Yara Noureddine

Martine Da Rocha

Clémence Médina

Mohamed Zouine

Joffrey Meijias

Michael Quentin

Pierre Abad

Bruno Favery

Stéphanie Jaubert-Possamai

Sébastien Thomine

Michaël Quentin

Stephanie Jaubert
email: stephanie.jaubert@inrae.fr

miR167-ARF8, an auxin responsive module involved in the formation of galls induced by root-knot nematodes in tomato

Keywords: microRNAs, Root-knot nematodes, Tomato, Arabidopsis thaliana, Galls knot nematodes, cyst nematodes, galls, syncytium, microRNAs, siRNAs root-knot nematodes, galls, microRNAs, tomato, auxin, ARF8

Root-knot nematodes (RKN), genus Meloidogyne, are microscopic plant parasitic worms which infect roots of more than 5 000 cultivated plant species and cause massive crop yield losses worldwide. Within host root, RKN induce the formation of root galls by inducing the dedifferentiation of root vascular cells into giant and multinucleated feeding cells. The formation of feeding cells can be split into two phases: successive nuclear divisions during the first ten days post infection (dpi), then from 10 to 21 dpi feeding cells nuclei undergo extensive endoreduplication and plant growth. These feeding cells supply water and nutrients essential for nematode development. The formation of these feeding cells is the result of an extensive reprogramming of gene expression in targeted root cells as shown by transcriptome analyses. However, few data are available on the regulation of gene expression in these structures. The objective of my PhD thesis was to study small non-coding RNAs, microRNAs, that are key regulators of gene expression. microRNAs act at the post-transcriptional level by inducing the degradation or inhibition of the translation of targeted messenger RNAs (mRNAs). During my PhD thesis, sequencing of small RNAs from tomato (Solanum lycopersicum) galls induced by Meloidogyne incognita RKN and uninfected roots identified 174 microRNAs that are differentially expressed in galls at 7 and/or 14 dpi. mRNAs targeted by microRNAs in tomato galls were then identified by integrating microRNA sequencing data with data from transcriptome analysis and from a specific sequencing of cleaved mRNAs named degradome approach. This integrative analysis built a microRNA-gene regulatory network acting during the formation of galls and feeding cells in tomato roots. Three microRNAs families, miR167, miR398 and miR408, were selected for functional analyses. MiR167 family targets the auxin-response factors ARF8A and ARF8B. These ARFs belong to auxin signaling pathway, a key hormone in plant-RKN interaction. Using tomato lines expressing the two ARF8 promoters fused to GUS reporter gene, I showed a strong activity of both ARF8 promoters in galls at 7 and 14 dpi, confirming the transcriptomic analyzes. Moreover, we analyzed the effect of a CRISPR deletion within ARF8A and ARF8B coding sequences on the infection by M. incognita. Both CRISPR lines showed a significantly increased resistance to nematode infection correlated with defects in feeding cell formation. Altogether, these result showed that ARF8A and ARF8B expression is required for successful tomato-RKN interaction. The two conserved microRNA families, miR398 and miR408, are upregulated in tomato and Arabidopsis thaliana galls. miR398 and miR408 and their targets have been previously described to be involved in the copper signaling pathway. MIR398 and MIR408 expression is activated in response to copper starvation by the SPL7 transcription factor and mature miR398 and miR408 repress expression of genes encoding copper binding proteins non-essential for plant development. By using Arabidopsis lines expressing transcriptional fusion with GUS reporter gene, I showed that both MIR408 and SPL7 were expressed within nematode induced feeding cells. Moreover, infection assays with mir408 and spl7 mutants or lines expressing mutated targets resistant for miR398 cleavage showed an increased resistance of these lines to nematode infection. Finally, watering plants with copper sulfate, at concentration below toxic concentrations for the plants or for the nematodes, induced a strong resistance to nematode infection. Altogether, these results demonstrate the role of the copper signaling through activation of miR398 and miR408 by SPL7 in the formation of giant feeding cells. To conclude, the work presented in this thesis demonstrates the important role of three microRNAs families and their targets in the formation of nematode-induced feeding cells.

Resumé

Les nématodes à galles, du genre Meloidogyne, sont des vers microscopiques parasites des plantes qui infectent les racines de plus de 5 000 espèces de plantes et causent des pertes de rendement massives. Ces nématodes induisent la formation de galles en induisant la dédifférenciation de cellules racinaires en cellules nourricières géantes. La formation des cellules nourricières peut être divisée en deux phases : des mitoses successives sans cytokinèse au cours des dix premiers jours après infection (jai) puis de 10 à 21 jai, une phase d'endoreduplication et de forte croissance cellulaire. La formation de ces cellules est le résultat d'une reprogrammation massive de l'expression génique dans les cellules racinaires ciblées, comme le montrent les analyses transcriptomiques de galles. L'objectif de ma thèse était d'étudier des petits ARN non codants, les microARN, qui sont des régulateurs clés de l'expression génique chez les eucaryotes. Ces microARN agissent en induisant la dégradation ou l'inhibition de la traduction des ARN messagers (ARNm) ciblés. Au cours de ma thèse, le séquençage des petits ARN de galles de tomate induites par le nématode Meloidogyne incognita et de racines non infectés a permis d'identifier 174 microARN qui sont différentiellement exprimés dans les galles à 7 et/ou 14 jai. Les ARNm ciblés par les microARN dans ces galles ont été identifiés en intégrant les données de séquençage des microARN avec les données de transcriptome et d'un séquençage spécifique des ARNm clivés appelé dégradome. Cette analyse intégrative a permis la construction d'un réseau de régulation de l'expression génique agissant lors de la formation des cellules nourricières chez la tomate. Trois familles de microARN, miR167, miR398 et miR408, ont été sélectionnées pour les analyses fonctionnelles. La famille miR167 ciblent les transcrits d'ARF8A et ARF8B, codant des facteurs de réponse à l'auxine appartiennent à la voie de signalisation de l'auxine, un régulateur majeur dans l'interaction plante-nématode. En utilisant des lignées de tomates exprimant les deux promoteurs ARF8 fusionnés au gène rapporteur GUS, nous avons montré une forte activité des deux promoteurs dans les galles à 7 et 14 jai, confirmant les analyses transcriptomiques. Nous avons analysé l'effet d'une délétion CRISPR au sein des séquences codantes ARF8A et ARF8B sur l'infection par M. incognita. Ces deux lignées présentent une résistance accrue à l'infection en raison de défauts de formation des cellules nourricières. L'ensemble des résultats a montré que l'expression d'ARF8A et d'ARF8B est nécessaire pour la formation des cellules nourricières. Les familles de microARN conservées, miR398 et miR408, sont surexprimées dans les galles de tomate et d'Arabidopsis thaliana. Ces microARN et leurs cibles sont impliqués dans la signalisation du cuivre. Lors de carence en cuivre, l'expression des gènes MiR398 et MIR408 est activée par le facteur de transcription SPL7, réprimant alors l'expression de gènes codant des protéines liant le cuivre non essentiel au développement des plantes. En utilisant des lignées d'A. thaliana exprimant une fusion transcriptionnelle avec le gène GUS, nous avons montré que MIR408 et SPL7 étaient exprimés dans des cellules nourricières. Des tests d'infection réalisés avec des mutants mir408 et spl7, ou des lignées exprimant des cibles mutées résistantes au clivage par miR398 ont montré une résistance accrue de ces lignées aux nématodes. L'apport de sulfate de cuivre, à une concentration inférieure aux concentrations toxiques, a induit une forte résistance à l'infection. L'ensemble de ces résultats montrent le rôle de l'homéostasie du cuivre dans la formation de cellules géantes via le module SPL7, miR408 et miR398. Pour conclure, les travaux présentés dans ce travail de thèse démontrent le rôle de ces trois familles de microARN et de leurs cibles dans la formation de cellules nourricières géantes induites par les nématodes.

Mots clés : microARN, Nématodes à galles, Tomate, Arabidopsis thaliana, Galles

Acknowledgments

These three years of my PhD wouldn't be this perfect without the presence of amazing persons all along this journey. I would like first to thank my amazing chef Stéphanie Jaubert-Possamai. Steph you are a great person both professionally and personally. I feel so lucky to have you as my mentor and that I worked with you, because I have learnt a lot from you. You were the one that believed in me after my internship, and the one that made me progress in my work. You are one of the patient and caring person that I have ever known, and you made me fall in love in research. I could never thank you enough for being beside me all along this thesis, and we both know that it wasn't easy all the time. I will always be grateful to have worked with you. I wish you all the best in the world! I am lucky enough to have not one amazing chef but two. I would like to thank the director of my thesis Bruno Favery. Bruno thank you for your investment in my thesis. You were always present for everything I needed, for every idea to discuss and my endless questions. Believe it or not, I have never see Bruno not smiling, always in a good mood and that is contagious! I have learnt a lot from you, you are a great chef and students are definitely lucky to work with you, because I am.

I would like to thank all the members of the jury: Paola Furla, Nicolas Bouché, Maria Manzanares-Dauleux, Sébastian Thomine and Christine Lelandais. Thank you for you for accepting to evaluate my thesis and for your time! I would also to thank the members of my thesis committee Patrick Laufs and Christine Lelandais for all the discussions that we had. I would like to thank Pierre Abad. Thank you Pierre for having me in the IPN team. Thank you for all the discussions that we had and your time to read my thesis/article, and of course for the best panbagnat I have ever had! I would like to thank Laetitia Zurletto, Michael Quentin and Sophie Mantelin. Laetitia you are superwoman, I have always admired your work, thank you for encouragement all along my thesis! Michael, thank you for your time invested during my thesis, for all the discussions/ideas, it helped me progress a lot! Sophie, thank you for all the discussions that we had, I hope you are happy here in the south! A special thanks for Martine da Rocha for all your bioinformatics work. We couldn't have done it without you! I would like to thank all the interns that worked with me during my thesis: Latifa Salaka, Mathilde Petitpas, Karma Jammoul and Claire Vigne. I wish you girls all the best in your journey! My journey in the lab and France would be the same without my two amazing friends; Salma, no matter what I say I will never be able to thank you enough. You are the big sister that I never had. You are always here to hear me nagging, taking about the same things over and over, but above all to help me whenever I need it. I have never thought that I will have a great friend as you. I am just simply lucky, and I am thankful that I shared all the good/bad days with you. I am so proud of you and I wish that you will find happiness because you deserve it. Rouba, you are one of the best people that I have ever known. You are the calm and reasonable friend that everyone need in their life. Thank you for being always next to me, helping me to get through everything. I will always miss and cherish all the time that we had together! Lots of love for you and Walid and best of luck in your journey in Denmark! Hope to see you soon.

I would like to thank the PhD students that became my friends and family in the lab. Rahim, I am really glad that I shared this journey with you. Thank you for being you! You are a great colleague and an amazing friend. I wish you all the happiness in the world with Ilona (and the tardigrades:p). Keep on dreaming big (don't forget the virus) and I hope that lizards will be the only obstacle in your life. Salomé, you are a one true special friend. I have always wondered how you are able to be positive all the time. I wish you all the best in your thesis, I am sure that it will be great! Hugs for you, Mélina and Moka. Dries, you are one of a kind person and I am really glad that I know you, I wish you all the best in the rest of your thesis and also in your America-bicycle-tour! Have fun a lot! Arnaud, the fresh PhD student, I hope that we didn't traumatize you. Good luck on your thesis and I hope that you will get the chair:p A special thanks to the two amazing girls that shared me the office and supported me while writing the thesis:p Claire C., Glad to have you back in the team! You are an awesome person, don't ever change! Morgane, I hope that you liked working with nematodes! Thank you for your help with all the seeds that we collected! A special thanks for Karine Mullet, for supporting me with all my endless orders but above all for being the nicest and most caring person. I wish you all the best! A big thanks for Nathalie Marteu. I know I drove you crazy with all the J2 that I ordered all the time. You are great person and I am glad that I have met you in the institute. Laurent Lapeyre, thank you for all the laughs that we shared and of course for the parsley that you get me, I made the best Tabbouleh with it! I would like to thanks all the other members of IPN team especially Phillippe Castagnone, Caroline Caporalino, Daniel Esmenjaud, Etienne Danchin, Simon Saucet, Cyril Van-Ghelder, Ulysse Poitier, Christian Wdziekonski I would like also to thank my colleagues that aren't in the lab anymore. Laura, even that you didn't stay in the lab for so long, I was really glad that I knew you, because I have made a one amazing friend. I wish you all the best for you and Maxime, and can't wait to see you again! Joffrey, I am thankful that you were in the lab during my internship and my two first years of my thesis. I have learned a lot from you, and this journey wouldn't be the same without you.

