QTL: Quantitative Trait Loci RVD: Repeat Variable Di-residues

Keywords: CBB, Common Bacterial Blight of Bean EBE, Effector Binding Element Multi-Locus Sequence Analysis

Je souhaite tout d'abord remercier Nicolas Chen pour m'avoir donné l'opportunité d'effectuer mon stage au sein de l'équipe Emersys, et pour son encadrement. Merci pour ta bienveillance et tes nombreux encouragements. Merci à Sophie Cesbron qui m'a également encadré, principalement pour les manipulations en laboratoire et l'analyse des données. Merci de m'avoir donné du temps quand j'avais besoin d'un coup de main. Merci pour ton soutien et ton humour lorsque je constatais avec tristesse que je devais refaire les manipulations à causes d'une panne. Je remercie également Enora Dupas, Killian Grilloton et Damien Le Goff qui m'ont accompagné lors des longues manipulations en laboratoire. Merci pour votre bonne humeur à toute épreuve, vos conseils, votre soutien et surtout votre humour. On se retrouve en novembre pour de nouvelles aventures. Merci Martial Briand pour ton aide inestimable pour les analyses bio-informatiques. J'ai apprécié ton implication et ta réactivité lors du projet. Je remercie également Marie-Agnès Jacques d'avoir gardé un oeil sur le projet et d'avoir apporté des remarques constructives lors de nos réunions. Merci à ma tutrice Florence Val pour m'avoir suivi lors de ce stage. Vos conseils ont été essentiels au bon déroulement de mon stage. Je tiens à remercier Servane Le Grand, stagiaire Ecofun avec qui j'ai partagé le même bureau pendant plusieurs mois. Je n'oublie pas ta bonne humeur, tes encouragements et les très bons moments partagés avec toi parce qu' "il vaut mieux briller comme le trésor d'un pirate abandonné". Je remercie également ma grand-mère, Lucas Paupy et mes camarades PPE connues sous le nom des Jeannes-Yvonnettes qui ont été d'un soutien infaillible au cours de stage. Merci pour vos encouragements et vos appels quotidiens. Enfin, je tiens à remercier l'ensemble de l'équipe Emersys pour m'avoir guidé et pour votre sympathie.

1

I. Introduction

I.1. Economic, environmental, and nutritional importance of beans

The common bean, Phaseolus vulgaris, is cultivated on all continents as a food-producing or commercial crop. Only these seeds (dry or fresh bean) and its immature pods (French bean) are consumed. In 2019, 57.3 million tonnes of beans in all forms were produced worldwide on 34.9 million hectares (FAO, 2019). Most of the production is represented by the dry beans (28.9 million tonnes) and the fresh beans (27.0 million tonnes). As a result, the bean is the most widely consumed seed vegetable in the world [START_REF] Broughton | Beans (Phaseolus spp.) -model food legumes[END_REF]. The majority of dry bean production is located in Asia (49,7%), Africa (24,4%) and America (24,4%) (FAO, 2019). The main producers are Burma (20,2%), India (18,4%), and Brazil (10,0%). Fresh beans are mainly produced in China (80%) while green beans are mainly grown in the USA (54.1%).

The bean is a crop of interest in the current context of a growing world population as it represents an important source of nutrients, vitamins, and minerals essential for human nutrition [START_REF] Broughton | Beans (Phaseolus spp.) -model food legumes[END_REF][START_REF] Petry | Review: The potential of the common bean (Phaseolus vulgaris) as a vehicle for iron biofortification[END_REF]. In Mexico and Brazil, the bean is the primary source of protein since the seed is rich in protein (over 20%) [START_REF] Broughton | Beans (Phaseolus spp.) -model food legumes[END_REF]).

Thus, beans play an important role in developing countries and are an interesting alternative to meat products.

Bean cultivation also meets another challenge, that of developing sustainable agriculture through better management of nitrogen and phosphorus inputs. The bean can establish a symbiosis with bacteria of the genus Rhizobium, which fix atmospheric nitrogen in nodules and turn it into ammonia directly useable by the plant [START_REF] Masson-Boivin | Establishing nitrogen-fixing symbiosis with legumes: how many rhizobium recipes?[END_REF][START_REF] Mora | Nitrogen-fixing rhizobial strains isolated from common bean seeds: Phylogeny, physiology, and genome analysis[END_REF]. The bean can also interact with a fungus of the genus Glomus, which supplies phosphorus to the plant via endomycorrhizal uptake [START_REF] He | Nitrogen Transfer Within and Between Plants Through Common Mycorrhizal Networks (CMNs)[END_REF][START_REF] Scheublin | Arbuscular mycorrhizal fungi colonize nonfixing root nodules of several legume species[END_REF]. Therefore, bean cultivation is relatively cheap and requires little input [START_REF] Rubiales | Introduction: Legumes in Sustainable Agriculture[END_REF].

I.2. Common bacterial blight of bean: impact and significance

Common bacterial blight of bean (CBB) is a phytobacterial disease first described in 1893 [START_REF] Zaumeyer | A monographic study of bean diseases and methods for their control[END_REF]. It is one of the most devastating diseases of beans, causing a yield loss of usually 10% to 45%. In case of heavy infection of bean plants, the yield loss can be up to 75% [START_REF] Belete | Common Bacterial Blight (Xanthomonas axonopodis pv. phaseoli) of Beans with Special Focus on Ethiopian Condition[END_REF][START_REF] Boersma | Impact of common bacterial blight on the yield, seed weight and seed discoloration of different market classes of dry beans (Phaseolus vulgaris L.)[END_REF][START_REF] Fininsa | Relationship between common bacterial blight severity and bean yield loss in pure stand and bean-maize intercropping systems[END_REF][START_REF] Gillard | The performance of dry bean cultivars with and without common bacterial blight resistance in field studies across Canada[END_REF]Rodriguez De Luque and Creamer, 2014). The disease is present in all bean production areas and is favoured by humid heat. Consequently, large yield losses are recorded in countries with tropical or subtropical climates such as East Africa or South America, affecting the local economy [START_REF] Belete | Common Bacterial Blight (Xanthomonas axonopodis pv. phaseoli) of Beans with Special Focus on Ethiopian Condition[END_REF][START_REF] Fourie | Distribution and severity of bacterial diseases on dry beans (Phaseolus vulgaris L.) in South Africa[END_REF][START_REF] Karavina | A review of the occurrence, biology and management of common bacterial blight[END_REF]. The disease is transmitted by seeds. Thus, it represents a major economic drawback for the seed industry.

I.3. Diversity of Xanthomonas strains causing CBB

The pathogen responsible for CBB was first identified in 1897 as Bacillus phaseoli [START_REF] Smith | Description of Bacillus phaseoli n. sp[END_REF] and later as Xanthomonas phaseoli [START_REF] Dowson | On the generic names Pseudomonas, Xanthomonas and Bacterium for certain bacterial plant pathogens[END_REF]. In 1978, EPPO distinguished between socalled fuscans bacteria and non-fuscans bacteria (EPPO, 1978). The so-called fuscans bacteria are able to produce a brown pigment on a tyrosine-rich medium [START_REF] Darrasse | Genome sequence of Xanthomonas fuscans subsp. Fuscans strain 4834-R reveals that flagellar motility is not a general feature of xanthomonads[END_REF]. More recently, the strains responsible for the disease were classified into two pathovars present in two species: Xanthomonas phaseoli pv. phaseoli (Xpp) and Xanthomonas citri pv. fuscans (Xcf) [START_REF] Constantin | Genetic characterization of strains named as Xanthomonas axonopodis pv. dieffenbachiae leads to a taxonomic revision of the X. axonopodis species complex[END_REF]. The Xpp pathovar contains strains belonging to the GL1 lineage, while the Xcf pathovar is divided into three genetic lineages called GL2, GL3 and fuscans, where only strains belonging to the fuscans lineage can produce the brown pigment (Figure 1). Strains belonging to GL1 are phylogenetically distant from the other genetic lineages.

I.4. Disease cycle

Primary infection results from contaminated seed or infected plant debris [START_REF] Zaumeyer | The bacterial blight of beans caused by Bacterium phaseoli[END_REF].

