opines found in the transformed tissues. Opines will be discussed in I.4.2. A typical Ti or Ri plasmid (figure 3A) includes one or two T-DNAs, a virulence region, an origin of replication, an opine uptake and catabolism region and a conjugative transfer region. On the T-DNA, there are generally genes for the production of indole-3-acetic acid (IAA, an auxin), cytokinins and opines. Genes from the plast gene family (see below) are also present on T-DNAs, their functions are not yet clearly defined. After transfer and integration into the plant genome, the T-DNA genes are expressed and lead to local growth. Specific opines will be produced by the transformed plant tissues and can be used by the original bacterial strain which contains specific opine uptake and catabolism genes.

A. tumefaciens Ti plasmid replication and T-DNA transfer involve a cell-to-cell communication system called quorum sensing (QS). A bacterial population can "sense" its cell density by secretion and detection of small signal molecules (Fuqua et al., 1994) and regulate its gene expression accordingly.

I.3. Agrobacterium T-DNA transfer mechanism

Agrobacterium T-DNAs can be transferred into plant cells by a special mechanism. This T-DNA transfer system is classified as a bacterial type IV secretion system (figure 4). The ability of Agrobacterium to transfer T-DNAs into plants is widely used in plant genetic engineering and has also been extended to other organisms like animals, fungi and yeast [START_REF] Kunik | Genetic transformation of HeLa cells by Agrobacterium[END_REF][START_REF] Bulgakov | Agrobacterium mediated transformation of sea urchin embryos[END_REF][START_REF] De Groot | Agrobacterium tumefaciens mediated transformation of filamentous fungi[END_REF][START_REF] Michielse | Agrobacterium mediated transformation of the filamentous fungus Aspergillus awamori[END_REF][START_REF] Wolterink-Van, Loo | Interaction of the Agrobacterium tumefaciens virulence protein VirD2 with histones[END_REF].

The transformation process consists of different steps.

I.3.1 Attachment of Agrobacterium to plant surfaces

A. tumefaciens binds to many surfaces including those of plants, fungi, soil particles, even polyesters and plastics [START_REF] Matthysse | Attachment of Agrobacterium to plant surfaces[END_REF]. There are two types of binding to these surfaces: unipolar polysaccharide-dependent polar attachment and unipolar polysaccharide-independent attachment (both polar and lateral). Attachment of A. tumefaciens to plants is mediated by the unipolar polysaccharides. Cellulose is another important element for surface attachment. A. tumefaciens carries a cellulose synthase gene, celA, regulated by celB, celG and c-di-GMP [START_REF] Matthysse | Attachment of Agrobacterium to plant surfaces[END_REF]. It is known that cellulose fibrils bind tightly to other cellulose fibrils and cellulose synthesis therefore results in a strong attachment of A. tumefaciens to cellulose-rich plant surfaces [START_REF] Matthysse | Attachment of Agrobacterium to plant surfaces[END_REF]. VirB on the pTi plasmid may also be involved in this attachment process. Both VirB2 which is part of the shaft of the pilus structure and VirB5 which is located in the pilus tip could help bacterial attachment [START_REF] Christie | Mechanism and structure of the bacterial type IV secretion systems[END_REF].

I.3.2 Virulence gene activation

Most Ti and Ri plasmids carry virulence (vir) genes close to the T-DNA region (figure 3A). Their function is to enable T-DNA transfer from Agrobacteria into plant cells. virA, virB, virC, virD, virE, virF and virG genes are included in this group. Several "minor" vir genes, like virH and virM have also been described.

The vir system can be activated when some components like sugars and phenols reach critical concentrations. Under natural conditions, a wounded plant tissue can lead to an acidic pH and create a high nutrient environment that favors vir gene expression [START_REF] Wolanin | Histidine protein kinases: key signal transducers outside the animal kingdom[END_REF]. This can be sensed by the VirA protein located on the surface of the bacterium.

VirA protein is a membrane-bound receptor and histidine kinase that can activate VirG by transferring its phosphate to VirG. Phosphorylated VirG is a transcription factor that activates virB, virC, virD, virE and virF transcription [START_REF] Brencic | Detection of and response to signals involved in host-microbe interactions by plant-associated bacteria[END_REF].

I.3.3 Type IV secretion systems (T4SSs) in A. tumefaciens

VirD2 is an endonuclease which cuts a single stranded T-DNA fragment (T-strand) from the Ti/Ri plasmid and covalently links itself with the 5' end of the T-strand thus forming the T-DNA protein complex (T-complex) [START_REF] Herrera-Estrella | VirD proteins of Agrobacterium tumefaciens are required for the formation of a covalent DNA protein complex at the 5' terminus of T-strand molecules[END_REF][START_REF] Ward | VirD2 protein of Agrobacterium tumefaciens very tightly linked to the 5' end of T-strand DNA[END_REF][START_REF] Young | Association of the VirD2 protein with the 5' end of T-strands in Agrobacterium tumefaciens[END_REF][START_REF] Durrenberger | Covalently bound VirD2 protein of Agrobacterium tumefaciens protects the T-DNA from exonucleolytic degradation[END_REF][START_REF] Gelvin | Traversing the Cell: Agrobacterium T-DNA's journey to the host genome[END_REF]. The T-complex can be secreted out of Agrobacterium through the T4SS.

The T4SSs are translocation systems that are present in most bacteria and some archaea [START_REF] Baron | From bioremediation to biowarfare: on the impact and mechanism of type IV secretion systems[END_REF][START_REF] Alvarez-Martinez | Biological diversity of prokaryotic type IV secretion systems[END_REF][START_REF] Llosa | Bacterial type IV secretion systems in human disease[END_REF][START_REF] Backert | Pathogenesis of Helicobacter pylori infection[END_REF][START_REF] Nagai | Type IVB secretion systems of Legionella and other Gram-negative bacteria[END_REF][START_REF] Fischer | Assembly and molecular mode of action of the Helicobacter pylori Cag type IV secretion apparatus[END_REF]. These systems are able to translocate DNA and proteins from bacteria to bacteria or to other organisms. Two main groups of T4SSs are the conjugation machines and the effector translocator systems. The first ones enable single strand DNA to translocate within or between bacteria so that antibiotic resistance genes or virulence genes can disseminate among different bacterial species [START_REF] Alvarez-Martinez | Biological diversity of prokaryotic type IV secretion systems[END_REF]. The effector translocator systems are responsible for transport of proteins or single strand DNA-protein complexes from bacteria into eukaryotic cells. The VirB/VirD4 system that delivers the T-complex from A. tumefaciens to plant cells is one of the best well-known effector translocator systems.

A general VirB/VirD4 system contains several Vir proteins which are VirB1 to VirB11, coded by a single virB operon and a VirD4 protein coded by a separate virD operon (figure 4B). The VirB/VirD4 system consists of different components with different functions (figure 4A). 1. VirD4 is a Type IV coupling protein (T4CP), i.e. an ATPase related to the SpoIIIE/FtsK DNA translocases.

It binds with DNA and protein substrates and facilitates translocation. 2. VirB3, VirB4, VirB6, VirB8 and VirB11 form the inner membrane complex (IMC), and help the T-complex to transfer across the inner membrane. 3. VirB2, VirB7, VirB8, VirB9 and VirB10 form the envelope-spanning outer membrane complex (OMC) located between inner membrane and periplasm, it helps the Tcomplex to pass the periplasm and outer membrane. 4. VirB2 and VirB5 form the conjugative pilus, enabling the T-complex to contact with the plant surface [START_REF] Christie | Mechanism and structure of the bacterial type IV secretion systems[END_REF].

I.3.4 T-complex entry into the plant cell

The mechanism of T-complex transfer through the plant cell membrane is still unknown. In the plant cell, the T-DNA was found as a T-complex bound to VirD2 with the full length of DNA covered with VirE2. One proposal for T-complex transfer is that VirE2 may sit in the plant plasma membrane, forming a channel through which the T-DNA can pass [START_REF] Duckely | The VirE2 protein of Agrobacterium tumefaciens: the Yin and Yang of T-DNA transfer[END_REF].

Once the T-complex enters the plant cell, it needs to travel to the nucleus. VirD2 plays a very important role in this process since the C-terminal of VirD2 contains a nuclear localization signal sequence which enables proteins to target the eucaryotic nucleus [START_REF] Herrera-Estrella | A bacterial peptide acting as a plant nuclear targeting signal: the amino-terminal portion of Agrobacterium VirD2 protein directs a βgalactosidase fusion protein into tobacco nuclei[END_REF][START_REF] Howard | The VirD2 protein of A. tumefaciens contains a C-terminal bipartite nuclear localization signal: implications for nuclear uptake of DNA in plant cells[END_REF]Tinland et al., 1992b;[START_REF] Rossi | The VirD2 protein of Agrobacterium tumefaciens carries nuclear localization signals important for transfer of T-DNA to plant[END_REF][START_REF] Citovsky | Nuclear import of Agrobacterium VirD2 and VirE2 proteins in maize and tobacco[END_REF][START_REF] Mysore | Role of the Agrobacterium tumefaciens VirD2 protein in T-DNA transfer and integration[END_REF]. The cytoskeletal structures and the molecular motors of the plant cell may also help Tcomplex transport [START_REF] Gelvin | Traversing the Cell: Agrobacterium T-DNA's journey to the host genome[END_REF]. Some plant proteins like importin α, vir-interacting protein 1 (Vip1) and vir-interacting protein 2 (Vip2) also play a role in helping T-complex transfer (figure 5) [START_REF] Gelvin | Traversing the Cell: Agrobacterium T-DNA's journey to the host genome[END_REF]. In the plant cytoplasm, Vip1 directly binds with VirE2 (figure 5). The plant defense signaling mitogen-activated protein kinase is able to activate the phosphorylation of Vip1 allowing phospho-Vip1-T-complex import into the nucleus [START_REF] Magori | The role of the ubiquitin-proteasome system in Agrobacterium tumefaciens-mediated genetic transformation of plants[END_REF].

In the plant nucleus, once the T-complex is close to the chromosome, it is necessary to uncoat the T-complex. VirF has been demonstrated to interact with plant homologs of yeast Skp1 protein [START_REF] Schrammeijer | Interaction of the virulence protein VirF of Agrobacterium tumefaciens with plant homologs of the yeast Skp1 protein[END_REF]. The loss of virF reduces virulence of A. tumefaciens and can be complemented by expression in plant cells showing that VirF has a function in the plant cell (Otten et al. 1985;[START_REF] Schrammeijer | Interaction of the virulence protein VirF of Agrobacterium tumefaciens with plant homologs of the yeast Skp1 protein[END_REF]. Later studies showed that VirF is an F-box protein which helps to release single strand T-DNA from the T-complex [START_REF] Magori | The role of the ubiquitin-proteasome system in Agrobacterium tumefaciens-mediated genetic transformation of plants[END_REF].

VirF can be stabilized by VirD5. The Vip1-VirE2-VirD2 protein complex can be polyubiqitinated by VirF thus leading to its degradation. After this process, single strand T-DNA is naked and ready to be inserted into the plant genome.

I.4. Agrobacterium T-DNA genes

The T-DNA located genes can be divided into different groups: hormone synthesis genes, opine synthesis genes, rol genes and plast genes. The rol genes (rol for "root locus") were initially identified by their effects on hairy root induction [START_REF] White | Molecular and genetic analysis of the transferred DNA regions of the root-inducing plasmid of Agrobacterium rhizogenes[END_REF]. The rolA, rolB and rolC are necessary and sufficient for the induction of hairy roots by A. rhizogenes. rolD gene encodes an ornithine cyclodeaminase (OCD) enzyme which catalyzes the conversion of ornithine to proline.

I.4.1 Hormone synthesis genes

Several Agrobacterium T-DNAs contain hormone synthesis genes like iaaM, iaaH and ipt. iaaM and iaaH code for two steps in auxin synthesis. First, the amino acid tryptophan (Trp) is converted to indole-3-acetamide (IAM) by tryptophan-2-monooxygenase (IaaM, coded by iaaM), subsequently, indole-3-acetamide hydrolase (IaaH, coded by iaaH), converts indole-3-acetamide into indole-3-acetic acid (IAA) (figure 6A).

Cytokinin is formed by isopentenyltransferase (Ipt, coded by ipt) which adds a prenyl moiety from dimethylallyl pyrophosphate (DMAPP) or hydroxymethylbutenyl pyrophosphate (HMBPP) to ATP/ADP/AMP to produce isoprene cytokinins (figure 6B).

I.4.2 Opine synthesis genes and opines

Opines are produced by Agrobacterium-infected plants and are found in or around plant tumors or hairy roots. They are low molecular weight molecules which can be separated into two groups: agrocinopines and secondary amine derivatives (Baek et al., 2003). Agrocinopines are sugarphosphodiesters and can be used by Agrobacterium as carbon and phosphorus sources. The amine derivatives are synthesized by condensation of a sugar or a keto acid with an amino acid.

Thus they are a source of carbon and nitrogen. Over 20 types of opines have been characterized [START_REF] Dessaux | Opines and opine-like molecules involved in plant-Rhizobiaceae interactions[END_REF]. The chemical structures of the major opines are shown in figure 7.

Agrobacterium Ti or Ri plasmids can be classified based on the opines they induce (table 1). Two examples can be cited. On the octopine type plasmid pTiAch5, ocs codes for the octopine synthase enzyme Ocs that leads to octopine, octopinic acid, histopine and lysopine synthesis in tumors [START_REF] Otten | Properties of D(+)-lysopine dehydrogenase from crown gall tumour tissue[END_REF][START_REF] Dessaux | Opines and opine-like molecules involved in plant-Rhizobiaceae interactions[END_REF][START_REF] Flores-Mireles | Agrobacterium tumefaciens can obtain sulphur from an opine that is synthesized by octopine synthase using S-methylmethionine as a substrate[END_REF]. On the agropine type Ri plasmid pRiA4, mas2' codes for mannopine synthase 2' (Mas2') that converts glutamine and sugar into DFG. DFG can be reduced to mannopine by the mas1' encoded mannopine synthase 1' (Mas1'). Finally, mannopine is cyclized to agropine by agropine cyclase (Ags) encoded by ags.

As described before, RolD encodes an ornithine cyclodeaminase which converts ornithine to proline and may therefore be considered as an opine synthesis gene if the proline is used by Agrobacterium. However, this has not been shown sofar. Unlike the other rol genes, rolD was only discovered in the agropine Ri plasmid pRiA4 [START_REF] Christey | Use of Ri-mediated transformation for production of transgenic plants[END_REF]. The neighboring orf17 and orf18 genes represent two presumably inactive halves of a rolD repeat. rolD-expressing tobacco plants show inhibition of root formation and early flowering [START_REF] Trovato | The plant oncogene rolD encodes a functional ornithine cyclodeaminase[END_REF]. Interestingly, some opine synthesis genes are not only found in Agrobacterium but also in plants and fungi. Only a few of these genes are intact. These will be described in Chapter III.

I.4.3 plast genes

In the first paper mentioning the plast family [START_REF] Levesque | Common evolutionary origin of the central portion of the Ri TL-DNA of Agrobacterium rhizogenes and the Ti T-DNAs of Agrobacterium tumefaciens[END_REF] 11 highly diverged plast proteins were identified. They have sizes of 250-280 amino acids and are encoded by the following genes: 5' end of orf8 and iaaM, orf11 (rolB), orf12 (rolC), orf13 and orf14 (on the TL-DNA from A.

rhizogenes strain A4 strain), gene 5, 7, 5' end of iaaM, 6a, 6b, orf21 (3') (from the TL and TR-DNA from the A. tumefaciens 15955 octopine strain). Later, other T-DNA genes could be added: rolBTR (a rolB variant on the TR-DNA of A. rhizogenes A4, [START_REF] Bouchez | Identification of a putative rolB gene on the TR-DNA of the Agrobacterium rhizogenes A4 Ri plasmid[END_REF]), e (from the A.

tumefaciens C58 nopaline strain, [START_REF] Broer | Identification of the Agrobacterium tumefaciens C58 T-DNA genes e and f and their impact on crown gall tumour formation[END_REF], and lso from the A. tumefaciens AB2/73 strain (Lippia strain) [START_REF] Otten | A T-DNA from the Agrobacterium limited-host range strain AB2/73 contains a single oncogene[END_REF]. Although the blastp search method could not confirm inclusion in the plast family of gene 7, rolC and the 5' part of iaaM, the reiterative psi-blast method did [START_REF] Otten | Sequence and functional analysis of the left-hand part of the T-region from the nopaline-type Ti plasmid pTiC58[END_REF]. These studies also showed that several genes from the largely unexplored left part of the C58 T-DNA were part of the plast family: genes b (a 5-like gene), c', d (a 3'-like gene) and e. In the same study, weak homology between gene 7 and orf18 of the A. tumefaciens 15955 TR-DNA (also called gene 4') was reported. Since 1999, additional plast genes were reported, most being similar to already described ones. The fact that a broad range of Agrobacterium strains had already been investigated suggested that the majority of Agrobacterium plast genes might have been identified. In 2005, the plast gene family (called the RolB/RolC glucosidase family in data banks) was considered to be specific for Agrobacterium species and plants [START_REF] Studholme | Protein domains and architectural innovation in plant-associated Proteobacteria[END_REF].

However, plast genes from other organisms were reported subsequently. These included genes from Laccaria bicolor (Mohajjel-Shoja, 2010) and Rhizobium mesoamericanum (Chen et al., 2014).

More recent searches (unpublished, this thesis) identified plast genes in Bradyrhizobium sp., Mesorhizobium plurifarium, Rhizobium leguminosarum, Burkholderia sp., Pisolithus microcarpus and Laccaria amethystina.

An alignment of the central parts of 50 plast proteins [START_REF] Helfer | Functional diversity and mutational analysis of Agrobacterium 6B oncoproteins[END_REF] The morphological effects and possible functions of the different plast genes are the following.

I.4.3.1 rolB rolB (orf11) genes code for proteins ranging from 28 to 31 kDa in different strains with about 60% similarity among each other (Mohajjel-Shoja, 2010). The rolB gene induces roots in leaf explants of many plant species [START_REF] Cardarelli | Agrobacterium rhizogenes T-DNA genes capable of inducing hairy root phenotype[END_REF][START_REF] Spena | Independent and synergistic activity of rolA, B and C loci in stimulating abnormal growth in plants[END_REF][START_REF] Capone | Induction and growth properties of carrot roots with different complements of Agrobacterium rhizogenes T-DNA[END_REF]. In transgenic plants it leads to necrosis [START_REF] Schmülling | Single genes from Agrobacterium rhizogenes influence plant development[END_REF], rolB-expressing roots grow faster than normal roots [START_REF] Altabella | Effect of the rol genes from Agrobacterium rhizogenes on polyamine metabolism in tobacco roots[END_REF]. The rolB gene also modifies flower induction from thin cell layers in tobacco [START_REF] Altamura | The plant oncogene rolB stimulates the formation of flower and root meristemoids in tobacco thin cell layers[END_REF]. Apparently, the rolB effects are not grafttransmissible [START_REF] Hansen | Phenotypic effects of overexpression of Agrobacterium rhizogenes T-DNA ORF13 in transgenic tobacco plants are mediated by diffusible factor(s)[END_REF]. The rolB gene is induced by auxin [START_REF] Maurel | Auxin regulates the promoter of the root-inducing rolB gene of Agrobacterium rhizogenes in transgenic tobacco[END_REF][START_REF] Capone | Different promoter regions control level and tissue specificity of expression of Agrobacterium rhizogenes rolB gene in plants[END_REF][START_REF] Capone | Expression in different populations of cells of the root meristem is controlled by different domains of the rolB promoter[END_REF] and sucrose [START_REF] Nilsson | Getting to the root: the role of the Agrobacterium rhizogenes rol genes in the formation of hairy roots[END_REF] and expressed in phloem parenchyma [START_REF] Altamura | Histological analysis of the expression of Agrobacterium rhizogenes rolB-GUS gene fusion in transgenic tobacco[END_REF]. It becomes active at the end of the globular stage of tobacco zygotic embryos (Chichiricco et al., 1992;[START_REF] Di Cola | RolB expression pattern in the early stages of carrot somatic embryogenesis[END_REF], and in root pericycle cells during outgrowth of lateral roots [START_REF] Nilsson | Getting to the root: the role of the Agrobacterium rhizogenes rol genes in the formation of hairy roots[END_REF].

