Les nématodes à galles (RKN), Meloidogyne spp. sont des petits vers parasites qui infectent les racines des plantes où ils induisent la formation de sites nourriciers. Les RKN sont des endoparasites à large gamme d'hôtes, englobant les principales espèces de plantes cultivées. Meloidogyne javanica, M. graminicola et M. incognita sont les principales espèces parasitant le riz (Oryza sativa). Le succès infectieux des RKN repose sur la production de protéines effecteurs de virulence, secrétées dans leurs glandes oesophagiennes et libérées dans les cellules de la plante hôte par leur stylet. La caractéristique principale des RKN est leur capacité à déréguler des cellules du parenchyme vasculaire pour induire la formation de cellules géantes multinucléées, à haute activité métabolique. Les processus moléculaires sous-jacents restent encore mal connus, alors que l’identification d’effecteurs de virulence et de leurs cibles végétales pourrait fournir de nouvelles perspectives pour le contrôle des RKN. Ainsi, les objectifs de cette étude étaient (1) d’évaluer le rôle de protéines de Meloidogyne sécrétées (MSP) au cours des interactions riz - RKN et (2) d'identifier des cibles des MSP parmi les principales protéines Hub d’Arabidopsis thaliana impliquées dans l'immunité des plantes, afin d'évaluer la fonction putative des MSP dans les cellules hôtes. Pour la première partie de notre étude, nous avons sélectionné trois MSP exprimées dans les glandes oesophagiennes et possiblement sécrétées. L’analyse de l’expression des gènes par RT-qPCR a montré que MSP2 est fortement exprimé dans les premiers stades du cycle du nématode, tandis que MSP18 et MSP19 sont activés au cours du parasitisme dans les racines du riz. Les essais de localisation subcellulaire dans les cellules d'oignon ont identifié le noyau (pour MSP2) et le cytoplasme (pour MSP7 et MSP18) comme compartiments cellulaires ciblés par les protéines du nématode. Des plants de riz (O. sativa Nipponbare) transgéniques ont été produits pour évaluer le rôle des MSP au cours des interactions riz-RKN. Des lignées de riz surexprimant MSP18 ont permis un taux de reproduction plus élevé de M. javanica et M. graminicola. Au contraire, des retards de développement et de reproduction de M. javanica ont été observés sur des lignées de riz exprimant des micro-RNAs capables de supprimer l’expression des gènes MSP2 ou MSP19. Ces données ont montré que MSP2, MSP18 et MSP19 peuvent être des gènes importants pour le parasitisme ou le développement du nématode. Les tests d'expression transitoire dans le tabac (Nicotiana benthamiana) ont montré que MSP18 peut interférer avec la mort cellulaire programmée déclenchée par INF1, ce qui suggère que MSP18 pourrait supprimer les voies de défense des plantes pour faciliter l’infection. Dans une deuxième partie de ce travail, des analyses systématiques en double-hybride chez la levure ont été menées pour vérifier les interactions protéine-protéine entre 6 MSP et 18 protéines Hub d’A. thaliana. Chez la levure, la protéine du nématode MSP400 interagit avec trois protéines Hub, l’Anaphase-Promoting-complex 8 (At-APC8) et les facteurs de transcription At-TCP14 et At-TCP15. L'interaction physique de MSP400 avec At-APC8, un régulateur clé du cycle cellulaire de la plante, a été confirmée in planta par complémentation bimoléculaire de fluorescence (BiFC). Ces résultats démontrent pour la première fois qu'un effecteur de nématode est capable d'interagir directement avec une protéine régulatrice du cycle cellulaire chez la plante, révélant un nouveau mécanisme utilisé par les RKN pour commander la machinerie du cycle de la cellule hôte et induire ainsi la formation du site d'alimentation. Les données obtenues dans cette étude élargissent considérablement notre connaissance des acteurs moléculaires qui contribuent à la pathogénicité des nématodes, mettant en évidence les différents mécanismes exploités par les RKN pour promouvoir la sensibilité