Indeed, the CBB agents can overwinter and survive for up to 30 years in infected seeds as it can be found on both sides of the seed coat and on the surface of the embryo [START_REF] Saettler | Common bacterial blight[END_REF][START_REF] Darrasse | Niches and routes of transmission of Xanthomonas citri pv. fuscans to bean seeds[END_REF]. The survival time in the seed depends mainly on the storage temperature and humidity [START_REF] Marques | Survival and viability of Xanthomonas axonopodis pv. phaseoli associated with bean seeds stored under controlled conditions[END_REF]. Locally, the spread of bacteria can mainly occur through direct contact, splashing, transport via animals (insects), farm workers or farm equipment [START_REF] Belete | Common Bacterial Blight (Xanthomonas axonopodis pv. phaseoli) of Beans with Special Focus on Ethiopian Condition[END_REF][START_REF] Saettler | Diseases caused by bacteria[END_REF][START_REF] Zaumeyer | A monographic study of bean diseases and methods for their control[END_REF]. Upon physical contact with the host plant, bacteria settle on the surface of the leaves and produce a biofilm (composed of adhesins and polysaccharides) protecting them from the various stresses of the phyllosphere [START_REF] Jacques | Xanthomonas axonopodis pv. phaseoli var. fuscans Is Aggregated in Stable Biofilm Population Sizes in the Phyllosphere of Field-Grown Beans[END_REF]. Then, the bacteria enter through openings such as stomata, hydathodes and wounds before colonising the plant tissues and multiplying in the intercellular space and vessels [START_REF] Rudolph | Infection of the plant by Xanthomonas[END_REF][START_REF] Zaumeyer | The bacterial blight of beans caused by Bacterium phaseoli[END_REF]. Once the population reaches a threshold of about 10 6 bacteria, symptoms can appear [START_REF] Weller | colonization and distribution of X.phaseoli and X.phaseoli var. fuscans in field-grown Navy Beans[END_REF]. The CBB V.R Wallen, Agriculture Canada pathogen causes symptoms on the various aerial organs of bean plants [START_REF] Zaumeyer | A monographic study of bean diseases and methods for their control[END_REF] (Figure 2). On leaves, small greasy spots on the underside appear and develop into large, dry, brown necroses surrounded by chlorosis [START_REF] Goodwin | Water Stress in Leaves of Phaseolus vulgaris Infected with Xanthomonas campestris pv. phaseoli[END_REF]. The necroses may coalesce and invade the whole leaf. On the stem, reddish streaks appear and may crack and develop into canker or soft rot [START_REF] Belete | Common Bacterial Blight (Xanthomonas axonopodis pv. phaseoli) of Beans with Special Focus on Ethiopian Condition[END_REF][START_REF] Gilbertson | Common bacterial blight of bean[END_REF]. The disease induces the appearance of circular greasy spots on the pod which may turn reddish brown. If infection occurs during pod or seed formation, seeds may fail to develop or rot. In case of less severe infection, seeds develop normally but may be slightly wrinkled or show yellow-brown spots [START_REF] Karavina | A review of the occurrence, biology and management of common bacterial blight[END_REF][START_REF] Mabagala | The effect of populations of Xanthomonas campestris pv. phaseoli in bean reproductive tissues on seed infection of resistant and susceptible bean genotypes[END_REF]. Seedlings from infected seeds may be affected in their germination and vigour as the disease is vertically transmitted [START_REF] Belete | Common Bacterial Blight (Xanthomonas axonopodis pv. phaseoli) of Beans with Special Focus on Ethiopian Condition[END_REF].

I.5. Disease management

Few chemical or biological control methods are available. Copper-based treatments can be applied during the vegetative development of the plant, but their effectiveness is limited (Schwart et al., 2005;[START_REF] Weller | Chemical control of common and fuscous bacterial blights in Michigan navy (pea) beans[END_REF]. In some countries, antibiotics such as streptomycin or kasugamycin are used (Liang et al., 1992;[START_REF] Webster | Bacterial blights of snap beans and their control[END_REF]. Alternative methods such as the application of essential oils or the use of antagonistic micro-organisms (Pseudomonas sp., Rahnella aquatilis, Bacillus spp., or Rhodococcus fascians) have been investigated but remain little used in fields [START_REF] Giorgio | Rhizobacteria isolated from common bean in southern Italy as potential biocontrol agents against common bacterial blight[END_REF][START_REF] Goncalves Da Silva | Estudo de mecanismos de biocontrole do crestamento bacteriano do feijoeiro por bactérias[END_REF][START_REF] Sallam | Biological control of common blight of bean (Phaseolus vulgaris) caused by Xanthomonas axonopodis pv. phaseoli by using the bacterium Rahnella aquatilis[END_REF][START_REF] Zanatta | Bioassay for selection of biocontroller bacteria against bean common blight (Xanthomonas axonopodis pv. phaseoli)[END_REF]. Resistant varieties have also been obtained by interspecies crossing, but the resistances showed variable heritability and expression level depending on the environment, the epidemic pressure, and the genetic background [START_REF] Singh | Breeding common bean for resistance to diseases: A review[END_REF][START_REF] Miklas | Common bean breeding for resistance against biotic and abiotic stresses: From classical to MAS breeding[END_REF]. In addition, the expressed resistances may not be effective against the various strains causing CBB as the pod and leaves have different resistance systems [START_REF] Aggour | Comparison of leaf and pod disease reactions of beans (Phaseolus vulgaris L.) inoculated by different methods with strains of Xanthomonas campestris pv. Phaseoli (Smith) dye[END_REF][START_REF] Duncan | Interaction of Common Bacterial Blight Bacteria with Disease Resistance Quantitative Trait Loci in Common Bean[END_REF][START_REF] Viteri | Progress in breeding Andean common bean for resistance to common bacterial blight[END_REF]. Therefore, bean genotypes were constructed by pyramiding to ensure resistance to various strains in different conditions and tissues (Miklas et al., 2000b;[START_REF] Mutlu | Backcross breeding for improved resistance to common bacterial blight in pinto bean ( Phaseolus vulgaris L .)[END_REF][START_REF] Viteri | Response of 21 common beans of diverse origins to two strains of the common bacterial blight pathogen, Xanthomonas campestris pv. phaseoli[END_REF]. However, the presence of several QTLs in the same genotype does not necessarily lead to an addition of beneficial effects as there may be epistasis between the QTLs (O'Bouly et al., 2007). Moreover, the genes involved in the establishment of resistance are not always compatible with the desired agronomic traits. Therefore, prophylactic control remains the most effective option against Xcf and Xpp. Seed lots must be sanitarily selected as seeds are the first major source of inoculum. Identification of Infected seed lots can be done by isolation on Petri dishes, specific PCR test or pathogenicity test [START_REF] Audy | Detection of the bean common blight bacteria, Xanthomonas campestris pv. phaseoli and X.c.phaseoli var. fuscans, using the polymerase chain reaction[END_REF], Boureau et al., 2013). Furthermore, crop residues should also be buried or burned as they are a source of primary inoculum [START_REF] Gilbertson | Survival of Xanthomonas campestris pv. phaseoli and Pectolytic Strains of X. campestris in Bean Debris[END_REF][START_REF] Karavina | A review of the occurrence, biology and management of common bacterial blight[END_REF]. Agricultural equipment must be disinfected to prevent dispersal by contact. Seed can also be produced in disease-free areas or areas not conducive to disease development, such as the western states of America [START_REF] Gilbertson | Common bacterial blight of bean[END_REF].

Overhead irrigation should be avoided since it promotes the spread of bacteria by splashing [START_REF] Akhavan | Bean common bacterial blight: Pathogen epiphytic life and effect of irrigation practices[END_REF]. Host weed should also be removed to limit the contamination. Finally, a 3-year rotation system between each bean crop and a crop combination (maize, sorghum) could reduce the risk of disease emergence on beans [START_REF] Efsa Panel On | Scientific Opinion on the pest categorisation of Xanthomonas axonopodis pv. phaseoli and Xanthomonas fuscans subsp. fuscans[END_REF][START_REF] Fininsa | Effect of intercropping bean with maize on bean common bacterial blight and rust diseases[END_REF][START_REF] Fininsa | Effect of primary inoculum sources of bean common bacterial blight on early epidemics, seed yield and quality aspects[END_REF][START_REF] Nassary | Intensification of common bean and maize production through rotations to improve food security for smallholder farmers[END_REF].

I.6. Analysis of the diversity of CBB agents

In 2010, a study of the diversity of these strains was carried out in the lab by Nadia Mhedbi-Hajri, using MLST (Multi-Locus Sequence Typing) and MLSA (Multi-Locus Sequence Analysis)

approaches. MLST was developed in 1988 by Maiden et al. to study the genetic diversity of bacterial strains of the species Neisseria meningitidis [START_REF] Martin | Multilocus sequence typing: A portable approach to the identification of clones within populations of pathogenic microorganisms[END_REF]. MLST is a tool that allows differentiation of strains below the species level and can be used in epidemiological surveillance to monitor the spread of pathogens (Ravelomanantosa et al., 2016;[START_REF] Gonçalves | Genetic diversity of Curtobacterium flaccumfaciens revealed by multilocus sequence analysis[END_REF]. This method was later adapted as MLSA for phylogenetic analyses [START_REF] Naser | Application of multilocus sequence analysis (MLSA) for rapid identification of Enterococcus species based on rpoA and pheS genes[END_REF]. MLSA provides a framework for species definition and allows the study of genetic distance between strains and the observation of recombination events. Both approaches are based on the search for polymorphism within the nucleotide sequences of several housekeeping genes (generally four to eight) which are highly conserved constitutive genes necessary for the survival of bacteria. MLST is based on DNA sequence comparison for each of the amplified housekeeping genes. Each different sequence represents a different allele.

The set of sequences obtained for a strain corresponds to a characteristic profile represented by a coding called Sequence Type. The principle is similar for MLSA except that the sequences obtained are concatenated and compared to each other using phylogeny. The resolution power of these two methods can allow infra-species analyses but is often not sufficient for bacteria with little or no polymorphism (so-called monomorphic bacteria) or for the study of populations over a small geographical or temporal area [START_REF] Maiden | Multilocus Sequence Typing of Bacteria[END_REF][START_REF] Achtman | Evolution, Population Structure, and Phylogeography of Genetically Monomorphic Bacterial Pathogens[END_REF]. In 2004, the MLVA (Multi-Locus VNTR (Variable Number Tandem Repeat) Analysis) approach was developed by [START_REF] Schouls | Multiple-Locus Variable-Number Tandem Repeat Analysis of Dutch Bordetella pertussis Strains Reveals Rapid Genetic Changes with Clonal Expansion during the Late 1990s[END_REF]. This approach provides the possibility to study more recent events and to discriminate more precisely between strains [START_REF] Schouls | Multiple-Locus Variable-Number Tandem Repeat Analysis of Dutch Bordetella pertussis Strains Reveals Rapid Genetic Changes with Clonal Expansion during the Late 1990s[END_REF]. This method is based on the typing of VNTRs which are microsatellite sequences repeated in tandem.