Further promoter studies identified a promoter region (ACTTTA) that binds the DOF transcription factor NtBBF1 in tobacco [START_REF] Baumann | The DNA binding site of the Dof protein NtBBF1 is essential for tissue-specific and auxin-regulated expression of the rolB oncogene in plants[END_REF]. Initially it was reported that RolB increases auxin sensitivity in excised organs and protoplasts of tobacco [START_REF] Cardarelli | Agrobacterium rhizogenes T-DNA genes capable of inducing hairy root phenotype[END_REF][START_REF] Shen | Hairy roots are more sensitive to auxin than normal roots[END_REF]Shen et al., , 1989;;[START_REF] Barbier-Brygoo | Perception of the auxin signal at the plasma membrane of tobacco mesophyll protoplasts[END_REF][START_REF] Maurel | Single rol genes from the Agrobacterium rhizogenes T(L)-DNA alter some of the cellular responses to auxin in Nicotiana tabacum[END_REF][START_REF] Schmülling | Hormonal content and sensitivity of transgenic tobacco and potato plants expressing single rol genes of Agrobacterium rhizogenes T-DNA[END_REF]. In 1991, it was proposed that RolB has glucosidase activity acting on indoxyl-beta-glucoside suggesting it could liberate auxin from conjugated auxin forms (Estruch et al., 1991b), but this has been questioned by others [START_REF] Nilsson | Hormonal characterization of transgenic tobacco plants expressing the rolC gene of Agrobacterium rhizogenes TL-DNA[END_REF][START_REF] Schmülling | Hormonal content and sensitivity of transgenic tobacco and potato plants expressing single rol genes of Agrobacterium rhizogenes T-DNA[END_REF][START_REF] Delbarre | The rolB gene of Agrobacterium rhizogenes does not increase the auxin sensitivity of tobacco protoplasts by modifying the intracellular auxin concentration[END_REF]. In 1996 it was proposed that RolB has tyrosine phosphatase activity and is localized in the plasma membrane [START_REF] Filippini | A plant oncogene as a phosphatase[END_REF]. This was partly based on the presence of a CX5R motif. However, mutation of this motif did not abolish the necrotic effect in leaf patches [START_REF] Mohajjel-Shoja | Contribution to the study of the Agrobacterium rhizogenes plast genes, rol B and rol C, and their homologs in Nicotiana tabacum[END_REF]. A rolB-like gene, rolBTR, was found on the TR-DNA of A4, and found to differ from rolB by the lack of the CX5R motif and its lack of root induction, transgenic plants show a different phenotype as rolB plants [START_REF] Lemcke | Gain of function assays identify non-rol genes from Agrobacterium rhizogenes TL-DNA that alter plant morphogenesis or hormone sensitivity[END_REF]). An extensive RolB study in 2004 [START_REF] Moriuchi | Nuclear localization and interaction of RolB with plant 14-3-3 proteins correlates with induction of adventitious roots by the oncogene rolB[END_REF] found an interaction between RolB from A. rhizogenes strain 1724 and the tobacco Nt-14-3-3-omega II protein, and localized RolB in the nucleus.

A rolB-like gene in N. glauca (NgrolB) was found to be mutated, but upon re-activation induced several abnormalities that differed from those of RirolB genes from A. rhizogenes [START_REF] Aoki | Synergistic function of rolB, rolC, ORF13 and ORF14 of TL-DNA of Agrobacterium rhizogenes in hairy root induction in Nicotiana tabacum[END_REF][START_REF] Aoki | Resurrection of an ancestral gene: functional and evolutionary analyses of the Ngrol genes transferred from Agrobacterium to Nicotiana[END_REF].

I.4.3.2 rolC rolC genes (also called orf12) code for proteins of about 20 kDa with more than 65% similarity among strains [START_REF] Mohajjel-Shoja | Contribution to the study of the Agrobacterium rhizogenes plast genes, rol B and rol C, and their homologs in Nicotiana tabacum[END_REF]. rolC is required for hairy root induction. 35S-rolC tobacco and potato plants show dwarfism, reduction of apical dominance, pale green leaves, and malesterile flowers [START_REF] Spena | Cell-autonomous behavior of the rolC gene of Agrobacterium rhizogenes during leaf development: a visual assay for transposon excision in transgenic plants[END_REF][START_REF] Schmülling | Single genes from Agrobacterium rhizogenes influence plant development[END_REF][START_REF] Fladung | Transformation of diploid and tetraploid potato clones with the rolC gene of Agrobacterium rhizogenes and characterization of transgenic plants[END_REF]) and stem fasciation [START_REF] Nilsson | Expression of the Agrobacterium rhizogenes rolC gene in a deciduous forest tree alters growth and development and leads to stem fasciation[END_REF]. The rolC effect is cell-autonomous [START_REF] Spena | Cell-autonomous behavior of the rolC gene of Agrobacterium rhizogenes during leaf development: a visual assay for transposon excision in transgenic plants[END_REF]. rolC is expressed in phloem cells and induced by sucrose [START_REF] Yokoyama | The rolC promoter of Agrobacterium rhizogenes Ri plasmid is activated by sucrose in transgenic tobacco plants[END_REF][START_REF] Nilsson | Getting to the root: the role of the Agrobacterium rhizogenes rol genes in the formation of hairy roots[END_REF].

It was reported that rolC releases cytokinin from conjugates by a glucosidase activity (Estruch et al., 1991a), is located in the cytoplasm (Estruch et al., 1991b) and alters cytokinin and gibberellin metabolism [START_REF] Nilsson | Hormonal characterization of transgenic tobacco plants expressing the rolC gene of Agrobacterium rhizogenes TL-DNA[END_REF], but the glucosidase activity was strongly questioned in 1996 [START_REF] Nilsson | Expression of the Agrobacterium rhizogenes rolC gene in a deciduous forest tree alters growth and development and leads to stem fasciation[END_REF]. Because of the rolC-induced phenotype and its expression in the phloem, it has been proposed that rolC could create a sink for sucrose (Nilsson and Olsson, 1997, overview). The gene has been expressed in many plant species and may be used for modification of flowers [START_REF] Casanova | Influence of rol genes in floriculture[END_REF]. An active rolC gene was found in N. glauca [START_REF] Aoki | Synergistic function of rolB, rolC, ORF13 and ORF14 of TL-DNA of Agrobacterium rhizogenes in hairy root induction in Nicotiana tabacum[END_REF] where it is expressed in the vascular system [START_REF] Nagata | Different expression patterns of the promoters of the NgrolB and NgrolC genes during the development of tobacco genetic tumors[END_REF] and in N.

tabacum [START_REF] Meyer | Horizontal gene transfer: regulated expression of tobacco homologue of the Agrobacterium rhizogenes rolC gene[END_REF][START_REF] Mohajjel-Shoja | Contribution to the study of the Agrobacterium rhizogenes plast genes, rol B and rol C, and their homologs in Nicotiana tabacum[END_REF].

I.4.3.3 orf13

orf13 genes were found on T-DNAs from A. rhizogenes and in some plants like N. glauca, N.

tabacum and the ancestor of tobacco N. tomentosiformis [START_REF] Fründt | Evidence for the ancient transfer of Ri plasmid T-DNA genes between bacteria and plants[END_REF]Chen et al., 2014).

DNA sequence homology among these orf13 genes is about 70-90% [START_REF] Fründt | Evidence for the ancient transfer of Ri plasmid T-DNA genes between bacteria and plants[END_REF]. The orf13 gene does not belong to the rol genes involved in hairy root induction but seems to enhance rooting by rolA, B and C [START_REF] Capone | Induction and growth properties of carrot roots with different complements of Agrobacterium rhizogenes T-DNA[END_REF] and the necrotic rolB response (Aoki and Syono, 1999a). Plants expressing a 35S-orf13 gene are dwarfs, have irregular leaves, and curly roots [START_REF] Lemcke | Gain of function assays identify non-rol genes from Agrobacterium rhizogenes TL-DNA that alter plant morphogenesis or hormone sensitivity[END_REF]. The phenotypic effects of the 8196-orf13 gene are grafttransmissible [START_REF] Hansen | Phenotypic effects of overexpression of Agrobacterium rhizogenes T-DNA ORF13 in transgenic tobacco plants are mediated by diffusible factor(s)[END_REF]. In tomato orf13 induced stunted leaves with "spikes" on the abaxial side, changes in phyllotaxis and fasciation, and increased cell division in the SAM [START_REF] Stieger | The orf13 T-DNA gene of Agrobacterium rhizogenes confers meristematic competence to differentiated cells[END_REF]. The 8196-orf13 expression is high in roots and induced by wounding [START_REF] Hansen | Wound-inducible and organ-specific expression of ORF13 from Agrobacterium rhizogenes 8196 T-DNA in transgenic tobacco plants[END_REF], A4-orf13 root expression seems lower [START_REF] Udagawa | Expression analysis of the NgORF13 promoter during the development of tobacco genetic tumors[END_REF] and can be induced by wounding or ageing. It has been proposed that 8196 Orf13 binds to the Retinoblastoma protein with an LxCxE motif. This motif is not found in other plast proteins. An orf13 gene was found in N. glauca and a 35S-Ngorf13 gene leads to round and dark-green leaves in tobacco (Aoki and Syono, 1999a).

An N. tabacum torf13 gene was also shown to be active, it causes cell proliferation on carrot disks [START_REF] Fründt | Evidence for the ancient transfer of Ri plasmid T-DNA genes between bacteria and plants[END_REF].

I.4.3.4 orf14

The orf14 gene of A. rhizogenes has also been reported to enhance rolB-induced rooting [START_REF] Capone | Induction and growth properties of carrot roots with different complements of Agrobacterium rhizogenes T-DNA[END_REF]. A 35S-A4-orf14 construct did not modify the normal tobacco phenotype (Aoki and Syono 1999a). The orf14 gene was also found in N. glauca and weakly promotes root induction by rolB as does the A. rhizogenes orf14 gene [START_REF] Aoki | Synergistic function of rolB, rolC, ORF13 and ORF14 of TL-DNA of Agrobacterium rhizogenes in hairy root induction in Nicotiana tabacum[END_REF]. N. tabacum contains several orf14 genes (see Chapter I), their possible activities remain to be tested.

I.4.3.5 Gene 6a

Gene 6a is part of the A. tumefaciens T-DNAs. The octopine and nopaline-type 6a genes were found to play a role in opine secretion [START_REF] Messens | Genetic basis for opine secretion from crown gall tumor cells[END_REF] and were therefore named ons, for octopine/nopaline secretion. Unfortunately, this very interesting result was not followed up by further research on the precise mechanism of transport.

I.4.3.6 Gene 6b

The 6b gene has been found on T-DNAs from A. tumefaciens and A. vitis. This gene causes tumors on certain plant species by an unknown mechanism [START_REF] Hooykaas | The Agrobacterium tumefaciens T-DNA gene 6b is an oncogene[END_REF]. It was recently also discovered in N. otophora as part of the TE cT-DNA (Chen et al., 2014). The 6b gene causes very strong morphological changes when overexpressed in plants. An important part of my thesis concerns the plast gene 6b. An overview of the studies concerning this gene will therefore be presented in Chapter II.

I.4.3.7 Gene 5

Gene 5 is found on T-DNAs from several A. tumefaciens strains like C58 (nopaline strain) and Ach5 (octopine strain). The Ach5 5 gene was reported to direct synthesis of indole-3-lactate [START_REF] Körber | T-DNA gene 5 of Agrobacterium modulates auxin response by autoregulated synthesis of a growth hormone antagonist in plants[END_REF]. Unfortunately, no further research has been done with this gene. It does not lead to a phenotype when expressed under 35S promoter control in tobacco [START_REF] Otten | A T-DNA from the Agrobacterium limited-host range strain AB2/73 contains a single oncogene[END_REF].

I.4.3.8 Gene e

The protein encoded by gene e has high similarity to RolB from A. rhizogenes and to p5 from A.

tumefaciens [START_REF] Broer | Identification of the Agrobacterium tumefaciens C58 T-DNA genes e and f and their impact on crown gall tumour formation[END_REF]. The same authors reported that e was involved in tumor formation but this was later questioned since a test of gene e on N. glauca, N. rustica and N.

tabacum failed to induce tumors [START_REF] Otten | Sequence and functional analysis of the left-hand part of the T-region from the nopaline-type Ti plasmid pTiC58[END_REF].

I.4.3.9 lso

The lso gene (for Lippia strain oncogene) was initially found in A. tumefaciens strain AB2/73 [START_REF] Otten | A T-DNA from the Agrobacterium limited-host range strain AB2/73 contains a single oncogene[END_REF]. It is able to induce small tumors on different plant species. Plants over-expressing lso have a dwarf phenotype with wrinkled leaves and reduced root growth, the phenotype is not graft-transmissible [START_REF] Schmidt | Etude d'un oncogène de la souche AB2/73 d'Agrobacterium tumefaciens[END_REF].

I.4.3.10 5' end of iaaM and orf8 genes

The iaaM and orf8 genes have been found in A. tumefaciens, A. rhizogenes and A. vitis. The iaaM gene codes for the synthesis of indoleacetamide (IAM) from tryptophan, this activity is well documented (Van Onkelen et al., 1985;[START_REF] Umber | The T-DNA oncogene A4-orf8 from Agrobacterium rhizogenes A4 induces abnormal growth in tobacco[END_REF]. The iaaM gene is associated with an iaaH gene that codes for the conversion of IAM into IAA. The orf8 gene is highly similar to the iaaM gene, but is not associated with an iaaH gene, and does not code for IAM production. The original plast paper [START_REF] Levesque | Common evolutionary origin of the central portion of the Ri TL-DNA of Agrobacterium rhizogenes and the Ti T-DNAs of Agrobacterium tumefaciens[END_REF] noticed that the N-terminal part of IaaM (about 250 amino acids) is part of the plast family and resembles RolB. iaaM genes in other organisms, like Pseudomonas or Rhizobium, do not contain this rolB-like extension [START_REF] Otten | Biological activity of the rolB-like 5' end of the A4-orf8 gene from the Agrobacterium rhizogenes TL-DNA[END_REF]. Expression of the RolB part of A4-Orf8 under 35S promoter control leads to strong accumulation of hexose sugars and starch in tobacco, whereas the equivalent region from Ach5-IaaM has no such activity [START_REF] Otten | Biological activity of the rolB-like 5' end of the A4-orf8 gene from the Agrobacterium rhizogenes TL-DNA[END_REF][START_REF] Umber | The rolB-like part of the Agrobacterium rhizogenes orf8 gene inhibits sucrose export in tobacco[END_REF][START_REF] Umber | The T-DNA oncogene A4-orf8 from Agrobacterium rhizogenes A4 induces abnormal growth in tobacco[END_REF]. 35S-A4-Norf8 plants retain sucrose in the source leaves and convert it to massive amounts of starch.

I.4.3.11 Gene 3' Gene 3' from A. tumefaciens octopine strain Ach5 induces very small tumors on K. tubiflora [START_REF] Otten | Sequence and functional analysis of the left-hand part of the T-region from the nopaline-type Ti plasmid pTiC58[END_REF]. In the A. vitis strain AB4 [START_REF] Otten | Agrobacterium vitis nopaline Ti plasmid pTiAB4: relationship to other Ti plasmids and T-DNA structure[END_REF] and in A. tumefaciens strain 82.139 [START_REF] Drevet | Oncogene arrangement in a shooty strain of Agrobacterium tumefaciens[END_REF] and on the TB-region of the A. vitis octopine/cucumopine strain Tm4), c', d and e, the latter three from A. tumefaciens nopaline strain C58 [START_REF] Otten | Sequence and functional analysis of the left-hand part of the T-region from the nopaline-type Ti plasmid pTiC58[END_REF]. They do not seem to have a notable role in tumor induction, but it will be interesting to study their capacity to modify plant growth by over-expression in different host plants.

I.4.4 Genes unrelated to the first three groups

I.4.4.1 rolA rolA encodes a small protein of about 11 kDa [START_REF] Nilsson | Getting to the root: the role of the Agrobacterium rhizogenes rol genes in the formation of hairy roots[END_REF]. rolA transgenic tobacco plants have wrinkled leaves, show inhibition of plant growth and abnormal flowers [START_REF] Trovato | Recent advances on rol genes research: a tool to study plant differentiation[END_REF]. The phenotype of rolA is graft transmissible from both rootstocks and scions to the untransformed plant part [START_REF] Guivarch | Tissue-specific expression of the rolA gene mediates morphological changes in transgenic tobacco[END_REF]. rolA transgenic Arabidopsis thaliana has much lower levels of phytohormones (auxin, cytokinin, gibberellic acid and abscisic acid) compared to WT plants [START_REF] Dehio | Stable expression of a single-copy rolA gene in transgenic Arabidopsis thaliana plants allows an exhaustive mutagenic analysis of the transgene-associated phenotype[END_REF]. The mRNA of rolA has an untranslated intron region. In the 5' end of rolA, this transcribed but untranslated intron region is important for rolA function since the splicing of this part abolishes the rolA phenotype in Arabidopsis [START_REF] Magrelli | Splicing of the rolA transcript of Agrobacterium rhizogenes in Arabidopsis[END_REF]. This intron was later found to be a prokaryotic promoter which allows rolA expression in A.

rhizogenes [START_REF] Pandolfini | The spliceosomal intron of the rolA gene of Agrobacterium rhizogenes is a prokaryotic promoter[END_REF].

I.4.4.2 Gene c

Gene c is found on the A.tumefaciens pTiC58 T-DNA and A. vitis pTiTm4 TB-DNA and code for a 523 amino acid protein [START_REF] Otten | Sequence and functional analysis of the left-hand part of the T-region from the nopaline-type Ti plasmid pTiC58[END_REF]. Recently, homologs have been found in other organisms (Chen et al., 2014) but the function remains unknown.

I.4.4.3 Gene 3

Gene 3 (also called ORF3n) is located on the left part of the T-DNA from A. rhizogenes strain HRI [START_REF] Lemcke | Gain of function assays identify non-rol genes from Agrobacterium rhizogenes TL-DNA that alter plant morphogenesis or hormone sensitivity[END_REF]. A 35S::3 transgenic tobacco is clearly modified. The stem of this transgenic tobacco is about 20-30% shorter than WT tobacco. The leaf tip, base and sepals all have various levels of necrosis. The flowering time of these plants is about 2-3 weeks later compared to the WT plant [START_REF] Lemcke | Gain of function assays identify non-rol genes from Agrobacterium rhizogenes TL-DNA that alter plant morphogenesis or hormone sensitivity[END_REF].

I.5. Stable horizontal gene transfer

Horizontal gene transfer (HGT) is the abnormal transfer of genes between different species as opposed to vertical transfer, i.e. normal gene transfer to the next generation within the same species. HGT has been shown to play an important role in the evolution of many organisms and occurs between bacteriophages and bacteria, among different types of bacteria, from bacteria to eukaryotes like fungi or plants and within eukaryotes from the chloroplast and mitochondrial genomes to the nuclear genomes.

I.5.1 HGT from Agrobacteria to plants

About 30 years ago, sequences with homology to the T-DNA of the A. rhizogenes pRi plasmid were found in the genome of Nicotiana glauca; these were named "cellular T-DNA" (cT-DNA) sequences [START_REF] White | Sequence homologous to Agrobacterium rhizogenes T-DNA in the genomes of uninfected plants[END_REF][START_REF] Furner | An Agrobacterium transformation in the evolution of the genus Nicotiana[END_REF]. Southern blot analysis showed that the N.

glauca cT-DNA consisted of an imperfect repeat. Initially, only part of this cT-DNA was sequenced, to be completed several years later [START_REF] Furner | An Agrobacterium transformation in the evolution of the genus Nicotiana[END_REF][START_REF] Aoki | Sequence of the cellular T-DNA in the untransformed genome of Nicotiana glauca that is homologous to ORFs 13 and 14 of the Ri plasmid and analysis of its expression in genetic tumors of N. glauca x N. langsdorffii[END_REF][START_REF] Suzuki | Tobacco plants were transformed by Agrobacterium rhizogenes infection during their evolution[END_REF]. The N. glauca cT-DNA contains the NgrolB, NgrolC, NgORF13, NgORF14 and Ngmis genes (figure 9) [START_REF] Suzuki | Tobacco plants were transformed by Agrobacterium rhizogenes infection during their evolution[END_REF]. NgrolB, NgrolC, NgORF13 and NgORF14 were found to be expressed in genetic tumors formed by hybrids between N. glauca and N. langsdorffii [START_REF] Aoki | Sequence of the cellular T-DNA in the untransformed genome of Nicotiana glauca that is homologous to ORFs 13 and 14 of the Ri plasmid and analysis of its expression in genetic tumors of N. glauca x N. langsdorffii[END_REF]. Later, the Ngmis gene was found to encode an enzymatically active mikimopine synthase when expressed in E. coli, but no mikimopine was found in N. glauca plants [START_REF] Suzuki | Tobacco plants were transformed by Agrobacterium rhizogenes infection during their evolution[END_REF].