Thank you for all the help and I hope that you will always be happy wherever you are! YongPan, I am proud to say that you are my friend. Even that we are miles apart we were able to stay always in touch. You were the joy in the lab, the hard worker, the friend, and everyone loved you! We miss you a lot! I would like also to thank other members in the institute, especially Janice De-Almeida, Ariane Fazari, the IPO team especially Harald Keller, Laila Giordano, Maelle Jaouannet I have always appreciated our morning walks from the bus stop to the lab! I would like to thank my future teaching team in the university; Isabelle Garcia and Eric Boncompagni! Thank you for this opportunity! I can't wait to experience teaching with you next semester! I would like also to thank my Lebanese family in Nice. Hawraa, you were the one who helped me to settle down in Nice and I couldn't have done it without you. You are my big sister that I know I can depend on her in everything. You are simply a great person and I am really proud for the person you have become. I will always be next to you and you can always count on me. I love you and Mimi so so much! Mariam, the person that make everything better! I will always carry with me our laughs, the music that only the two of us love it:p, the hangouts.. you will never know how precious you are to me and how much I love you! I wish you all the best and the love in the world, because you deserve it! Rana, you were a big part of my life when I first came to Nice. Thank you for everything that you have done for me. I hope you will succeed in your work and to find happiness and success in everything you do! I would like to thank my friends in Nice especially, Hiba and Ahmad Haidar and your amazing girls! Zeinab Chreif I wish you all the best for you and your husband, and your little mazing boys! Amani Wehbe, thank you for helping me to discover Nice! Soumaya, Mohammad Makkiyeh, Hassan Daher all you guys are awesome! I would like to thank all my friends back in my hometown. Rafif, it has been more than 15 years that we know each other and you have been there through thick and thin. I am lucky to have always my childhood best friend right next to me. I love you so much and best of luck in Milano. Amar, my other childhood friend, I have always enjoyed our video call on Sunday and our coffee mornings! Best of luck on your thesis, you will always shine! Rime Tannous, I miss your singing. Pauline, Good luck on your journey in Paris! A big thanks to my favorite group in the Lebanese University, that no matter what are the circumstances in life we are still close as ever. Lots of love to Marie Joelle, Hussein, Bassel, Malak, Nour, Souad, Rima, Lara, Mayssa, Abbas (x2), Mohammad, Mourad. I will always carry our memories wherever I go. Nadine, I miss you so much! Firas, you are one of the closest/greatest friend I have ever had. Thank you for always being there for me! I miss you so much. Canada is lucky to have you! Ali Tarhini, best of luck in everything you do! You know that I miss you so much, thank you for being a big part of my life! A big thanks to my favorite cousins. You are all a big part of my childhood and I have the best memories with you. I wish you all the best and I love you so much. Mayssa (I miss you so much my childhood friend/cousin), May, Rita, Ziad, Hamada, Ali, Hussein, Wissam. Lots of love to Ghassan and Ranime. You are great parents to your little angels Melissa and Elsa. I love you so much! Aya, thank you for your positivity and your angelic voice! Roha, I miss you so much. I wish you all the best for you and your beautiful family. A special thanks to my auntie Samia and my grandparents. Thanks for being in my life since day one! And now I arrive to the most important person in my life. Rana, you are the sunshine of my life. I am really proud for the person you have become, you are one of the strongest, confident, smartest person I have ever known, and I am glad that above all this you are my sister and my best friend! Wherever you will go and whatever you will do, you will always be my little sister that will always look after her. Thank you for supporting me and being on my side, I love you so much! Ali, my little brother who isn't little anymore! Even that I am 10 years older than you, I have never thought that we will be this close. I am always proud to tell people about you because simply you are one of a kind. I have always enjoyed our time together especially watching (mostly arguing) football! You can always count on me; I love you so much. I hope that you and Rana will achieve everything you want in life! Mom and Dad, I could never find the words to thank you enough! You were the one to give me the opportunity to come to France and follow my dreams. I could have never achieved all of this without you believing in me. You have put all your dreams away to make me achieve mine. I am the luckiest one to have you as my parents. Some people don't believe in heroes, because they have never met you! You are my super heroes, and I hope that I will always make you proud of me. I know the circumstances in Lebanon, force us to be miles apart, but I promise you that I will make your dreams come true! I love you to the moon and back! Thanks to all the people that shared this journey with me!

Preamble

Nematodes are roundworms living in various ecosystems. Caenorhabditis elegans is the most famous of the 26 000 nematode species described. Its genome was entirely sequenced in 1998, and this model organism, with its rapid reproduction and ease of generating mutants, enables the study of many cellular and molecular mechanisms. Studies with C. elegans allows major advances in the study of organ development and programmed cell death (S. Brenner, H. R. Horvitz and J. E. Sulston, Nobel Prize in Physiology or Medicine, 2002) and of the RNA interference (RNAi)gene silencing by double-stranded RNA (A. Fire and G. Mello, Nobel Prize in Physiology or Medicine, 2006).

Despite these studies, the knowledge available on other nematodes, in particular animal and plant parasitic nematodes, remains limited. This is due to the difficulty of manipulating and genetically modifying these organisms. Sedentary endoparasitic nematodes are one of the most important crop pests that induce massive crop yield losses worldwide. Among them, root-knot nematodes (RKN) of the genus Meloidogyne are capable of infecting thousands of plant species and are considered as the most damaging genus. RKN are able to transform root cells into hypertrophied, hypermetabolic and multinucleate feeding cells that supply nutrients, essential for their development. Limitations of RKN control methods leads to an attempt for better understanding the development of the disease and the molecular dialogue between plants and RKN. The formation of the giant feeding cells implies a reprogramming of root cell gene expression. The aim of this thesis is to deepen our knowledge on the role of the small noncoding microRNAs, key regulators of plant gene expression, in the formation of giant feeding cells induced by M. incognita.

In the following introduction, I will first present the common characteristics of nematodes, then focus on RKN and feeding cell formation, and I will detail the reprogramming of plant gene expression that occurs within the feeding cells. Then I will present the plant small RNAs by focusing on the microRNAs and their role in plant development and plant response to abiotic and biotic stresses.

Introduction 1. Generalities

Nematodes are round unsegmented worms. Nematodes etymology includes two ancient Greek words: "nêma", meaning, "thread", and "oidès" meaning, "like". While different species of nematodes vary in size, from 0.1 millimeters in length up to 1 meter, they all tend to be thin (less than 100 micrometers thick). These pluricellular organisms are classified in the Nematoda phylum. Until now, more than 26 000 species of nematodes have been described [START_REF] Zhang | Animal biodiversity: An update of classification and diversity in 2013[END_REF] including the best-known nematode: the model organism Caenorhabditis elegans. Their development is characterized by an embryonic phase followed by larval stages, which developed through cuticular molts in adult stage.

Nematodes are among the simplest organized animals. They are covered with a thick permeable cuticle, composed mainly of collagens, that enables the diffusion of water and gas exchange and compensates the lack of respiratory system (Figure 1A-B). Thanks to their cuticle that protects internal organs, nematodes are able to withstand extreme environments [START_REF] Decraemer | Structure and classification[END_REF]. The nematode cuticle allows growth and expansion between molting periods. Nematodes have digestive, nervous and reproductive systems. The digestive system consists of a mouth connected to an esophagus (pharynx) which ends in an intestine and a rectum [START_REF] Hussey | Monoclonal antibodies to secretory granules in esophageal glands of meloidogyne species[END_REF]). The nervous system controls mainly the somatic musculature and sensory perception through the chemoreceptor organs: amphids and phasmids. This system is composed of a nerve ring connected to several ganglia that is the coordinating center for the nervous system. The muscular apparatus is composed of longitudinal muscles under the cuticle and hypodermis that allow the nematode to move by undulations.

Nematodes occupy very diverse ecological niches on earth. They are spread in most ecosystems such as oceans and seas, fresh water, water films in the ground or in plants.

Nematodes have varied lifestyles: parasitic and free living (Perry and Moens, 2011a). Free living nematodes, such as C. elegans (Figure 1C), reside in both aquatic and terrestrial habitats [START_REF] Masler | Free-Living Nematodes[END_REF]. They can feed on bacteria like Cephalobus pseudoparvus [START_REF] Blanc | Nutrition on bacteria by bacterial-feeding nematodes and consequences on the structure of soil bacterial community[END_REF], fungi like Aphelenchus avenae [START_REF] Li | Interactions between fungal-feeding nematodes and fungi and their effects on soil nitrogen mineralization[END_REF] or other nematodes like Mononchus aquaticus [START_REF] Grootaert | CULTIVATION AND LIFE CYCLE OF MONONCHUS AQUATICUS[END_REF]. Parasitic nematodes infect various types of organisms including humans, insects, and plants (Figure 1D-F).

Nematodes have been distributed into five major clades in the phylogeny of the phylum Nematoda based on the sequences of small subunit of ribosomal RNA [START_REF] Blaxter | The evolution of parasitism in Nematoda[END_REF]. Each of these clades contains species with diverse lifestyles and all includes parasitic species.

Plant-parasitic nematode

Plant parasitism appeared independently in three clades: in Dorylaimida (clade 1), Diptherophorina (clade 2) and in Tylenchomorpha (clade 4) [START_REF] Holterman | Phylum-wide analysis of SSU rDNA reveals deep phylogenetic relationships among nematodes and accelerated evolution toward crown clades[END_REF][START_REF] Blaxter | The evolution of parasitism in Nematoda[END_REF]. The Dorylaimida and Triplonchida are virus vectors. The Tylenchomorpha are the most important clade of plant-parasitic nematodes (PPN), both in term of species number and in terms of damage caused to the plant they parasitize. This clade contains the Heteroderidae family that is responsible for most of the economic damage. These major crop pests are currently the subject of a large number of studies aimed at elucidating the mechanisms of interaction between the nematode and the plant. All PPN have a mouth stylet, a hollow retractable needle (Figure 2). Nematodes use the stylet to perforate the cell wall of root cells, aspirate the cellular content and inject the esophageal secretions. The stylet is connected to the pharynx that is connected to the intestine. PPN are classified according to their life styles and feeding habits. PPN that stay outside the root and feed externally by inserting their stylets into root cells are classified as ectoparasites. Those that penetrate host root to feed from inner cell types are classified as endoparasites. According to their feeding habit, they are subclassified further into sedentary or migratory parasites (Figure 3).

Ectoparasitic nematodes

Ectoparasitic nematodes have a rather long thin stylet that enables them to dig deep plant cells. Migratory ectoparasites feed all along the root from multiple sites (e.g. genus Xiphinema, Trichodorus, Longidorus, Macrotrophurus, Tylenchorhynchus) and sedentary ectoparasites puncture a single specific root site (e.g. Rotylenchus). Several of these nematodes cause significant indirect damage (Figure 4A, 4B) through transmission of plant viruses like Xiphinema spp. which delivers the Grapevine Fanleaf Virus (GFLV) and leads to severe damages to grapevine [START_REF] Andret-Link | Ectoparasitic Nematode Vectors of Grapevine Viruses[END_REF].

Endoparasitic nematodes

Endoparasitic nematodes spend most of their life cycle within host roots. They have a rather short and robust stylet to penetrate into the root. Endoparasitic nematodes are also subclassified according to their feeding habits: the migratory species move into the root or even the shoot and frequently cause cell necrosis like Radopholus spp. and Pratylenchus spp. The sedentary species, e.g. root-knot nematodes (RKN) and cyst-nematodes (CN), which feed from a single feeding structure and are the most damaging groups for agriculture (Jones et al., 2013) (Figure 4C,4D). Sedentary endoparasitic nematodes establish an elaborate relationship with their host by setting up a complex feeding structure that provides nutrients required for nematode development. Infection by endoparasitic nematodes generally causes defects in root development, a reduction in water uptake, defects in leaf expansion, and in photosynthesis rate. Moreover, damages to plant tissues leave the plant more vulnerable to diseases caused by plant viruses, fungi and oomycetes [START_REF] Jones | Root-knot Nematodes and Giant Cells[END_REF].

Heteroderidae family

Among sedentary endoparasitic nematodes, the Heteroderidae family includes the most damaging species worldwide. Damages are estimated at more than 100 billion dollars per year [START_REF] Trudgill | Apomictic, polyphagous root-knot nematodes: Exceptionally successful and damaging biotrophic root pathogens[END_REF]. Heteroderidae is composed of two subfamilies: the cyst nematode (CN) (e.g. Heterodera and Globodera spp.) and the root-knot nematode (RKN) (Meloidogyne spp.) (De [START_REF] Ley | Systematic Position and Phylogeny[END_REF]. RKN can feed on almost all vascular plants [START_REF] Jones | Root-knot Nematodes and Giant Cells[END_REF] whereas CN show a more specific host preference [START_REF] Mimee | Life cycle of the golden cyst nematode, Globodera rostochiensis, in Quebec, Canada[END_REF]. RKN and CN have very different host ranges and generate varied symptoms on the host roots. They have a close development cycle and establish a close relationship with their host by inducing the formation of root feeding site.

Cyst nematodes

The CN includes eight genera [START_REF] Turner | Cyst nematodes[END_REF] but Heterodera and Globodera are the two most important economically genera. CN induce the formation of a feeding cell, named syncytium, within host root from which they withdraw nutrients required for their development. The second juvenile stage (J2) (Figure 5A) penetrates the root and migrates intracellularly to reach the vascular central cylinder causing cell necrosis on its pathway. The J2 selects one root cell into which it injects a cocktail of secretion produced in the esophageal glands. The targeted cell expands within the vascular tissue by a partial cell wall dissolution which will merge with the adjacent cells via cytoplasm fusion (Golinowski et al., 1996b;[START_REF] Belkhadir | Brassinosteroid signaling pathway[END_REF]. This unique feeding structure is formed by the fusion of up to 200 root cells. Then the J2 moults in stage 3 (J3) and stage 4 (J4) juveniles then finally to the adult stage (male or female). CN have a sexual reproduction or "amphimixis". After fertilization and egg development, the CN female dies and hardens forming a cyst visible at the root level (Figure 5C). This cyst contains up to 1 000 eggs from which new J2s will emerge.

Root-knot nematodes

RKN genus is formed by more than 90 obligatory endoparasites species (Jones et al., 2013).

Infection by RKN larvae (Figure 5B) cause characteristic host root deformation named "knot" or "gall" (Figure 5D). Because of their extreme polyphagy, with a host spectrum including more than 5 000 plant species, and their wide geographical distribution, RKN are a major problem for worldwide agriculture [START_REF] Castagnone-Sereno | Genetic variability and adaptive evolution in parthenogenetic root-knot nematodes[END_REF]Blok et al., 2008;[START_REF] Abad | Plant Nematode Interaction: A Sophisticated Dialogue[END_REF]. There are three types of reproduction in RKN species. The most polyphagous Meloidogyne species, e.g. M. arenaria, M. incognita, M. javanica and M.

enterolobii, reproduce by mitotic parthenogenesis. The female produces a progeny, with no male participation in the reproduction [START_REF] Castagnone-Sereno | Diversity and evolution of root-knot nematodes, genus meloidogyne: New insights from the genomic era[END_REF]. Some species (e.g.