During DNA replication, errors occur, especially through replication slippage (Figure 3), resulting in the addition or deletion of one or few repeats [START_REF] Kornberg | ENZYMATIC SYNTHESIS OF DEOXYRIBONUCLEIC ACID, XVI. OLIGONUCLEOTIDES AS TEMPLATES AND THE MECHANISM OF THEIR REPLICATION[END_REF][START_REF] Streisinger | Frameshift Mutations and the Genetic Code[END_REF]. These errors are the source of inter-individual variation. These variable size sequences are amplified and sequenced to obtain the number of repeats of the motif for each locus. For each strain, the concatenation of the number of repeats obtained for each locus provides a Sequence Type or haplotype (Figure 4).

I.7. The role of TALEs in the pathogenicity of Xanthomonas

The pathogenicity of Xanthomonas is partially based on a type three secretion system (T3SS) that allows the injection of type three effectors (T3E) into the plant cell [START_REF] Büttner | Regulation and secretion of Xanthomonas virulence factors[END_REF][START_REF] Timilsina | Xanthomonas diversity, virulence and plant-pathogen interactions[END_REF]. T3Es induce physiological changes in the plant. These changes aim to facilitate infection by the bacterium either by disrupting the plant's defence systems and/or by altering the metabolism of plant cells in favour of the bacterium's development.

Among the Xanthomonas T3Es are effectors named TALEs (Transcription Activator-Like Effectors) that induce the expression of susceptibility genes in the plant to promote infection [START_REF] Boch | TAL effectors--pathogen strategies and plant resistance engineering[END_REF]. Once injected into the cytoplasm of the plant cell by the T3SS, TALEs are addressed to the nucleus where they can bind specifically to the promoter region of susceptibility gene to activate its transcription [START_REF] Boch | Xanthomonas AvrBs3 family-type III effectors: discovery and function[END_REF]) (Figure 5). TALEs have a particular structure that is essential to their function (Figure 6). The N-terminal part of these proteins has a secretion signal allowing them to be injected into the plant cell via the T3SS (Nga-sze [START_REF] Mak | The crystal structure of TAL effector PthXO1 bound to its DNA target[END_REF] preferentially binds thymine. As a result, all of these RVDs together form a characteristic sequence which can bind specifically to a DNA sequence in the plant called the Effector Binding Element (EBE). The deciphering of the code formed by the RVDs has made it possible to generate bioinformatics tools to predict the potential targets of TALEs [START_REF] Moscou | A Simple Cipher Governs DNA Recognition by TAL Effectors[END_REF]Gruau et al., 2013).

Therefore, TALEs behave as transcription factors as they induce the transcription of susceptibility genes in the plant [START_REF] Cohn | Xanthomonas axonopodis virulence is promoted by a transcription activator-like effectormediated induction of a SWEET sugar transporter in cassava[END_REF]. For example, in rice, genes of the OsSWEET family coding for sugar exporters to the apoplasm are targeted by TALEs. Their overproduction would increase the source of carbon available to the bacterium.

In 2017, the TALEs diversity of 17 Xcf-Xpp strains was studied [START_REF] Ruh | a) Xanthomonas adaptation to common bean is associated with horizontal transfers of genes encoding TAL effectors[END_REF]) (Figure 7).

Analysis of the PacBio genomes of these strains revealed the existence of four tal genes named tal18H, tal18G, tal20F, and tal23A, respectively. According to [START_REF] Ruh | a) Xanthomonas adaptation to common bean is associated with horizontal transfers of genes encoding TAL effectors[END_REF], sequence analysis of these TALEs revealed recent horizontal gene transfers between Xcp and Xpp.

I.8. Mechanisms of plant resistance to TALEs

Up to now, four main defence mechanisms blocking the action of TAL effectors have been identified [START_REF] Bogdanove | TAL effectors: Finding plant genes for disease and defense[END_REF][START_REF] Schornack | Engineering Plant Disease Resistance Based on TAL Effectors[END_REF] (Figure 8). The first one corresponds to the binding of TALEs to resistance proteins (Figure 8, (E)). For example, the Bs4 protein specifically recognizes the N-terminal part of the TALE avrBs4 in sweet pepper. The second mechanism involves mutation of the messenger RNA, thus preventing its recruitment by TALEs (Figure 8, (B)). Third, the TALE-targeted EBE can be found in the promoter region of a resistance gene named Executor such as Bs3 in sweet pepper (Figure 8, (D)) [START_REF] Römer | Plant pathogen recognition mediated by promoter activation of the pepper Bs3 resistance gene[END_REF]. The last mechanism involves the modification of the sequence of the TALE-targeted EBE preventing the specific binding of the TALE to the target DNA sequence (Figure 8, (C)) [START_REF] White | Host and pathogen factors controlling the rice-Xanthomonas oryzae interaction[END_REF]. This mode of action is interesting since there is a natural diversity of EBE variants that could be used as sources of resistance (Hutin et al., 2015a).

I.9. Objectives of the project and the internship

The CBB project, which is mainly supported by VMK and HMC, is divided into four workpackages. The final objective is to identify sources of resistance to the pathogens responsible for CBB. The internship represents the second workpackage of the project. Its first objective is to characterise a global diversity of Xanthomonas strains responsible for CBB that have been isolated from bean production areas. This diversity analysis will be carried out via the development of an MLVA approach on the entire collection. The second objective of the internship is to study the diversity of the TALE effectors of these strains in order to identify new TALE profiles. Based on the results of the two diversity analyses, 10 strains representing the highest diversity will be sequenced by PacBio and the target sequences of the TALE genes of these strains will be determined in silico. This will lead to the identification of new sources of resistance.

II. Materials and Methods

II.1. Bacterial strains and growth conditions

During this project, a collection of 116 Xanthomonas strains was used: 26 strains were provided by Vilmorin Mikado (VMK), 19 by HM Clause (HMC) including two from the International Center for Tropical Agriculture (CIAT), 28 were preserved by the French Collection of Plant-Associated Bacteria (CIRM-CFBP), and 23 strains were stored in the host laboratory (IRHS, INRAE Beaucouzé) (Annex 1). The VMK strains were isolated from their production areas in France while the HMC strains were isolated from their production areas in the USA (Figure 9). The strains stored at CIAT come from Colombia. The strains preserved in the laboratory were collected during many isolation campaigns: one in Cameroon in 2009 and the others in Brazil. The 28 CFBP strains, of which 17 were sequenced in PacBio, were isolated at diverse locations in the world at different dates and were chosen to represent the diversity of strains responsible for CBB. All strains were grown at 28°C for two to three days, either in Trypticase soy (TS) broth (17.0 g.L - 1 pancreatic digest of casein; 3.0 g.L - 1 enzymatic digest of soy bean; 5.0 g.L - 1 NaCl; 2.5 g.L - 1 K2HPO4; 2.5 g.L - 1 glucose; qsp 1L H2O) or on

Trypticase Soy Agar (TSA) medium (TS supplemented with agar at 15 g.L - 1 )

II.2. Bacterial suspensions and DNA extraction

To access bacterial genomic DNA, it was first planned to use boiled bacterial suspensions.

However, due to low bad PCR amplification with bacterial suspensions, it was later decided to perform genomic DNA extractions. Bacterial suspensions were made from fresh cultures on TSA suspended in 5 mL of sterile distilled water, adjusted at OD600nm=0,1 (1x10 8 CFU.mL -1 ), boiled for 20 mins, cooled on ice for 10 mins and centrifuged at 10 000g during 10 min. Genomic DNA extraction was performed from 24h cultures in TS broth. Bacterial DNA was extracted using the Wizard® Genomic DNA Purification Kit (Promega), following the manufacturer's recommendations (Annex 2). Quality and quantity of DNA were assessed using a NanoDrop ND-100 Spectrophotometer. Both bacterial suspensions and DNA extractions were stored at -20°C.

II.3. Construction of phylogenetic trees based on the amplification of housekeeping genes

Sequencing of the gyrB housekeeping gene was performed on the whole collection in order to identify the genus/species of each strain, and discriminate strains from the GL1, fuscans, and GL2/GL3 lineages (Figure 10). Sequencing of the rpoD housekeeping gene was also conducted on GL2/GL3 strains to discriminate strains belonging to lineage GL2 from those belonging to lineage GL3, using strain 6546 (GL1) as outgroup. For both genes, PCR amplification was performed using specific primers (Figure 10). PCR products were sequenced by Genoscreen. The resulting sequences were run on NCBI WGS database to confirm the genus or the species of each strain. These sequences were also used to draw phylogenetic trees using the Neighbour-joining approach and the Jukes-Cantor model on Geneious (100 bootstraps and 50% support threshold).