Additional studies showed that other Nicotiana species also contained cT-DNA sequences. These included cT-DNA fragments from N. tomentosa, N. kawakamii, N. tomentosiformis and N.

otophora (figure 10). Partial cT-DNA sequences were also reported from N. tabacum (Meyer et al., 1995, summary in Chapter I). In 2012, after a systematic search in 127 plant species, a cT-DNA with genes rolB, rolC, ORF13, ORF14 and mis was found in Linaria vulgaris (Matveeva et al., 2012) (figure 11A). This cT-DNA is organized as a single, imperfect direct tandem repeat. None of the cT-DNA genes seems to be expressed (Matveeva et al., 2012).

More recently, cT-DNAs were found in Ipomoea batatas (sweet potato) which is an important food crop (figure 11B). Two different T-DNA regions called ibT-DNA1 and ibT-DNA2 were described.

I.5.2 HGT among other organisms

HGT is one of the main causes of microrganism genome diversity. Based on deep-sequencing from 116 prokaryotic genomes, it has been found that about 14% of ORFs were introduced by recent HGT [START_REF] Nakamura | Biased biological functions of horizontally transferred genes in prokaryotic genomes[END_REF]. Most of the genes involved in HGT among bacteria code for antibiotic resistance, utilization of different substrates, bacteriocins, immunity to bacteriophages, production of exopolysaccharide and biogenic amines [START_REF] Rossi | Horizontal gene transfer among microorganisms in food: current knowledge and future perspectives[END_REF]. Antibiotic resistant genes have spread from bacteriophages to bacteria through mobile elements and have been strongly selected [START_REF] Muniesa | Potential impact of environmental bacteriophages in spreading antibiotic resistance genes[END_REF].

Plasmid-derived HGT from bacteria to fungi has become more and more likely since in experimental conditions, Agrobacterium is able to mediate the transformation of the filamentous fungi [START_REF] Michielse | Agrobacterium mediated transformation of the filamentous fungus Aspergillus awamori[END_REF]. Soil bacteria are quite often associated with fungi and some fungal genes were found to have homology to bacterial genes [START_REF] Haq | The interactions of bacteria with fungi in soil: emerging concepts[END_REF][START_REF] Zhang | The mycosphere constitutes an arena for horizontal gene transfer with strong evolutionary implications for bacterial-fungal interactions[END_REF].

Tryptophan-dependent IAA biosynthesis in plants might be derived from HGT involving bacteria (Huang et al., 2014). The mitochondrial genome of the angiosperm Amborella contains six genome equivalents of foreign mitochondrial DNA, indicating the HGT of entire genomes from one angiosperm to another [START_REF] Rice | Horizontal transfer of entire genomes via mitochondrial fusion in the angiosperm Amborella[END_REF]. Recently, it was shown that about one in six of Hypsibius dujardini (water bear or tardigrade) genomic sequences were derived from bacteria, plants, fungi or Archaea according to deep-sequencing data [START_REF] Boothby | Evidence for extensive horizontal gene transfer from the draft genome of a tardigrade[END_REF]. These two examples demonstrate the big potential of HGT between plants and even between animals with other organisms.

In my thesis, I will describe five new cT-DNA sequences from the genus Nicotiana (Publication Chen et al. 2014, Chapter I). We have shown that the evolution of cT-DNA sequences in the genus Nicotiana is much more complex than previously assumed.

II. Leaf formation, and effects related to hormones and sugar

Among the cT-DNA genes that we discovered in N. otophora (Chen et al., 2014), TE-6b (from the N. otophora-specific TE region) has homology to the 6b gene from A. tumefaciens. The Agrobacterium 6b gene was found to incite tumors on N. glauca and K. tubiflora and can therefore be considered as an oncogene [START_REF] Hooykaas | The Agrobacterium tumefaciens T-DNA gene 6b is an oncogene[END_REF]. The 6b genes from different Agrobacterium strains show strong and highly specific morphogenetic effects in transgenic plants, collectively called the "enation syndrome" [START_REF] Helfer | The Agrobacterium oncogene AB-6b causes a graft-transmissible enation syndrome in tobacco[END_REF]. Enations are abnormal additional leaf blades formed on the lower (abaxial) leaf side. Another remarkable property of 6btransformed plants is their capacity to transmit the enation phenotype to normal plants by grafting.

In order to compare the N. otophora TE-6b gene with other 6b genes, we decided to obtain a more precise description of the morphological changes of the aerial part of the plant using an

II.1. Leaf formation

Leaf formation can be considered to occur in three steps: 1. leaf primordium initiation; 2. leaf differentiation; 3. leaf expansion and maturation [START_REF] Dkhar | What determines a leaf's shape?[END_REF]. Each will be discussed separately.

1. Leaf primordium initiation (figure 12A). PIN-FORMED1 (PIN1) is a transmembrane protein which is responsible for auxin transport. In the epidermis cells surrounding the SAM, the polarized PIN1 is highly expressed. The PIN1 is responsible for auxin gradient formation in the area beside the centrally located SAM where auxin has the highest concentration. A provascular system which is the beginning of a leaf primordium, is formed here. Another very important factor is KNOX1 which plays a role in stem cell differentiation (figure 12B) [START_REF] Dkhar | What determines a leaf's shape?[END_REF]. In the KNOX1 mutant of maize, SAM development is strongly repressed [START_REF] Kerstetter | Loss of function mutations in the maize homeobox gene, knotted1, are defective in shoot meristem maintenance[END_REF].

2. Leaf cell proliferation and differentiation (figure 12B). Proximodistal, adaxial/abaxial, and mediolateral polarities are created once a leaf primordium is formed. In Arabidopsis, the KAN, HD-ZIPIII gene family and YAB are the major contributors. AS1, AS2, TAS3, ARF3, ARF4, and miRNAs (miR165, miR166) are also involved in this process. These genes work together and many of them can inhibit expression of the others [START_REF] Dkhar | What determines a leaf's shape?[END_REF]. For example, it was shown that in the abaxial domain, miR165/miR166 repress expression of the HD-ZIPIII gene family [START_REF] Zhou | Overexpression of miR165 affects apical meristem formation, organ polarity establishment and vascular development in Arabidopsis[END_REF][START_REF] Kim | MicroRNA-directed cleavage of ATHB15 mRNA regulates vascular development in Arabidopsis inflorescence stems[END_REF]. show expression level changes of major adaxial/abaxial leaf polarity genes (Huang et al., 2014).

The functional relation between the different genes is shown in figure 12B.

II.2 Auxin and cytokinin metabolism, transport and signaling

Auxins and cytokinins are two of the most important phytohormones and have been implied in various aspects of plast gene activity.

II.2.1 Auxin

Auxin has a general effect on plant wound response, root growth and development, apical dominance, fruit growth and development, flowering and ethylene biosynthesis. TAA codes for a transaminase which catalyzes the conversion of tryptophan to indole-3-pyruvate [START_REF] Zhao | Auxin biosynthesis[END_REF].

Indole-3-pyruvate is then converted into IAA by a flavin monooxygenase encoded by YUCCA [START_REF] Tao | Rapid synthesis of auxin via a new tryptophan dependent pathway is required for shade avoidance in plants[END_REF][START_REF] Stepanova | TAA1-mediated auxin biosynthesis is essential for hormone crosstalk and plant development[END_REF]. As mentioned in II.1, transmembrane protein PIN1 determines auxin flux. The AUX1/LAX gene is responsible for auxin import (figure 13). The F-box protein TIR1 is an auxin receptor and is part of an SCF complex which leads to the proteolytic degradation of the AUX/IAA repressor protection in the presence of auxin [START_REF] Salehin | SCF TIR1/AFB -based auxin perception: mechanism and role in plant growth and development[END_REF].

The auxin response factor (ARF) can be repressed by AUX/IAA when the auxin concentration is low. ARF is responsible for activating auxin-regulated gene expression [START_REF] Ulmasov | Activation and repression of transcription by auxinresponse factors[END_REF].

II.2.2 Cytokinin

Cytokinin is an important factor in plant cell division and differentiation. It works together with auxin and other factors like sugar to direct plant growth. Cytokinin biosynthesis involves two steps.

First, an N 6 -isopentenyladenine (iP) ribotide is formed by adding a prenyl moiety from dimethylallyl diphosphate to ATP/ADP. This step is catalyzed by isopentenyltransferase (IPT).

Cytochrome P450 enzyme CYP735A1/2 then hydroxylates the isoprenoid side chain of iP ribotide yielding trans-zeatin cytokinin [START_REF] Sakakibara | Cytokinins: activity, biosynthesis, and translocation[END_REF]. In cytokinin signaling, a histidine kinase (AHK) transfers a phosphate group to a histidine-containing phosphotransfer (AHP) protein which directly regulates the response regulators (RRs) in order to get the cytokinin effect (figure 13).

II.3 The combined role of sucrose and auxin on plant cell division and expansion

Sucrose is one of the most important carbon sources and a source of energy. It is transported from source (photosynthetic leaves) to sink (roots, flowers, seeds and meristem) tissues. Sucrose concentration can strongly influence plant activity. Generally, high sucrose levels inhibit photosynthesis in source tissues and stimulate growth and storage in sink tissues. Conversely, low sucrose levels stimulate photosynthesis in source leaves [START_REF] Wang | Regulation of cell division and expansion by sugar and auxin signaling[END_REF]. There are two sucrose degradation pathways. One is by hydrolysis into glucose and fructose catalyzed by invertase, the other is by degradation into UDP-glucose and fructose by sucrose synthase.

II.3.1 Sugar and auxin affect plant cell division

Sugar works not only as carbon source but also as a signaling compound which directly regulates plant gene expression (reviewed by [START_REF] Gibson | Control of plant development and gene expression by sugar signaling[END_REF][START_REF] Eveland | signaling, and plant development[END_REF]. In plant cells, glucose is able to crosstalk with plant hormones like auxin, cytokinin, abscisic acid and ethylene [START_REF] Gazzarrini | Genetic interaction between ABA, ethylene and sugar signaling pathways[END_REF][START_REF] Hartig | Crosstalk between auxin, cytokinins, and sugars in the plant cell cycle[END_REF]. Activation of glucose-induced auxin biosynthesis and transport genes has differential effects on auxin receptors [START_REF] Mishra | Glucose and auxin signaling interaction in controlling Arabidopsis thaliana seedlings root growth and development[END_REF].

Interestingly, exogenous glucose enhances defects in lateral root initiation and root hair elongation in auxin sensitive mutants, indicating glucose may affect root development through auxin-based gene regulation [START_REF] Mishra | Glucose and auxin signaling interaction in controlling Arabidopsis thaliana seedlings root growth and development[END_REF][START_REF] Andrea | signalling, and plant development[END_REF].

The plant cell cycle is divided into different phases: gap0 (G0), gap1 (G1), DNA synthesis (S), gap2 (G2) and mitosis (M). Sugar and auxin work together to regulate the plant cell cycle (figure 14). In G1 phase, glucose (Glc) and auxin both induce cyclinD (CycD) gene expression. Auxin independently induces the cyclin-dependent kinase A-1 gene (CDKA) and activates the CycD/CDKA complex by phosphorylation and repression of the retinoblastoma related (RBR)

protein [START_REF] Wang | Regulation of cell division and expansion by sugar and auxin signaling[END_REF]. Activated CycD/CDKA can phosphorylate RBR in order to release adenovirus E2 promotor binding factor A/B (E2FA/B) and the dimerization partner A (DPA) [START_REF] Wang | Regulation of cell division and expansion by sugar and auxin signaling[END_REF]. The E2FA/B-DPA complex initiates S phase gene expression. In S phase, glucose promotes phosphorylation of the CycA3/CDKA complex and leads to expression of S phase genes. Auxin accelerates S phase kinase associated protein 2A (SKP2A) degradation and stablilizes the E2FC (E2 promoter binding factor C)-DPB complex which represses the expression of S phase genes [START_REF] Wang | Regulation of cell division and expansion by sugar and auxin signaling[END_REF]. Recent data show that G2/M transition is regulated by TPR domain suppressor of stimpy (TSS) which is repressed by glucose [START_REF] Skylar | Metabolic sugar signal promotes Arabidopsis meristematic proliferation via G2[END_REF].

II.3.2 Sugar and auxin effect on plant cell expansion

Cell expansion is an important progress which includes the accumulation of osmotically active solutes, water influx into the cell, cell wall loosening and reconstruction [START_REF] Dkhar | What determines a leaf's shape?[END_REF][START_REF] Wang | Regulation of cell division and expansion by sugar and auxin signaling[END_REF]. The final plant cell size is defined by the end of cell expansion.

Sugars are osmotically active solutes and their intercellular increase can lead to increased osmotic pressure. The hydrolysis of sugar into glucose and fructose doubles the osmotic contribution to the osmotic pressure in the vacuole. This in turn leads to cell size increase under the condition of cell wall plasticity. Sugars not only work as osmotically active solutes, but also as signals to modulate cell expansion. One possibility is that sugar influences cell plasticity by downstream regulation of wall associated kinases [START_REF] Wang | Regulation of cell division and expansion by sugar and auxin signaling[END_REF].

Results

Chapter S1. TA: covered by 15 TW142 contigs. TB: a single TW142 contig covers the complete region, in addition, seven small contigs cover parts of TB. TC: two TW142 contigs overlap, four additional ones cover small regions. TD: two TW142 contigs overlap, an additional small one covers a small part. S2 and Figure 6a.

II.2 N. setchellii cT-DNA

Initially, we tested cT-DNAs in all species of the Tomentosa section species except for N.

setchellii for which no seeds were available at the Institut du Tabac de Bergerac. Our results

showed that it would be very interesting to test this rare species for cT-DNA. According to our evolutionary model for cT-DNAs in Nicotiana (Chen et al., 2014, figure 9) and earlier data on the Nicotiana species evolution [START_REF] Knapp | Nomenclatural changes and a new sectional classification in Nicotiana (Solanaceae)[END_REF], N. setchellii would not be expected to contain TA, TB, TD and TE but only TC. Seeds were obtained from the US Department of Agricultural

Research Service and genomic DNA was tested by PCR using primers flanking the insertion sites of TA, TB, TC, TD and TE. Results showed that these insertion sites were all intact, except that TC could not be amplified (table 4). Therefore, TC was tested in more detail. Two fragments, 2372 to 2375 (c1) and 2420 to 2413 (c2) (Chen et al., 2014, table 3) inside the TC region were tested, but only abnormal amplification fragments were obtained. It could be that the region in which TC was inserted in N. tomentosiformis has been deleted in N. setchellii, either before or after TC insertion. It seems therefore that N. setchellii does not contain any of the known cT-DNA sequences. However, other cT-DNAs might be present. Only a full sequence analysis using genomic sequencing will give a definitive answer.

II.3 TE-6b

An intact 6b gene was earlier found in the N. otophora cT-DNA TE region (Chen et al., 2014). It was very surprising to find a 6b gene since cT-DNAs are normally derived from A. rhizogenes strains and the 6b gene has never been found in such strains. Also 6b genes are known to produce remarkable growth effects. In order to learn more about the unusual growth induction properties of the 6b genes, a detailed study of a dex inducible T-6b gene was carried out (Chapter II, [START_REF] Chen | Morphological analysis of the 6b oncogene-induced enation syndrome[END_REF]. Additional data on TE-6b will therefore be presented at the end of Chapter II.

II.4 mas2'

A mas2' gene was found in the N. tomentosiformis cT-DNA TB region. A 35S::mas2' construct led to the production of DFG in a transient expression assay in N. benthamiana (Chen et al., 2014, Chapter I). The expression level of mas2' in N. tomentosiformis is very low (figure 15). However, we discovered by chance that in some N. tabacum cultivars, the mas2' gene is highly expressed and therefore carried out an extensive study on these cultivars. This work will be presented in

Chapter III as a manuscript to be submitted, entitled: "High mas2' gene expression and opine synthesis in wild-type Nicotiana tabacum".

Chapter II: Morphogenetic properties of 6b genes from Agrobacterium and

Nicotiana otophora

I. Introduction

One of the Agrobacterium T-DNA plast genes is the 6b gene. In 1988 it was noticed that this gene induces tumors on Nicotiana glauca and Kalanchoe tubiflora [START_REF] Hooykaas | The Agrobacterium tumefaciens T-DNA gene 6b is an oncogene[END_REF]. Our group has been interested in the 6b gene for several years. We found earlier that different 6b

genes have different effects on plant growth. A-6b from Ach5, C-6b from C58 and T-6b from Tm4, when coinfected with the Ach5 cytokinin (ipt) gene, induced tumors with shoots, green tumors and tumors with a necrotic surface respectively [START_REF] Tinland | Agrobacterium tumefaciens 6b genes are strain-specific and affect the activity of auxin as well as cytokinin genes[END_REF]. T-6b from Tm4 cotransferred with rolA, B, and C from A. rhizogenes caused unorganized calli on carrot roots and very large roots on N. rustica [START_REF] Tinland | Agrobacterium tumefaciens T-DNA gene 6b stimulates rol-induced root formation, permits growth at high auxin concentrations and increases root size[END_REF]. In order to study T-6b effects on normal plants, this gene was placed under control of a hsp70 heat-shock promoter from Drosophila melanogaster. It was found that heat shock induced hsp-T6b protoplasts have higher sensitivity to hormones compared to normal protoplasts (Tinland et al., 1992). In 2003, we reported that the AB-6b gene induced enations and that the enation syndrome was graft-transmissible [START_REF] Helfer | The Agrobacterium oncogene AB-6b causes a graft-transmissible enation syndrome in tobacco[END_REF]. Subsequently, the T-6b gene was placed under a dexamethasone (dex) inducible promotor to study its effects on normally growing plants [START_REF] Grémillon | New plant growth-modifying properties of the Agrobacterium T-6b oncogene revealed by the use of a dexamethasone-inducible promoter[END_REF]. It was found that T-6b induced cell expansion like indole-3-acetic acid (IAA), but T-6b expression did not increase IAA levels, nor did it induce an IAA responsive gene [START_REF] Clément | The Agrobacterium vitis T-6b oncoprotein induces auxin-independent cell expansion in tobacco[END_REF]. Interestingly, in the T-6b tobacco roots, large amounts of sugars and phenolics accumulated [START_REF] Clément | Abnormal accumulation of sugars and phenolics in tobacco roots expressing the Agrobacterium T-6b oncogene and the role of these compounds in 6b-induced growth[END_REF]. In 6b roots, the dominant phenolic compound is chlorogenic acid (CGA). When the synthesis of CGA was blocked by 2-aminoindan-2-phosphonic acid (AIP) in T-6b tobacco, CGA accumulation was abolished, but the 6b phenotype and sugar accumulation remained unmodified [START_REF] Clément | Abnormal accumulation of sugars and phenolics in tobacco roots expressing the Agrobacterium T-6b oncogene and the role of these compounds in 6b-induced growth[END_REF]. The contribution of other groups to the study of 6b genes is presented in the introduction of our recent paper in Planta [START_REF] Chen | Morphological analysis of the 6b oncogene-induced enation syndrome[END_REF].

Recently

III. Additional data III.1 Introduction of the TE-6b gene in N. tabacum

Since the publication of this work, we have started to investigate the possible activity of the 6b gene from N. otophora, TE-6b (Chapter II). As a first step, the TE-6b gene was put under 2x35S

promoter control and introduced into N. tabacum, which lacks this gene. Different accessions of N. otophora exist in different seed banks. We chose accession TW95 from the US Department of Agricultural Research Service since it was used to obtain the deepsequencing data [START_REF] Sierro | The tobacco genome sequence and its comparison with those of tomato and potato[END_REF]. The TE-6b gene from N. otophora was put under 2x35S

promotor control using the pCK GFP S65C and pBI121.2 vectors (materials and methods) and transferred to N. tabacum cultivar Samsun nn. About 40 independent TE-6b regenerants were cultured in vitro, their leaves showed various abnormalities: narrow leaf blades, thicker veins, parallel running secondary veins and ectopic leaves growing on older leaves (figure 17). The TE-6b RNA expression levels from 31 independently regenerated R0 plants (TE-6b-1 to TE-6b-31)

were measured by qPCR and found to be variable as expected (figure 17).