M. hapla) reproduces by facultative meiotic parthenogenesis or amphimixis. Only few species have a strictly sexual reproduction.

M. incognita life cycle

M. incognita is the model species used to understand the molecular processes of plant-RKN interaction, for which the genome has been fully sequenced and annotated [START_REF] Abad | Genome sequence of the metazoan plantparasitic nematode Meloidogyne incognita[END_REF]Blanc-Mathieu et al., 2017). M. incognita life cycle (Figure 6) can be completed from three to eight weeks depending on the environmental conditions and host plants (Escobar et al., 2015, reviewed in Favery et al., 2016). The RKN life cycle starts with the first stage juvenile (J1) (Figure 6A) that develops inside the egg from which the J2 (Figure 6B) hatches and migrates within the soil towards host roots. Roots secreted compounds are detected by J2's chemosensory sensilla "amphids" and attract the J2 to the root (Perry and Moens, 2011). J2s

were shown to be attracted by compounds from the seeds [START_REF] Tsai | Regulation of Root-Knot Nematode Behavior by Seed-Coat Mucilage-Derived Attractants[END_REF], volatiles compounds [START_REF] Williamson | Assessing Attraction of Nematodes to Host Roots Using Pluronic Gel Medium[END_REF] or compounds from root exsudates [START_REF] Oota | Identification of Naturally Occurring Polyamines as Root-Knot Nematode Attractants[END_REF]. J2 penetrates at the elongation zone of the host root and migrates intercellularly towards the root apex, turns around to enter the central cylinder and migrates to the differentiation zone. When it reaches vascular cylinder, J2 injects secretions though the stylet within five to seven root parenchyma vascular cells. In response to these nematode signals, targeted cells dedifferentiate into giant, multinucleated, hypermetabolic feeding cells named giant cells (GCs) forming a feeding site that supplies nutrients required for nematode development (Escobar et al., 2015) (Figure 6C-D). Additionally, the neighboring cells surrounding the giant cells start to divide. Formation of giant feeding cells and division of neighboring cells result in the formation of a root-knot called gall, which is a characteristic symptom of RKN infection. Formation of this feeding site enables J2 sedentarization and development through three consecutive molts (J3, J4) towards adult stage. Since Meloidogyne reproduce by parthenogenesis, males are not necessary for reproduction and leave the roots.

M. incognita males are thought to have a role in the control of nematode population when environmental conditions are not optimized (Figure 6E). The femalesafter asexual reproductionproduce eggs that they lay at the root surface in a gelatinous matrix called "egg mass" (Figure 6F), enabling a new cycle to proceed.

RKN control strategies

Until recently, the use of chemicals nematicides nematode were the nematode control strategies. Due to their high toxicity, chemicals like methyl bromide and nematicides based on carbamate have been banned in Europe since 2010. Other chemicals, belonging to fluoroalkenyl thioether group, have been shown to be effective against RKN with a lower impact on the environment but can disrupt other organisms [START_REF] Kearn | Fluensulfone is a nematicide with a mode of action distinct from anticholinesterases and macrocyclic lactones[END_REF]. Biological control is an alternative strategy that is ecofriendly and uses predators, parasites or pathogen of nematodes such as the bacteria Pasteuria penetrans or nematophagous fungi. However, there are still few commercial biological products successfully used in the field [START_REF] Timper | Utilization of Biological Control for Managing Plant-Parasitic Nematodes[END_REF]. While crop rotations are limited due to the wide host range of RKN, the use of plants with natural resistance genes, like Mi genes in tomato, is the most effective method to control RKN infestation and reduce yield losses [START_REF] Ammiraju | The heat-stable root-knot nematode resistance gene Mi-9 from Lycopersicon peruvianum is localized on the short arm of chromosome 6[END_REF]. However, some virulent population of nematodes and/or new species (e.g. M. enterolobii) emerged all over the world that bypass these natural resistances [START_REF] Jacquet | Variation in resistance to the root-knot nematode Meloidogyne incognita in tomato genotypes bearing the Mi gene[END_REF]. Development of novel and durable strategies to control RKN, it is therefore necessary. A better fundamental knowledge of the disease development and molecular mechanisms of susceptible plant-RKN interaction should leads to develop new strategies to control RKN.

Molecular mechanisms of plant-RKN susceptible interaction

RKN effectors

The formation of the feeding site is essential for RKN survival, and its formation is induced by molecules mainly synthetized in nematode salivary esophageal glands and secreted into the host plant via the stylet (Figure 7A). The esophageal gland secretions are released in spherical granules that vary in composition and size depending on nematode development stage and species [START_REF] Hussey | Secrets in secretions: Genes that control nematode parasitism of plants[END_REF]. They play a main role in different parasitism phases including invasion and the formation of the feeding site [START_REF] Davis | Getting to the roots of parasitism by nematodes[END_REF][START_REF] Mitchum | Nematode effector proteins: An emerging paradigm of parasitism[END_REF]. Molecules synthetized in esophageal glands and secreted in plants are known as "effectors" [START_REF] Toruño | Plant-Pathogen Effectors: Cellular Probes Interfering with Plant Defenses in Spatial and Temporal Manners[END_REF]. Effectors are "the molecules of pathogens/parasites secreted in the host, which modify the structure and/or function of the host cell and which promote parasitism" [START_REF] Hogenhout | Emerging concepts in effector biology of plant-associated organisms[END_REF]. Effectors produced from the two subventral glands are expressed in the first steps of infection while those secreted during parasitism are produced by the dorsal gland [START_REF] Mitchum | Nematode effector proteins: An emerging paradigm of parasitism[END_REF][START_REF] Nguyen | A root-knot nematode small glycine and cysteine-rich secreted effector, MiSGCR1, is involved in plant parasitism[END_REF] (Figure 7B, 7C). Some effectors may also be produced in other secretory organs, including amphids like Mh-TTL2 effector [START_REF] Gleason | Identification of two Meloidogyne hapla genes and an investigation of their roles in the plant-nematode interaction[END_REF] or secreted through the cuticle like Mi-MIF2 effector [START_REF] Zhao | A MIF-like effector suppresses plant immunity and facilitates nematode parasitism by interacting with plant annexins[END_REF]. So far, most of PPN effectors that have been identified are proteins or peptides [START_REF] Mitchum | Nematode effector proteins: An emerging paradigm of parasitism[END_REF][START_REF] Quentin | Plant parasitic nematode effectors target host defense and nuclear functions to establish feeding cells[END_REF][START_REF] Vieira | Plant-parasitic nematode effectorsinsights into their diversity and new tools for their identification[END_REF].

The identification of RKN effectors, understanding their functions and identifying their targets in planta remain a crucial challenge to better understand nematode parasitism and giant cell formation. Many approaches were developed to identify potential effectors involved in parasitism such as candidate gene strategy and the direct by proteomics analyses of esophageal secretions. Whole genome sequenced of two RKN M. incognita and M. hapla [START_REF] Abad | Genome sequence of the metazoan plantparasitic nematode Meloidogyne incognita[END_REF][START_REF] Opperman | Sequence and genetic map of Meloidogyne hapla: A compact nematode genome for plant parasitism[END_REF] facilitated effector identification through bioinformatics analyses (Truong et al., 2015). Unlike C. elegans, PPN can't be genetically transformed. Therefore, the functional characterization of RKN effectors can still be a difficult step to bypass. Indeed, RKN effectors manipulate host cellular processes through interactions with plant target to favor parasitism. Currently, more than ten plant proteins targeted by RKN effectors have been identified and shown to contribute to feeding site formation in different plant species (reviewed [START_REF] Mejias | Plant Proteins and Processes Targeted by Parasitic Nematode Effectors[END_REF].

RKN effectors are major players in manipulating the physiology of the host plant. They can be classified depending on their role in parasitism.

Effectors involved in the migration and cell wall degradation

Cell wall is the first obstacle encountered by the free-living J2s. RKN needs to weaken the boundaries between the cells in order to facilitate their penetration and the inter-cellular migration through root tissue. The secretions of these cell wall degradation/modification enzymes (CWDE) helps i) the nematode along the penetration into the root and the vascular system and ii) the modification of cell wall during the formation of the feeding site [START_REF] Bohlmann | The plant cell wall in the feeding sites of cyst nematodes[END_REF]. Dozens of effectors secreted by RKN were shown to be CWDE including pectate lyases (Jaubert et al., 2002b), β-1.4-endoglucanase [START_REF] Rosso | Isolation of a cDNA encoding a β-1,4-endoglucanase in the root-knot nematode Meloidogyne incognita and expression analysis during plant parasitism[END_REF][START_REF] Ledger | Characterization of a new β-1,4endoglucanase gene from the root-knot nematode Meloidogyne incognita and evolutionary scheme for phytonematode family 5 glycosyl hydrolases[END_REF] and endo-1,4-β-xylanase [START_REF] Mitreva-Dautova | A symbiont-independent endo-1,4-β-xylanase from the plant-parasitic nematode Meloidogyne incognita[END_REF]. Finally, M. incognita genome sequence revealed 81 genes encoding CWDE [START_REF] Abad | Genome sequence of the metazoan plantparasitic nematode Meloidogyne incognita[END_REF]. These genes are mainly expressed in the subventral oesophageal glands (Jaubert et al., 2002a;[START_REF] Davis | Getting to the roots of parasitism by nematodes[END_REF][START_REF] Davis | Degradation of the Plant Cell Wall by Nematodes[END_REF]. Interestingly, it seems that these functions necessary for parasitism have been acquired several times independently through the phylum of phytoparasitic nematodes via horizontal transfers of genetic material of bacterial origin [START_REF] Danchin | Multiple lateral gene transfers and duplications have promoted plant parasitism ability in nematodes[END_REF][START_REF] Danchin | The Transcriptomes of Xiphinema index and Longidorus elongatus Suggest Independent Acquisition of Some Plant Parasitism Genes by Horizontal Gene Transfer in Early-Branching Nematodes[END_REF].

Effectors involved in the suppression of defense reactions

RKN facilitate their infection by suppressing plant immunity with effectors [START_REF] Quentin | Plant parasitic nematode effectors target host defense and nuclear functions to establish feeding cells[END_REF]. Mi-CRT protein produced in esophageal glands, was the first RKN proteins which secretion has been established in planta [START_REF] Jaubert | In planta secretion of a calreticulin by migratory and sedentary stages of root-knot nematode[END_REF]. Using A. thaliana lines overexpressing Mi-CRT, [START_REF] Jaouannet | The root-knot nematode calreticulin Mi-CRT is a key effector in plant defense suppression[END_REF] have demonstrated that this effector is able to suppress the defense induced by the bacterial PAMP effector elf18 [START_REF] Jaouannet | The root-knot nematode calreticulin Mi-CRT is a key effector in plant defense suppression[END_REF].

The silencing of this effector by RNAi leads to a decrease in infection rate in A. thaliana.

A similar study in 2016, suggested a role for Mi-Msp40 effector as an ETI suppressor [START_REF] Niu | Msp40 effector of root-knot nematode manipulates plant immunity to facilitate parasitism[END_REF]. The overexpression of Mi-Msp40 in A. thaliana suppressed also the callose deposition in infected plants and increased susceptibility towards RKN infection. In addition, some RKN effectors have been shown to interact with host proteins to scavenge the ROS. The M. javanica effector Mj-TTL5 activates host ROS-scavenging system in A. thaliana to eliminate H 2 O 2 by interacting with ferredoxin: thioredoxin reductase catalytic (FTRc), a component of host antioxidant system [START_REF] Lin | A novel nematode effector suppresses plant immunity by activating host reactive oxygen species-scavenging system[END_REF]. RKN effectors can also participate in the final redox state of giant cells. Recently, effectors of M. graminicola Mg-MO289 and M. incognita Mi-PDII have been demonstrated to regulate redox state [START_REF] Zhao | The root-knot nematode effector MiPDI1 targets a stress-associated protein (SAP) to establish disease in Solanaceae and Arabidopsis[END_REF][START_REF] Song | The Meloidogyne graminicola effector MgMO289 targets a novel copper metallochaperone to suppress immunity in rice[END_REF]. Mi-PDII targets a stress-associated protein (SAP) to fine-tune SAP-mediated responses at the interface of redox-signaling, defense and stress acclimation in Solanaceae and A. thaliana [START_REF] Zhao | The root-knot nematode effector MiPDI1 targets a stress-associated protein (SAP) to establish disease in Solanaceae and Arabidopsis[END_REF].

Effectors involved in the formation of giant cells

The formation of the feeding site and its maintenance is a critical step of plant-RKN interaction. Until now, only few RKN effectors have been demonstrated to be involved in feeding cell formation by i) their presence in the giant cell and/or ii) of the giant cell defect associated with the inactivation of the effector or its plant target.