II.4. MLVA analysis

Prior to my internship, a list of 82 primers was compiled from various publications studying Xanthomonas strains closely related to Xcf or Xpp or previously designed by the laboratory to amplify strains from the fuscans lineage [START_REF] Arrieta-Ortiz | Genomic Survey of Pathogenicity Determinants and VNTR Markers in the Cassava Bacterial Pathogen Xanthomonas axonopodis pv. Manihotis Strain CIO151[END_REF][START_REF] Ferreira | Xanthomonas citri pv. Viticola affecting grapevine in Brazil: Emergence of a successful monomorphic pathogen[END_REF]L. Bui Thi Ngoc et al.;2009). For each primer, the size of the amplicons was determined with Geneious (v.9.1.8) on the genome of 79 Xcf-Xpp strains including 17 PacBio genomes sequenced previously [START_REF] Ruh | a) Xanthomonas adaptation to common bean is associated with horizontal transfers of genes encoding TAL effectors[END_REF]. In total, 15 primers were selected for the course based on their polymorphism and their ability to amplify microsatellite sequences in all 17 strains (Annex 3). Seven primers were selected from Arrieta-Ortiz et al. number of tandem repeats were identified using Geneious (v.9.1.8) on the 17 sequenced strains.

To validate the 15 VNTR primers, PCRs were first performed on the 17 reference strains, using either bacterial suspensions or DNA extractions (Figure 11). The PCR products were visualized on 1.2% agarose gel electrophoresis in Tris-borate EDTA buffer. The amplified sequences were sequenced and then analysed on Geneious to verify the number of VNTR repeats and their sequence.

Subsequently, for each VNTR, the forward primer was labelled using four dyes (FAM, HEX, ATTO550, ATTO565) (Table 1). Then, PCRs using those labelled primers were run on the entire collection. PCRs were conducted in the same way as described above except that only DNA extractions were used as genetic material. Then, PCR products were multiplexed by four according to the fluorescent dyes used (FAM, HEX, ATTO550, ATTO565), then diluted by 32 in ultrapure water (Table 1). Next, 2,4µL of each multiplexed PCR product was mixed with 9.35µL of Hi-Di formamide and 0.15µL of Genscan 500 Liz internal line size standard for four-capillary electrophoresis using an ABI PRISM 3130 genetic analyzer.

Electrophoregrams were analysed using Peakscanner and each Liz peak was checked by eye.

The size of the VNTR amplicon was read by eye and artefacts, or small peaks, were corrected.

The number of tandem repeats (N) for each strain was calculated using the following equation: Using these data, Minimum Spanning Trees (MST) were drawn using BioNumerics (v.7.6). For each MST, Simpson's diversity index associated with each VNTR was calculated with BioNumerics (v.7.6). To test the representativity of the dataset, rarefaction curve was drawn for each MST on R studio (v.1.3.1073) (Annex V).

II.5. Analysis of TALE diversity

Prior to this internship, a primer pair (XapF2 and XapR) was designed by the lab to amplify all the TALE-encoding genes from the 17 reference strains (Figure 12). Another pair of primer (tal-859-F and Xfutal-3204-R) was designed by the lab during the internship (Figure 12). PCRs were performed on the 17 reference strains to verify that the obtained PCR profiles match the number and size of TALE-encoding genes retrieved from the PCR products were run on a 1% agarose gel electrophoresis before reading the amplicon size. The number of TALEs and their size were determined by eye. Subsequently, PCRs were carried out on the whole collection using the same procedure. To verify the identity of the 144 strains in the collection, the gyrb gene was amplified and sequenced for all strains in the collection. The gyrB gene is a housekeeping gene that codes for the B subunit of DNA gyrase. This gene has been used in various studies to differentiate

III. Results

III

Xcf and Xpp from each other and from other pathovars [START_REF] Young | A multilocus sequence analysis of the genus Xanthomonas[END_REF]Ah-You et al., 2008, Bui Thi Ngoc et al., 2010;[START_REF] Hamza | Genetic and pathological diversity among Xanthomonas strains responsible for bacterial spot on tomato and pepper in the southwest Indian Ocean region[END_REF][START_REF] Mhedbi-Hajri | Evolutionary history of the plant pathogenic bacterium Xanthomonas axonopodis[END_REF]. To identify these strains, Blastn of the gyrB sequences were run on NCBI database. The gyrB gene could not be amplified for 18 strains in the collection. Therefore, it was not possible to determine the identity of these strains which were excluded from the rest of the project. Among the 126 remaining strains, four strains from Brazil (BRA78, BRA100, BRA157, and BRA192) were found to be Xanthomonas campestris pv. zinnia or pv. esculenti, two strains from Cameroon (17.9 and 19.1) belonged to Xanthomonas euvesicatoria pv. euvisecatoria, and nine other strains from Cameroon (10.1.1a,11.1.a,12.6.1a,13.2.a,14.1,16.8.1b,18.3.1,20.2.2,and 15.11.2) were identified as Stenotrophomonas acidaminiphila. These 15 strains were also excluded from the rest of the project. All other strains in the collection were identified as Xcf or Xpp. In total, this analysis confirmed that 111 strains out of the initial 144 belonged to Xcf or Xpp, 78 belonging to Xcf while 33 were identified as Xpp.

Within Xcf, phylogeny using the gyrB housekeeping gene allowed to differentiate between lineage fuscans from GL2 and GL3 (Figure 13a). However, it was not discriminating enough to distinguish strains belonging to lineage GL2 from those belonging to lineage GL3. Therefore, to discriminate between the GL2 and GL3 strains, the rpoD housekeeping gene was used to construct a second phylogenetic tree (Figure 13b). This housekeeping gene encodes the RNA polymerase σ factor and was used in other studies to investigate phylogenetic relationships between Xanthomonas strains [START_REF] Young | A multilocus sequence analysis of the genus Xanthomonas[END_REF][START_REF] Hajri | A «repertoire for repertoire» hypothesis: Repertoires of type three effectors are candidate determinants of host specificity in Xanthomonas[END_REF]. This time, the phylogenetic tree allowed to differentiate the GL2 strains from the GL3 strains. Four strains from the collection (6992, 6993, 6994, and 6996) belonged to the GL3 lineage while nine strains (LSV-29, BRA 13, SNES28, 6533, 6988, 6991, 6989, 6990, and 6532) belonged to the GL2 lineage. Up to now, the GL2 and GL3 strains have been described only in Reunion, except for the 6994 (GL3) strain from Tanzania. During this analysis, no GL3 strain from another country was identified, which suggests that GL3 strains are effectively endemic to the region of the world comprised between Réunion and Tanzania. On the contrary, the phylogenetic analysis allowed the identification of new GL2 strains isolated in Brazil, China, and Tunisia. To our knowledge this is the first description showing that GL2 strains are not restricted to Reunion.

III.1.b. MLVA analysis

III.1.b.1. Tests on a set of 17 strains

In order to study the diversity of Xcf and Xpp strains more in depth, an MLVA scheme was build, using 15 VNTR primers designed before this internship (see Materials and Methods).

Prior to MLVA analysis, a first step was to verify the ability of each primer to amplify the targeted VNTR sequences. For this, the target VNTR sequences were identified by searching for the primers on the genomic sequences of 17 strains previously sequenced by PacBio [START_REF] Ruh | a) Xanthomonas adaptation to common bean is associated with horizontal transfers of genes encoding TAL effectors[END_REF]. This analysis validated 14 out of the 15 primers. Indeed, the VNTR Karine_vntr5

was rejected as at least one of the tandem repeats was found to be included in the forward primer sequence, which would probably lead to multiple PCR amplification products (Figure 14). The second purpose of this step was to determine the amplicon size, the offset size, the tandem repeat number, and the motif for each strain and VNTR.

Moreover, each VNTR was amplified for each of the 17 PacBio strains via PCR. Amplicon sizes were congruent with their corresponding genomic sequences. Then, each amplicon was sequenced from both sides, which allowed to confirm that the sequence of the repeats was identical to the genomic ones. In some cases, variations of one or two repeats were observed between the amplicon and the genomic sequence, which can be explained by assembly errors between the forward and the reverse sequences of these amplicons.

Together, these analyses on the 17 PacBio strains validated the set of primers and provided the necessary information (offset size and repeat sequence) to proceed to the analysis on the whole collection (Table 2).

III.1.b.2. MLVA on the whole strain collection

MLVA analysis using four-capillary electrophoresis provided the size of the amplicon for each strain and VNTR, and thus the number of tandem repeats sequence. For each lineage, Minimum Spawning Trees (MST) were drawn based on the number of tandem repeats of each strain per VNTR (Figure 15). For this, only VNTRs showing the same offset size for all strains within a lineage were used. The discriminatory power of each locus was evaluated by the Simpson diversity Index (Table 3). In addition, to evaluate the representativity of the strain diversity according to the dataset used, rarefaction curves were plotted for each MST (Table 3).

III.1.b.2.i. Diversity of the fuscans strains

The fuscans MST based on the analysis of ten VNTRs showed a high degree of strain diversity (Figure 15 a). Indeed, except for a few strains, most haplotypes were composed of a single strain. The rarefaction curve revealed that more than 95% of the haplotypes were detected with a set of eight markers. Simpson's diversity index showed that the VNTRs Karine_vntr12 (0.93), XaG1_72 (0.87), and XaG1_70 (0.74) had the strongest discriminatory power for the analysis of fuscans strain diversity (Table 3). In contrast, VNTR 7764v1 had a null Simpson diversity index, which corresponds to a monomorphic VNTR for this lineage. Therefore, this VNTR can be removed from the MLVA analysis when studying the diversity of fuscans strains.