The seeds from TE-6b-1 to TE-6b-31 were harvested and sown on M0222 medium with kanamycin in order to identify single locus plants. 19 were single locus plants: 1, 2, 4, 5, 7, 8, 9, 10, 12, 13, 14, 16, 19, 20, 23, 24, 26, 28, and 31. Kanamycin resistant plants were transferred from the plates to soil in order to visually select for homozygous plants. Typical R0 TE-6b tobacco plants are presented in figure 19. A first investigation on the morphology of these plants showed some abnormalities; although they did not appear systematically on all plants, we believe they are nevertheless related to the expression of the TE-6b transgene. A striking phenotype is seen on the TE-6b-1 plant: two leaves share a common petiole and part of the midvein (figure 19A). Relatively thick and transparent secondary veins were found on the TE-6b-5 plant (figure 19B, lower). An abnormal petiole wing with small triangle leaf like structure was found on TE-6b-5 plant (figure 19C, right). The TE-6b-8 plant had narrow and thicker leaves (figure 19D). Chlorotic leaves were present on the TE-6b-13 plant (figure 19E).

Dark green leaves with a wrinkled surface were very common in TE-6b plants with high expression. Such plants also showed stunted growth as for example the TE-6b-21 (figure 19F)

and the TE-6b-5 (figure 20) plants. N. rustica stems were scratched with a needle and infected with Agrobacterium carrying an empty vector, a 2X35S::TE-6b or a 2X35S::T-6b construct. 75 days after infection, pictures were taken of the wounded stems (figure 22). The areas infected by Agrobacterium with the 2X35S::TE-6b construct showed the same wound reaction as the one infected by the Agrobacterium with the empty vector. However, the area infected by Agrobacterium with the 2X35S::T-6b construct clearly showed tumor outgrowth. This experiment shows that the N. rustica stem is not sensitive to the TE-6b gene. Nevertheness, we can not exclude that the TE-6b gene could still act as an oncogenic gene on other plant species.

III.2 Conclusion and perspectives

III.2.1 Phenotypic comparison between T-6b and TE-6b plants

Since the TE-6b work is still going on, and homozygous F2 seeds from single copy TE-6b plants

are not yet available, a phenotypic comparison between 2x35S::TE-6b and 2x35S::AB-6b plants [START_REF] Helfer | The Agrobacterium oncogene AB-6b causes a graft-transmissible enation syndrome in tobacco[END_REF] or dex T-6b plants [START_REF] Grémillon | New plant growth-modifying properties of the Agrobacterium T-6b oncogene revealed by the use of a dexamethasone-inducible promoter[END_REF][START_REF] Clément | The Agrobacterium vitis T-6b oncoprotein induces auxin-independent cell expansion in tobacco[END_REF][START_REF] Clément | Abnormal accumulation of sugars and phenolics in tobacco roots expressing the Agrobacterium T-6b oncogene and the role of these compounds in 6b-induced growth[END_REF][START_REF] Chen | Morphological analysis of the 6b oncogene-induced enation syndrome[END_REF] is still in its early stage. However, some modifications of the TE-6b plants are similar as those of dex T-6b plants, most notably stunted growth, dark-green leaves, leaf wrinkling, chlorotic leaves and parallel running secondary veins, ectopic meristems and modified petiole wings [START_REF] Chen | Morphological analysis of the 6b oncogene-induced enation syndrome[END_REF]. The precise anatomical structures of these phenotypes need further analysis.

III.2.2 A hypothesis on leaf blade outgrowth from free ends of vascular strands

In the petioles of a normal tobacco plant, petiole wings are formed at the lateral ends of the normal vascular system (figure 23 A andB). In the T-6b tobacco plant, several observations showed the inhibition of petiole wing outgrowth in the presence of EVS (figure 23 C andD).

Interestingly, we noticed that leaf blade (petiole wing) formation could still occur, at the free ends of the string-like series of parallel running vascular strands. Four examples illustrate this observation (figure 23 E-L). A handcut cross section was made from an ectopic leaf petiole which grew on an asymmetric T-6b leaf (figure 23E). Figure 23E shows that EVS were only formed on the left side of the petiole and the petiole wing grew out exactly at the end of these EVS. Another example shows a T-6b stem with a subpetiolar ridge and two narrow laminae growing out from the free ends of the EVS series (figure 23G). In a third case, a small leaf blade was growing on the abaxial side of a midvein and was associated with a few small vascular strands on the abaxial side of the midvein (figure 23I). Finally, a hand section from a cupshaped leaf showed leaf lamina tissue formed between two individual well separated vascular strands (figure 23K). According to these different observations, we propose that the free ends of EVS are necessary for the outgrowth of leaf laminae. However, free ends of vascular strands do not always induce leaf blade outgrowth. In many cases, only EVS were found on T-6b petiole or stem without any leaf tissue. The exact mechanism of the relation between free EVS ends and leaf blade outgrowth still remains to be studied. An important question that should also be addressed is the polarity of the EVS.

Introduction

Agrobacterium is well known for its capacity to transfer part of its DNA (T-DNA) to plant cells, under natural conditions this leads to crown gall tumors and hairy roots that synthesize small molecules used by the bacterium for its growth, the so-called opines (Ian et al., 1979;Vladimirov et al., 2015). Several types of opine synthesis genes have been described. Generally, they code for enzymes that combine two common metabolites into a product that can then be taken up by Agrobacteria and degraded into the constituent parts to serve as carbon, nitrogen and energy sources for the bacterium (Hansen et al., 1991). Among the different types of opines, the Amadori-type opines have been found in both tumors and hairy roots. Synthesis occurs in several steps, in the first one glutamine or glutamate is conjugated with glucose to N-1-deoxy-D-fructosyl-L-glutamine (DFG, commonly called santhopine) and N-1-deoxy-D-fructosyl-L-glutamate (DFGA) respectively, in the second step DFG is converted into mannopine (MOP) and DFGA into mannopinic acid (MOA), and finally mannopine is cyclised into agropine (AGR) and mannopinic acid into agropinic acid (AGA) [START_REF] Hong | Functional role of the Ti plasmid-encoded catabolic mannopine cyclase in mannityl opine catabolism by Agrobacterium spp[END_REF]Kim et al., 2001). Other members of this group are chrysopine (the spiropyranosyl lactone of DFG), and N-1-deoxy-D-fructosyl-5-oxo-L-proline (DFOP). Mannopine/agropine synthesis genes have been identified on T-DNAs from different A.

tumefaciens strains: the octopine/agropine strains A6 and Ach5, the succinamopine strain Bo542, the chrysopine strain Chry5, and on T-DNAs from different A. rhizogenes strains: the agropine strains A4 and 1855 and the mannopine strain 8196. The mas2' gene codes for DFG synthesis, mas1' for MOP synthesis and ags for AGR synthesis. Recently, mas2', mas1' and ags genes have been detected in the genomes of N. tomentosiformis and related species, among which N.

tabacum. Nicotiana species of the Tomentosa section carry several T-DNAs that result from horizontal gene transfer through ancient Agrobacterium infections, these cellular T-DNAs are called cT-DNAs. One of these, called the TB region, carries TB-mas2', TB-mas1' and TB-ags, organized in the same way as on pTi and pRi plasmids. The mas1' and mas2' genes are divergently transcribed and controlled by a so-called dual (divergent) promoter region situated between the two initiation codons, ags is controlled by its own promoter. In N. tomentosiformis, only TB-mas2' is intact; it encodes an active enzyme as shown by the appearance of a DFG-like compound upon over-expression in the cT-DNA-less N. benthamiana (Chen et al., 2014). Here we investigate the expression of TB-mas2' in different tobacco cultivars and show the presence of a DFG-like compound in roots of cultivars with high mas2' expression.

Results

DNA and protein sequences of mas regions and Mas proteins

The structure and organization of the mas region of the N. tomentosiformis TB region (KJ599827, Chen et al., 2014) were compared (figure 1) to those of A. rhizogenes (A4 and 8196) and A.

tumefaciens (Ach5 and Bo542). The N. tomentosiformis sequence mostly resembles the A. rhizogenes A4 sequence (86% identity). The intergenic region is partially similar to those of A. rhizogenes strains A4 and 8196, but very different from those of the A. tumefaciens strains Ach5 and Bo542 (see also below). The predicted Mas1' and Mas2' protein structures were compared to the published Agrobacterium proteins (including protein sequences from A. larrymoorii) (figure 2).

mas2' gene expression in different Nicotiana species

TB sequences with mas/ags genes have been found in N. tomentosiformis and N. tabacum (cultivars K326, Basma/Xanthi and TN90), N. kawakamii and N. tomentosa but not in the closely related species N. otophora (Chen et al. 2014). The latter contains an unrelated cT-DNA (TE)

with mutated and presumably inactivated mas/ags genes. In a preliminary genome-wide RNAseq expression study using a mixture of root and leaf RNAs from N. tomentosiformis and 162 cultivars of N. tabacum (table 1) we found that most had very low mas2' expression levels like the N. tabacum parent N. tomentosiformis (low expression or LE cultivars). Since the proportions of leaf and root tissues in the tobacco extracts used for RNAseq analysis were not precisely controlled, mas2' expression was further investigated by RT-qPCR analysis Sequences from N. tabacum LE and HE cultivars for which enough data were available showed no differences in the dual promoter region (results not shown). Thus, the differences in mas2' gene expression levels in LE and HE cultivars are not due to promoter differences but possibly to sequence differences outside the dual promoter or to epigenetic differences. Since epigenetic differences can lead to non-Mendelian inheritance patterns, this led us it to study the Mendelian genetics of the LE and HE phenotype.

Inheritance of LE and HE phenotypes

In order to study the inheritance of the LE/HE phenotypes, HE cultivars Petico, Burley 49 and Russian Burley were each crossed with LE cv. BB16, each combination in both directions. Levels of mas2' expression in roots of F1 seedlings growing in vitro, as measured by RT-qPCR, were intermediate between those of the parents (figure 7a).

In F2 seedlings, mas2' expression segregated as expected for a single Mendelian factor, results

for Russian BurleyxBB16 and BB16xRussian Burley are shown in figure 7b. This result suggests that the difference between LE and HE plants is not due to an epigenetic effects on mas2', but does not fully exclude it.

5-Azacytidine treatment of LE cultivars

Gene methylation can lead to extinction of gene expression, but methylation can be removed by 5-azacytidine treatment leading to reactivation of gene expression, this has also been demonstrated for the Ach5 mas region (van Slogteren et al., 1984). An attempt was made to reactivate the TB-mas2' gene of the LE cv. BB16 by plating seeds on 0, 1, 2 and 10 μM 5azacytidine in vitro, followed by RT-qPCR analysis of mas2' gene expression in roots. At 2 μM 5-azacytidine seedlings grew slower, at 10 μM growth was fully inhibited. In spite of the clear effect of the 5-azacytidine on growth, no increase in mas2' expression was found at any concentration (not shown), strongly indicating that the difference between LE and HE plants is not due to methylation.

Tissue-specific regulation of the TB-mas2' promoter

In order to visualize the TB-mas2' expression pattern in whole plants, the TB-mas dual promoter (figure 1a) was cloned upstream of the promoter-less GUS gene from pBI101 (Materials and Methods) yielding pTB-mas2'-GUS, and transferred to N. benthamiana by agro-transformation.

The A4-mas promoter region linked to the GUS gene (pA4-mas2'-GUS) was used as a control.

Several independent transformants were regenerated for each construct. GUS staining of F1 seedlings growing in vitro was variable in intensity as expected for independent transformants (figure 8), nevertheless similar with regard to tissue specificity for both constructs. Expression was mostly limited to the sub-apical root area (figure 8u*-y), but in plants with strong expression leaf veins were also stained, in several cases erratic staining along the roots indicated possible induction by wounding, this remains to be investigated. In one exceptional pA4-mas2'-GUS line the apical end of the root was stained (figure 8i, w).

*

These data confirm the root-specific expression found by RT-qPCR (see above) and indicate strongly that the TB-mas2' gene promoter has retained its tissue-specific regulation after horizontal transfer from an A. rhizogenes strain to the N. tomentosiformis ancestor.

DFG presence in different tobacco cultivars

Different LE and HE tobacco cultivars were tested for the presence of DFG in their roots by paper electrophoresis. Since DFG is not commercially available, it was isolated from Chry5 tumors and also prepared by chemical synthesis (Materials and Methods). In HE cultivars, but not in LE cultivars, a spot could be found migrating at the DFG position (figure 9).

Discussion

Earlier it was shown that the mas2' gene from the TB cT-DNA of N. tomentosiformis placed under control of a 2x35S promoter codes for an active enzyme and leads to a product that co-migrated with DFG in paper electrophoresis (Chen et al., 2014). The TB-mas region is most closely related to the mas regions from A. rhizogenes A4 and 8196, and less so to those of A. tumefaciens Ach5, Chry5 and Bo542. The predicted structural properties of the Mas proteins and the absence of DNA similarity for the dual promoter region indicate two different types of mas regions, possibly related to functional requirements for opine synthesis in and secretion from tumors and hairy roots. The TB-mas2' gene is little or not expressed in N. tomentosiformis and several N. tabacum cultivars, but in 25 out of 162 tobacco cultivars expression was found to be surprisingly high.

Several of these high expressing (HE) cultivars are closely related, like Burley 21, Burley 49 and Burley 64 or Zerlina, BUS63, Skroniowsky70 and Bursan, as defined by molecular markers (Julio and Dorlhac de Borne, unpublished). Since N. tomentosiformis is the paternal ancestor of N.

tabacum and already contained the TB region (Chen et al., 2014) we assume that TB-mas2' has been activated at some point in N. tabacum evolution. Alternatively, N. tabacum inherited an active mas2' gene from a N. tomentosiformis ancestor with high activity, and later lost this activity in the ancestor(s) of the LE cultivars. Crosses between HE cultivars and an LE cultivar (in both directions) showed intermediate mas2' expression, suggesting that each mas2' allele controls its own expression level in cis. In the F2 generation, the HE and LE phenotypes segregated as expected for a Mendelian factor. Since the promoter sequences of LE and HE cultivars are identical, the difference is likely due to a sequence outside the promoter region, which remains to be identified. Transformation of N. benthamiana with pTB-mas2'-GUS and pA4-mas2'-GUS reporter genes and analysis of different independently transformed lines showed specific mas2' expression in the root tisp and leaf veins, suggesting that the promoter properties of the mas2'

gene have changed little since its stable transfer into N. tomentosiformis. It may be interesting to transform an LE cv. and a closely related HE cv. with the pTB-mas2'-GUS construct, we predict this will lead to similar ranges of GUS expression because the results from LExHE crosses indicate that low expression is due to differences acting in cis. Studies on the Ach5 TR mas2'

promoter (Teeri et al., 1989;Leung et al., 1991;Saito et al., 1991) showed highest expression in roots and leaf vascular tissues. Remarkably, although the Ach5 mas2' promoter sequence is very different from the A4 and TB sequences, both ensure expression in roots and vascular system.

Further studies are needed on the regulation of the TB-mas2' and other mas dual promoters, these should identify the regions that ensure their tissue-specific expression and the corresponding transcription factors. The high expression of TB-mas2' in roots of HE cultivars leads to DFG synthesis as it does in crown gall tumors and hairy roots. This is the first demonstration of opine synthesis in a naturally transformed wild-type species. Although N. glauca contains an intact mikimopine synthase (mis) gene as shown by expression in E. coli [START_REF] Suzuki | Tobacco plants were transformed by Agrobacterium rhizogenes infection during their evolution[END_REF]) it has so far not been shown to contain mikimopine (Suzuki et al., 2001). N.

tomentosiformis contains an intact octopine synthase-like gene (ocl) on its TC region, and N.

otophora an intact vitopine synthase-like gene (vis) on its TE region (Chen et al., 2014). Possibly, as in the case of DFG, opines synthesized by mis, ocl and vis are synthesized in a restricted number of cells that remain to be identified. The very high mas2' expression in some tobacco cultivars is either fortuitous, or could have been selected naturally or by tobacco growers. The production of DFG merits further study, since it could have measurable consequences. One of these could be modification of root metabolism by sequestering glucose and glutamine into DFG.

It will be important to establish the transport and turnover of these substances in HE cultivars and the impact on their growth under C and N-limiting conditions. Another consequence could be a modification of the root micro-flora by DFG secretion. DFG can be produced by rotting plant material [START_REF] Anet | Chemistry of non-enzymic browning. II. Reactions between amino Acids, organic acids, and sugars in freeze-dried apricots and peaches[END_REF] and is degraded by many micro-organisms (Baek et al., 2003). Stable introduction of Agrobacterium mas genes into plants to create artificial symbioses [START_REF] Guyon | Transformed plants producing opines specifically promote growth of opine-degrading agrobacteria[END_REF]reviewed in Savka et al., 2002) has shown that mannopine synthesis can lead to changes in bacterial populations in the rhizosphere. Many members of the Rhizobiaceae family contain genes for DFG degradation (Baek et al., 2005), and could allow them to utilize DFG released from HE tobacco cultivars. The possible effects of TB-mas2' expression on root and plant growth and on root micro-flora might be studied by silencing its expression or by studying pairs of very closely related LE/HE couples like Boheme Andorre (LE)/Vuelta Abajo (HE)

or Paraguay Périgueux (LE)/Jaune de Belmont (HE).

Materials and Methods

Sequence analysis

DNA sequences were analyzed using the NCBI blastn program with the following settings:

Database: nr/nt, optimized for "somewhat similar sequences". For comparison with Nicotiana whole genome sequences the "whole-genome shotgun contigs" (wgs) setting was used with the WGS projects AWOK, AWOJ and AYMY (N. tabacum) and ASAG (N. tomentosiformis). Protein analysis was done with the NCBI blastp program with the nr database. Trees were constructed by using the NCBI/blastn program, with the "align two or more sequences" setting, then using "distance tree of results" with the following settings: Tree method: Fast Minimum Evolution, Max

Seq Difference: 0.85, Distance: Grishin (protein).

Reporter gene construction

The reporter genes pTB-mas2'-GUS and pA4-mas2'-GUS were constructed by PCR amplification of the mas dual promoter sequences with appropriate primers, using as targets total N. tomentosiformis DNA and A4 total DNA. Primers contained a HindIII site at the promoter proximal end and a BamHI site at the distal end. PCR fragments were cloned into pBI101 (Jefferson et al., 1987). Positive clones were checked by sequencing, and introduced into LBA4404 (Hoekema et al., 1983) by transformation. The resulting strains were used for transformation of N. benthamiana.

RT-qPCR analysis

RT-qPCR analysis was done according to Chen et al., 2014. DFG analysis by paper electrophoresis DFG analysis was done by extraction of plant tissues with 80% ethanol and separation of neutral sugars and DFG by paper electrophoresis, reducing sugars were revealed by silver nitrate staining (Chen et al. 2014).

DFG synthesis

To a solution of D-glucose (1.35 g, 7.49 mmol, 3.5 equiv.) in AcOH (15 mL) was added Lglutamine (300 mg, 2.05 mmol, 1 equiv.). The reaction mixture was stirred at 95°C. Aliquots (0.5 mL) were collected every 30 sec over 7 min and then every 5 min until 30 min and then frozen at -80°C. Analysis by paper electrophoresis in acidic medium (H2O-AcOH-HCOOH 910-60-30) and silver nitrate staining revealed that the sample at 3 min contained enough DFG for further analysis. This sample was then purified by preparative electrophoresis. Upon elution with the same acid medium and lyophilization, a solid was obtained and analyzed. NMR spectroscopy revealed that the product corresponded to the predicted structure of DFG and that the pyranose was the major form in water solution. For seedling material seeds were germinated in vitro on M0237 with 1% sucrose.

GUS staining

During the last few years, we fully sequenced the TA, TB, TC and TD cT-DNAs from N.

tomentosiformis (Chen et al., 2014). On the TE region of N. otophora [START_REF] Sierro | The tobacco genome sequence and its comparison with those of tomato and potato[END_REF] we identified a TE-6b plast gene. This gene is biologically active as shown by expression in N.

tabacum. A detailed anatomical study was carried out on the 6b model gene from A. vitis Tm4 [START_REF] Chen | Morphological analysis of the 6b oncogene-induced enation syndrome[END_REF], in order to serve as a basis for the future analysis of the TE-6b gene effect. The mas2' gene of the TB region was shown to be highly expressed in roots of some N.

tabacum cultivars and lead to the synthesis of desoxyfructose-glutamine (Publication 3, to be submitted). In the following part, I discuss these results and propose some possibilities for future studies.