Only seven RKN effectors have been shown to be secreted into the giant cells. Among them five target the nucleus of giant cells: M. incognita Mi-EFF1 [START_REF] Jaouannet | A root-knot nematode-secreted protein is injected into giant cells and targeted to the nuclei[END_REF] and Mi-EFF18 [START_REF] Mejias | The root-knot nematode effector MiEFF18 interacts with the plant core spliceosomal protein SmD1 required for giant cell formation[END_REF], M. javanica Mj-NULG1 [START_REF] Lin | A Novel Effector Protein, MJ-NULG1a, Targeted to Giant Cell Nuclei Plays a Role in Meloidogyne javanica Parasitism[END_REF] and M. graminicola Mg-GPP [START_REF] Chen | A novel Meloidogyne graminicola effector, MgGPP, is secreted into host cells and undergoes glycosylation in concert with proteolysis to suppress plant defenses and promote parasitism[END_REF] and Mg16820 [START_REF] Naalden | The Meloidogyne graminicola effector Mg16820 is secreted in the apoplast and cytoplasm to suppress plant host defense responses[END_REF]. Knock down of Mg-GPP effector in planta in transgenic rice plants using RNAi reduced the number of female produced, suggesting that Mg-GPP plays a role in parasitism. Interestingly, subcellular localization assays showed that Mg-GPP can translocate from the endoplasmic reticulum to the nucleus. Furthermore, this effector suppress the cell death induced by ETI proteins Gpa2/RBP-1 in rice [START_REF] Chen | A novel Meloidogyne graminicola effector, MgGPP, is secreted into host cells and undergoes glycosylation in concert with proteolysis to suppress plant defenses and promote parasitism[END_REF]. The nuclear effector of M. incognita MiEFF18 has been demonstrated to target the plant core spliceosomal protein SMD1, a major component of the spliceosome, a complex involved in pre-mRNA splicing and alternative splicing [START_REF] Mejias | The root-knot nematode effector MiEFF18 interacts with the plant core spliceosomal protein SmD1 required for giant cell formation[END_REF]. A.

thaliana smd1b mutants showed altered susceptibility to M. incognita infection and the giant cells formed on these mutants displayed developmental defects. This study revealed the importance of the regulation of gene expression by alternative splicing during the giant cell formation. Mi-16D10 effector have been shown to interact with two SCARECROW-like transcription factors, AtSCL6 and AtSCL21, that play a role in root development [START_REF] Huang | A root-knot nematode secretory peptide functions as a ligand for a plant transcription factor[END_REF]. The repression of Mi-16D10 in A. thaliana by RNAi in planta induces an important resistance towards RKN infection [START_REF] Shivakumara | RNAi-induced silencing of an effector confers transcriptional oscillation in another group of effectors in the root-knot nematode, Meloidogyne incognita[END_REF]. [START_REF] Leelarasamee | The root-knot nematode effector MiPFN3 disrupts plant actin filaments and promotes parasitism[END_REF] identified a M. incognita effector, Mi-PFN3, encoding a profilin, an actin-binding protein.

Interestingly, the protoplasts of leaves overexpressing the effector Mi-PFN3 showed disrupted actin filaments, a process observed during giant cell formation. Mi-7E12 could be involved in the fragmentation of vacuoles observed in Giant cells (dos Santos de Lima e [START_REF] Dos Santos De Lima E Souza | Ectopic expression of a Meloidogyne incognita dorsal gland protein in tobacco accelerates the formation of the nematode feeding site[END_REF].

RKN also secrete some effectors that mimics plant peptide hormones such as C-TERMINALLY ENCODED PEPTIDE (CEP)-like and INFLORESCENCE DEFICIENT IN ABSCISSION (IDA)-like peptides [START_REF] Bird | Signatures of adaptation to plant parasitism in nematode genomes[END_REF]. IDA peptides are signaling peptides that control cell separation during cell division within the root apical meristem. The Mi-IDL1 effector is a peptide capable of mimicking the proteins of the IDA family [START_REF] Kim | The root-knot nematode Meloidogyne incognita produces a functional mimic of the Arabidopsis INFLORESCENCE DEFICIENT in ABSCISSION signaling peptide[END_REF]. The repression of Mi-IDL1 in planta using RNAi showed fewer and smaller galls at 35 and 42 dpi compared to the control plants. These peptide mimics would play a role in the manipulation of hormone balance and in the formation of the feeding site.

3.2 RKN-induced feeding site

Morphology and ontogenesis of RKN-induced feeding site

RKN are able to induce the dedifferentiation of several root vascular cells into a sophisticated feeding structure that functions as a metabolically sink to supply water and nutrients required for nematode development. Giant feeding cells are multinucleate and hypertrophied [START_REF] Bird Af | The ultrastructure and histochemistry of a nematode-induced giant cell[END_REF][START_REF] Huang | Wall modifications in developing giant cells of Vicia faba and Cucumis sativus induced by root knot nematode, Meloidogyne javanica[END_REF]) (Figure 8A, 8B). In A. thaliana, the volume of the giant feeding cell increases continuously and can reach a volume more than 400 times higher than normal root cell (Figure 8C) (Cabrera et al., 2015). Giant feeding cells are metabolically highly active. They are characterized by a dense cytoplasm containing abundant organelles and the multiplied fragmented vacuoles [START_REF] Berg | Development of the root-knot nematode feeding cell[END_REF]. Giant cells with the same phenotype are induced in more than 5 000 plant species which suggest that nematode manipulate conserved mechanisms. Elucidating all the processes involved in the transformation of initial root cells into a feeding cell remains a challenge.

The first sign of giant cell formation is nuclear division observed in cells around the J2 "head". Cell plate alignment appeared to proceed normally, but cytokinesis was unsuccessful and binucleated cells formed subsequently in Impatiens balsamina [START_REF] Jones | Early stages of nematode-induced giant-cell formation in roots of Impatiens balsamina[END_REF]. No wall breakdown was evident then or later. The number of nuclei increases by repeated mitoses without cytokinesis which leads to the multinucleated state of the giant cells [START_REF] Jones | Early stages of nematode-induced giant-cell formation in roots of Impatiens balsamina[END_REF]Caillaud et al., 2008c). The number of nuclei per giant cell increases rapidly until 10 days post infection (dpi), with a further small increases in the number of nuclei being observed at 15 dpi. The highest number of nuclei found in any single giant cell was 134 (in pea), with numerous giant cell having more than 75 nuclei each [START_REF] Starr | Dynamics of the Nuclear Complement of Giant Cells Induced by Meloidogyne incognita[END_REF]. Similar increase in the number of nuclei has been reported in giant cells from various plant hosts e.g pea, lettuce, tomato and broad bean [START_REF] Starr | Dynamics of the Nuclear Complement of Giant Cells Induced by Meloidogyne incognita[END_REF]. The formation of giant cells can be divided into two phases: a first phase of successive mitoses without cytokinesis followed by a phase of endoreduplication (DNA replication without nuclear division) (de Almeida Engler and Gheysen, 2013) (Figure 9) .

The nuclei of feeding cells are large with an irregular lobed shape and with large conspicuous nucleoli and an increase in ploidy (Escobar and Fenoll, 2015). The increase in the number of nuclei and the endoreduplication enable the isotropic growth of the giant cells. Giant cells are characterized by the development of cell wall ingrowths, typical of transfer cells that are in contact with the xylem and phloem vessel elements to increase the surface area of the associated membrane. Cell wall ingrowths (CWI) enable the nematodes to withdraw water and nutrients from the sap for its own development (Figure 10A) (Jones and Northcote, 1972b;Sobczak and Golinowski, 2011;[START_REF] Rodiuc | On the track of transfer cell formation by specialized plant-parasitic nematodes[END_REF]. The sieve elements around giant cells are interconnected by plasmodesmata (PDs) (Figure 10B, 10C) [START_REF] Jones | Root-knot Nematodes and Giant Cells[END_REF][START_REF] Bartlem | Vascularization and nutrient delivery at root-knot nematode feeding sites in host roots[END_REF]. Giant cells are symplastically isolated from the surrounding tissue, and nutrient uptake depend on transport across membrane [START_REF] Hoth | Differential vascularization of nematode-induced feeding sites[END_REF].

Neighboring cells vs Giant cells

Cells surrounding the feeding giant cells and the nematode are named "neighboring cells" (NC) (Figure 11A). When feeding cells are induced, neighboring cells re-enter into cell division [START_REF] Berg | Development of the root-knot nematode feeding cell[END_REF]. The division of neighboring cells probably participates to the growth of giant cells by preventing the tissue around from breaking but also by initiating a de novo vascular differentiation process important to provide the elements necessary for the development and functioning of giant cells [START_REF] Bartlem | Vascularization and nutrient delivery at root-knot nematode feeding sites in host roots[END_REF]. A transcriptome analysis of microdissected giant cell and neighboring cells induced by M. incognita in Medicago truncatula showed that 740 genes are differentially expressed in neighboring cells at 7 dpi of which 498 genes are common with giant cells. The upregulated genes in neighboring cells are involved in the 'cell', 'signaling' and 'cell-wall' related pathway, suggesting that genes DE in neighboring cells are involved in cell to cell communication and signaling [START_REF] Damiani | Plant genes involved in harbouring symbiotic rhizobia or pathogenic nematodes[END_REF]. Among the genes specifically expressed in the neighboring cells and not in the giant cells are genes encoding small signaling peptides, named phytosulfokines (PSK), and their receptor PSKR1 [START_REF] Rodiuc | Evolutionarily distant pathogens require the Arabidopsis phytosulfokine signalling pathway to establish disease[END_REF]) (Figure 11B). In the absence of functional PSKR1, giant cells arrested their development and failed to fully differentiate. This result indicates that the observed restriction of PSK signaling to cells surrounding giant cells contributes to the isotropic growth and maturation of giant cells [START_REF] Rodiuc | Evolutionarily distant pathogens require the Arabidopsis phytosulfokine signalling pathway to establish disease[END_REF].

Analysis of gene expression in galls and giant cells

One of the first method used to study the expression of "candidate" genes, believed to be involved in gall and giant cell formation was the transformation of plants with gene promoters fused to a GUS (-glucuronidase) [START_REF] Gheysen | Gene expression in nematode feeding sites[END_REF]. This strategy was used to study the spatio-temporal expression of genes encoding key cell-cycle regulators: CYCLINS (CYCs) and CYCLIN DEPENDANT KINASES (CDKs) in A. thaliana [START_REF] Niebel | Induction of cdc2a and cyc1At expression in Arabidopsis thaliana during early phases of nematode-induced feeding cell formation[END_REF][START_REF] De Almeida Engler | Molecular Markers and Cell Cycle Inhibitors Show the Importance of Cell Cycle Progression in Nematode-Induced Galls and Syncytia[END_REF]. In A. thaliana lines expressing GUS under the control of the CDK CDC2a and the mitotic cyclin CYC1 promoters showed a GUS signal in early stages (3 dpi) of gall formation indicating the early activation of these two genes. The use of inhibitors of the cell cycle such as oryzalin affected giant cell normal development demonstrating that the cell cycle is a key process in giant cell formation (de Almeida [START_REF] De Almeida Engler | Molecular Markers and Cell Cycle Inhibitors Show the Importance of Cell Cycle Progression in Nematode-Induced Galls and Syncytia[END_REF]. Though this candidateapproach was very useful to understand the role of known genes in giant cell formation, it was restricted to genes already described and doesn't enable the identification novel genes without a priori. In order to identify genes involved in giant cell formation without a priori, other approaches have been developed, such as promoter trap and transcriptomic analyses.

Promoter trap strategy consists on the random insertion of an Agrobacterium transfer DNA (T-DNA) carrying a GUS reporter and a selectable marker gene in the genome of a plant like A. thaliana [START_REF] Favery | RPE, a plant gene involved in early developmental steps of nematode feeding cells[END_REF]. The mRNA of the reporter gene is transcribed only when the T-DNA is inserted within a transcriptionally active gene. In this case, the expression of the reporter gene reflects the expression of the gene in which the T-DNA was inserted. The screening of thousands of A. thaliana insertion lines following RKN infection and GUS staining of the galls resulted in the identification of several genes involved in giant cell formation such as the RPE gene coding a key enzyme of the pentose phosphate pathway [START_REF] Favery | RPE, a plant gene involved in early developmental steps of nematode feeding cells[END_REF] and the MAP65-3 encoding a microtubule associated protein (Caillaud et al., 2008c). One of the interest of this approach as also the use of collections of T-DNA insertion lines, is to have access to insertion mutants in these genes in order to assess their role in the process studied. This strategy enabled to demonstrate the essential role of RPE and MAP65-3 in the formation of giant cells in A. thaliana [START_REF] Favery | RPE, a plant gene involved in early developmental steps of nematode feeding cells[END_REF]Caillaud et al., 2008c).