des plantes. / Root-knot nematodes (RKN), Meloidogyne spp. are small parasitic worms that infect plant roots where they induce the formation of highly specialized nutrient feeding sites. RKN are endoparasites with a wide host range encompassing major plant crops, impairing effective specific control. Meloidogyne javanica, M. graminicola, and M. incognita are the principal RKN species responsible for rice (Oryza sativa) production losses. Successful plant infection is likely achieved by nematode effector proteins produced in their esophageal gland cells and released into the host plant cells through their stylet. In particular, one of the striking features of RKN is their ability to deregulate vascular parenchyma cells to induce the formation of multinucleated giant cells with a high metabolic activity in the roots. The molecular processes underlying plant-RKN interactions still remain poorly understood. Identification of nematode virulence effectors and their plant targets may provide new insights for developing control strategies towards RKN. Thus, the aims of this study were to (1) assess the role of Meloidogyne secreted proteins (MSP) in rice – RKN interactions and (2) identify MSP targets among the major Arabidopsis thaliana Hub proteins involved in plant immunity, to assess the putative MSP function into host cells. For the first part of our study, we selected three Meloidogyne-genus specific proteins expressed in esophageal glands and predicted to be secreted. Gene expression analysis by RT-qPCR showed that MSP2 is highly expressed in the early stages of the nematode cycle, while MSP18 and MSP19 are up-regulated during parasitism in rice roots. Subcellular localization assays in onion cells identified the nucleus (for MSP2) and cytoplasm (for MSP7 and MSP18) as the main cellular compartments targeted by nematode proteins. Transgenic rice (O. sativa Nipponbare) plants expressing the MSP cDNAs or artificial micro-RNAs (amiRNAs) able to silence MSP genes were used to assess the role of MSPs during rice-RKN interactions. Homozygous transgenic lines were inoculated with pre-parasitic juveniles (J2) and (i) the number and developmental stage of nematodes present in roots after 21 days, (ii) the number of egg masses laid after 28 days and, (iii) the number of next-generation hatched J2 after 45 days were assessed. Rice lines overexpressing MSP18 allowed a higher reproduction rate of M. javanica and M. graminicola. On the contrary, impaired M. javanica development and reproduction was observed in rice lines expressing amiRNAs against MSP2 or MSP19 genes. These data showed that MSP2, MSP18, and MSP19 genes might be important genes for nematode parasitism or development. Transient expression assays in tobacco (Nicotiana benthamiana) revealed that MSP18 interfered with the INF1-triggered programmed cell death, suggesting that MSP18 could suppress the plant defense pathways to facilitate nematode parasitism. In the second part of this work, systematic yeast-two-hybrid paired assays were conducted to check for protein-protein interactions between 6 MSP and 18 A. thaliana Hub proteins. In yeast, the nematode MSP400 protein interacts with three Hub proteins, the Anaphase-Promoting-Complex 8 (At-APC8) and the transcription factors At-TCP14 and At-TCP15. Physical interaction of MSP400 with At-APC8, a key plant cell cycle regulator, was confirmed in planta by bimolecular fluorescence complementation (BiFC) assays. These results demonstrated for the first time that a plant parasitic nematode effector is able to directly interact with a cell cycle regulatory protein, revealing a novel mechanism utilized by RKN to control the host cell cycle machinery and thereby induce feeding site formation. The data obtained in this study significantly broaden our knowledge of the molecular players contributing to nematode pathogenicity, highlighting the different mechanisms exploited by RKN to promote plant susceptibility.