I. Nicotiana genus cT-DNAs

I.1 Assembling the TC and TE regions from N. otophora

Genomic contigs covering the N. otophora genome have been published [START_REF] Sierro | The tobacco genome sequence and its comparison with those of tomato and potato[END_REF]. The contigs from the TE region were identified on the basis of homology to the T-DNAs from A.

tumefaciens, A. rhizogenes and A. vitis (Chen et al., 2014, figure 6A-C). Putative TE insertion site sequences were aligned with N. tomentosiformis and N. sylvestris contigs (Chen et al., 2014, figure 6D-E). However, since the TE region clearly contains repeats, complete assembly will require careful, manual analysis of reads as well as PCR experiments followed by sequencing, as we have done for TA, TB, TC and TD.

The assembly of the TC region from N. otophora might be easier since the TC from N.

tomentosiformis has already been fully assembled (Chen et al., 2014). Nevertheness, the attribution of polymorphisms in the TC repeats still needs to be done and as for TE, will require sequencing of repeat-specific PCR products.

I.2 Nicotiana genus cT-DNA structure

Within the Nicotiana genus, only cT-DNAs from N. glauca [START_REF] Aoki | Resurrection of an ancestral gene: functional and evolutionary analyses of the Ngrol genes transferred from Agrobacterium to Nicotiana[END_REF] and N. tomentosiformis (Chen et al., 2014) have been fully assembled. We tested whether TA, TB, TC and TD from N.

tomentosiformis were present in N. tomentosa, N. otophora and N. kawakamii (Chen et al., 2014, table 3), but the complete cT-DNAs from these species have not yet been sequenced. The deepsequencing data from N. benthamiana and N. otophora were published last year [START_REF] Sohn | Cell-autonomous-like silencing of GFP-partitioned transgenic Nicotiana benthamiana[END_REF][START_REF] Sierro | The tobacco genome sequence and its comparison with those of tomato and potato[END_REF]. No cT-DNA was found in N. benthamiana (Blast data not shown). N.

otophora contains TC and TE (Chen et al., 2014). TA, TB and TD are absent from N. setchellii, the situation for TC remains unclear because the expected TC insertion site could not be amplified (Chapter I, II.2). In order to show whether TC is absent or not, southern analysis with TC probes can be used. Apparently cT-DNAs from N. tabacum are variable among different cultivars since some of them contain a full TA region whereas others show deletions. It still remains to be investigated how this TA part was lost during evolution. It would be interesting to obtain the assembled cT-DNAs of the other Nicotiana species since a better knowledge of the divergence between the cT-DNA repeats would improve our understanding of Nicotiana species evolution. At the same time, it will be important to obtain more data on the overall evolution of the Nicotiana species. Such data not only concern normal sequence divergence, but will probably also reveal HGT by interspecific hybridization which is common in Nicotiana.

Apart for the cT-DNAs of the Nicotiana genus, cT-DNAs were also found in L. vulgaris and I.

batatas. We expect that additional cT-DNAs will be discovered in other genera as plant genomic sequences continue to accumulate. New natural transformants may reveal new types of T-DNAs like the TE region we found in N. otophora. Large scale Agrobacterium sequencing involving hundreds of natural isolates may allow us to find the strains that caused the original transformations leading to the natural transformants. If such strains can be found, they could provide the sequences needed to "repair" the mutated cT-DNA genes, and allow a comparison between the properties of the original T-DNA genes and their present-day plant-borne copies.

I.3 Functional analysis of intact ORFs from cT-DNAs

Several intact ORFs in the cT-DNAs are expressed in N. tomentosiformis (Chapter I, figure15) as well as I. batatas (Kyndta et al., 2015) and might code for particular functions. In order to learn more about the function of these different intact ORFs, it would be important to knock out their expression, e.g. by RNAi, or to remove them completely by CRISPR, and to study the effects of cT-DNA removal on plant growth or opine synthesis. Reporter gene studies with promoters from active cT-DNA genes and comparison with the promoter properties from the corresponding genes from Agrobacterium would allow us to establish whether the regulation of these genes underwent significant changes after the regeneration of the natural transformants. Cloning these genes under a strong or inducible promoter and expressing them in a plant species without cT-DNA like N. benthamiana will be a further essential approach to study their characteristics.

I.4 Functional analysis of cT-DNA insertion sites in plants

One question that we did not address in our cT-DNA study was whether the insertions of cT-DNAs affected the function of original genes located at the insertion sites. The first step would be to investigate the expression of the intact regions in those species that are closely related to plants with a particular cT-DNA but lack this cT-DNA, for example by comparing N. otophora which lacks TA with N. tomentosiformis. The possible functions of these genes might then be studied by removal with CRISPR. However, it is quite possible that these functions are redundant or that the effects are too slight to be noticed. One might also try to repair the cT-DNA interrupted regions by CRISPR technology, by removing the cT-DNA and adding back the plant sequences that were deleted during the insertion process.

II. Further study of the 6b genes II.1 Mechanism of 6b gene activity 6b gene is a member of the plast gene family whose basic function is still unknown. Although mechanisms have been proposed for several plast genes, i.e. gene 5 [START_REF] Körber | T-DNA gene 5 of Agrobacterium modulates auxin response by autoregulated synthesis of a growth hormone antagonist in plants[END_REF], rolB [START_REF] Cardarelli | Agrobacterium rhizogenes T-DNA genes capable of inducing hairy root phenotype[END_REF][START_REF] Spena | Independent and synergistic activity of rolA, B and C loci in stimulating abnormal growth in plants[END_REF][START_REF] Capone | Induction and growth properties of carrot roots with different complements of Agrobacterium rhizogenes T-DNA[END_REF][START_REF] Maurel | Auxin regulates the promoter of the root-inducing rolB gene of Agrobacterium rhizogenes in transgenic tobacco[END_REF][START_REF] Capone | Expression in different populations of cells of the root meristem is controlled by different domains of the rolB promoter[END_REF], rolC [START_REF] Meyer | Horizontal gene transfer: regulated expression of tobacco homologue of the Agrobacterium rhizogenes rolC gene[END_REF][START_REF] Mohajjel-Shoja | Contribution to the study of the Agrobacterium rhizogenes plast genes, rol B and rol C, and their homologs in Nicotiana tabacum[END_REF] and orf13 [START_REF] Fründt | Evidence for the ancient transfer of Ri plasmid T-DNA genes between bacteria and plants[END_REF][START_REF] Lemcke | Gain of function assays identify non-rol genes from Agrobacterium rhizogenes TL-DNA that alter plant morphogenesis or hormone sensitivity[END_REF][START_REF] Stieger | The orf13 T-DNA gene of Agrobacterium rhizogenes confers meristematic competence to differentiated cells[END_REF], we believe these results need to be confirmed before they can be considered as conclusive. Within the plast family, 6b gene causes the strongest plant modifications, and is therefore a good choice to approach this basic function. The different 6b variants known sofar have different properties as was shown by tumor induction with six 6b genes placed in the same expression cassette [START_REF] Helfer | The Agrobacterium oncogene AB-6b causes a graft-transmissible enation syndrome in tobacco[END_REF]. It would be interesting to obtain plants with inducible versions of each of these variants and to observe the morphogenetic effects at different expression levels. The properties of the other plast genes also merit further analysis as they could tell us more about the biological role of these genes. It will be especially important to study their expressions in their natural context, i.e. in tumor or hairy root tissues.

Our anatomical studies on inducible dex-T-6b tobacco plants showed that 6b can induce ectopic photosynthetic cells leading to leaf primordia and ectopic vascular strands. It remains to be investigated where and how these cells start their development. In the case of the ectopic photosynthetic cells, they appear on the abaxial side of young leaves and could result from The C terminal side of T-6b amino acid sequence contains a highly acidic region (EEGEDDDNEIGDEGEAGGAE), similar regions were found in other 6b proteins [START_REF] Helfer | The Agrobacterium oncogene AB-6b causes a graft-transmissible enation syndrome in tobacco[END_REF]. In R. leguminosarum, a hypothetical plast protein (locus WP_041936630) of 238 amino acids contains 22 glutamic acids in the beginning of its C terminal region. This acidic region may be of special interest and could be mutated in various ways to determine the importance of the number of acidic residues, their nature, and their position in the protein. The acidic region could interact with basic partners but it is also possible that the acidic amino acids play a role in opine synthesis, if either synthesis of the 6b protein or its degradation could affect the pool of these amino acids.

Basically four mechanisms were proposed for 6b activity (Chen and Otten, 2015, Introduction).

Our group proposed sucrose uptake since large amounts of sucrose, glucose and fructose accumulate in 6b plants and isolated root fragments were shown to take up sucrose upon 6b induction [START_REF] Clément | Abnormal accumulation of sugars and phenolics in tobacco roots expressing the Agrobacterium T-6b oncogene and the role of these compounds in 6b-induced growth[END_REF]. It is well known that sucrose is able to affect cell cycle and cell expansion (Reviewed in [START_REF] Wang | Regulation of cell division and expansion by sugar and auxin signaling[END_REF]. However, the precise link between 6b and sucrose uptake is still lacking. Since sucrose is essential for plant growth, it is difficult to obtain sucrose transporter mutants. Nevertheless, it might be possible to engineer plants with an inducible loss of sucrose uptake in order to study the capacity of the 6b protein to complement Further studies on the 6b mechanisms proposed by other groups will be required, e.g. it still has to be confirmed that the 6b protein has ADP-ribosylation activity on AGO1 and SERRATE (SE) proteins in the presence of the ADP-ribosylation factor [START_REF] Wang | Molecular insights into plant cell proliferation disturbance by Agrobacterium protein 6b[END_REF]. The latter 6b studies are among the most advanced and should be extended to TE-6b, other 6b genes like AB-6b, C-6b, S-6b, A-6b, Bo542-6b, AKE10-6b and to other plast genes.

Recently, plast protein-encoding genes were found in ectomycorrhizal fungi like Laccaria bicolor and Pisolithus microcarpus, and in nodulating Rhizobium strains like R. leguminosarum, R.

mesoamericanum, Bradyrhizobium and Mesorhizobium plurifarium. This finding is very striking for two reasons. First of all, the sporadic appearance of plast gene in there organisms suggests that their presence results from HGT. Secondly, it indicates that plast genes can play roles in two very different types of plant-associated organisms. Studies with knock-out mutants would therefore be very important. In addition, the regulation of these genes merits further study.

II.2 Comparison of TE-6b with T-6b

2x35S TE-6b single copy homozygous tobacco plants derived from different R0 plants have recently been obtained. These plants will be studied in detail and compared to dex-T-6b plants [START_REF] Chen | Morphological analysis of the 6b oncogene-induced enation syndrome[END_REF]. Special emphasis should be given to the anatomical details of leaves and roots. A dex-inducible TE-6b version will also be important to test since this allows the induction of TE-6b in a normally growing plant and could avoid problems of regeneration of plants with too high TE-6b expression levels. An inducible dex-TE-6b plant should also be useful to study sucrose uptake and accumulation.

Removal of TE-6b from N. otophora by CRISPR could show whether the TE-6b gene has an effect on the growth of N. otophora. The introduction of the gene in the N. otophora ancestor could have caused sufficiently strong growth changes to lead to speciation. However, the TE-6b gene is maybe not the only TE gene with growth-modifying properties. Other intact genes (vis, rolC, orf13 and orf14) could also be candidates. To answer this question, it will be necessary to first remove TE by CRISPR and to restore the original insertion region.

II.3 Can the 6b gene be used as a genetic tool to enhance plant regeneration?

In plant genetic engineering, a common technological problem is poor regeneration efficiency during transgenic plant production. Several genes like WUS [START_REF] Laux | The WUSCHEL gene is required for shoot and floral meristem integrity in Arabidopsis[END_REF][START_REF] Zheng | AtWuschel promotes formation of the embryogenic callus in Gossypium hirsutum[END_REF] and BBM (Boutilier et al., 2002;[START_REF] Florez | Enhanced somatic embryogenesis in Theobroma cacao using the homologous BABY BOOM transcription factor[END_REF] can be used to increase the regeneration properties of transgenic tissues especially by using inducible promoters. The 6b gene is able to induce calli (shown by infection of N. rustica stems) and more importantly, leaf primordial [START_REF] Chen | Morphological analysis of the 6b oncogene-induced enation syndrome[END_REF]. Based on these two activities, it seems reasonable to propose the 6b gene as a tool to transiently increase the regeneration efficiency of transgenic plants.

III. Further studies on the mas2' gene

According to mas2' gene expression levels, N. tabacum cultivars can be separated into two groups (HE and LE) (Publication 3, to be submitted). In order to know more about the function of the mas2' gene, it is necessary to knock out this gene in an HE plant and to compare the growth of the mas2'-less plant with that of the original cultivar. It has been shown that the secretion of mannopine, mannopinic and agropinic acid by L. corniculatus plants transformed with A.

rhizogenes strain 8196 can lead to the selection of mannopine-utilizing microorganisms in the rhizosphere [START_REF] Guyon | Transformed plants producing opines specifically promote growth of opine-degrading agrobacteria[END_REF]. It was also found in the rhizopheres of transgenic A. thaliana which is able to make octopine, the density of octopine-using bacteria increased [START_REF] Mondy | An increasing opine carbon bias in artificial exudation systems and genetically modified plant rhizospheres leads to an increasing reshaping of bacterial populations[END_REF]. It is possible that the high expression of the mas2' gene in some N. tabacum cultivars leads to a similar selection pressure. It would therefore be interesting to study this possibility, especially if a mas2' knock out version of these HE cultivars will become available that could serve as an isogenic negative control.

In order to study the bacterial use of DFG in HE tobacco cultivars, it will be useful to test Agrobacterium strains with DFG utilizing capacity and their corresponding mutants. The further study of the N. benthamiana lines expressing the mas2' promoter-gus reporter gene could reveal conditions that lead to even higher mas2' expression and DFG synthesis. Furthermore, an inducible system to increase DFG synthesis in an LE line or to decrease DFG synthesis in an HE line would be useful to study the metabolic consequences of DFG synthesis in plants, and especially in roots.

The synthesis of DFG by HE cultivars remains to be placed in the context of amino acid and sugar metabolism and transport in roots. DFG may be produced locally in regions of high mas2' expression (the subapical root region) and from there exported or secreted. It may also be converted to other componds. Experiments with labeled precursors will be necessary to study such metabolic effects. Sofar, we do not know which precursors are used by the Mas2' enzyme, and it will be necessary to study its enzymatic properties with the purified enzyme.

Materials and methods

I.2.2 Agrobacterium tumefaciens strain LBA4404

Strain LBA4404 was used for N. tabacum transformation and N. benthamiana infiltration. This strain contains a disarmed Ti plasmid pAL4404 (Hoekema et al., 1983) which carries the virulence genes, but no T-DNA. It is rifampicin and streptomycine resistance. (Hoekema et al., 1983).

I.3 Vectors

I.3.1 Cloning vector: pCK GFP S65C

The pCK GFP S65C vector contains an ampicillin resistance gene and a gfp gene under the 2x35S promoter (figure 24A). Genes of interest were amplified by PCR and used to replace the gfp gene of this plasmid. Colonies with the right construct were identified by miniprep DNA analysis, and sequencing. Subsequently, a HindIII fragment with the target gene was inserted into vector pBI121.2. [START_REF] Carrington | Bipartite signal sequence mediates nuclear translocation of the plant potyviral NIa protein[END_REF].

I.3.2 Transformation vector: pBI121.2 binary vector

This plasmid contains T-DNA left and right borders (LB and RB) (figure 24B). The T-DNA contains a nptII gene, a 2x35S-gus gene and a multiple cloning site. A bacterial nptII gene outside the T-DNA allows for selection of the plasmid on kanamycin, both in E. coli and A.

tumefaciens. The plasmid can be placed in different Agrobacterium backgrounds that provide the virulence functions. This vector was used for the TB-mas2' and TE-6b gene clone. (Jefferson et al., 1987).

I.3.3 Reporter gene vector: pBI101 binary vector

Vector pBI101 is similar to pBI121.2 except for the part on the right of the multiple cloning site.

Instead of tNOS, gus and p35S from pBI121.2, pBI101 contains only gus and tNOS (figure 24C).

This allows the insertion of promoter sequences in order to test their properties. This vector was used to test the promoter characteristics of TB-mas2' and A4-mas2'. (Jefferson et al., 1987). the gene of interest was first grown on solid YEB medium with rifampicin (100 mg/L) and kanamycin (25 mg/L) for 2 days at 28 °C, then one colony was used to start a liquid culture in 5 mL YEB with the same antibiotics at 28 °C. After 48 h, the bacterial culture was centrifuged at 6000 rpm for 5 min at RT. The supernatant was removed and the pellet was washed with MgSO 4

(10 mM). The bacterial pellet was suspended in MgSO 4 (10 mM) and the bacterial concentration was adjusted to an optical density of 0.5 at 600 nm. N. benthamiana leaves were infiltrated with this bacterial suspension using a 1 mL needleless syringe.

II.1.2 Tobacco transformation

II.1.2.1 Preparation of Agrobacteria suspension

The Agrobacteria were cultured in 100 mL YEB medium for one day and centrifuged 10 min at 6000 rpm. The pellet was twice washed by 10 mM MgSO 4 and resuspended in the same solution at OD 600 =1.0.

II.1.2.2 Tobacco leaf transformation

Fresh leaves from green-house grown plants (N. tabacum cv. Samsun nn or N. benthamiana)

were used. 70% ethanol was used to wash the leaves for 10 seconds. Then they were immerged into 200 mL 5% sodium hypochlorite with 500 μL of a 10% Tween 20 solution and shaken gently for 20 min. Sterile water was used to wash the leaves 3 times for 5 min. Finally, the leaves were placed on sterile paper and disks with 2 cm diameter were removed and shortly suspended in the Agrobacteria suspension. These disks were then put on M0222 (Duchefa) medium with 1% sugar and 0.8% agar for 24 h in the culture room. The next day they were washed with liquid M0222 medium with 350 mg/L cefotaxime and transferred on M0222 medium with 350 mg/L cefotaxime, 150 mg/L kanamycin and hormones (0.05 mg/L NAA, 2 mg/L BA). The protein extracts were run on the gel in migration buffer at 100 V for 1 h. The protein in the gel was then transferred onto a nitrocellulose membrane at 500 mA, 80 V for 2 h on ice. The membrane was washed in TBS buffer with 5% degreased milk powder for 5 min and blocked in the same buffer for 1 h at RT. Subsequently it was incubated with primary antibody at 4 °C overnight and washed 3 times for 5 min in TBS buffer. A secondary incubation was done by incubating the membrane with secondary antibody for 1 h at RT. Finally the membrane was washed 3 times in TBS and treated with Lumi-Light PLUS Western Blotting kit (Roche) and exposed using an autoradiography film.

II.1.5 Opine analysis

Plant material was extracted and opines were detected by paper electrophoresis as described in Chen et al., 2014.

II.2 Bacterial techniques II.2.1 Bacterial competent cell preparation

An E. coli single colony was inoculated in 5 mL LB medium and incubated overnight at 37 °C with moderate agitation (about 250 rpm). 500 mL LB medium was inoculated with 1 mL of this preculture and incubated overnight at 37 °C with shaking until OD 595nm =0.375. The following steps should be done on ice or at 4 °C. The medium with E. coli cells was transferred into tubes and centrifuged for 10 min at 5000 rpm, the pellet was washed in 500 mL H 2 O. This washing step was repeated once. Cells were left on ice for 30 min. The medium was centrifuged again for 10 min at 5000 rpm and the pellet was resuspended in 20 mL of 15% glycerol. This suspension was aliquoted in 100 μL tubes with 50 μL final volume and stored at -80 °C.

II.2.2 Plasmid DNA extraction

E. coli strains were harvested after 8 h culture at 37 °C. The cells were transferred into 1.5 mL tubes and centrifuged at 11000 rpm for 5 min. The supernatant was removed from the tubes and 100 μL solution was added for resuspending the pellets by vortexing. After 2 min, 200 μL solution was added and mixed well. Then, 150 μL solution was added and the tubes were centrifuged for 5 min at 11000 rpm. 400 μL supernatant was transferred into new tubes and 400 μL phenol/chloroform (v/v=1:1) was added and mixed. The tubes were centrifuged at 11000 rpm for 5 min and 300 μL top phase was transferred into new tubes. 600 μL ethanol was added into tubes at RT, mixed well and spun at 11000 rpm for 5 min.