To identify the genes that are differentially expressed (DE) in response to RKN infection, transcriptomic analysis was developed (Table 1 and2). The first large-scale transcriptomic studies of plant-nematode interaction were realized by using microarrays. Microarrays are based on a hybridization of labelled nucleic acid samples to a very large set of nucleotide probes attached to a solid support [START_REF] Zvara | Microarray technology[END_REF]. Transcriptome analysis using A. thaliana whole genome microarrays was performed to study the gene expression in dissected galls induced by M. incognita [START_REF] Hammes | Nematode-induced changes of transporter gene expression in Arabidopsis roots[END_REF][START_REF] Jammes | Genome-wide expression profiling of the host response to root-knot nematode infection in Arabidopsisa[END_REF]. [START_REF] Jammes | Genome-wide expression profiling of the host response to root-knot nematode infection in Arabidopsisa[END_REF] showed that, 15% of protein-encoding genes were DE between uninfected roots and galls tissue at 7 and/or 14 dpi. These transcriptomic studies were developed in A. thaliana but also in other plants like tomato and Medicago truncatula [START_REF] Fuller | Differential gene expression in Arabidopsis following infection by plant-parasitic nematodes Meloidogyne incognita and Heterodera schachtii[END_REF][START_REF] Ibrahim | Analysis of Gene expression in soybean (Glycine max) roots in response to the root knot nematode Meloidogyne incognita using microarrays and KEGG pathways[END_REF][START_REF] Damiani | Plant genes involved in harbouring symbiotic rhizobia or pathogenic nematodes[END_REF][START_REF] Bagnaresi | Solanum torvum responses to the root-knot nematode Meloidogyne incognita[END_REF] (Table 1). Development of New Generation Sequencing (NGS) facilitated transcriptome analysis for model and non-model plants. NGS identified 22% of genes encoding proteins DE in A. thaliana galls at 3, 5 and 7 dpi (Yamaguchi et al., 2017). NGS data of galls or whole infected roots are now available for M. truncatula [START_REF] Postnikova | Transcriptome Analysis of Resistant and Susceptible Alfalfa Cultivars Infected With Root-Knot Nematode Meloidogyne incognita[END_REF], tomato [START_REF] Shukla | Transcriptome analysis of root-knot nematode (Meloidogyne incognita)-infected tomato (Solanum lycopersicum) roots reveals complex gene expression profiles and metabolic networks of both host and nematode during susceptible and resistance responses[END_REF][START_REF] Balestrini | Transcriptomic Responses to Water Deficit and Nematode Infection in Mycorrhizal Tomato Roots[END_REF], sweet potato [START_REF] Lee | Transcriptome analysis of root-knot nematode (Meloidogyne incognita)-resistant and susceptible sweetpotato cultivars[END_REF], eggplant [START_REF] Zhang | Transcriptome analysis of eggplant root in response to root-knot nematode infection[END_REF], N. tabacum (Li et al., 2018a), cotton [START_REF] Kumar | Transcriptome analysis of a nematode resistant and susceptible upland cotton line at two critical stages of Meloidogyne incognita infection and development[END_REF], rice [START_REF] Kyndt | Transcriptional reprogramming by root knot and migratory nematode infection in rice[END_REF][START_REF] Petitot | Transcriptomic and histological responses of African rice (Oryza glaberrima) to Meloidogyne graminicola provide new insights into root-knot nematode resistance in monocots[END_REF][START_REF] Zhou | Transcriptome Analysis of Rice Roots in Response to Root-Knot Nematode Infection[END_REF], cucumber [START_REF] Li | Transcriptomic and Histological Analysis of the Response of Susceptible and Resistant Cucumber to Meloidogyne incognita Infection Revealing Complex Resistance via Multiple Signaling Pathways[END_REF], Solanum torvum [START_REF] Sato | Transcriptomic Analysis of Resistant and Susceptible Responses in a New Model Root-Knot Nematode Infection System Using Solanum torvum and Meloidogyne arenaria[END_REF] and mulberry [START_REF] Shao | Transcriptome analysis of resistant and susceptible mulberry responses to Meloidogyne enterolobii infection[END_REF] (Table 2). These studies showed a similar wide reprogramming of gene expression within the roots in response to RKN infection in multiple plant species. This RKN ability to induce similar giant cells in more than 5 000 plant species suggests that the plant molecular mechanisms manipulated by the RKN are conserved across the plant kingdom. Transcriptomic analysis of Portillo et al. (2013) in tomato compared to Barcala et al. (2010) in A. thaliana showed that 132 and 379 genes are differentially expressed genes at 3 and 7 dpi, respectively, in both plants. Using Gene Ontology (GO) algorithm to categorize the tomato and A. thaliana genes based on their functions, the authors showed that only 15 transcription factor families are coregulated in both plants. They are involved in metabolism and defense processes such as MYB and WRKY transcription factor families or hormone signaling pathway like Auxin Response Factors (ARF) (Portillo et al., 2013).

Galls are mixed tissues composed of giant cells, neighboring cells, external root cell layers and nematode. In order to identify the genes specifically expressed in giant cells laser microdissection were developed [START_REF] Fosu-Nyarko | Functional characterization of transcripts expressed in early-stage Meloidogyne javanica-induced giant cells isolated by laser microdissection[END_REF]Barcala et al., 2010;[START_REF] Ji | Transcriptional analysis through RNA sequencing of giant cells induced by Meloidogyne graminicola in rice roots[END_REF]Portillo et al., 2013), that enabled to identify differences in expression patterns between whole gall and giant cells. For example, the comparison between genes expressed at 7 dpi in giant cells and in galls in rice roots infected by M. graminicola identified transcripts of genes coding proteins involved salicylic acid (SA), gibberellic acid (GA) and abscisic acid biosynthesis, induced in whole gall while they are downregulated in giant cells [START_REF] Kyndt | Transcriptional reprogramming by root knot and migratory nematode infection in rice[END_REF][START_REF] Ji | Transcriptional analysis through RNA sequencing of giant cells induced by Meloidogyne graminicola in rice roots[END_REF].

These transcriptomic analyses identified a wide range of genes DE between infected roots or galls and uninfected roots and shed light on the molecular mechanisms putatively involved in the giant cell formation. However, functional validations of these genes are necessary to understand their functions and the processes in which they are involved.

Key processes involved in formation of giant cells

Infection of plants by RKN supposes a tightly controlled reprogramming of plant cell machinery to enable proper development of feeding cells. Here, I will detail key processes involved in the formation of giant cells: modification of the cellular metabolism, cell cycle and cytoskeleton rearrangement, cell wall reorganization, defense suppression and modification of the phytohormone balance.

Metabolism

Giant cells are the unique source of nutrients for the nematodes. When parenchyma cells dedifferentiate to giant cells, their metabolism becomes over active [START_REF] Machado | Metabolic profiling in the roots of coffee plants exposed to the coffee root-knot nematode, Meloidogyne exigua[END_REF].

In addition to dense cytoplasm, cell wall ingrowths proliferate increasing the surface area of the transport of nutrients into the feeding cells (Jones and Northcote, 1972a;[START_REF] Berg | Development of the root-knot nematode feeding cell[END_REF]Sobczak et al., 2011;[START_REF] Rodiuc | On the track of transfer cell formation by specialized plant-parasitic nematodes[END_REF]. Several studies focused on the regulation and the transport of water and solutes during the giant cell formation [START_REF] Hoth | Differential vascularization of nematode-induced feeding sites[END_REF]Hofmann et al., 2010;Absmanner et al., 2013b;[START_REF] Rodiuc | On the track of transfer cell formation by specialized plant-parasitic nematodes[END_REF]. Metabolomic analysis of galls induced by M. incognita in M. truncatula showed an increased content in amino acids, in sucrose, glucose, malate and fumarate (Baldacci-Cresp et al., 2012). Moreover, many genes encoding proteins involved in key metabolism pathways of sugar, amino acid and water transporters are DE in galls [START_REF] Jammes | Genome-wide expression profiling of the host response to root-knot nematode infection in Arabidopsisa[END_REF][START_REF] Hammes | Nematode-induced changes of transporter gene expression in Arabidopsis roots[END_REF]Barcala et al., 2010;[START_REF] Marella | The amino acid permeases AAP3 and AAP6 are involved in root-knot nematode parasitism of arabidopsis[END_REF]. As mentioned above, a key enzyme in the oxidative pentose phosphate pathway, RPE, has been showed to be essential for the early steps of giant cell formation in A. thaliana [START_REF] Favery | RPE, a plant gene involved in early developmental steps of nematode feeding cells[END_REF]. Transcriptome analysis of tomato roots infected by M. incognita showed that genes encoding carbohydrate and sugar transporters (19 genes), lipid transporters (7 genes), aquaporins (13 genes), and peptide, nitrate and amino acid transporters (18 genes) were DE in galls mainly from 14 to 28 dpi [START_REF] Shukla | Transcriptome analysis of root-knot nematode (Meloidogyne incognita)-infected tomato (Solanum lycopersicum) roots reveals complex gene expression profiles and metabolic networks of both host and nematode during susceptible and resistance responses[END_REF]. An upregulation of genes encoding water channel proteins, such as aquaporins, e.g. AtPIP2.5, supports the idea of massive water import into the Giant cells [START_REF] Favery | The Endosymbiosis-Induced Genes ENOD40 and CCS52a Are Involved in Endoparasitic-Nematode Interactions in Medicago truncatula[END_REF][START_REF] Hammes | Nematode-induced changes of transporter gene expression in Arabidopsis roots[END_REF].

Interestingly, the M. incognita effector Mi8D05 has been shown to interact with tomato aquaporin tonoplast intrinsic protein 2 (TIP2) showing that this process is also directly targeted by the nematode [START_REF] Xue | The 8D05 parasitism gene of Meloidogyne incognita is required for successful infection of host roots[END_REF]. Loss of function of some genes coding amino acid transporters like in the Arabidopsis mutants app3 and app6, induced a decrease in the proportion of RKN juveniles that matured into females [START_REF] Marella | The amino acid permeases AAP3 and AAP6 are involved in root-knot nematode parasitism of arabidopsis[END_REF].

In A. thaliana, genes encoding main enzymes that cleave the sucrose, invertase CINV1 and sucrose synthases (SUS), SUS1 and SUS2, are upregulated in giant cells and galls at 3 dpi (Barcala et al., 2010). A. thaliana Knock-out (KO) mutant sus1/sus4, cinv1 and cinv1/cinv2 infected by M. javanica showed an increase in number of galls per root reflecting an increased susceptibility to RKN [START_REF] Cabello | Altered sucrose synthase and invertase expression affects the local and systemic sugar metabolism of nematode-infected Arabidopsis thaliana plants[END_REF]. High-performance liquid chromatography (HPLC) was used to identify key sucrose transporter, SUT/SUC and SWEET, tonoplast monosaccharide transporter (TMT) and vacuolar glucose transporter (VGT) in tomato leaves and roots during early infection (from 12 to 72 hours post infection) by M. incognita (Li et al., 2018b). The expression of three SUTs, 17 SWEETs, two TMTs and one VGT1 showed an upregulation in roots at different times of nematode infection confirmed by qPCR, suggesting that transport of water and solutes occured within the giant cell formation. Since SUT1 gene in tomato has high homology with SUC2 gene in A. thaliana, the authors used Arabidopsis KO, suc2, to evaluate its role in nematode infection [START_REF] Hackel | Sucrose transporter LeSUT1 and LeSUT2 inhibition affects tomato fruit development in different ways[END_REF][START_REF] Gong | Arabidopsis AtSUC2 and AtSUC4, encoding sucrose transporters, are required for abiotic stress tolerance in an ABA-dependent pathway[END_REF]. suc2 KO mutants did not affect the invasion of nematode at early stage of infection but delayed RKN development with a lower proportion of fourth-stage juveniles (J4s) than in the wild-type at 15 dpi. In contrast, the downregulation of SUT1, 2, 3, 4 and 5 were shown in galls at 3, 7 and 14 dpi in rice galls (Xu et al., 2021b). Overexpressing GNS2 that encodes a callose synthase led to an increase in callose deposition, a reduced PD permeability and a decreased sucrose content in rice galls at 7 dpi induced by M. graminicola (Xu et al., 2021b). These results suggest that callose negatively affected sucrose supply and that plasmodesmata may mediate sucrose transport in galls.

Cell wall modification

During their formation, giant cells undergo considerable growth due to extensive cell wall modifications. In A. thaliana, the volume of giant cells can increase by 100 fold from 3 to 40 dpi (Cabrera et al., 2015). The plant cell wall is a complex structure formed by high molecular weight polysaccharides like cellulose and hemicellulose, structural proteins and aromatic substances. The expansion of giant cells requires both loosening and thickening of the cell walls of giant cells [START_REF] Bohlmann | The plant cell wall in the feeding sites of cyst nematodes[END_REF]Escobar et al., 2015). Interestingly, CELL WALL DEGRADING ENZYMES (CWDEs) like cellulases, xylanases, pectate lyases, and expansins were found in M. incognita secretions (Jaubert et al., 2002a;[START_REF] Ledger | Characterization of a new β-1,4endoglucanase gene from the root-knot nematode Meloidogyne incognita and evolutionary scheme for phytonematode family 5 glycosyl hydrolases[END_REF][START_REF] Bellafiore | Direct identification of the Meloidogyne incognita secretome reveals proteins with host cell reprogramming potential[END_REF]. Beyond the CWDEs produced by the RKN, microarrays analysis in A. thaliana and soybean [START_REF] Jammes | Genome-wide expression profiling of the host response to root-knot nematode infection in Arabidopsisa[END_REF]Barcala et al., 2010;[START_REF] Ibrahim | Analysis of Gene expression in soybean (Glycine max) roots in response to the root knot nematode Meloidogyne incognita using microarrays and KEGG pathways[END_REF] showed that most of genes involved in cellulose synthesis and cell wall structural proteins, are upregulated, in plant-nematode interaction. Arabidopsis cell wall-related mutants were used to investigate the role of cell wall components in RKN parasitism [START_REF] Bozbuga | Host-specific signatures of the cell wall changes induced by the plant parasitic nematode, Meloidogyne incognita[END_REF]. Beta-galactosidase-5 (bgal5) and mannan synthesis related 1 (msr1) KO mutants, involved in the synthesis of galactan and mannan respectively, were infected by M. incognita. These two mutants showed a negative impact on RKN development: roots of mutant plants stained with acid fuchsin showed fewer nematodes than wild type plants at 21 dpi and smaller juveniles in bgal5, indicating that they are essential for giant cells function.

Expansins are proteins well known to be involved in plant cell wall expansion (Sobczak et al., 2011). In tomato, the gene coding for expansin LeEXPA5 is expressed in neighboring cells [START_REF] Gal | Expression of a plant expansin is involved in the establishment of root knot nematode parasitism in tomato[END_REF]. In transgenic tomato roots silenced by antisense construct for LeEXPA5, the number of eggs per egg masses produced by nematodes was significantly reduced at 42 dpi compared to the control lines. Moreover, cross sections of galls showed a significant reduction in the diameter of giant cells with smaller galls in LeEXPA5-silenced lines compared to control plants. This suggested that LeEXPA5 is essential for a successful nematode infection.