Since most of the strains were well distinguished from each other, including strains from single isolation campaigns (Brazil mainly in 2014, Cameroon in 2009), this VNTR set seems to be appropriate to be used as an epidemiologic monitoring tool for the fuscans lineage.

On this MST, some Brazilian strains (BRA 74,BRA 158,BRA 105,BRA 243,and BRA 244) formed a network suggesting that specific diversification of these strains occurred in Brazil. The same was true for some Cameroonian strains (15.1, 16.8.2, 7767R, and 17.1.2) forming another network within the MST. However, the rest of the Cameroonian and Brazilian strains were dispersed in the MST, which was also the case for the strains from other countries. This suggests that multiple exchanges occurred between countries. For example, a Cameroonian strain (16.3.1) and a French strain (i72066.3) were in the same haplotype. In fact, most strains from Cameroon were closely related to French strains, suggesting that multiple exchanges of fuscans strains occurred between France and Cameroon. Eleven VNTRs allowed to discriminate all GL2 strains from each other as all haplotypes were composed of a single strain (Figure 15 e). Similarly to fuscans strains, the rarefaction curve showed that 95% of the haplotypes were identified with a set of 8 loci. The Simpson's diversity index showed that two VNTRs were monomorphic and can be removed from the set of VNTRs used (Table 3). Again, this analysis indicates that this primer set can be used for epidemiological analyses on the GL2 lineage.

Despite the limited number of GL2 strains studied, the MST showed that the French strains were grouped together. Tunisian strains were also grouped together. This suggests that these strains have evolved independently of each other within each country.

III.1.b.2.iii. Diversity of GL3 strains

Eleven VNTRs were used to discriminate the four GL3 strains (Figure 15 g). All these strains appeared individualized on the MST. Therefore, the VNTR set allowed to discriminate the strains efficiently. However, the results were not representative of the GL3 lineage since only four strains could be analysed. The rarefaction curve showed that six loci are sufficient to represent 95% of the diversity (Table 3). This data set appears to be sufficient to differentiate GL3 strains. Furthermore, Simpson's diversity index showed that a set of seven out of eleven primers can be used since four VNTRs were monomorphic (Table 3). These results need to be verified on a larger number of GL3 strains before concluding about the possible use of this primer set for epidemiological analyses.

III.1.b.2.iv. Diversity of GL1 strains

The MST was obtained from the analysis of seven VNTRs (Figure 15 c). The diversity of the GL1 strains seems to be lower than the diversity of the fuscans strains (Figure 15 b). Indeed, a central haplotype representing four strains can be observed. This lower diversity can be explained by the fact that most of the strains come from HMC production areas in the USA.

Another way to explain this lower diversity is that the VNTR primer set used was more restricted than for the other lineages. According to the rarefaction curve, five loci explained 95% of the diversity of GL1 strains compared to 8 for fuscans strains. (Table 3). Simpson's diversity index was relatively high for all VNTRs, except for VNTR Karine_vntr4 which is monomorphic for this lineage. Similarly to the fuscans strains, these results suggest that this set of VNTRs can be used to conduct epidemiological monitoring within the GL1 lineage. The structure of the MST suggests that the United States of America is the origin country of the GL1 strains. Therefore, the diversity of GL1 strains could be explained by migration and mutation in other countries of the American strains. Since most of the strains were supplied by HMC from the USA, this remains a hypothesis to be confirmed by conducting an analysis with more GL1 strains isolated in other countries and on a shorter time scale.

III.2. Study of the TALE diversity

The TALE PCR primers had already been developed before the internship, and a first step was to develop the protocol to amplify TALE-encoding genes from Xcf and Xpp strains.

II.2.a. Development of PCR on TALE genes

A first TALE PCR was performed to test the XapF2 and XapR primers on the 17 PacBio strains.

To compare the efficiency of the PCR on different sources of genetic material, the PCR was performed on DNA suspensions of the 17 PacBio strains and on the extracted DNA of four strains (Figure 16). These four strains were selected among the 17 PacBio strains so that each lineage was represented (4885g (fuscans), 6988g (GL2), 6546g (GL1), 6994g (GL3)). The gel revealed that it was not possible to amplify the TALE sequences using bacterial suspensions (Figure 16). The hypothesis is that the DNA present in the bacterial suspensions was not clean enough, which could hinder PCR amplification. The following TALE PCRs were therefore conducted using DNA extractions.

However, the TALE profiles of the four strains obtained with the XapF2 and XapR primers were not as expected. Indeed, one amplicon was observed for strain 6546 instead of two and no amplicon was identified for the strain 6994 instead of one [START_REF] Ruh | a) Xanthomonas adaptation to common bean is associated with horizontal transfers of genes encoding TAL effectors[END_REF]. In addition, the amplicons appeared weak in intensity. Therefore, another pair of TALE primers (tal-859-F and Xfutal-3204-R) was tested. The hybridization temperature (Tm) of the primer pair was determined by PCR on the four strains via a temperature gradient (Figure 17). First, the TALE profiles observed on those gels corresponded to those described by [START_REF] Ruh | a) Xanthomonas adaptation to common bean is associated with horizontal transfers of genes encoding TAL effectors[END_REF].

Therefore, these primers were used for the rest of the internship (Figure 17). Also, these two gels showed variable results depending on the strain. Indeed, for strains 6988 and 6546, the best Tm was the highest (69°C), while the best Tm for strains 4885 and 6994 was the lowest (63°C). To amplify TALE genes for each strain regardless of the Tm, the next PCRs were carried out using touch-down PCR. This method consists in decreasing the Tm at each PCR cycle, starting from the highest Tm (69°C) to the lowest (63°C). A test was carried out on the four strains previously used to confirm the effectiveness of the touch-down (Figure 18). This test allowed to validate the protocol as the TALE profiles identified were those expected.

III.2.b. Analysis of the TALEs over the whole collection

Touch-down PCR was performed on the whole strain collection. Fifteen TALE profiles were identified, eight of which were already described in [START_REF] Ruh | a) Xanthomonas adaptation to common bean is associated with horizontal transfers of genes encoding TAL effectors[END_REF]. The different profiles were analysed lineage by lineage (Table 4) (Annex VI).

III.2.b.1. TALE diversity of fuscans strains

Within the fuscans lineage, four profiles were observed (Table 4) (Figure 15 b). The most represented TALE profile found in 44 of the 63 strains comprised two TALEs of 23 and 18 repeats, respectively. This TALE profile corresponded to the one of CFBP 4885 carrying tal23A and tal18H haplotypes [START_REF] Ruh | a) Xanthomonas adaptation to common bean is associated with horizontal transfers of genes encoding TAL effectors[END_REF]. This suggests that this profile was the initial one.

Deletion event of tal23A or both tal18H and tal23A in some strains would have occurred resulting in the emergence of these two new profiles. Strain CFBP 6165 had a specific TALE profile (one TALE of 22 repeats and one of 18 repeats). Its genome showed that it bears tal22B and tal18H. This strain was highly divergent from the others, which could explain why its TALE profile was different from all other fuscans strains.

III.2.b.2. TALE diversity of GL2 strains

In GL2 strains, three profiles were identified (Table 4) (Figure 15 f). The TALE profile of CFBP 6988, CFBP 6989, and CFBP 6990 strains had already been identified as comprising tal18G, tal20F, and tal23A haplotypes (Figure 7). The tal18G has been shown to be specific to the GL2 lineage [START_REF] Ruh | a) Xanthomonas adaptation to common bean is associated with horizontal transfers of genes encoding TAL effectors[END_REF]. The TALE profile of strain CFBP6991 was also previously determined as carrying tal18G, tal20F, and tal22C haplotypes (Figure 7). The last TALE profile has never been described before within the GL2 lineage as it had two TALEs of 23 and 18 repeats, respectively. According to the TALE profiles of the GL2 strains described above and the specificity of tal18G for the GL2 lineage, this profile may be described as comprising tal18G and tal23A haplotypes. It is the most represented profile. It was found in five out of the nine strains suggesting that this profile was the initial one. Addition event tal20F and a potential deletion of the first repeat within the tal23A sequence in some strains would have occurred resulting in the emergence of the two other profiles described above [START_REF] Ruh | a) Xanthomonas adaptation to common bean is associated with horizontal transfers of genes encoding TAL effectors[END_REF]. In [START_REF] Ruh | a) Xanthomonas adaptation to common bean is associated with horizontal transfers of genes encoding TAL effectors[END_REF], tal18G and tal20F were shown to be paralogous genes resulting from duplication.

Therefore, profile analysis suggests that tal20F results from duplication of tal18G with an addition of two repeats since only tal18G and tal23A were identified in the majority profile.

Also, the MST showed that all strains had the same TALE profile except for the four French strains which presented two different profiles. This suggests that at least one recombination event occurred only in France. This remains a hypothesis to be confirmed by conducting the same analysis on a larger number of GL2 strains.

III.2.b.3. TALE diversity of GL3 strains

Within the GL3 line, no new TALE profile was discovered as only one strain was added to the latest analysis by [START_REF] Ruh | a) Xanthomonas adaptation to common bean is associated with horizontal transfers of genes encoding TAL effectors[END_REF] (Table 4) (Figure 15 h). This profile comprised one TALE of 23 repeats. This TALE profile corresponded to the one of CFBP 6992 carrying tal23A haplotype [START_REF] Ruh | a) Xanthomonas adaptation to common bean is associated with horizontal transfers of genes encoding TAL effectors[END_REF]. These results indicate that no differentiation of TALEs occurred within this lineage.