II.2.3 Transformation of E. coli bacteria

1 μL ligation solution was added to 50 μL competent E. coli cells and put on ice for 15 min. The mixture was treated by heating at 42 °C for 90 s or by electroporation. The mixture was transferred into 1.5 mL LB medium and shaken for 1 h at 37 °C. The pre-cultured medium was centrifuged at 8000 rpm for 3 min and plated on antibiotic plates. After incubating at 37 °C overnight, singlecolonies were used for further analysis.

References Figure 5. Les structures chimiques des opines produites par galles de la couronne et les chevelus racinaires

Les structures chimiques de octopine, l'acide octopinique, lysopine, histopine, nopaline, acide nopalinic, succinamopine, lactame succinamopine, leucinopine, lactame leucinopine, mannopine, l'acide mannopinique, désoxy fructosyl-glutamine (DFG), désoxy fructosyl-5-oxoproline, l'acide agroponic, agropine, chrysopine, cucumopine/mikimopine, cucumoplne/lactame miklmopine, agrocinopine A, B, C et D, rideopine, vitopine/heliopine (adapté de [START_REF] Dessaux | Opines and opine-like molecules involved in plant-Rhizobiaceae interactions[END_REF]. Sequencing and functional analysis of cT-DNAs in Nicotiana Summary:

The bacterium Agrobacterium tumefaciens is well-known for its utilisation in plant genetic engineering where it serves as a gene vector. This bacterium and the related species Agrobacterium rhizogenes are phytopathogens that induce tumors and hairy roots respectively on susceptible plants like grapevine or fruit trees. Their phytopathogenicity is due to horizontal transfer of bacterial genes to the plant host, from a plasmid called the Ti (tumor-inducing) or Ri (root-inducing) plasmid. The subject of my Thesis concerns two particular aspects of this bacterium.

1. Their capacity to stably transform several plant species in nature, thereby yielding naturally transformed plants, especially in the genus Nicotiana. We have shown by deep sequencing of the Nicotiana tomentosiformis genome and by analysis of other recently published Nicotiana sequences the presence of five different Agrobacterium-derived sequences (cT-DNAs), totalling 65 kb, some of which carry intact genes. We have shown that two of them (TB-mas2' from N. tabacum and TE-6b from N. otophora) have biological activity. A detailed comparative study has allowed us to better understand the evolution of these cT-DNAs (Chen et al., 2014). The mas2' gene is well-known, it codes for the synthesis of desoxyfructosyl-glutamine (DFG) in tumors or roots induced by Agrobacterium. Recent work in our group has shown that the TB-mas2' gene is highly expressed in some N. tabacum cultivars and leads to the accumulation of detectable amounts of DFG. This work is presented as a manuscript to be submitted.

2.

A second part of the Thesis describes new properties of the T-6b gene, which is part of the DNA transferred by A. vitis strain Tm4 and leads to abnormal growth caracterized by the appearance of enations, so far the mode of action of this gene is unknown. The 6b gene is part of the so called plast family (for phenotypic plasticity), with different and often remarkable growth effects on plants. The T-6b gene wasearlier placed under control of a dexamethasone-inducible promoter, and tobacco plants transformed with this construct have now been studied in detail, at different times after the start of induction. A large number of changes was analyzed, both at the morphological and anatomical level, these include various unprecedented morphological changes, like for example the appearance of shoot primordia at the base of trichomes, or the appearance of ectopic vascular strands parallel to the normal strands with a regular development leading to complex but predictable structures [START_REF] Chen | Morphological analysis of the 6b oncogene-induced enation syndrome[END_REF]. The TE-6b gene from N. otophora was placed under strong and constitutive promoter control and introduced into tobacco, where it was found to cause new types of morphological change, different from those observed for T-6b. The latter results are preliminary and will be presented as a complement to the work on T-6b. They indicate that the introduction of the TE-6b gene in the N. otophora ancestor could have caused a change in growth pattern, and might have favored the appearance of a new species.

Keywords: Agrobacterium, N. tomentosiformis, cT-DNA, natural transformation, horizontal gene transfer, 6b, leaf polarity, plast genes

I

  am very happy and proud to have Dr. Paul Hooykaas, Dr. Denis Faure, Dr. Marie Claire Lett and Dr. Wenhui Shen as my jury members. Thank you so much for your coming and I hope you enjoy my presentation and our discussion. I have to thank the China Scholarship Council (CSC) for their financial support during four years. Introduction ............................................................................................ II.2 Auxin and cytokinin metabolism, transport and signaling ................................... II.2.1 Auxin .................................................................................................................................... II.2.2 Cytokinin .............................................................................................................................. II.3 The combined role of sucrose and auxin on plant cell division and expansion ... II.3.1 Sugar and auxin affect plant cell division ............................................................................. II.3.2 Sugar and auxin effect on plant cell expansion ................................................................... Results .................................................................................................. Chapter : Deep sequencing of Nicotiana tomentosiformis reveals multiple T-DNA inserts ........................................................................... I. Publication 1 .......................................................................................................... II. Additional data ...................................................................................................... II.1 Expression levels of ORFs from N. tomentosiformis cT-DNAs ........................... II.2 N. setchellii cT-DNA ........................................................................................... II.3 TE-6b .................................................................................................................. II.4 mas2' .................................................................................................................. Chapter II: Morphogenetic properties of 6b genes from Agrobacterium and Nicotiana otophora .......................................................................... I. Introduction ............................................................................................................ II. Publication 2 ......................................................................................................... III. Additional data ..................................................................................................... III.1 Introduction of the TE-6b gene in N. tabacum ................................................... III.2 Conclusion and perspectives ............................................................................. III.2.1 Phenotypic comparison between T-6b and TE-6b plants ................................................... III.2.2 A hypothesis on leaf blade outgrowth from free ends of vascular strands .......................... Chapter III: The characteristics of the Nicotiana tabacum mas2' gene .. I. Introduction .......................................................................................................... II. Publication 3 ....................................................................................................... Discussion and perspectives ............................................................ I. Nicotiana genus cT-DNAs ................................................................................... I.1 Assembling the TC and TE regions from N. otophora ....................................... I.2 Nicotiana genus cT-DNA structure .................................................................... I.3 Functional analysis of intact ORFs from cT-DNAs ............................................. I.4 Functional analysis of cT-DNA insertion sites in plants ..................................... II. Further study of the 6b genes ............................................................................. II.1 Mechanism of 6b gene activity ......................................................................... II.2 Comparison of TE-6b with T-6b ........................................................................ II.3 Can the 6b gene be used as a genetic tool to enhance plant regeneration? .... III. Further studies on the mas2' gene ....................................................................Materials and methods ...................................................................... I. Materials .............................................................................................................. I.1 Plant materials ................................................................................................... I.1.1 N. tabacum ......................................................................................................................... I.1.2 N. otophora ......................................................................................................................... I.1.3 N. setchellii ......................................................................................................................... I.1.4 N. benthamiana .................................................................................................................. I.1.5 Other Nicotiana species ...................................................................................................... I.2 Bacteria ............................................................................................................. I.2.1 Escherichia coli Top 10 strain ............................................................................................. I.2.2 Agrobacterium tumefaciens strain LBA4404 ....................................................................... I.3 Vectors .............................................................................................................. I.3.1 Cloning vector: pCK GFP S65C ......................................................................................... I.3.2 Transformation vector: pBI121.2 binary vector ................................................................... I.3.3 Reporter gene vector: pBI101 binary vector ....................................................................... II. Methods .............................................................................................................. II.1 Plant techniques ............................................................................................... II.1.1 Leaf patch infection ........................................................................................................... II.1.2 Tobacco transformation .....................................................................................................

  ... II.1.3 Nucleic acid analysis .........................................................................................................

Figure 1 .

 1 Figure 1. Phylogenetic tree of the Rhizobiaceae family based on sequence analysisStrains forming species-level clades in different colors based on average nucleotide identity, with a 95% cut-off level. This tree was constructed using a concatenated alignment of the most discriminative amino acid positions of 384 proteins conserved in the chromosomes of all completely sequenced genomes. Abbreviations: R, Rhizobium; A, Agrobacterium; Al, Allorhizobium; S, Sinorhizobium; E, Ensifer; N, Neorhizobium. The scale bar represents the estimated number of amino acid changes per site for a unit of branch length.[START_REF] Ormeño-Orrillo | Taxonomy of rhizobia and agrobacteria from the Rhizobiaceae family in light of genomics[END_REF].

  bacterial conjugation, bacteria can gain or lose plasmids. Because of this, plasmid sequences should not be included in phylogenetic studies. In 2001, Young reclassified the Rhizobiaceae family based on 16S rDNA analysis. They proposed to transfer A. radiobacter, A. rhizogenes, A. rubi, A. undicola and A. vitis to Rhizobium while maintaining A. tumefaciens. In 2015, Ormeño-Orrillo classified the Rhizobiaceae family according to in silico DNA-DNA hybridization (DDH) and average nucleotide identity (ANI) based on whole genome sequencing (figure 1). The results showed that A. rhizogenes and A. vitis belong to the Rhizobium and Allorhizobium genus respectively, and should therefore be called Rhizobium rhizogenes and Allorhizobium vitis. Since the old names A. rhizogenes and A. vitis are still widely used, I will use them in this thesis.

Figure 2

 2 Figure 2. Agrobacterium and associated plant syndrome (A) Electron microscopy image of A. tumefaciens strain C58 (Bar: 1μm) (from http://bacmap.wishartlab.com/organisms/79). (B) Crown gall caused by A. tumefaciens (from http://brokenwillow.com/gallery/crown-gall). (C) Hairy roots caused by A. rhizogenes (from http://www.cals.ncsu.edu/course/pp728/Rhizobium/Rhizobium_rhizogenes.htm).

Figure 3 .

 3 Figure 3. Scheme of Ti/Ri plasmid with an example of the Ri plasmid from A. rhizogenes strain A4 (A) A general scheme of a Ti or Ri plasmid from A. tumefaciens or A. rhizogenes. Arrows indicate the T-DNA transfer direction. (B) Genes present on the two A. rhizogenes strain A4 T-DNA regions: TL and TR. Arrows indicate orientation (5' to 3') of ORFs. Genes which belong to the plast family are marked in red. LB: left border; RB: right border (adapted from Mohajjel-Shoja 2010, with some modifications).

Figure 4 .

 4 Figure 4. Scheme depicting elements of the A. tumefaciens VirB/VirD4 type IV secretion system (T4SS) (A) Different elements of the T4SS system. T4CP: type IV coupling protein; IMC: inner membrane complex; OMC: outer membrane complex; IM: inner membrane; P: periplasm; OM: outer membrane. (B) Lower: virD operon codes for VirD4 protein and virB operon codes for 11 VirB proteins. Upper: VirB/VirD4 subunits localized in the inner membrane (IM) or delivered to the periplasm (P). (Christie et al., 2014).

Figure 5 .

 5 Figure 5. T-complex transfer from Agrobacterium into the plant cellVirulence proteins from Agrobacteria and plant proteins are both involved in T-complex translocation. The result of this process is the integration of T-DNA into the plant genome[START_REF] Gelvin | Traversing the Cell: Agrobacterium T-DNA's journey to the host genome[END_REF].

Figure 6 .

 6 Figure 6. iaaM, iaaH and ipt enzymatic functions (A) Trp is converted to IAM by tryptophan-2-monooxygenase coded by iaaM; IAM is converted to IAA by indole-3acetamide hydrolase coded by iaaH. (B) A prenyl group is added to AMP in the presence of isoprenyl transferase coded by ipt to form the cytokinin zeatin.

Figure 7 .

 7 Figure 7. Chemical structures of opines produced by crown galls and hairy rootsChemical structures of octopine, octopinic acid, lysopine, histopine, nopaline, nopalinic acid, succinamopine, succinamopine lactam, leucinopine, leucinopine lactam, mannopine, mannopinic acid, deoxyfructosyl-glutamine (DFG), deoxyfructosyl-5-oxoproline, agropinic acid, agropine, chrysopine, cucumopine/mikimopine, cucumoplne/miklmopine lactam, agrocinopine A, B, C and D, rideopine, vitopine/heliopine (adapted from[START_REF] Dessaux | Opines and opine-like molecules involved in plant-Rhizobiaceae interactions[END_REF].

gene 3 '

 3 replaces gene 6a between the 6b and nos genes. No transgenic plants expressing the 3' gene have yet been regenerated. I.4.3.12 Other plast genes Less well-studied plast genes include: gene 4' (from the TR-region of A. tumefaciens octopine strains), 7 (from the TL-region of A. tumefaciens octopine strains), b (found in A. tumefaciens C58

Figure 9 .

 9 Figure 9. Map of cT-DNA from N. glauca compared with the A. rhizogenes pRi1724 TL-DNA (A) Genes located on right part of the A.rhizogenes pRi1724 T-DNA and its right border (RB). The arrows indicate regions with homology to cT-DNA in N. glauca. (B) cT-DNA in N. glauca with indications of open reading frames and imperfect inverted repeats (left and right arm). (Adapted from Suzuki et al., 2002).

Figure 10 .

 10 Figure 10. Different Nicotiana species, some of which contain cT-DNA sequences (marked by yellow triangles)

  ibT-DNA1 was found to contain four ORFs homologous to the iaaM, iaaH, C-protein (C-prot), and agrocinopine synthase (acs) genes. ibT-DNA2 contains at least five ORFs with significant homology to the orf14, orf17n, rolB/rolC, orf13, and orf18/orf17n genes of A. rhizogenes. The ibT-DNA1 insertion is located in an Ipomoea gene which could have been functional before T-DNA insertion. acs, iaaM, iaaH, C-prot, rolB and rolC genes all have very low RNA expression levels in different Ipomoea tissues(Kyndta et al., 2015). The different plant species with cT-DNAs are natural GMOs, and studies on their origin, evolution and properties are only just starting.

Figure 11 .

 11 Figure 11. Natural GMOs (A) Linaria vulgaris (from https://gobotany.newenglandwild.org/species/linaria/vulgaris/). (B) Ipomoea batatas (from http://www.simplyappalachian.com/article/2015/10/sweet-sweet-potatoes).

  inducible T-6b gene (publicationChen et al., 2015, Chapter II). For a better understanding of the "enation syndrome" effects, I will first present an overview of what is known about shoot apical meristem (SAM) development and leaf initiation.

  KAN and the ARF gene family are very important for abaxial domain formation. Besides, recent research has identified a new participant: the PUF RNA-binding protein family (APUM23). It also plays a role in leaf polarity formation. Mutations of APUM23

3 .

 3 Leaf expansion and maturation. Transcription coactivator ANGUSTIFOLIA3 (AN3) and transcription factor GROWTH-REGULATING FACTOR5 (GRF5) are key factors in this stage[START_REF] Horiguchi | The transcription factor AtGRF5 and the transcription coactivator AN3 regulate cell proliferation in leaf primordia of Arabidopsis thaliana[END_REF].

Figure 12 .

 12 Figure 12. A model of genes involved in leaf polarity development in Arabidopsis (A) Leaf primordium initiation. P: primodium. (B) Genes involved in leaf maturation. miR165/miR166 and KAN inhibit the expression of HD-ZIPIII; AS1/2 and KAN downregulate each other; YAB promotes expression of KAN, HD-ZIPIII and ARF3/4 but inhibits KNOX1; the APUM23 function is not yet defined (adapted from Dkhar, 2014).

Figure 13 .

 13 Figure 13. Auxin and cytokinin metabolism, transport and signaling Different genes and their function involved in the auxin and cytokinin pathway. Orange round box: auxin; green round box: cyctokinin; +: stimulation; -: inhibition. (Schaller et al., 2015).

Figure 14 .

 14 Figure 14. Roles of glucose and auxin in regulating the plant cell cycleDifferent genes the expression of which is regulated by auxin and glucose. In G1, auxin induces both CycD and CDKA expression. Glc independently promotes CycD expression. Phosphorylated CycD/CDKA transfers the phosphor groups to RBR so that the DPA/(E2FA/B) complex is released and favors initiation of S phase. In S, Glc stimulates phosphorylation of the CycA3/CDKA complex through release of CycA3 resulting in S phase gene expression. In contrary, auxin inhibits S phase gene expression by inhibiting phosphorylation of DPB/E2FC through repression of the SCF/SKP2 complex. Glc and auxin both induce G2 to M transition. Glc and auxin show transcriptional regulation, Glc and auxin protein level regulation.[START_REF] Wang | Regulation of cell division and expansion by sugar and auxin signaling[END_REF].

  : Deep sequencing of Nicotiana tomentosiformis reveals multiple T-DNA inserts I. Publication 1

Figure S1 .

 S1 Figure S1. Alignment of cT-DNAs from N. tomentosiformis ITB646 with contig sequences from N. tomentosiformis TW142 (Sierro et al., 2013) TA to TD were blasted against the TW142 genomic sequences from the whole-genome shotgun contigs (wgs) collection of the NCBI data bank (Sierro et al., 2013). The highly similar contigs from repeated regions outside the cT-DNAs were artificially reduced in number and are indicated by "various". cT-DNA repeats are indicated by double-headed arrows. Single arrows indicate extensions of the cT-DNA regions. The TW142 contig numbers indicated on the maps are detailed in TableS1. TA: covered by 15 TW142 contigs. TB: a single TW142 contig covers the complete region, in addition, seven small contigs cover parts of TB. TC: two TW142 contigs overlap, four additional ones cover small regions. TD: two TW142 contigs overlap, an additional small one covers a small part.

Figure S2 .

 S2 Figure S2. TA region sequences in other Nicotiana species The N. tomentosiformis TA region was used as a query against genomic contig collections from different Nicotiana species. (a) N. tabacum cv. K326. (b) N. tabacum cv. TN90. (c) N. tabacum cv. Basma/Xanthi. (d) N. otophora. Contigs are not detailed. Asterisk: contig from the N. otophora TC region with similarity to TA sequences.

Figure S3 .

 S3 Figure S3. TB region sequences in other Nicotiana species The N. tomentosiformis TB region was used as a query against genomic contig collections from different Nicotiana species. (a) N. tabacum cv. K326. (b) N. tabacum cv. TN90. (c) N. tabacum cv. Basma/Xanthi. (d) N. otophora. Contigs are not detailed. Boxed region in (d): TE contigs with similarity to TB sequences, contig numbers refer to those in TableS2and Figure6a.

Figure S5 .

 S5 Figure S5. TD region sequences in other Nicotiana species The N. tomentosiformis TD region was used as a query against genomic contig collections from different Nicotiana species. (a) N. tabacum cv. K326. (b) N. tabacum cv. TN90. (c) N. tabacum cv. Basma/Xanthi. (d) N. otophora. Contigs are not detailed. TD is absent in N. otophora.

Figure 15 .

 15 Figure 15. Expression levels of ORFs in N. tomentosiformis cT-DNAs detected by qPCR (A)-(D) Samples from leaf 1 to leaf 4. *: EF2 (a reference gene from N. tabacum) value is set at 1, out of scale in the figures. The expression levels were calculated relative to the EF2 value. Samples were taken when the N. tomentosiformis plant was at the 7 leaf stage.

II. Publication 2 Figure

 2 Figure S1. a Modification of 6b plant after induction at 4-leaf stage. Non-induced plant. b Induced plant. One month after induction by a single spray with 3 μM dexamethasone the plant is strongly modified and blocked for further growth. c Rare normalization after induction at the 4-leaf stage. After one month, the lower part of the plant is strongly modified, the upper part has normalized. Bars = 10 cm (a, b), 5 cm (c).

Figure S2 .

 S2 Figure S2. Chlorosis in L4 of a 6b plant, 2 months after induction From apex to base three types of modification appear: intercostal chlorosis (IC), homogeneous abaxial dark green tissue (DG) and abaxial dark green tissue around II and III veins (DGV). Bar = 1 cm.

Figure S3 .

 S3 Figure S3. a Early stage of EPCs along veins: even distribution of EPCs in a narrow zone on both sides of a II vein. b Later stage: EPCs are growing preferentially along the outer edges of the narrow zones. Arrow: enlarged area shown in inset. Inset: random small cell groups within EPC area. c Same stage as in (b): ridges with EPCs along midvein and II veins start to emerge from the leaf blade. d Later stage: enation outgrowth along midvein

Figure S7 .