Submitted by Maria Beatriz Vieira (mbeatriz.vieira@gmail.com) on 2017-04-27T12:35:26Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) tese_helen_saliba_rodrigues.pdf: 2012428 bytes, checksum: 82f289bcaf302235aecfab0417855a28 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-05-02T18:17:14Z (GMT) No. of bitstreams: 2 tese_helen_saliba_rodrigues.pdf: 2012428 bytes, checksum: 82f289bcaf302235aecfab0417855a28 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-05-02T18:17:56Z (GMT) No. of bitstreams: 2 tese_helen_saliba_rodrigues.pdf: 2012428 bytes, checksum: 82f289bcaf302235aecfab0417855a28 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-05-02T18:18:13Z (GMT). No. of bitstreams: 2 tese_helen_saliba_rodrigues.pdf: 2012428 bytes, checksum: 82f289bcaf302235aecfab0417855a28 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-02-26 / Sem bolsa / O objetivo do trabalho foi avaliar o efeito do tratamento químico na qualidade fisiológica de sementes melancia, melão e abóbora e no controle de Meloidogyne javanica em plantas destas espécies. Foram utilizados três lotes de sementes para cada cultura (Fator 1) que foram submetidos aos seguintes tratamentos (Fator 2): semente sem tratamento (TS1) e combinação dos princípios ativos: abamectina (nematicida) nas doses de 0,075g (TS 2); 0,150g (TS 3); 0,300g (TS 4) e 0,600g (TS 5) ia1000 sementes-1, combinados com tiabendazol (fungicida) e tiametoxam (inseticida), nas doses recomendadas de 0,08003g ia e 0,0192g ia por 1000 sementes, respectivamente. O volume de calda utilizado foi de 4,9 mL por 1000 sementes. Após os tratamentos as sementes foram secas a temperatura ambiente por um período de 24 horas e avaliadas pelo teste de germinação, primeira contagem, teste de frio, envelhecimento acelerado, massa fresca, massa seca da parte aérea e raiz, comprimento da parte aérea e raiz, índice de velocidade de emergência e emergência em campo. Em avaliação complementar visando avaliar a eficiência no controle de M. javanica, sementes tratadas foram semeadas e obteve-se mudas que foram transplantadas para vasos com capacidade de 1,8 litros, que receberam 10 mL de suspensão de M. javanica contendo 5.000 mil ovos. Aos 55 dias após a semeadura avaliou-se o número de galhas, número de ovos e fator de reprodução do nematoide. O tratamento de sementes com abamectina até a dose 0,600g combinado com 0,0803g de tiabendazol e 0,192g de tiametoxam controla a infestação M. javanica em raízes de plantas de melancia, melão e abóbora. Para melancia, a dose de 0,600g de abamectina+F+I, proporcionou acréscimos na germinação, envelhecimento acelerado, comprimento de plântula e índice de velocidade de germinação das sementes. A dose de 0,075g de abamectina +F+I mostrou ser a que apresentou melhor desempenho pelo teste de frio em sementes de melão e abóbora. A dose 0,600g de abamectina +F+I, favoreceu à produção de massa fresca em plantas de abóbora. / The aim of this work was evaluate the effect of chemical treatment on physiological seed quality and on the control of Meloidogyne javanica in pumpkin, melon and watermelon plants. Three lots of seeds were used for each crop. The treatments were constituted for untreated seed (ST1) and combination of active ingredients: abamectin (nematicide) at doses of 0.075g (ST 2); 0.150 g (ST 3); 0.300g (ST4) and 0,600g (ST 5) ia1000 seed-1, combined with thiabendazole (fungicide) and thiamethoxam (insecticide) at dose 0,08003g n 0,0192g per 1000 seeds, respectively. The spray volume used was 4.9 ml per 1000 seeds. The seeds were put to dry in temperature environment for 24 hours. The evaluations were carried out by germination test, first count, cold test, accelerated aging, fresh weight, and root and shoot dry weight, shoot and root lengths, index of seedling emergence and emergence. In additional assessment to evaluate the efficiency in the control of M. javanica, where the treated seeds were sown seedlings in trays and transplanted to pots of 1.8 liters, which were applied 10 mL of M. javanica suspension with 5.000 eggs. At 55 days after sowing were evaluated the number of galls, egg number and reproduction factor. Seed treatment with abamectin until 0.600g combined with 0,0803g of thiabendazole and 0,192g of thiamethoxam has controlled the infestation M. javanica of watermelon, melon and pumpkin roots. The dose of 0.600g abamectin + F + I, improved to germination, accelerated aging, Seedling length and index of seed emergence. The dose 0.075 g of abamectin + F + I improved the performance of melon and pumpkin seeds by cold test. The dose 0.600g of abamectin + F + I, promoted the production of dry weight in pumpkin plants.