III.2.b.3. TALE diversity of GL1 strains

For the GL1 lineage, a large diversity of TALE profiles was observed as ten profiles were identified of which seven were new (Table 4) (Figure 15 d). This large diversity of TALE profile suggests that several recombination and/or horizontal gene transfers occurred. For example, it was shown in [START_REF] Ruh | a) Xanthomonas adaptation to common bean is associated with horizontal transfers of genes encoding TAL effectors[END_REF] that recent horizontal transfers of genes occurred between fuscans and GL1 strains as both lineages carrying the tal18H gene on the same plasmid. Most of the GL1 strains studied showed a TALE profile including tal18H except for some strains in which tal18H was absent. As hypothesized in [START_REF] Ruh | a) Xanthomonas adaptation to common bean is associated with horizontal transfers of genes encoding TAL effectors[END_REF], it is likely that tal18H was deleted in these strains.

The most represented profile found in nine of the thirty-two strains comprised two TALEs of 18 and 19 repeats, respectively. This TALE profile corresponded to the one of CFBP 6982 carrying tal18H and tal19E haplotypes, which is similar to TALE profile of CFBP 6546 carrying tal18H and tal19I [START_REF] Ruh | a) Xanthomonas adaptation to common bean is associated with horizontal transfers of genes encoding TAL effectors[END_REF]. This suggests that this profile was the initial one. Deletion event of tal18H, tal19E (or tal19I), or both tal18H and tal19E (or tal19I) in some strains would have occurred resulting in the emergence of three TALE profiles (only tal18H, only tal19E (or tal19I), or no tal) of which two new.

Ten other strains showed a profile with two TALEs, one of 18 repeats and a second of variable size depending on the strain. This suggests that repeats have been added or deleted within the tal19E or tal19I sequence. This was confirmed for tal21C which have a similar sequence to those of tal19E and tal19I [START_REF] Ruh | a) Xanthomonas adaptation to common bean is associated with horizontal transfers of genes encoding TAL effectors[END_REF]. This remains a hypothesis for the other tal.

Among the seven new profiles, three new tal were discovered (Figure 15 d). The tal17 gene was only identified in BRA241 strain. Two others new tal were identified separately in BB013a

and CFBP 7428 strains, respectively. According to the amplicon size, these tal have a low repeat number. However, the identification of the exact number of repeats remains to be determined.

IV.

Discussion

During this study, we have characterized the diversity of Xanthomonas strains responsible for CBB and their TALEs. For this, an MLVA analysis was conducted on a collection of 144 strains chosen to represent a worldwide diversity. The results were presented by lineage via MSTs.

The analysis of TALE diversity was conducted on the same collection, using specific PCR. The two diversity analyses were both summarised on MSTs.

IV.1. Identification of the strain

First, the lineages of all the strains in the collection were identified, using PCRs on housekeeping genes. Contrary to expectations, the gyrb housekeeping gene could not be amplified on 18 strains in the collection suggesting that these strains are not Xanthomonas.

Another hypothesis is that the PCR failed. To confirm either hypothesis, another PCR on gyrb gene and PCRs on other Xanthomonas specific housekeeping genes could be performed. In addition, strains belonging to other pathovars and species than Xcf and Xpp were identified.

Since these strains were isolated from bean plants suspected of having CBB, questions arise about their impact on CBB. It is likely that their presence positively or negatively affects the level of infection of bean plants via biological interactions such as competition, mutualism, or commensalism between bacterial species/pathovars. All other strains in the collection were identified as Xcf (33 strains) or Xpp (78 strains).

IV.2. The distribution of GL2 and GL3 strains

Phylogenetic analysis on housekeeping genes shows a worldwide distribution of GL2 strains since they have been isolated in Asia, South America, and Africa. This invalidates the hypothesis that the GL2 lineage is endemic to Réunion. However, few GL2 strains have been found compared to GL1 and fuscans strains. Few GL3 strains have been also found and are only present in Reunion and Tanzania. This suggests that bean domestication had an impact on strain selection which was favourable to the dispersal of GL1 and fuscans strains.

IV.3. MLVA analysis to identify preventive strategies against Xcf and Xpp new introductions?

Then, MLVA analysis was performed on all the strains of the collection. The MLVA approach is However, to validate the good representativity of the data when performing an epidemiological monitoring via MLVA analysis, the VNTR sets used for the GL2 and GL3 lineages need to be tested on a larger number of strains than during the internship.

Despite the large scale of time and space separating the different isolation campaigns of the strains studied, the MLVA analysis made it possible to highlight exchanges of strains between countries, especially for the fuscans lineage. As Xcf and Xpp strains can survive in seed, it is likely that these exchanges are directly related to the transport of seed from one production area to another.

IV.4. A first approximation of the TALE diversity

The objective of the TALE analysis is to analyse molecular interactions between bacteria and beans plant in order to identify strategies for the establishment of resistance to Xcf and Xpp.

For this purpose, PCRs were performed on the genes encoding the TALEs. The analyses carried out during this internship gave a first approximation of the profiles. However, these analyses are biased for three main reasons. First, it was no possible to distinguish two TALEs with the same number of repeats but with divergent sequences within the same strain. Secondly, this approach does not detect variants with identical numbers of repeats but with mutations within the repeats. Thirdly, if a mutation occurred at the primers binding site, the genes encoding the TALEs could not be amplified. These three biases can only be corrected by sequencing the strains.

IV.5. The tal23A gene common to all strains?

The analysis of TALE profiles showed a low diversity of TALE profiles for GL1 strains, which had only four profiles, and GL3 strains, which had only tal23A. The al23A is found in the majority of Xpp strains suggesting that the tal23A gene has been the target of horizontal transfer between these lines. This hypothesis had already been assumed in [START_REF] Ruh | a) Xanthomonas adaptation to common bean is associated with horizontal transfers of genes encoding TAL effectors[END_REF]. GL1

strains have been described as having only variants of this tal23A such as tal21D, tal19I, and tal19E.

IV.6. New TALE profiles

The analysis of the TALEs also allowed to discover new tal such as tal17. Strains containing no tal were described. Two hypotheses can be made. Firstly, it is possible that the tal genes of these strains have been deleted suggesting that the pathogenicity of these strains is based on effectors other than TALEs. The second hypothesis is that the PCR did not amplify the TALE genes because the primer binding site was mutated. The verification of these hypotheses can only be done by sequencing these strains.

IV.7. A variety of TALEs with 18 repetitions

In [START_REF] Ruh | a) Xanthomonas adaptation to common bean is associated with horizontal transfers of genes encoding TAL effectors[END_REF], two TALEs of 18 repeats (tal18G which is chromosomic and tal18H carried by a plasmid) were identified. It was shown that tal18H is present in both fuscans and GL1

suggesting that recent horizontal gene transfers have occurred between these two lineages whereas tal18G is only present in GL2. During the profile analysis, we therefore considered that strains with a TALE of 18 repeats were associated with tal18G in GL2 and tal18H in fuscans and GL1. This hypothesis remains to be verified.

Furthermore, in [START_REF] Ruh | a) Xanthomonas adaptation to common bean is associated with horizontal transfers of genes encoding TAL effectors[END_REF], tal18G and tal20F were shown to be paralogous resulting from tandem duplication within GL2 strains. The results of the TALE profiles suggest that tal18G is the origin of tal20F. Indeed, tal18G would be duplicated in tandem before acquiring two repeats.

IV.8. Choice of strains to be sequenced

In order to confirm the above hypotheses, the genome of ten strains will be sequenced by PacBio. The sequencing will allow the acquisition of new TALE sequences, which will be used to predict their target DNA sequences in the host plant genome. The prediction of these targets will make it possible to search for resistance in the natural diversity of plants in order to construct plants genetically resistant to a diversity of Xcf and Xpp strains.

The choice of these strains was based on the two diversity analyses carried out during this internship (Figure 15). The strains were chosen to cover the widest diversity, with a focus on tal genes (Table 5). Considering the high intra-lineage and tal diversity of GL1 strains, six of the ten strains to be sequenced were chosen from this lineage. Up to now, only the genome of strains from Réunion has been sequenced for GL2. Thus, three GL2 strains from different countries and phylogenetically distant were chosen to be sequenced. No GL3 strains will be sequenced as only one new strain with a tal profile already described has been discovered.

Concerning the fuscans lineage, one strain with a new tal profile (1008017) will be sequenced.

V. Conclusion

The different analyses carried out during this internship allowed to characterize the diversity of a collection of Xcf-Xpp strains from different bean production areas. These analyses showed a high diversity of GL1 strains compared to strains from other lineages. Moreover, these results showed that GL2 strains have a worldwide distribution contrary to what was previously assumed. The internship also allowed the development of an MLVA analysis on Xcf-Xpp strains which could be used for epidemiological monitoring of Xcf-Xpp strains. The diversity of the TALEs of each strain was also determined for each lineage. All results support and complement the results of [START_REF] Ruh | a) Xanthomonas adaptation to common bean is associated with horizontal transfers of genes encoding TAL effectors[END_REF] concerning horizontal gene transfer between the two pathovars Xcf and Xpp. In addition, interesting TALE profiles including new tal genes have been identified

in different strains that will be sequenced.