 S7 Figure S7. a Curvature of petioles and midveins. Non-induced plant. b Induced plant. Hyponastic curvature (HC) of the petioles and epinastic curvature (EC) of the midvein three days after induction of a 6-leaf 6b plant. Bars = 2 cm (a, b).

Figure S8 .

 S8 Figure S8. a A 6b plant induced at the 4-leaf stage, one month after induction. Several leaves have long thin petioles and complex curvatures with a consistent pattern (see also figure S9). b Tightly coiled epinastic leaf without gutter-like structure and with a small terminal double blade. N: normal blade, E: enation blade. c Strongly epinastically folded leaf with dark green ridges without a gutter-like structure and a very short leaf blade with a hyponastic tip. Ad: adaxial side. Note the angular petiole structure also seen in figure S25b. d-f Leaf with a gutterlike structure and complex folding pattern, shown from three different angles. g Interpretation of leaf shown in (d-f), oriented as in (e), see also figure S9. 1. Straight part. 2. Epinastic part with dark green ridges. 3. Hyponastic part with gutter-like structure. 4. Epinastic part with double leaf blade. 5. Hyponastic part with leaf apex. Torsion along the longitudinal axis leads to additional complexity. h A creased leaf without gutter-like structure. i Same leaf as in (h), leaf blade removed. The folding is due to an epinastic petiole-midvein structure with torsion along the longitudinal axis. Bars = 2 cm (a), 1 cm (b-f, h, i).

Figure S9 .

 S9 Figure S9. Complex curvature of an L10 leaf, with five different regions: 1. Long straight region growing upwards from a hyponastic bend (not shown) close to the stem. 2. Epinastic region with dark green ridges (R), on the edge small leaflets (L, see figure. 8) emerge. 3. Hyponastic region with gutter-like structure (Gu). 4. Epinastic region with normal (N) and enation (E) leaf blades. 5. Short hyponastic region: apical end of normal leaf blade. Bar = 1 cm.

Figure S11 .

 S11 Figure S11. a Cross section of petiole, EVSs are found close to the lower surface and in more interior positions. b Thin section of petiole with EPCs (EP) and EVSs (EV) along the abaxial surface. c-f A series of cross sections

Figure

  Figure S12. a A late leaf with narrow enations along the midvein, broadening at the leaf apex (on the left). b Another late leaf with partial enations along the midvein. c Cross section through midvein with narrow enations; they arise from a few isolated EVSs at the central "bottom" part of the abaxial side. Bars = 1 mm (a), 5 mm (b), 2 mm (c)

Figure S14 .

 S14 Figure S14. a A long GST tube on an old 6b plant, the petiole has yellowed but GST tubes remain dark green. b A GST tube situated on a longitudinal ridge (R), which ends shortly to the left of the tube. c-f Four consecutive cross sections of a petiole region with three GST tubes, tube 1 emerges in (d), 2 in (e) and 3 in (f). The GST tubes are not connected to the EVSs. g-i Scanning electron microscope pictures of a GST tube. g Overview, with connection (C) between GST (G) and tube (T) indicated by arrows. h Detail of connection between GST and tube with large cells (LC) from the GST base and small cells (SC) from the tube. i Detail of basal part of GST tube. S: stoma. Bars = 5 mm (a), 2 mm (b), 1 mm (c-g), 0.1 mm (h), 0.5 mm (i).

Figure S15 .

 S15 Figure S15. a Importance of GST for outgrowth of GST tubes. At an early stage of GST tube growth, the trichome on one of a group of GST tubes (arrow) was removed and subsequent growth was monitored. b, c Later growth stages, the GST-less tube (arrow) grows as well as the other tubes. Bar = 5 mm.

Figure S16 .

 S16 Figure S16. a A group of small leaflets developing from GST tubes. b A well-developed leaflet with II veins, seen from the side. c Same leaf as in (b), abaxial side. Narrow enation outgrowths. G: GST. d Same leaf as in (b), adaxial side. The leaf blade can be seen to emerge as a small ridge along the GST tube. G: GST. e Bifurcated GST tube, both parts are similar in size, each carries a GST. f Bifurcated GST tube, one branch is longer than the other. g Branched structure with a broad base. h GST tube carrying two leaflets (L) at its base. i Detail of isolated leaflets from structure in (h). Bars = 1 mm (a-e, g, i), 2 mm (f, h).

Figure S17 .

 S17 Figure S17. a Part of double leaf, seen from the adaxial side of the enation blade (EAd). Two corniculate tubes (T) emerge from the end of the enation part of the composite II vein (II). EM: enation blade margin. MAb: midvein abaxial side. NAb: normal blade, abaxial side. b Same region, seen from the abaxial side of the normal blade (NAb), with corniculate structures at the end of the enation part of the composite II vein (itself with small enations); the normal part of the II vein (marked "II") continues on the normal blade. T: tubes, EAb: abaxial side enation blade, EM: enation leaf margin, C: connection between enation leaf and normal leaf. c Detail of the corniculate tubes, these carry small trichomes but lack GSTs. d Abaxial side of double leaf. Corniculate tubes (arrows) are seen at the distal ends of the enation part of each composite II vein. e Detail of corniculate tubes at the margin (M) of an enation leaf. MV: midvein, II: secondary vein with small enations continuing on normal leaf blade. f Two small corniculate tubes (arrow) at an early stage of double leaf development. M: margin of enation leaf, MV: midvein, II: secondary vein. g Long corniculate tubes (arrows), MV: midvein. Bars = 1 cm (a, b, d), 2 mm (c, f), 3 mm (e), 5 mm (g).

Figure S18 .

 S18 Figure S18. a Leaf with a hypoascidium (adaxial surface on the outside), the line marked (b) indicates the cross section shown in (b), the arrows marked (c) to (g) indicate cross sections shown in c-g. b Cross section along the line shown in (a). M: main vein, E: one of the EVSs. c-g Different cross sections of the petiole shown in (a). At the proximal side (c, d), the vascular system consists of a normal vascular system at the adaxial surface and a group of EVSs below it. Towards the leaf base (e, f), several isolated EVSs appear, surrounded by ridges. g Outgrowth of green lamina between two adjacent EVSs, leading to a cavity and a conical structure. Bars = 1 cm (a, b), 1 mm (c-g).

Figure S19 .

 S19 Figure S19. a Five-branched pin on 6b plant induced at 4-leaf stage, two months after induction. L: leaflets. Insert: structure at different angle. b Tip of the branched pin shown in (a), L: leaflets. SP: secondary pin. c-e Structure shown in (b) after removal of the leaflets, seen from three different angles. LP: leaflet petiole. PT: pin tip. SP: secondary pin. f Section through a pin, showing a central vascular system. The vascular system does not contain mature xylem vessels and is flattened on one side, indicating residual polarity (inset: overview). g Branched complex pin, some branches develop thin structures at their tips (T), others remain blunt (B), some carry leaf-like structures (L). Bars = 5 mm (a, g), 2 mm (b-e), 0.1 mm (f), 0.4 mm (inset f). See also figure. 9c, d.

Figure S20 .

 S20 Figure S20. a, b Minute leafy structures (L) at petiole positions below normal-looking buds (B). c, d Two different views of an empty site (E) without a petiole, the bud (B) is normal. The surface structure at the empty site looks different. e Another empty site (E), seen from the side. B: bud, W: abnormal petiole wing. The empty site is covered by long thin trichomes (T) similar to those on pins. Bars = 5 mm (a, e), 1 cm (b-d).

Figure S21 .

 S21 Figure S21. a Transverse stem section at the site of a subpetiolar ridge. On both sides, decurrent petiole wings are present (W). The EVS (E) series turns out towards the stem surface on both sides, leading to shallow EVS ridges (ER). b Another subpetiolar ridge (S), with a very small petiole wing (W), without EVS ridges or subpetiolar wings. c Subpetiolar ridge with outgrowth of narrow laminae (subpetiolar wings: SW). d Scheme showing polarity of subpetiolar structures: blue: abaxial side, red: adaxial side. LG: leaf gap, V: normal vascular system of petiole, E: EVS, W: petiole wing, SW: subpetiolar wing, S: subpetiolar ridge. Bars = 1 mm (a, b), 0.5 mm (c). See also figure. 10 d, e.

Figure S22 .

 S22 Figure S22. a A very small subpetiolar outgrowth (O) growing out from the stem, situated somewhat below the tip of a structure with small ridges (R) progressively separating towards the base of the stem. Side view. b Viewed from above, (c)-(f): positions of cross sections shown in c-f. c-f Cross sections as indicated in (b), no EVSs above the outgrowth, below it they show a regular pattern. Section in (d) shows the small leafy outgrowth (O). Bars = 5 mm (a, b), 1 mm (c-f)

Figure S24 .

 S24 Figure S24. a, b Accessory buds. Two different plants (a and b) show an upward shift of the normal bud (B) with respect to the petiole (P) and an accessory bud (A). c Upward shifted bud (B) with ridge (R) running from the bud down to the petiole (P). d Swelling (SW) above the lower petiole (P). Bud (B) is shifted upward with respect to the upper petiole (P*). Bars = 0.5 cm.

Figure S25 .

 S25 Figure S25. Phenotypes of leaky dex-T-6b tobacco plantsa Rootless in vitro plant, with numerous pins carrying long thin trichomes. b Plant with long ribbon-like "leaves". c, d Two views of a leaf that is strongly folded at the base, but normal at the apex. e Plant with small hypoascidia at its base. f Leaf with small epiascidia along the midvein, and small irregular cell groups within the lamina. Boxed: area shown in (g). g Detail from epiascidium boxed in (f). Bars = 5 mm (a), 2 cm (b-d), 1 cm (e, f), 2 mm (g).

Figure S26 .

 S26 Figure S26. a Leaf of wild-type tobacco scion grafted on dex-T-6b rootstock before induction. b Leaf of wild-type tobacco grafted on dex-T-6b rootstock, one week after induction, with EPCs around III veins. c RT-PCR analysis of RNAs from different leaves of wild-type scion and dex-T-6b rootstock. Part of the 6b coding region (coordinates 32-577) was amplified by PCR. 1-3: 6b rootstock at day 0. Two of three leaves show weak leaky expression. 4-6: three different leaves of wild-type scion at 2 dpi. No amplification products. 7-9: three different leaves of 6b rootstock at 2 dpi. Each shows strong 6b expression. 10-12: Three leaves of wild-type scion at 5 dpi. Two contain 6b mRNA. 13-14: Two 6b rootstock leaves at 5dpi, both contain 6b mRNA. L: 100 bp ladder (from 100 to 1000 bp). The PCR products migrate at the expected size of 546 bp.

Figure 16 .

 16 Figure 16. Provisional map of N. otophora TE region This map is based on the manual assembly of various small contigs. Unfortunately, this map is not yet fully assembled. Arrow: direction of transcription. GAP: an unknown region between left and right part. : truncated gene.

Figure 17 .

 17 Figure 17. TE-6b leaves from in vitro plants cleared up by immersion in 70% ethanol (A) WT N. tabacum leaf. (B)-(D) transgenic TE-6b N. tabacum leaves. (B) A narrow TE-6b leaf. The angles between midvein and secondary veins are smaller compared to the WT leaf (B and C). (D) Abnormal leaves growing directly from the leaf surface. Black arrow: normal leaf. Red arrow: abnormal leaf. Bars: 1 mm.

Figure 18 .

 18 Figure 18. TE-6b expression levels in 31 R0 plants measured by qPCR 1 cm diameter leaf disk samples were taken from leaves about 5 cm wide. Values were calculated relative to the one from TE-6b-1, chosen as 1.

Figure 19 .Figure 20 .

 1920 Figure 19. Phenotypes of different R0 TE-6b plants (A) TE-6b-1. Two leaves sharing one petiole and part of the midvein. Arrow: divergence point of midvein. (B) Upper: a leaf from WT plant. Lower: a leaf from TE-6b-5 plant with transparent secondary veins. (C) Left: a petiole with its petiole wing from WT plant. Right: A petiole with abnormal petiole wing from TE-6b-5. (D) TE-6b-8. A narrow and thick leaf. (E) TE-6b-13. A chlorotic leaf. (F) TE-6b-21. A dark green leaf with a wrinkled surface. Bars from A-C: 1 cm. Bars from D-F: 3 cm.

Figure 21 .

 21 Figure 21. Expression levels of TE-6b in 9 TE-6b-5 F1 plants tested by qPCR Plants 5 A-I correspond to A-I plants in figure 20. According to figure 20, A, B and C should be homozygous plants. However, plant C shows low TE-6b expression level. Values were calculated relative to the TE-6b-5 plant A, chosen as 1. *: plants with a more severe phenotype.

Figure 22 .

 22 Figure 22. N. rustica stem infected by Agrobacterium carrying a 2X35S::TE-6b construct From top to bottom, scratched N. rustica stem infected by Agrobacterium with empty vector, 2X35S::TE-6b construct (two different areas) and 2X35S::T-6b construct. Picture was taken 75 days after infection.

Figure 23 .

 23 Figure 23. Leaf blade outgrowth induced by free ends of vascular strands (A) Cross section of a petiole from WT tobacco plant. Publication 2, figure 6b. (B) Scheme of A. Outgrowth of the lamina occurs at the free ends of the vascular system. (C) Cross section of a petiole from a T-6b tobacco plant with additional vascular strands (EVS). Petiole without wing. Publication 2, figure 6d. (D) Scheme of C. Crosses indicate loss of petiole wings. (E) Cross section of an abnormal petiole with its asymmetric wing. Publication 2, figure S13e. (F) Scheme of E. (G) Subpetiolar ridge of stem with outgrowth of small laminae. Publication 2, figure S21c. (H) Scheme of G. (I) Cross section through midvein with narrow enations; they arise from EVSs at the central "bottom" part of the abaxial side. Publication 2, figure 12c. (J) Scheme of I. (K) Cross section from the base of a cup shaped leaf from a T-6b tobacco plant. Publication 2, figure 18b. (L) Scheme of K. Orange: vascular system; dark green: adaxial leaf side; light green: abaxial leaf blades (B, D, F, H, J and L). Bars = 2 mm (A, I), 1 mm (C), 1 cm (E, K), 0.5 mm (G).

Figure 1 .

 1 Figure 1. Alignment of N. tomentosiformis mas region (delimited by the stop codons of mas1' and mas2') with the corresponding regions from different Agrobacterium strains Arrows indicate direction of transcription. Note that no similarity is found between the intergenic regions from TB and those of Ach5 and Bo542.

Figure 3 .

 3 Figure 3. PHOBIUS analysis of Mas proteins. a. Mas1' proteins. b. Mas2' proteins A. rhizogenes and N. tomentosiformis Mas proteins on the one hand, and A. tumefaciens Mas proteins on the other hand constitute two groups. A. lar: A. larrymoorii. Symbols: Grey: transmembrane, green: cytoplasmic, blue: non-cytoplasmic, red: signal peptide.

Figure 4 .

 4 Figure 4. RNAseq analysis of the mas region from LE cv. Bahia (a) and HE cv. Vuelta Abajo (b) 100 nt reads mapped on the mas region show a very strong increase in mas2' expression in cv. Vuelta Abajo compared to cv. Bahia.

Figure 6 .

 6 Figure 6. The mas dual promoter region from the N. tomentosiformis TB-region (top strand) was compared to similar regions (lower strand) from A4 (a) and 8196 (b) Left end of sequence: start codon mas2', right end: start codon mas1'. Identity values TB/ A4: 77% and TB/8196: 69%.

Figure 7a .

 7a Figure 7a. mas2' expression in roots of F1 seedlings of different crosses between LE cv. BB16 and HE cvs Burley 49 (B49), Russian Burley (RUS) and Petico (PET) measured by RT-qPCR. Parent levels are shown on the right. Results are expressed as fractions of the expression level in cv. Russian Burley.

Figure 7b .

 7b Figure 7b. mas2' gene expression in individual F2 seedlings resulting from the Russian BurleyxBB16 (RxB) and the reverse BxR crosses Values are expressed as fractions of the BxR-28 value. 10 out of 37 F2 plants have very low mas2' expression, corresponding to the expected proportion of 1:4.

Figure 8 .

 8 Figure 8. GUS staining of representative F1 seedlings from different transgenic N. benthamiana lines expressing the pA4-mas2'-GUS reporter construct (a-k, root tip details: u*-w), or the pTB-mas2'-GUS reporter construct (l-u, root tip details: x-y) Although expression is variable, all lines show GUS staining in the subapical regions of roots and in some cases in the leaf veins.

Figure 9 .

 9 Figure 9. Opine analysis of roots of LE cultivars Kentucky 17 (K17), NC2327 (NC) and BB16 and HE cultivars Russian Burley (RUS), Petico (PET) and Burley 49 (BU49) Extracts were separated by paper electrophoresis and stained with silver nitrate for reducing compounds. HE cultivars show a spot migrating at the position of the DFG standard. Arrow: DFG position. NS: position of neutral sugars.

1H

  NMR (500 MHz, D 2 O, 25°C): δ 4.02-3.98 (m, 2H, H 2 , H 5 ), 3.88-3.85 (m, 1H H 4 ), 3.77-3.71 (m, 3H, H 3 , H 5 , CH), 3.32-3.25 (m, 2H, CH 2 ), 2.50-2.48 (m, 2H, CH 2 ), 2.20-2.13 (m, 2H, CH 2 ).13 C NMR (125 MHz, D 2 O, 25°C): δ 177.8 (C=O), 172.7 (C=O), 95.3 (C-1), 69.8 (C-3), 69.3 (C-4), 68.8 (C-2), 62.6 (CH), 63.8 (C-5), 52.5 (CH 2 ), 31.0 (CH 2 ), 24,8 (CH 2 ). HRMS (ESI, positive mode): calcd for C 11 H 21 N 2 O 8 (M+H + ) 309.1292 found: 309.1312.Transformation and tissue cultureFor transformation, N. benthamiana leaf fragments from greenhouse-grown plants were surfacesterilized. Agrobacterium cultures were grown overnight in liquid YEB medium, washed twice with 10 mM MgSO 4 and diluted to an OD600 of 0.5 in 10 mM MgSO 4 . Leaf disks were dipped into the Agrobacterium suspension, and placed on solid M0237 (Duchefa) medium with 1% sucrose, 0.05 mg/L NAA, 2 mg/L BAP (MH medium), left for 24 hrs and washed in liquid MH with 350 mg/L claforan. Disks were then cultured for one month on MH with 350 mg/L claforan and 150 mg/L kanamycin. Shoots were transferred to M0327 medium without hormones with 1% sucrose, 350 mg/L claforan and 150 mg/L kanamycin for root induction. Plantlets of ten independent transformants were transferred to the greenhouse, and seeds were harvested from selfed plants.

  additional rounds of division at the level of the spongy mesophyll. The first stages of this abnormal development should be studied at the level of the cell cycle, using different cell cycle markers. The extra vascular strands were easily recognized in the petioles and midveins. Their initiation and subsequent development needs more investigation. The remarkable structures observed in T-6b tobacco plants may be interest for developmental biology. The study of easily accessible ectopic leaf primodia at large trichome positions may reveal the mechanisms that govern their development and the subsequent initiation of an associated vascular system. It may be possible to induce these ectopic primodia in leaf explants in vitro thereby enhacing the possibilities for experimental approaches.

  such transporters. The expression of the 6b gene in other biological systems like mosses, algae, yeast, bacteria or xenopus oocytes linked with sucrose transport studies might be a promising way to study this gene. It could receal what kind of orgamisms are sensitive to its activity and provide simple experimental systems.

Figure 24 (

 24 Figure 24 (A) pCK GFP S65C vector (B) pBI121.2 vector (C) pBI101 vector Multiple cloning sites are shown above the vectors. (A) p35S: CaMV-35S promoter; TL: translational enhancer; gfp: green fluorescent protein coding gene; polyA-35S: polyadenylation signal of CaMV 35S gene. (B) pNOS:

Figure 7 .Figure 6 .

 76 Figure 7. Les arbres phylogénétiques des protéines de type « plast » Tableau 2. Les numéros d'accès correspondant aux protéines de type « plast » dans la figure 5 Ar: A. rhizogenes; At: A. tumefaciens; Av: A.vitis; Ng: N. glauca; Nt: N. tabacum; Lv: Linaria vulgaris. Protéines plasmatiques Numéro d'accès

  .. II.1.4 Protein analysis .................................................................................................................

II.1.4

.1 Protein extraction from plants .