Cell cycle regulation

Successive mitosis without cytokinesis generate multiple nuclei and lead to the multinucleate status of giant cells [START_REF] Jones | Early stages of nematode-induced giant-cell formation in roots of Impatiens balsamina[END_REF]). An increase of DNA content has been also confirmed within these multiple nuclei, due to endoreduplication cycles that occur in later stage of giant cell development [START_REF] Jones | Root-knot Nematodes and Giant Cells[END_REF]de Almeida Engler and Gheysen, 2013). Transcriptomic analyses and cell biology studies showed that genes encoding mitotic cyclins (e.g. CYCA1.2) and cyclin dependent genes (CDKs) involved in the transition phases of the cell cycle are expressed in the early developing galls at 3 dpi in tomato [START_REF] Niebel | Induction of cdc2a and cyc1At expression in Arabidopsis thaliana during early phases of nematode-induced feeding cell formation[END_REF]Barcala et al., 2010), A. thaliana [START_REF] Jammes | Genome-wide expression profiling of the host response to root-knot nematode infection in Arabidopsisa[END_REF] and rice [START_REF] Ji | Transcriptional analysis through RNA sequencing of giant cells induced by Meloidogyne graminicola in rice roots[END_REF]. Moreover, two cellcycle switch protein CCS52A1 and CCS52B genes involved in the switch from the mitotic cycle to endoreduplication [START_REF] Fülöp | Arabidopsis anaphase-promoting complexes: Multiple activators and wide range of substrates might keep APC perpetually busy[END_REF] are expressed in A. thaliana galls at 7 dpi [START_REF] Favery | The Endosymbiosis-Induced Genes ENOD40 and CCS52a Are Involved in Endoparasitic-Nematode Interactions in Medicago truncatula[END_REF][START_REF] Jammes | Genome-wide expression profiling of the host response to root-knot nematode infection in Arabidopsisa[END_REF][START_REF] Almeida Engler | CCS52 and DEL1 genes are key components of the endocycle in nematode-induced feeding sites[END_REF].

In order to identify the function of these DE genes, several studies used chemical inhibitors of the cell cycle (e.g. colchicine , which inhibits microtubule polymerization and thus assembly of the mitotic spindle) or mutants disrupted for key regulators of cell cycle resulted in defects in giant cell formation [START_REF] Wiggers | The effects of colchicine on number of giant cell nuclei and nematode development in Pisum sativum infected by Meloidogyne incognita[END_REF][START_REF] De Almeida Engler | Molecular Markers and Cell Cycle Inhibitors Show the Importance of Cell Cycle Progression in Nematode-Induced Galls and Syncytia[END_REF]de Almeida Engler and Gheysen, 2013). The RNAi silencing of A. thaliana cell cycle gene, CDKA.1, increases resistance towards RKN (Van De Cappelle et al., 2008). Overexpressing KIP-RELATED PROTEIN 6 (KRP6) in A. thaliana lines, an inhibitor of CDK, accelerates the entry into mitosis but delays mitosis progression confirming a role in the activation of the mitotic cell cycle. Moreover, the overexpression of KRP6 lines infected by M. incognita showed an increase in mitotic activity in galls, resulted in more nuclei in giant cell and induces a strong decrease in the number of egg masses [START_REF] Vieira | The cyclin-dependent kinase inhibitor KRP6 induces mitosis and impairs cytokinesis in giant cells induced by plant-parasitic nematodes in Arabidopsis[END_REF][START_REF] Vieira | Plant Cyclin-Dependent Kinase Inhibitors of the KRP Family: Potent Inhibitors of Root-Knot Nematode Feeding Sites in Plant Roots[END_REF]. The overexpression of DP-E2F-like (DEL) in A. thaliana, a repressor of endoreduplication, leads to galls with smaller giant cells and less number of egg masses while the loss-function of this gene, del1, showed defects in giant cells containing little cytoplasm (De Almeida Engler et al., 2012). Similarly, the role of a negative regulator of DNA replication and transcription ARMADILLO BTB ARABIDOPSIS PROTEIN 1 (ABAP1) has been recently highlighted in A. thaliana galls at 7 and 14 dpi [START_REF] Cabral | The Armadillo BTB Protein ABAP1 Is a Crucial Player in DNA Replication and Transcription of Nematode-Induced Galls[END_REF]. The knockdown or overexpression of ABAP1 showed defects in giant cell formation and in nematode development indicating that the balance of ABAP1 is needed for proper gall development. WEE1, a gene encoding for a protein kinase involved in the G2-to-M transition through the inhibition of activity of CDKs, has been shown to be expressed in A. thaliana galls at 3 dpi and localized to giant cell nuclei [START_REF] Cabral | The plant WEE1 kinase is involved in checkpoint control activation in nematode-induced galls[END_REF].

The knockdown of WEE1 leads to increased mitotic activity in galls and represses the RKN infection and reproduction demonstrating a function for WEE1 in DNA replication in galls.

Cytoskeleton reorganization

RKN induce changes in the organization of actin and microtubule cytoskeleton during giant cell development [START_REF] Almeida Engler | Dynamic cytoskeleton rearrangements in giant cells and syncytia of nematode-infected roots[END_REF]Caillaud et al., 2008a;[START_REF] Engler | Plant actin cytoskeleton re-modeling by plant parasitic nematodes[END_REF][START_REF] Vinet | A "missing" family of classical orthogonal polynomials[END_REF]) (Figure 12). Transcriptomic analyses showed multiple genes encoding key factors of cytoskeleton reorganization differentially expressed in giant cells. An overexpression of α, β, and γ tubulins and actin genes like ACT2 and ACT7 was first observed in A. thaliana galls (De Almeida [START_REF] Almeida Engler | Dynamic cytoskeleton rearrangements in giant cells and syncytia of nematode-infected roots[END_REF]. Moreover, ACTIN DEPOLYMERIZING FACTOR 2 gene (ADF2) is overexpressed in A. thaliana giant cells at late stage of gall development 14 & 21 dpi [START_REF] Clément | Actin-Depolymerizing factor2-mediated actin dynamics are essential for root-knot nematode infection of arabidopsis[END_REF]. A role of cytoskeleton in giant cell formation was confirmed by using different pharmacological analyses. The treatment of A. thaliana infected roots with taxol, that stabilizes microtubules and makes them resistant to depolymerization, led to the arrest of proper giant cells development (De Almeida [START_REF] Almeida Engler | Dynamic cytoskeleton rearrangements in giant cells and syncytia of nematode-infected roots[END_REF]. In cucumber roots, the treatment with Cytochalasin D, inhibitor of the rate of actin polymerization, reduced RKN parasitism with smaller galls and giant cells and with a significant reduction in the number of J2s compared with untreated plants at 21 dpi [START_REF] Liu | The Infection of Cucumber (Cucumis sativus L.) Roots by Meloidogyne incognita Alters the Expression of Actin-Depolymerizing Factor (ADF) Genes, Particularly in Association with Giant Cell Formation[END_REF].

Regulators of the microtubule cytoskeleton are also important for the formation of giant cells.

The Microtubule-Associated Protein65-3 (MAP65-3) protein in A. thaliana plays a key role in cell division and mainly in the organization of microtubules during mitosis and cytokinesis. The map65-3 loss-function lines infected with M. incognita, showed defects in giant cells development: giant cells failed to establish a proper development and degenerate which leads to nematode death (Figure 13) (Escobar et al., 2011). In contrast, transgenic A. thaliana FRAGILE FIBER 2 (fra2) mutant, altered in microtubule-severing katanin protein and displaying a loose in organization of microtubule, were more susceptible to RKN infection with an increase of number of females compared to control plants [START_REF] Meidani | Cell Wall Modifications in Giant Cells Induced by the Plant Parasitic Nematode Meloidogyne incognita in Wild-Type (Col-0) and the fra2 Arabidopsis thaliana Katanin Mutant[END_REF]. This difference observed in these mutants map65-3 and fra2 towards RKN parasitism is probably due to cellulose production defects observed in fra2. The fra2 mutant cell walls are extremely fragile due to reduced cellulose production, thus could lead to an easiest nematode penetration. Interestingly, a M. incognita effector, Mi-PFN3, was showed to disrupt actin filament formation [START_REF] Leelarasamee | The root-knot nematode effector MiPFN3 disrupts plant actin filaments and promotes parasitism[END_REF]. The overexpression of this effector in A.

thaliana plants led to an increase of susceptibility against RKN with an increase in the number of gall. Moreover, the protoplasts of leaves overexpressing the effector MiPFN3 fused to a red fluorescent protein (RFP) showed disrupted actin filaments.

Defense response

Transcriptomic analyses showed that most defense-associated genes are repressed in galls and/or giant cells. WRKY transcription factors, PATHOGENESIS-RELATED (PR) and lipoxygenases genes, are key markers and/or regulators of plant defense pathways in plant species like A. thaliana, tomato, M. truncatula and cotton [START_REF] Jammes | Genome-wide expression profiling of the host response to root-knot nematode infection in Arabidopsisa[END_REF]Barcala et al., 2010;[START_REF] Damiani | Plant genes involved in harbouring symbiotic rhizobia or pathogenic nematodes[END_REF][START_REF] Shukla | Transcriptome analysis of root-knot nematode (Meloidogyne incognita)-infected tomato (Solanum lycopersicum) roots reveals complex gene expression profiles and metabolic networks of both host and nematode during susceptible and resistance responses[END_REF][START_REF] Kumar | Transcriptome analysis of a nematode resistant and susceptible upland cotton line at two critical stages of Meloidogyne incognita infection and development[END_REF]. The WRKY family of transcription factors, known to act downstream several plant hormones in the activation of plant defenses, are generally repressed [START_REF] Jammes | Genome-wide expression profiling of the host response to root-knot nematode infection in Arabidopsisa[END_REF]Escobar et al., 2011). However, WRKY25 has been shown to be induced in susceptible tomato upon M. javanica infection. Histological analysis of WRKY45:GUS lines showed WRKY45 expression at early stage of infection (5 dpi) but also through gall formation and maintenance (15 and 28 dpi) [START_REF] Chinnapandi | SlWRKY45 , nematode-responsive tomato WRKY gene, enhances susceptibility to the root knot nematode; M. javanica infection[END_REF]. The overexpression of this gene was associated with a decrease in SA-(PR1) and jasmonic acid (JA)-defense marker genes and with an accelerated nematode infection and an increased in number of females and giant cell size in galls at 28 dpi.

Pathogenesis-related (PR) proteins, PR-1 and PR-2 salicylic acid (SA) marker genes have been shown to be induced in A. thaliana infected roots at 9 dpi and repressed at 14 dpi [START_REF] Hamamouch | Expression of Arabidopsis pathogenesis-related genes during nematode infection[END_REF]. The expression of two lipoxygenases, LOX3 and LOX4, were analyzed in A. thaliana infected by M. javanica [START_REF] Ozalvo | Two closely related members of Arabidopsis 13-lipoxygenases (13-LOXs), LOX3 and LOX4, reveal distinct functions in response to plant-parasitic nematode infection[END_REF]. Gene reporter GUS constructs showed local induction of LOX3 expression all along galls formation while LOX4 promoter was activated by RKN infection, although the GUS signal weakened as galls formation progressed. Intriguingly, the infection of lox3 and lox4 loss-function T-DNA mutants showed a decrease in number of females for lox3 lines in comparison to control plants, whereas lox4 lines showed an increase of susceptibility, with a higher in number of females and egg masses compared to control plants. This increase of susceptibility was also accompanied with an increase of JA and ethylene responsive genes 2 and/or 5 dpi. These results suggested that LOX3 and LOX4 interfere with different pathways that might affect the plant response to nematode infection while LOX4 might control defense toward nematode infection.

Several hormones have been shown to be involved in defense response. Plant defense hormones, such as SA, JA and ethylene control various aspects of plant defense responses to PPNs. Recent reviews have described the complexity of JA-and SA-dependent pathway regulation during nematode infection, and the role of other phytohormones, such as gibberellin and abscisic acid (ABA), in regulating JA-or SA-dependent signaling (Gheysen and Mitchum, 2019;Sato et al., 2019). In tomato, transcriptomic analyses between RKN-resistant and susceptible tomatoes showed that several genes related to ethylene such as aminocyclopropane-1-carboxylic acid oxidase (ACO), ethylene-responsive transcription factors (ERFs), several genes involved in JA-synthesis and SA-responsive signaling were differentially expressed during disease development [START_REF] Shukla | Transcriptome analysis of root-knot nematode (Meloidogyne incognita)-infected tomato (Solanum lycopersicum) roots reveals complex gene expression profiles and metabolic networks of both host and nematode during susceptible and resistance responses[END_REF]. In A. thaliana galls, genes related to ethylene responses were mostly downregulated while only one gene related SA was induced at 3 dpi (Barcala et al., 2010). Many transcripts involved in JA biosynthesis were shown to be repressed in rice giant cells [START_REF] Ji | Transcriptional analysis through RNA sequencing of giant cells induced by Meloidogyne graminicola in rice roots[END_REF].