VI. Perspectives

Based on the analysis performed during this internship, the selected strains will be sequenced in Pacbio. To study the potential target sequences of TALEs in bean genome, the sequences of these strains will be analysed. This will allow to confirm the profiles determined during the

Figure 1 :

 1 Figure 1: Phylogenetic tree from 17 CFBP strains showing the four lineages of Xcf-Xpp

Figure 2 :

 2 Figure 2: Illustration of symptoms on leaf, pod and seed of bean caused by CBB.

Figure 3 :Figure 4 :

 34 Figure 3: Diagram illustrating the replication slippage mechanism of TR rearrangement. Repeat units are shown as block on nascent (light green) and template (dark green). Adapted from Zhou et al., 2014

Figure 6 :Figure 8 :

 68 Figure 6: TALE structure. Adapted from[START_REF] Mak | The crystal structure of TAL effector PthXO1 bound to its DNA target[END_REF]

Figure

  Figure 9: Isolation location of the strains of the collection.

Figure 10 :

 10 Figure 10: Protocol of housekeeping genes PCR

  (2013) (XaG1_12, XaG1_29, XaG1_65, XaG1_70, XaG1_72, XaG1_73, and XaG2_52) , 4 from Ferreira et al. (2019) (7764v1, 7764v13, 7764v18, and 7764v24), 1 from L. Bui Thi Ngoc et al. (2009) (Ngoc_4), and three from those previously designed by the laboratory (Karine_vntr1.4, Karine_vntr5, and Karine_vntr12) (Annex 4). Then, for each primer, the size of the offset, the motif, and the

Figure 12 :

 12 Figure 12: TALE PCR protocol

  Figure 13:gyrB (a) and rpoD (b) phylogenetic trees.

  .1. Study of the strain diversity III.1.a. Identification of the strains

Figure 14 :

 14 Figure 14: Example of VNTR analysis on Geneious. a) Validation of VNTR Xa_G1_12 on strain 7767. b) Rejection of VNTR Karine_vntr5 on strain 6546R

Figure 15 :Figure 15 :

 1515 Figure 15: Diversity and TALE MST for each lineage. (a and b: fuscans; c and d: GL1; e and f: GL2; g and h: GL3). Each circle represents a haplotype and the diameter is proportional to the number of strains in the haplotype.

Figure 16 :

 16 Figure 16: Eletrophoresis gel after a TALE PCR on extracted DNA (red frame) and bacterial suspensions with the XapF2 and XapR primers

Figure 18 :

 18 Figure 18: Electrophoresis gel after a touch-down TALE PCR

  generally used to perform epidemiological monitoring via epidemic clone identification and pathway reconstruction on recent or old epidemics. Thus, epidemiological monitoring allows to identify the main factors involved in the success of the invasion and to develop strategies aiming at controlling and preventing new introductions. Epidemiological monitoring could be conducted on Xcf and Xpp strains isolated on a small time and space scale as the MLVA scheme was implemented on Xcf and Xpp and validated for the four lineages during this internship. This could allow to reconstruct the different introductions routes of Xcf and Xpp strains and identify strategies at preventing new introductions and developing genetic resistances. In Pablo[START_REF] López-Soriano | Multilocus Variable Number of Tandem Repeat Analysis Reveals Multiple Introductions in Spain of Xanthomonas arboricola pv. pruni, the Causal Agent of Bacterial Spot Disease of Stone Fruits and Almond[END_REF], MLVA allowed to identify multiple introductions in Spain of Xanthomonas arboricola pv. pruni responsible for the bacterial spot disease of stone fruits and almond. The different routes of introduction were identified.

Table 1 :

 1 VNTRs labeled and multiplexed for four-capillary electrophoretic analyses

		RUN 1:		DYES:	RUN 2:	DYES:
		XaG1_29		FAM	Karine_vntr1.4	FAM
		XaG1_65		HEX	7764v24	HEX
		7764v13		ATTO550	XaG1_70	ATTO550
		XaG1_72		ATTO565	7764v18	ATTO565
		RUN 3:		DYES:	RUN 4 :	Dyes:
		XaG1_12		FAM	XaG2_52	FAM
		XaG1_73		ATTO550	Karine_vntr12	ATTO565
			PCR mix
		Product	Product concentration	Volume µl (1 sample)
		H 2 O			7,15
		Green taq buffer	5X	5		Amorces
		MgCl 2		25mM	3	Amorces	Séquence
		dNTP		10mM	2	XapR	TCGTTGAATGCCGGGAAATC
		Primer 1		20µM	0,375	XapF2	CATACAGAGGCTGCCTCAG
		Primer 2		20µM	0,375	tal-859-F	CCGTTACAGTTGGACAC
		Bétaine		50mM	5	Xfutal-3204-R	ATCAGGGCGAGATAACTGGG
		GoTaqFlexi		5U/µl	0,1
		DNA			2
		Total			25
					Programme
			cycle (x 10)			cycle (x20)
	94°C	94°C	70°C -> 65°C	72°C	94°C	70 -> 65	72°C	72°C	15°C
	5'	45''	45''	2'30"	45''	45''	2'30''	7'	∞

XaG1_12 XaG1_29 XaG1_65 XaG1_70 XaG1_72 XaG1_73 XaG2_52 7764v1 7764v13 7764v18 7764v24 karine 1.4 Karine GL1

		69			359	226	204		269	354		39
	GL2	69	378	179	372	256	158	256	269	384	287	39
	GL3	69	378	179	367	250	158	249	269	391	287	39
	fuscans	69		179	367	250	158	249	269	377	287	39
							motif 1
							motif 2

a b

Table 2 :

 2 Offset size of the amplicon for each VNTR

Table 3 :

 3 Rarefaction curve and Simpson diversity index for each MST

	Lineage	Simpson diversity index	Rarefaction Curve
	fuscans
			Strains number
	GL1
			number
			Strains
	GL2
			number
			Strains
	GL3
			number
			Strains

  Annex VI : TALE profiles for each strain(PartII)

	F (Diagnostic)
	F (Diagnostic)
	F	15.2.1
	F	15.2.2
	F	17.1.2
	F	18.2.1
	F	58-6.d2b
	F	716806-1
	F	717626.1
	F	Diag1718-006
	F	I 70296.1.0
	F	J81747.1.1.2
	F	J96757
	F	Xap i71755
	F	1007014
	F	1008017
	F	BRA 1
	F	J81747.1.1.1
	F	6165
	F	1815
	F	1845
	F	4834
	F	4885
	F	6166
	F	6167
	F	6960
	F	6965
	F	6975
	F	6976
	F	7766
	F	7767
	F	1008019
	F (Diagnostic)
	F	13.6.2a
	F	13.6.2b
	F	15.1
	F	15.2.1a
	F	15.5.1
	F	15.8.1
	F	1516-008
	F	16.3.1
	F	16.3.3
	F	16.8.2
	F	18.6.1
	F	18.6.2
	F	71723 1.1
	F	BRA 158
	F	BRA 193
	F	BRA 242
	F	BRA 243
	F	BRA 244
	F	BRA 74
	F	Diag
	F	i71720-3
	F	i72066.3
	F	J17547-1
	F	J81747.1.2
	F	J95292.6.1.1
	F	LSV-13
	F	LSV-17
	F	LSV-8
	F	SNES-22
	F	XCpf-631

Remerciements

TALE diversity analysis and to identify sources of resistance to Xcp-Xpp. The MLVA analysis will be carried out on a larger number of strains to verify that the VNTRs sets used on a small collection can be used for the analysis of a larger number of strains isolated in a small time and space scale. This MLVA tool will therefore be used for epidemiological monitoring of Xcf-Xpp strains in order to establish their evolutionary history and to identify prevention strategies against new introductions of these Xcf-Xpp strains.

VII. References

Annex I: List of strains in the collection (part I)

Red lines = gyrB amplification has failed Mots-clés : MLVA -diversité -TALE -Xanthomonas ABSTRACT Xanthomonas citri pv. fuscans (Xcf) and Xanthomoas phaseoli pv. phaseoli (Xpp) are the pathogens responsible for the common bacterial blight of bean. Four genetic lineages were identified from these two pathovars: fuscans, GL2, GL3, and GL1. The pathogenicity of these strains is based on a repertory of various effectors such as TAL effectors. However, the diversity of TALEs within these lineages were poorly described. An analysis of the diversity of the strains and TALEs was conducted on a collection of 144 Xcf-Xpp strains via an MLVA approach. Here, we showed that the GL1 strains present a high diversity of strains and TALEs and that the GL2 strains are not endemic to Reunion as previously assumed. Our results give a description of the diversity of Xcf-Xpp strains present in the different bean production areas when the collection was established. They allow to conclude which lineage and TAL effectors are the most present in these production areas. The development of MLVA analysis on these strains will make possible to carry out epidemiological monitoring on Xcf-Xpp allowing to identify the different routes of introduction of the strains and to set up control strategies. The analysis of the TAL effectors will allow to identify the sources of resistance to these effectors.

Keywords : MLVA -diversity -TALE -Xanthomonas


Jin Guo

Les jeux sérieux sont de plus en plus utilisés dans le domaine de la santé. Les recherches sont en majorité dédiées au développement de logiciels éducatifs pour la formation à la médecine, ou pour aider au rétablissement sérieux destiné à former des internes de médecine générale à la consultation médicale, en insistant sur les talents communicationnels à développer plutôt que sur la capacité à réaliser un diagnostic. Le jeu simule le processus de la consultation en permettant au joueur de tenir le rôle du générateur de scénario permet de faire varier le scénario en fonction du profil des patients et des performances du experts du domaine, permet de définir les objectifs présentation combine les deux premiers sous la forme d'un environnement graphique pour rendre le jeu plaisant à jouer et ergonomique. Cette architecture permet aux facilite la collaboration des acteurs impliqués dans le développement.