  .. II.1.5 Opine analysis ................................................................................................................... Bacterial competent cell preparation ................................................................................. II.2.2 Plasmid DNA extraction ..................................................................................................... II.2.3 Transformation of E. coli bacteria ......................................................................................

	NAA	naphthaleneacetic acid
	Abbreviations NCBI National Center for Biotechnology Information
	NtSIP	Nicotiana tabacum 6b interacting protein
	nm AGO OD 600 ANI ORF ARF PAGE ATP PCR BA PHAN BBM PHB BLAST PHV bp pRi CaMV pTi cT-DNA qPCR cv. RB Da RNA DDH rpm dex RT DNA RT-PCR EDTA SAM g GFP SDS	nanometer argonaute optical density at 600 nm average nucleotide identity open reading frame auxin response factor polyacrylamide gel electrophoresis adenosine triphosphate polymerase chain reaction 6-benzyl adenine phantastica baby boom phabulosa basic local alignment search tool phavoluta base pair root inducing plasmid cauliflower mosaic virus tumor inducing plasmid cellular T-DNA quantitative polymerase chain reaction cultivar right border Dalton ribonucleic acid DNA-DNA hybridization revolutions per minute dexamethasone room temperature deoxyribonucleic acid reverse transcription polymerase chain reaction ethylene diamine tetraacetic acid shoot apical meristem gram green flurescent protein sodium dodecyl sulphate
	GUS	β-glucuronidase
	h	hours
	HD-ZIPIII	homeodomain leucine zipper class III
	HGT	horizontal gene transfer
	Hyg	hygromycin
	IAA	indole-3-acetic acid
	IAM	indole-3-acetamide
	ipt	isopentenyl transferase
	Kan	kanamycin
	KAN	kanadi
	KNOX	knotted-like homeobox
	kb	kilo base
	kDa	kilo dalton
	LB	Left border
	μ	micro
	m	milli
	M	molar (mole/liter)
	min	minutes
	miRNA	microRNA

II.2 Bacterial techniques ......................................................................................... II.2.1 References ..........................................................................................

Table 1 . Different Ti or Ri plasmids based on opines they produce (adapted from Dessaux et al., 1998)

 1

	Plasmid type	Relevant opine products
	Ti Plasmids
	Octopine	Octopine, octopinic acid, lysopine, histopine, agropine, mannopine, agropinic and
		mannopinic acid
	Nopaline	Nopaline, nopalinic acid, agrocinopine A and B
	Agropine	Agropine, mannopine, agropinic and mannopinic acid, agrocinopine C and D,
		leucinopine, leucinopine lactam, L,L succinamopine
	Succinamopine	D,L succinamopine, succinamopine lactam, succinopine
	Lippia	Agrocinopine C and D
	Chrysopine/succinamopine Chrysopine, deoxyfructosyl-5-oxoproline (dfop), L,L succinamopine, L,L leucinopine
	Chrysopine/nopaline	Chrysopine, deoxyfructosyl-5-oxoproline (dfop), nopaline
	Octopine/cucumopine	Octopine, cucumopine
	Vitopine	Vitopine
	Ri Plasmids
	Agropine	Agropine, mannopine, agropinic and mannopinic acid
	Mannopine	Mannopine, agropinic and mannopinic acid
	Cucumopine	Cucumopine, cucumopine lactam
	Mikimopine	Mikimopine, mikimopine lactam

Figure 8. Phylogenetic trees of plast proteins Proteins

  were analyzed using four different multiple sequence aligments (MSA): Clustal Omega (suitable for medium to large fragments), Kalign (suitable for large fragments), MAFFT (suitable for medium to large fragments) and MUSCLE (suitable for medium fragments). Boxed sequences are from organisms other than Agrobacterium or from plants naturally transformed by Agrobacterium. The plants are indicated by the following prefixes: t:

	showed only very few
	conserved residues and two subgroups containing, on the one hand, the proteins encoded by orf14,
	rolC, orf13, 6a, 6b, and on the other hand those encoded by the 5' parts of iaaM and orf8, orf18, 7,
	rolB, rolBTR, lso, c', e, 5, b, d and 3'. The latest trees (figure 8) confirm the group with orf14, rolC,
	6a and 6b, but three (Clustal Omega, MAFFT and MUSCLE) out of four trees do not include orf13 in
	this group.
	Phylogenetic trees of plast proteins are shown in figure 8. In the following section I will present
	results obtained for different plast genes.

Nicotiana tabacum, Ng: Nicotiana glauca, Lv: Linaria vulgaris, IB: Ipomoea batatas. The codes of the Agrobacterium and plant plast genes are given in table 2. Green box: Bradyrhizobium; yellow box: Rhizobium mesoamericanum; red box: Mesorhizobium plurifarium; pink box: Burkholderia; blue box: Rhizobium leguminosarum; orange box: Pisolithus microcarpus; purple box: Laccaria bicolor.

Table S1 .

 S1 Comparison of TA, TB, TC and TD sequences from N. tomentosiformis ITB646 with contig sequences from N. tomentosiformis (TW142) reported bySierro et al. 2013. Size of TW142 contigs, number of identities and gaps are indicated. TC-6 belongs to TA.

	cT-DNA Number	Ntom_contig	Length contig (bp) identities	gaps
	in Fig. S1	(TW142)
	TA	11442	79974	7484/7499	0
		121015	4468	3904/3936	3
		61177	13341	2104/2114	9
		214639	1335	1333/1335	0
		214471	1205	1203/1205	0
		214369	1147	1146/1147	0
		213549	867	867/867	0
		212858	744	743/744	0
		212752	731	730/731	0
		164652	772	749/772	19
		164205	665	665/665	0
		210769	551	550/551	0
		208989	468	467/468	0
		208575	454	454/454	0
		208574	454	453/454	0
	TB	39652	33077	14332/14451	73
		214372	1150	1148/1150	0
		214268	1099	1096/1105	6
		208936	466	462/466	2
		208862	463	459/464	1
		205336	377	376/377	0
		181320	223	222/223	0
		181321	223	221/223	0
	TC	44398	58632	8878/9003	17
		63776	31931	5600/5649	12
		175608	211	211/211	0
		173099	207	206/207	0
		173095	207	204/207	0
		213549	867	405/531	7
	TD	37704	24314	14726/14803	21
		37703	27578	6757/6794	21
		195780	277	277/277	0

Table S2 . TE region contigs from N. otophora The

 S2 T-DNA and cT-DNA sequences from Table1(numbers 1-13) were used as queries in a blastn analysis using the N. otophora contig collection from the wgs AWOL project as database. Thirty-seven contigs with similarity to T-DNA but differing from the TC sequences were retrieved and used to search for overlapping AWOL sequences, yielding seven additional contigs (1, 9-13 and 24). Contigs 1 and 44 carry plant sequences that correspond to the TE insertion site.

	15 010609990.1				x	x	14,16,17,18,19
	16 010392196.1				x	x	14,15
	17 010770268.1					15,18,19
	18 010408703.1			x	x	x x 15,17,18,19,20
	19 010850947.1					17,18
	20 010502717.1			x	x	x x 18,21,22
	21 010502718.1		x x	x	x	20,22,23,24
	22 010662377.1			x	x	x	20,21,24
	23 010199038.1					x	21,24,25,26
	24 011101811.1					22,23
	25 010654058.1	x x x x	x	x	23,24,26,27,28
	26 010544408.1	x x x x	x	x	23,24,25,27,28,29
	27 010544409.1	x x x x x	x	25,26,28,29,30
	28 010654059.1	x x x	x	x x	25,26,27,29,30
	29 011080209.1	x x		x	x	26,27,28,30
	30 010135827.1	x x x x	x	x x	27,28,29,31,32
	31 010763171.1	x x x x	x	x x	30,32
	32 011107188.1	x x x x	x	x x	30,31,33
	33 010452552.1	x x x x x	x x	32,34,35
	34 010261697.1	x x x x	x x	33,35,36
	35 011115268.1	x x x x	x x	33,34
	36 010950854.1				x	37
	37 010717398.1	x x x x	x x x x x	36,38,39
	38 010730224.1	x x x x	x	x x	37
	39 010717399.1		x		x x	37
	40 010796941.1		x		x x	41,42,43
	41 010426942.1		x		x x	40,42,43
	42 010152082.1		x		x x	40,41,43
	43 010282125.1		x		x x	x	14,40,41,42,44
	44 010282126.1	x	x		x	14,43
	N° AWOL contig size (bp)	1 2 3 4 5 6 7 8 9 10 11 12 13 overlap with:
	1 010660148.1					2,3
	2 010173010.1					x 4
	3 010172804.1					x 4
	4 011050915.1					x 2,3,5
	5 010594028.1					x 4,6
	6 010991983.1					x 5,7,8
	7 011090788.1					x 6,8,9
	8 011090789.1					x 6,7,9
	9 010851895.1					7,8,10,11
	10 010864716.1					9,11,12
	11 010864715.1					9,10,12
	12 010185183.1					10,11,13,14
	13 010974325.1					12,14
	14 010609991.1		x		x	12,13,15,16,43,44

Table 3 . qPCR primers used in figure 15

 3

	Region ORF	Primer	Primer F sequences(5' 3')	Primer	Primer R sequences(5' 3')
			(F)		(R)
	TA	rolC	3106	CATGAGGTGTCTCGTGGACTC 3107	CGAATAGCATCCTCATAGCTGA
		orf13	3110	GCTGCTTTAAGGGAAATCCA	3111	CATATCTGCTGGTCCCTGGT
	TB	orf14	3114	GATCACCGCGGTAGTCAAGT	3115	AGTCCTGGGTCCGGATGT
		mas2' 3118	CTGAACGCAAGAGAGCACTG	3119	GAAGTGCCTGCAGAGATGAA
	TC	ocl	3122	CGATGAGAGGCCCAAAATAC	3123	CGACTTTTCCAACAGATTCGT
	TD	orf511 3124	AGTTCCCGATCAAACGTCAT	3125	GCTGCAACAGTAGCCGAAAT
		orf14	3126	ACGGATCGCCATAGTTGATT	3127	CCTTCTCTGAGGTATCCAACGTA

Table 4 . PCR primers and results of TA to TE insertion site amplification in N. setchellii

 4

	Region Primer	Primer F sequences(5' 3')	Primer	Primer R sequences(5' 3')	Result
		(F)		(R)
	TA	3017	CGCTGCATCAGTATTGCTCA	3012	GAGTGGCGTGCTGGAGTATC	+
		3017	CGCTGCATCAGTATTGCTCA	3013	CTCAGGTATTTGCAGGAACAC	+
	TB	3136	TCTCCCATGTACTAGCTATC	3144	GAAGATCACCTAACTGAG	+
		3136	TCTCCCATGTACTAGCTATC	3145	CGCTAATGGACTTCGTAC	+
		3137	GAGATATGCATACTATGTTA	3144	GAAGATCACCTAACTGAG	+
		3137	GAGATATGCATACTATGTTA	3145	CGCTAATGGACTTCGTAC	+
	TC	2420	CTGTCGAAAGCGATCAAGGC	2368	CCAGGCACCGTTGCTTCTTG	-
		2420	CTGTCGAAAGCGATCAAGGC	2369	GAAACACATACCGTCCATAG	-
		2372	GCCGAGAGATAACCGGCAAG	2375	GCCTTGATCGCTTTCGACAG	-
		2420	CTGTCGAAAGCGATCAAGGC	2413	AGGCGAAGAGGAGTTCAAAG	-
	TD	3147	ATTTCACTCCACATCACCTC	3150	CCCCATCACTAGAAATCTCC	+
		3148	AATCCCTGATGAGCGTCTAC	3150	CCCCATCACTAGAAATCTCC	+
	TE	3206	AGGAGGAACTGTTGCGTTGT	3203	TGTGAGTTAGTTTAAGTTGGGGC +
		3207	CACAGATATACCCAAGGTGCCA 3205	TGATCACGGAGAAGGGAAAGAG +

+: PCR fragment with correct size for an intact target region; -: no significant PCR fragment.

Table 1 . N. tabacum cultivars and Nicotiana species tested for TB-mas2' expression

 1 N. tabacum cultivars and species for which preliminary RNAseq data were obtained. Plants that strongly express TB-mas2' according to these data and which were then confirmed by RT-qPCR analysis are marked in bold. nt: high TB-mas2' expression in RNAseq experiments but not tested by RT-qPCR.

		Type	Variety		Type	Variety
	1	ANCESTRAL	AMBALEMA SR	85	BURLEY	Kentucky 17/BB16A
	2	BRUN	ARAPIRA	86	BURLEY	PULAWSKY 66
	3	BRUN	ARAPIRACA	87	BURLEY	RUSSIAN BURLEY
	4	BRUN	BAHIA	88	BURLEY	SHIROENSHU
	5	BRUN	Big Cuba	89	BURLEY	SKRONIOWSKI 70
	6	BRUN	BRASIL AND	90	BURLEY	START
	7	BRUN	BRASIL D	91	BURLEY	TENNESSEE 90
	8	BRUN	BRESIL BAHIA	92	BURLEY	TN86
	9	BRUN	BRESIL MATTA	93	BURLEY	WHITE BURLEY
	10	BRUN	CABOT	94	BURLEY	ZERLINA
	11	BRUN	CHEBLI	95	EXPERIMENTAL	BEINHART 1000-1

However, 25 N. tabacum cultivars showed surprisingly high mas2' expression levels (high expression or HE cultivars, marked in bold in Table

1

). As an example, RNAseq data for the LE cv. Bahia and HE cv. Vuelta Abajo are shown in figure

4a and b

respectively.

  Cultivar Samsun nn and cultivar Havana 425 were kindly provided by Dr. Fred Meins. The following cultivars were obtained from the Institut du Tabac de Bergerac (ITB).

	I.1.4 N. benthamiana
	I. Materials
	N. benthamiana plants were provided by the IBMP green house.
	I.1 Plant materials
	I.1.5 Other Nicotiana species
	I.1.1 N. tabacum
		Seeds from the Institut du Tabac de Bergerac
		Species	Accession name
		Nicotiana tomentosiformis ITB646
		Nicotiana sylvestris	ITB626
		Nicotiana kawakamii	ITB642
	Type	Variety Nicotiana tomentosa	ITB N ° ITB1015 Type	Variety	ITB N °
	BRUN	DRAGON VERT	652	BRUN	ARAPIRA
	BRUN I.2 Bacteria	PARAGUAY PERIGUEUX 48	765	BRUN	COULO
	BRUN	PETICO		BRUN	GALPAO
	BRUN KENTUCKY	KENTUCKY G	188	BRUN	MAZINGA
	BURLEY	BURLEY 49	175	BRUN	SANTA CRUZ
	BURLEY	KENTUCKY 14	211	BRUN	SANTA FE
	BURLEY	RUSSIAN BURLEY	226	BRUN	VUELTA ABAJO
	EXPERIMENTAL	KOKUBU	963	BRUN MARYLAND	AMARELLO MAURICE 2
	ORIENT	BASMA DRAMA 2	339	BRUN MARYLAND	MARYLAND BROAD LEAF
	ORIENT	KABA KOULAK	908	BURLEY	BURLEY 21
	ORIENT SEMI	BOHEME ANDORRE	359	BURLEY	BURLEY 64
	ORIENT SEMI	ROUM	929	BURLEY	BURLEY AMERICAIN
	VIRGINIE	CASH	19	BURLEY	BURSAN
	VIRGINIE	NC 2326	77	BURLEY	HARROW VELVET
		BB16	450	BURLEY	JAUNE DE BELMONT
	BURLEY	ZERLINA	241	BURLEY	JUDY'S PRIDE BURLEY (56)
	ORIENT	HERCEGOVINE	902	BURLEY	PULAWSKY 66
	VIRGINIE	POLALTA	447	BURLEY	SKRONIOWSKI 70
	VIRGINIE	STOLAC 17	101	BURLEY	START
	BURLEY	BUS63	158	BURLEY	WHITE BURLEY
	EXPERIMENTAL	TI657	978
	I.1.2 N. otophora
	N. otophora seeds are from the US Department of Agricultural Research Service, National Plant
	Germplasm System (inventory number PI235553) with accession name TW95.
	I.1.3 N. setchellii
	Seeds (inventory number PI555557 with the accession name TW121) were from the US
	Department of Agricultural Research Service, National Plant Germplasm System.

I.2.1 Escherichia coli Top 10 strain

E. coli Top 10 strain was used for plasmid amplification. Its genotype is: F-, mcrA, Δ (mrr-hsdRMS-mcrBC),

Φ80lacZΔM15, ΔlacX74, recA1, araD139, Δ (ara, leu) 7697, galU, galK, rpsL (StrR), endA1, nupG.

  The pellet was resuspended into 50 μL H 2 O. The plasmid DNA solution could be used for restriction enzyme test, PCR or sequencing.

		Plasmid DNA extraction buffers
	Solution	Solution	Solution
	25 mM Tris-HCl pH 8	200 mM NaOH	58.8 g/200 ml potassium acetate
	10 mM EDTA	1% SDS	23 ml acetic acid
	50 mM glucose

Acknowledgements

From the most beginning I would like to thank my supervisor professor Léon Otten for his four Chapter III: The characteristics of the Nicotiana tabacum mas2' gene

An intact TB-mas2' gene which encodes mannopine synthase was found on the TB region in N. tomentosiformis (Chen et al., 2014). Apparently its expression level in N. tomentosiformis is extremely low (figure 15). In order to study its capacity to encode a functional TB-Mas2' enzyme, it was put under 2x35S promoter control and expressed in N. benthamiana. It was found that the Mas2' enzyme from 35S::TB-mas2' lead to a product with the same electrophoretic mobility as DFG synthesized by the A4-Mas2' enzyme from A. rhizogenes A4 (Chen et al., 2014). Surprisingly, in some N. tabacum cultivars, the mas2' gene is very highly expressed according to RNAseq data (Publication 3, to be submitted). Such high expression might lead to detectable levels of DFG the production of which might affect normal plant growth by consuming sugars and amino acids. DFG synthesis could also have an effect on soil micro-organisms surrounding these plants and lead to a selection of DFG utilizing species. Such a selection has been shown to occur in Lotus corniculatus plants transformed with genes produce mannopine, mannopinic and agropinic acids from A. rhizogenes strain 8196 [START_REF] Guyon | Transformed plants producing opines specifically promote growth of opine-degrading agrobacteria[END_REF]. The increased density of octopine-using bacteria was found in the rhizosphere of transgenic A. thaliana which is able to synthesize octopine [START_REF] Mondy | An increasing opine carbon bias in artificial exudation systems and genetically modified plant rhizospheres leads to an increasing reshaping of bacterial populations[END_REF].

The following manuscript describes details about mas2' gene expression levels in different N. tabacum cultivars according to both RNAseq and qPCR data. Possible reasons of the strong variation in expression are studied. A comparison of the DFG produced by N. tabacum with chemically synthesized DFG will be presented. 3a). Conversely, TB-Mas2', A4-Mas2' and 8196-Mas2' contain TM domains, whereas Ach5-Mas1', Bo542-Mas1' and A. larrymoorii-Mas1' do not (figure 3b). No signal peptides were found.

The Ags proteins from A4 and Ach5 do not contain TM domains (not shown). Thus, these data reveal two types of mas regions in Agrobacterium T-DNAs, with possible functional consequences that remain to be investigated. using different tissues of plants grown under standard conditions in vitro. Expression was very low to undetectable for all tissues of cv. NC2326 and detectable in different tissues of cv. Russian Burley, with highest levels in the roots (figure 5, see also below) confirming the RNAseq data. The high level of mas2' expression in roots as compared to leaves was confirmed for two other HE cultivars, Petico and Burley 49 (results not shown). High expression in roots was then confirmed by RT-qPCR for 23 out of 25 HE cultivars (marked in Table 1). The difference between LE and HE cultivars might be due to their promoters. We therefore investigated the mas2' promoter region.

Sequence of mas dual promoter region

The TB-mas2' promoter is part of a bidirectional (or dual) promoter sequence also found in homology could be detected by blastn analysis even using the "somewhat similar sequences" setting.

Discussion and perspectives

About 1 month later, shoots had grown on the edge of the leaf disks. These shoots were then transferred on M0222 with corresponding antibiotics without hormones, in order to induce rooting. After 3 weeks, rooted plantlets were transferred on soil. Les gènes des T-DNA sont de quatre types.

II

1. Les gènes de synthèse d'opines, des petites molécules qui servent comme source de carbone, d'azote et de phosphore pour la croissance de la bactérie (tableau 1 et figure 5).