Exogenous applications of defense related hormones were used to understand their role during RKN infection. In rice, the exogenous application of ethylene (ethephon) and JA (methyl jasmonate-MeJA) induced a strong systemic defense response against M. graminicola with an increased resistance reflected by a decrease in numbers of galls associated to upregulation of PR1 gene [START_REF] Nahar | Brassinosteroids suppress rice defense against root-knot nematodes through antagonism with the jasmonate pathway[END_REF]. Foliar application of MeJA significantly reduced the level of M. graminicola infection in rice [START_REF] Verbeek | Jasmonate-Induced Defense Mechanisms in the Belowground Antagonistic Interaction Between Pythium arrhenomanes and Meloidogyne graminicola in Rice[END_REF]. SA soil drenching reduced gall formations in tomato infected by M. incognita [START_REF] Molinari | Systemic acquired resistance activation in solanaceous crops as a management strategy against root-knot nematodes[END_REF] while foliar treatment with MeJA significantly reduced the infection of RKN [START_REF] Fujimoto | Expression profile of jasmonic acid-induced genes and the induced resistance against the root-knot nematode (Meloidogyne incognita) in tomato plants (Solanum lycopersicum) after foliar treatment with methyl jasmonate[END_REF]. Tomatoes inoculated by M. incognita and treated with SA showed an upregulation in the expression of PR1 gene and a decreased number of galls [START_REF] Bozbuga | Expressions of Pathogenesis related 1 (PR1) Gene in Solanum lycopersicum and Influence of Salicylic Acid Exposures on Host-Meloidogyne incognita Interactions[END_REF].

The overexpression of NPR1, an activator of SA signalization, in tobacco also led to a decreased number of galls and egg masses induced by M. incognita [START_REF] Priya | Transgenic tobacco plants constitutively expressing Arabidopsis NPR1 show enhanced resistance to root-knot nematode, Meloidogyne incognita[END_REF]. Moreover, a M. incognita effector, Mi-CM-3, encoding a chorismate mutase, was shown to suppress plant immunity by regulating the SA pathway in infected Nicotiana benthamiana.

The transient expression of this effector causes a reduction in SA level and increased susceptibility to nematode infection (Wang et al., 2018b).

Hydrogen peroxide (H 2 O 2 ) and reactive oxygen species (ROS) act as signal molecules to activate plant immunity [START_REF] Forman | Signaling functions of reactive oxygen species[END_REF]. Moreover, treatment with JA of tomato seedlings infected by M. incognita showed a 40 % decrease in H 2 O 2 contents [START_REF] Bali | Jasmonic acid-induced tolerance to root-knot nematodes in tomato plants through altered photosynthetic and antioxidative defense mechanisms[END_REF].

In addition, JA treatment (100 nM) increased the activation of ROS-scavenging enzyme superoxide dismutase SOD activity in tomato treated and infected by M. incognita compared to JA-treated control [START_REF] Bali | Seed Priming with Jasmonic Acid Counteracts Root Knot Nematode Infection in Tomato by Modulating the Activity and Expression of Antioxidative Enzymes[END_REF].

Interestingly, ethylene signalization was shown to modulate the attraction of RKN to the plant. Root tomato exudates were significantly more attractive to the M. incognita in the knockdown erf-e2 mutant [START_REF] Dyer | Ethylene Response Factor (ERF) genes modulate plant root exudate composition and the attraction of plant parasitic nematodes[END_REF]. The repression of EIN2b involved in ethylene signalization led to an increase in resistance in rice towards M. graminicola [START_REF] Nahar | The jasmonate pathway is a key player in systemically induced defense against root knot nematodes in rice[END_REF]

Modulation of auxin and cytokinin phytohormones

Nematode interaction with their host implies the modulation of phytohormones balance to either suppress plant defenses (as described above) or promote the differentiation of their feeding sites (reviewed in Gheysen and Mitchum, 2019;[START_REF] Oosterbeek | Sedentary Plant-Parasitic Nematodes Alter Auxin Homeostasis via Multiple Strategies[END_REF]. Early studies highlighted the main role of auxin and cytokinin in giant cell formation [START_REF] Balasubramanian | Presence of indole compound in nematode galls[END_REF][START_REF] Dimalla | Cytokinins in the root-knot nematode, Meloidogyne incognita[END_REF]. The presence of cytokinin and auxin-like compounds in M. incognita secretions was evidenced by mass spectrometry analyses of induced secretions, suggesting that these hormones could play crucial functions in feeding cell development [START_REF] De Meutter | Identification of cytokinins produced by the plant parasitic nematodes Heterodera schachtii and Meloidogyne incognita[END_REF]. Auxin or indole-3-acetic acid (IAA), is a simple signaling molecule that plays a critical role in plant development and growth regulating cell division, elongation, and differentiation. Auxin is involved in a wide range of developmental processes e.g. organ differentiation and lateral root initiation [START_REF] Quint | Auxin signaling Marcel[END_REF][START_REF] Gutierrez | Auxin Controls Arabidopsis Adventitious Root Initiation by Regulating Jasmonic Acid Homeostasis[END_REF][START_REF] Guilfoyle | The PB1 domain in auxin response factor and aux/IAA proteins: A versatile protein interaction module in the auxin response[END_REF][START_REF] Di | The biosynthesis of auxin: how many paths truly lead to IAA?[END_REF][START_REF] Majda | The Role of Auxin in Cell Wall Expansion[END_REF]. Auxin triggers cell or tissue specific responses though a well-studied signaling pathway. Inside the cell, auxin is bound by the TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX PROTEIN (TIR1/AFB) receptors. TIR1 proteins bind three different ligands: an SCF-TIR1 ubiquitin ligase complex, auxin and AUX/IAA proteins. The binding of all these three ligands at the same time triggers the ubiquitination of AUX/IAA proteins and their subsequent degradation by the 26S proteasome. AUX/IAA proteins repress the transcription of auxin-induced genes by building multimers with other AUX/IAA proteins and with AUXIN RESPONSE FACTOR (ARF) transcription factors. After the auxin-induced degradation of Aux/IAAs, ARF transcription factors are free to activate or repress the expression of genes to which promoters they are bound (reviewed in [START_REF] Chandler | Auxin response factors[END_REF]Li et al., 2016b). Microarray analyses of A. thaliana gall transcripts showed an early activation of genes responsible for auxin homeostasis and auxin-responsive genes, while repressors of auxin responses were downregulated [START_REF] Hammes | Nematode-induced changes of transporter gene expression in Arabidopsis roots[END_REF][START_REF] Jammes | Genome-wide expression profiling of the host response to root-knot nematode infection in Arabidopsisa[END_REF]Barcala et al., 2010). The use of lines expressing the reporter GUS gene under the control auxin-responsive promoter GH3 or the synthetic auxin responsive promoter DR5 showed activation of these two promoters in galls induced by RKN (Hutangura et al., 1999;[START_REF] Karczmarek | Feeding cell development by cyst and rootknot nematodes involves a similar early, local and transient activation of a specific auxininducible promoter element[END_REF]Absmanner et al., 2013). Within A. thaliana galls, DR5:GUS lines showed a strong signal in giant cells but also in neighboring cells at 4 dpi (Figure 14) (Cabrera et al., 2014b). Mutants in components of the auxin efflux carrier PIN-FORMEDs involved in the root-specific auxin transport, pin2 and pin3, showed an increase of resistance towards RKN [START_REF] Mazarei | Expression of an Arabidopsis phosphoglycerate mutase homologue is localized to apical meristems, regulated by hormones, and induced by sedentary plant-parasitic nematodes[END_REF][START_REF] Kyndt | Redirection of auxin flow in Arabidopsis thaliana roots after infection by root-knot nematodes[END_REF]. PIN2 and PIN3 appear to be important for the delivery of auxin into the giant cells [START_REF] Grunewald | Manipulation of Auxin Transport in Plant Roots during Rhizobium Symbiosis and Nematode Parasitism[END_REF]. In A. thaliana, two ARFs, ARF7 and ARF19, positively regulate lateral root formation through activation of the plant-specific transcriptional regulators LATERAL ORGAN BOUNDARIES-DOMAIN, and in particular LBD16. The loss of function of LBD16, induced a defect in giant cell formation demonstrating its role in giant cell formation (Cabrera et al., 2014b). The expression of this LBD16 is also detected also in M.

javanica and M. arenaria galls [START_REF] Okushima | ARF7 and ARF19 regulate lateral root formation via direct activation of LBD/ASL genes in Arabidopsis[END_REF][START_REF] Lee | LBD18/ASL20 regulates lateral root formation in combination with LBD16/ASL18 downstream of ARF7 and ARF19 in Arabidopsis[END_REF]. The auxin-cytokinin balance is considered the main hormonal control system in plant [START_REF] Moubayidin | Cytokinin-auxin crosstalk[END_REF].

Auxin induces cell division in the meristems while cytokinin control cell division and differentiation [START_REF] Moubayidin | Cytokinin-auxin crosstalk[END_REF][START_REF] Bielach | Genetic approach towards the identification of auxin -cytokinin crosstalk components involved in root development[END_REF][START_REF] Schaller | Cytokinin and the cell cycle[END_REF]. The activation of cytokinin-regulated genes has also been show in early stages of gall formation, by using the cytokinin-responsive promoter AAR5 [START_REF] Lohar | Cytokinins play opposite roles in lateral root formation, and nematode and Rhizobial symbioses[END_REF]. ARR5 expression was specifically absent in mature giant cells, although dividing cells around the giant cells continued to express ARR5. In this study, the authors also showed that the overexpression of the CYTOKININ OXIDASES (CKX), gene coding for an enzyme that catalyzes the degradation of cytokinin, induced a resistance towards RKN. Even though these results support a role for cytokinin in gall formation, cytokinin signalization is still poorly described in plant response to RKN and the cytokinin responsive promoter TCS is not induced in galls. On the contrary, this cytokinin marker is activated in syncytia induced by cyst nematodes (Absmanner et al., 2013b) and the role of cytokinin is more described in syncytia [START_REF] Ali | An Arabidopsis ATPase gene involved in nematode-induced syncytium development and abiotic stress responses[END_REF].

Other phytohormones were also shown to be involved in formation of giant cells. In rice infected by M. graminicola, the application of low concentration of gibberellic acid (GA) enhanced nematode infection in rice [START_REF] Yimer | Gibberellin antagonizes jasmonate-induced defense against Meloidogyne graminicola in rice[END_REF]. Brassinosteroids are involved in response to M. graminicola [START_REF] Nahar | Brassinosteroids suppress rice defense against root-knot nematodes through antagonism with the jasmonate pathway[END_REF][START_REF] Kyndt | Interplay between Carotenoids, Abscisic Acid and Jasmonate Guides the Compatible Rice-Meloidogyne graminicola Interaction[END_REF]. The application of abscisic acid (ABA) increases the susceptibility of rice RKN infection. Strigolactones suppresses jasmonate accumulation and enhance M. graminicola infection in rice [START_REF] Lahari | Strigolactones enhance root-knot nematode (Meloidogyne graminicola) infection in rice by antagonizing the jasmonate pathway[END_REF].

MicroRNAs

As presented in the previous paragraphs, all transcriptome analyses performed in various plant species showed that the formation of galls and/or giant cells was the result of extensive reprogramming of plant cell transcription in host root. With the development of NGS, many genes with differentially expressed between galls and uninfected roots were identified in multiple plant species. On the contrary, how does this reprogrammation of gene expression occur in root cells remains poorly understood and the regulators involved in this process are mainly unknown. During my thesis, I focused my investigations on the role in formation of giant cells of a family of small non coding RNAs, the microRNAs. As a part of miRNAs are highly conserved within the plant kingdom, they can be a part of the conserved mechanisms manipulated by RKN to induce the formation of the feeding site.

Generalities

Following transcription of eukaryotic protein-coding genes, the primary RNA transcript is processed to one or several mature messenger RNAs (mRNA). mRNAs are exported from the nucleus to the cytoplasm, where they are translated to amino acid sequence to form protein.

Eukaryote cells have many sophisticated ways to control gene expression acting at different levels: chromatin, transcriptional level, RNA processing, RNA transport and translational controls. RNA interference (RNAi) known also as "gene silencing" was first discovered in plants in 1990[START_REF] Van Der Krol | Flavonoid genes in petunia: Addition of a limited number of gene copies may lead to a suppression of gene expression[END_REF][START_REF] Napoli | Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans[END_REF]. In order to enhance the purple color of petunia petals, a vector carrying the coding sequence of a gene involved in the biosynthesis of the purple pigment, was introduced to the plant. The overexpression of this gene caused a reverting phenotype with partially or completely white flowers due to the extinction of both exogenous and endogenous genes. In 1998, Dr Andrew Fire and Dr Craig Mello discovered in C. elegans a regulatory mechanism, named RNAi, that degrades mRNA from a specific gene. RNAi is activated when double-stranded RNA (dsRNA) molecules are detected in the cell. dsRNA was shown to have a silencing effect by targeting mRNA with complementary sequence [START_REF] Fire | Potent and specific genetic interference by double-stranded RNA in {$\$textless}i{$\$textgreater}Caenorhabditis elegans{$\$textless}/i{$\$textgreater}[END_REF][START_REF] Tabara | The dsRNA binding protein RDE-4 interacts with RDE-1, DCR-1, and a DExH-box helicase to direct RNAi in C. elegans[END_REF]. RNAi was found in plants, animals, and humans and is widely used as a method to repress gene's expression in functional analysis. Since the discovery of RNAi, different classes of small non coding RNAs (ncRNAs), involved in various processes regulation of gene expression, have been identified. ncRNAs are classified depending on their size, their biogenesis and their regulation process. Small ncRNAs have a length of 18 to 30 nucleotides (nt). In plants, small ncRNAs are subclassified based on the biogenesis and /or functions: microRNAs (miRNA), hairpin RNAs (hpRNA), secondary siRNAs, natural antisense transcript siRNAs (reviewed in Axtell, 2013a; Borges and Martienssen, 2015) (Figure 15). Small ncRNAs regulate gene expression at the transcriptional level before gene transcription by DNA methylation or at the posttranscriptional level by cleaving mRNAs or inhibiting their translation. "Post Transcriptional Gene Silencing" (PTGS) is driven by miRNA and siRNA [START_REF] Mallory | MicroRNA-directed regulation: to cleave or not to cleave[END_REF] while "Transcriptional Gene Silencing" (TGS) is driven by RNA [START_REF] Ekwall | The RITS Complex -A Direct Link between Small RNA and Heterochromatin[END_REF]. siRNAs have a very complex biogenesis and a mode of action. During my thesis I focused my studies on miRNAs, that I will be developed it in my introduction.