Les internes de médecine générale sont conduits, dans le cadre de leur Diplômes d' effectuer un stage de 6 mois dans le cabinet médical d'un alors aux pratiques de cette spécialité clinique, en assistant leur maître de stage dans la gestion du cabinet, les démarches cliniques et la communication avec les patients. Cette dernière activité introduit une situation inhabituelle trouver et des situations difficiles bien que prévisibles vont se présenter au cours de la consultation : intervention intempestive de l'interne, difficultés comportementales, manque de compétence technique, refus du patient de la présence de l'interne ou simple gène de sa part à évoquer formés avant l'accueil d'un in conserver la confiance de leur patient potentiellement dérangé par la présence de l'interne, et à pouvoir « rattraper » les consultations mal engagées. Cette formation se fait entre autre par l'utilisation de jeux de rôles dans lesquels les futurs maîtres de stage et les maîtres formateurs peuvent jouer successivement les rôles du médecin, de l'interne et du patient. La consultation est définie comme étant composée de cinq éparation à l'accueil du patient (consultation du dossier), accueil du patient et identification de la raison de sa venue.

anamnèse. Cette phase consiste à explorer les problèmes du patient pour identifier la perspective biomédicale (la maladie), la perspective du patient (le antécédents familiaux, antécédents personnels et sociaux, prise de médicaments, allergies etc.). Les actions du joueur sont enregistrées par le moteur de jeu, en particulier quand il fait un mauvais choix. A la fin de chaque phase de la consultation, un résumé s'affiche sur l'écran, qui récap processus, et donne des conseils visant à corriger ses actions pour la prochaine partie. Puis la prochaine phase peut démarrer.

  d'information est étayé par les examens physiques nécessaires et réalisables au sein du cabinet. communication des explications au patient : Cette phase consiste à fournir au patient le diagnostic et les informations adéquates en l'aidant à comprendre et à mémoriser ces informations, afin d'arriver à une bonne compréhension et à une prise de décision partagée.

Fig. 1 .

 1 Fig. 1. Exemple d'Information acquise sur le patient Le jeu permet par conséquent au joueur de parcourir ces concepts illustrés par le jeu et les synthèses de connaissance proposées après chaque phase de la consultation. Par exemple, l'interface du jeu offre au

  secret à découvrir par le joueur. Concrètement, le joueur peut choisir les questions à poser parmi différents thèmes prédéfinis, et le patient virtuel répondra à chaque question posée tel que défini par le scénario. Cette exploration destinée à découvrir la vie du patient est un des ressorts ludiques du jeu et s'intègre parfaitement à la partie plus pédagogique d'apprentissage de la consultation médicale.La structure de notre système se présente comme développements et les tests du SG entre les différentes équipes. L'indépendance du module de présentation permet un grand choix possible de modes de rendu pour le jeu. Par exemple les scénarios du jeu peuvent prendre utiliser un environnement moins riche graphiquement en ne représentant que le visage du patient. Des interactions retenu (actions sur des objets comme par exemple l'ordinateur du cabinet médical, proposées sous forme de click dans le jeu ou de menu sur une interface plus basique, etc.). Cette modularité est rendu possible par le fait que le coeur de la consultation est basé sur un dialogue es examens physiques dont la liste est finie. Les règles représentent la partie métier indépendante du scénario et du définies par des experts du domaine, comme des médecins, à définir les actions bénéfiques et négatives dans la conduite d'une consultation médicale, mais aussi l'influence de chaque type d'action sur l'évolution du définir une liste de scénarios représentant chacun un patient en consultation. Chaque scénario défini donc un patient avec son profil, ses problèmes (maladie, problèmes personnels), ses symptômes, et l'ensemble des dialogues et interactions possibles avec ce patient pendant la consultation.

Fig. 2 .

 2 Fig. 2. Structure du système

Fig. 4 .

 4 Fig. 4. Diagramme du moteur de jeu pour un scenario

Fig. 5 .

 5 Fig. 5. Le module des questions générales

  forme d'un Serious Game visant à scénariser une consultation médicale dans laquelle le joueur pourra jouer le rôle du médecin aussi bien dans le cas d'une consultation classique que dans celui d'une consultation Dans le premier cas (consultation classique), le logiciel es en médecine afin de se former à la conduite d'une consultation médicale, de préférence avant le début de leur stage. Dans le deuxième par les maîtres de stage pour anticiper certains pièges liés à l'accueil d'un interne dans leur cabinet.

	consultation médical. L'objectif pédagogique de ce jeu est
	de se familiariser avec le processus général d'une
	consultation en mettant l'accent sur les enjeux
	communicationnels de l'entrevue. A ce titre, le serious
	en permettant au joueur d' « affronter » plusieurs types de
	patients, chacun étant défini par un profil. Ce profil
	caractérise certains traits du patient, comme par exemple
	son humeur du jour, sa culture, ou son historique par
	rapport au médecin. L'interface offerte au joueur lui
	permet de définir sa stratégie de consultation parmi un
	ensemble d'actions possibles défini pendant la phase de
	conception du scénario.
	présente le fonctionnement général du jeu ainsi que son
	architecture. La première partie détaille certaines
	problématiques métiers liées à la consultation médicale et
	décrit les grands principes du jeu. La deuxième partie
	concluons sur l'état actuel du projet et le travail restant à
	accomplir.
	par la notion de « colloque singulier ». Le but principal
	d'un patient venant pour une consultation dans un cabinet
	médical est bien évidemment médical, mais le patient
	cherche aussi à établit une relation humaine avec son
	respect et recevoir des informations claires sur son
	problème et sa maladie, y compris en ce qui concerne la
	méthode de traitement et ses conséquences. D'un autre
	côté, le but du médecin est de guérir le patient ou du moins
	de le soulager. Il s'agit pour le médecin de bien écouter
	son patient et de converser avec lui en lui donnant un
	maximum d'explications, ceci afin d'établir une relation
	de confiance. Cette relation est essentielle dans la
	consultation, afin que les deux parties obtiennent les
	bonnes informations, en comprennent les enjeux et
	puissent prendre des décisions partagées.
	d'un médecin avec un patient virtuel au cours d'une

  Les éléments ludiques sont essentiels pour un « serious game » et il est souvent délicat de trouver le bon équilibre entre la partie apprentissage et la partie ludique. Pour motiver le joueur à mener correctement sa consultation, le intonations de voix, la musique de fond, etc.Pour augmenter la motivation du joueur, deux objectifs secondaire. La mission principale est simplement de réussir la consultation. Cela consiste à en respecter les différentes phases, et à maintenir l'état psychologique du patient dans de bonnes conditions. Si cela n'a pas été fait par le joueur, et si le patient virtuel n'a pas mis fin prématurément à la consultation, le jeu peu simuler un accident à la fin de l'entrevue. Cette possibilité dépend de la (mauvaise) performance du joueur. Par exemple, si le joueur a omis de poser des questions sur les éventuelles allergies du patient, le jeu symbolisera cette faute par un mauvais choix dans la prescription de médicaments. Dans tous les cas, à la fin de la partie, le score est affiché avec une liste des meilleurs scores. De nouveaux patients agenda selon ce score et le joueur pourra commencer une autre session avec un nouveau patient.L'objectif secondaire est de découvrir certains « secrets» du patient en lui posant des questions proposées par le module « questions générales ». Ces questions concernent la vie du patient, son travail, sa famille, etc. Dans les cas réels, elles sont destinées à établir une relation de confiance entre le médecin et son patient, à faciliter certains examens délicats, ou encore à aider au diagnostic.

de score basé sur le stress du patient, ainsi qu'un « secret » à découvrir sur le patient avant la fin de la partie. Ce système de motivation concret est épaulé par des mécanismes qui éveillent la curiosité du joueur en jouant sur la diversité des comportements (ou des réactions) du patient virtuel, et qui l'encourage à persévérer en rendant visibles les réactions positives ou négatives du patient (visibles graphiquement ou dans les dialogues)

[6]

. Il est à noter que le stress du patient est visualisé par le joueur comme un score mais qu'il est aussi utilisé par le moteur de jeu pour calculer les actions du patient. Ainsi un cours de la consultation à poser des questions parfois Inversement le joueur est doté d'actions lui permettant de faire baisser la tension du patient sous la forme par bon escient. Tout l'équilibre de la partie repose alors sur le maintien du stress du patient à un niveau acceptable tout en posant les questions difficiles nécessaires à la consultation et à la découverte du « secret » du patient. Bien entendu chaque profil de patient modélise une évolution du stress différente. Ainsi le joueur doit adapter ses actions à chaque type de patient, tant dans la forme de ses questions que dans leur séquence. Au début du jeu, le joueur peut organiser son agenda virtuel et consulter la liste des patients planifiés pour une consultation dans son cabinet. Initialement le joueur peut planifier deux patients dans son agenda. Ce nombre peut évoluer par la suite en fonction de la performance du joueur. Pendant la session de consultation, en plus du stress du patient, le joueur peut utiliser d'autres indicateurs de performance comme par exemple des icônes représentant l'humeur du patient, l'expression de son visage, des sons caractérisant des