microRNAs and PTGS

Biogenesis and maturation of microRNAs

miRNAs are small ncRNAs of 20 to 22 nt in plants encoded by MIR genes often organized in multigene family within plant genomes (Jones-Rhoades, 2012). MIR genes are transcribed by RNA polymerase II (Pol II) [START_REF] Kim | The role of Mediator in small and long noncoding RNA production in Arabidopsis thaliana[END_REF] to generate a single-stranded primary transcript (pri-miRNA) (Figure 16). Several core activators interact with RNA Pol II such as NEGATIVE ON TATA LESS2 (NOT2), the putative MYB domain-containing DNA-binding protein CELL DIVISION CYCLE 5 (CDC5) and the Elongator complex [START_REF] Wang | NOT2 Proteins promote polymerase II-dependent transcription and interact with multiple microRNA biogenesis factors in Arabidopsis[END_REF]Zhang et al., 2013;[START_REF] Fang | Transcription and processing of primary microRNAs are coupled by Elongator complex in Arabidopsis[END_REF]. The pri-miRNAs are polyadenylated at the 3' end, folds into a hairpin structure, thanks to a partial complementarity of its sequence and is then cleaved by the endonuclease DICER LIKE1 (DCL1) to generate a shorter stem loop precursors, the pre-miRNAs [START_REF] Szarzynska | Gene structures and processing of Arabidopsis thaliana HYL1dependent pri-miRNAs[END_REF]. These pre-miRNAs are processed by the dicing complex composed of DCL1 and other core factors (HYPONASTIC LEAVES1 (HYL1) and SERRATE (SE)) [START_REF] Kurihara | Arabidopsis micro-RNA biogenesis through Dicer-like 1 protein functions[END_REF][START_REF] Fukudome | Plant dicer-like proteins: double-stranded RNA-cleaving enzymes for small RNA biogenesis[END_REF]. The pri-miRNA is then cleaved, in the nucleus, by Dicer-like 1, in association with hyponastic leaves 1 and serrate, to produce a precursor miRNA (pre-miRNA). The pre-miRNA is, in turn, cleaved by DCL1 and its cofactors, thus generating a duplex composed of the mature miRNA and its complementary strand. The HUA ENHANCER 1 protein then adds a methyl group to the OH end of each strand of the miRNA duplex, to protect against degradation. The miRNA duplex is then actively transported from the nucleus to the cytosol through interaction with the HASTY (HST) exportin. One of the two strands of the duplex is then loaded onto the ARGONAUTE-1 protein, the main constituent of the multiprotein RNA-induced silencing complex (RISC). The AGO1-associated strand guides the RISC to target mRNAs by sequence complementarity, resulting in target cleavage or the inhibition of protein synthesis. DCL1, Dicer-like 1; HYL1, hyponastic leaves 1; SE, serrate; HEN1, HUA ENHANCER 1 protein; HST, hasty; AGO1, argonaute 1. [START_REF] Jaubert-Possamai | MicroRNAs, New Players in the Plant-Nematode Interaction[END_REF] Within the nucleus, DCL1 interacts with these cofactors to unit pre-miRNA in nuclear bodies, called dicing bodies [START_REF] Song | Arabidopsis primary microRNA processing proteins HYL1 and DCL1 define a nuclear body distinct from the Cajal body[END_REF][START_REF] Laubinger | Dual roles of the nuclear cap-binding complex and SERRATE in pre-mRNA splicing and microRNA processing in Arabidopsis thaliana[END_REF]. DCL1 cleaves pre-miRNA into miRNA-5p/miRNA-3p mature duplex [START_REF] Margis | The evolution and diversification of Dicers in plants[END_REF]. This miRNA duplex is stabilized by HUA ENHANCER 1 (HEN1) that deposits a methyl group onto the OH group of each strand to protect it from degradation [START_REF] Li | Methylation protects miRNAs and siRNAs from a 3′-end uridylation activity in Arabidopsis[END_REF][START_REF] Yu | Methylation as a crucial step in plant microRNA biogenesis[END_REF]. The duplex is then exported from the nucleus to the cytoplasm by HST [START_REF] Mee | Nuclear processing and export of microRNAs in Arabidopsis[END_REF].

Mode of action of microRNAs

ARGONAUTE-1 protein (AGO1) is the main protein of the RNA-induced silencing complex (RISC). AGO1 forms a complex with HEAT SHOCK PROTEIN (HSP90) that allows miRNA-5p/miRNA-3p to be incorporated [START_REF] Iki | In vitro assembly of plant RNA-induced silencing complexes facilitated by molecular chaperone HSP90[END_REF]. AGOs are the main silencing effectors that fold into a bilobal structure displaying a central groove for duplex binding [START_REF] Mallory | Form, function, and regulation of ARGONAUTE proteins[END_REF]. One strand of the miRNA-5p/miRNA-3p duplex, known as the guide strand, is loaded in AGO1 complex whereas the other strand is removed. The mature miRNA strand loaded in AGO1 guides RISC to a mRNA targeted by sequence complementarity resulting in the silencing of the corresponding gene [START_REF] Yu | The "how" and "where" of plant microRNAs[END_REF].

Plant gene silencing by miRNAs occurs at two levels (reviewed in [START_REF] Voinnet | Origin, biogenesis, and activity of plant microRNAs[END_REF][START_REF] Yu | The "how" and "where" of plant microRNAs[END_REF] depending on the degree of sequence complementary between miRNAs and targeted mRNA [START_REF] Hutvágner | A microRNA in a multiple-turnover RNAi enzyme complex[END_REF]Franco-Zorrilla et al., 2007). When sequence complementarity between the miRNA and its target is perfect or nearly perfect, the RISC cleaves the targeted mRNA. When it has a reduced degree of complementarity, RISC represses mRNA translation by preventing ribosome access to mRNAs. In plants, repression of translation is less observed than mRNA cleavage. AGO1 mediates miRNA target cleaved, followed by the degradation of the cleavage fragments by exonucleases such as EXORIBONUCLEASE 4 (XRN4) [START_REF] Souret | AtXRN4 degrades mRNA in Arabidopsis and its substrates include selected miRNA targets[END_REF]. The cleavage zone is generally located at the center the miRNA sequence at position 10 or 11 (Palatnik et al., 2007) (Figure 17).

Most MIR genes are organized as multigenic families. The different MIR genes of a miRNA families show difference in the sequence of the precursor but identical, or nearly identical, sequence of the mature miRNA and share often the same targets (Figure 17) (Palatnik et al., 2007). Several miRNAs families are conserved within the plant kingdom, e.g. miR156, miR169, miR166 that were identified in more than 40 different plant species [START_REF] Sunkar | Identification of novel and candidate miRNAs in rice by high throughput sequencing[END_REF]. However, the number of miRNAs specific for a plant species is higher than the number of conserved miRNAs [START_REF] Cui | The evolution of microRNAs in plants[END_REF]. Several miRNAs/targets pairs are conserved between plant species [START_REF] Cuperus | Evolution and Functional Diversification of MIRNA Genes[END_REF]Jones-Rhoades, 2012), e.g. miR159 and MYB33 transcription factor family in A. thaliana, tomato and rice [START_REF] Allen | MicroR159 regulation of most conserved targets in Arabidopsis has negligible phenotypic effects[END_REF]da Silva et al., 2017;[START_REF] Zhao | Suppression of microRNA159 impacts multiple agronomic traits in rice (Oryza sativa L.)[END_REF]. In some cases, the functions of these miRNAs and their targets can be also conserved. The regulation of two auxin response factors ARF6 and ARF8 by miR167 family are conserved in tomato and A. thaliana, and the cleavage by miR167 shows similar developmental functions in the two plants [START_REF] Liu | Down-regulation of AUXIN RESPONSE FACTORS 6 and 8 by microRNA 167 leads to floral development defects and female sterility in tomato[END_REF]Wang et al., 2015a).

miRNA/targets has complexes regulations and are generally integrated in a complex network. While the same miRNA can regulate several targets, e.g. miR164 that targets the two genes NAC1 and CUC2 in A. thaliana, several miRNAs can regulate the same targets. miRNAs can also trigger the production of siRNAs, known as phased secondary siRNAs (phasiRNAs) that silence mRNA at PTGS level (reviewed in [START_REF] Rogers | Biogenesis, turnover, and mode of action of plant microRNAs[END_REF].

Role of microRNAs

microRNAs are major regulators of gene expression in plants and their roles have been described in various processes from plant development to response to biotic and abiotic stresses but also in trans-kingdom communication between pathogens and their hosts [START_REF] Sunkar | Small RNAs as big players in plant abiotic stress responses and nutrient deprivation[END_REF]Weiberg et al., 2015;[START_REF] Couzigou | Plant microRNAs : key regulators of root architecture and biotic interactions[END_REF]. NGS analysis of small RNAs identify many microRNAs DE in various biological conditions suggesting a role for these microRNAs in these processes. However, functional validation is needed to test the functions of these genes, in particular by using mutants of these miRNAs and their target. In this chapter, I will present plant miRNAs for which a role has been biologically validated.

MicroRNAs in plant development

microRNAs regulate shoot, leaf and root developmental processes. Some microRNAs can act individually while other miRNAs regulate coordinately their targets during plant developmental processes.

Multiple microRNAs and their targets were shown to regulate leaf development. Leaf development mostly relies on the function of the shoot apical meristem (SAM). It begins with the initiation of leaf primordia then polarity establishment till the leaf acquired its final shape and size [START_REF] Laufs | Cellular parameters of the shoot apical meristem in Arabidopsis[END_REF][START_REF] Barton | Twenty years on: The inner workings of the shoot apical meristem, a developmental dynamo[END_REF]. miR394 and its target LEAF CURLING RESPONSIVENESS (LCR); miR396/GROWTH REGULATING FACTORS (GRFs) and miR319/TEOSINTE BRANCHED1, CYCLOIDEA and PROLIFERATING CELL NUCLEAR ANTIGEN BINDING FACTOR (TCP) are involved in the control of SAM [START_REF] Schoof | The stem cell population of Arabidopsis shoot meristems is maintained by a regulatory loop between the CLAVATA and WUSCHEL genes[END_REF][START_REF] Efroni | A protracted and dynamic maturation schedule underlies Arabidopsis leaf development[END_REF][START_REF] Schommer | Control of jasmonate biosynthesis and senescence by miR319 targets[END_REF][START_REF] Liu | Ectopic expression of miR396 suppresses GRF target gene expression and alters leaf growth in Arabidopsis[END_REF][START_REF] Baucher | A role for the miR396/GRF network in specification of organ type during flower development, as supported by ectopic expression of Populus trichocarpa miR396c in transgenic tobacco[END_REF]Li et al., 2016a). Other microRNAs control leaf polarity like miR164 /CUP-SHAPED COTYLEDON2 (CUC2), miR165/miR166 /HOMEODOMAIN LEUCINE ZIPPER (HD-ZIP) and miR390/TAS3-tasiRNA [START_REF] Peaucelle | Plants expressing a miR164-resistant CUC2 gene reveal the importance of post-meristematic maintenance of phyllotaxy in Arabidopsis[END_REF][START_REF] Huijser | The control of developmental phase transitions in plants[END_REF][START_REF] Moon | How a leaf gets its shape[END_REF][START_REF] Rubio-Somoza | MicroRNA networks and developmental plasticity in plants[END_REF][START_REF] Felippes | A single miR390 targeting event is sufficient for triggering TAS3-tasiRNA biogenesis in Arabidopsis[END_REF].

An example of coordination of several miRNAs is the interplay between miR396 and miR319 and their targets in the control of cell proliferation and leaf shape in A. thaliana [START_REF] Schommer | Repression of cell proliferation by miR319-regulated TCP4[END_REF], also with miR164 in the regulation of the shape and the size of the limbus (Rubio-Somoza and Weigel, 2011) and with miR156 in the switch from the juvenile to the adult phases of vegetative development [START_REF] Rodriguez | Control of cell proliferation by microRNAs in plants[END_REF]) (Figure 18). miR396 regulates leaf morphology by targeting GROWTH REGULATION FACTORS (GRF) transcription factor family [START_REF] Liu | Ectopic expression of miR396 suppresses GRF target gene expression and alters leaf growth in Arabidopsis[END_REF][START_REF] Baucher | A role for the miR396/GRF network in specification of organ type during flower development, as supported by ectopic expression of Populus trichocarpa miR396c in transgenic tobacco[END_REF]. GRFs are highly expressed in leaf meristematic tissue and positively regulate leaf size by promoting cell proliferation [START_REF] Kim | The AtGRF family of putative transcription factors is involved in leaf and cotyledon growth in Arabidopsis[END_REF]. Overexpression of miR396 induces the silencing of six GRF genes and leads to the formation of narrow leaves with a reduced size like the grf KO mutants [START_REF] Horiguchi | The transcription factor AtGRF5 and the transcription coactivator AN3 regulate cell proliferation in leaf primordia of Arabidopsis thaliana[END_REF][START_REF] Jeong | GROWTH-REGULATING FACTOR4 of Arabidopsis thaliana is required for development of leaves, cotyledons, and shoot apical meristem[END_REF]. The expression of MIR396 is activated by TCP4 which is targeted by miR319 that is involved in leaf shape development (Palatnik et al., 2007;[START_REF] Schommer | Repression of cell proliferation by miR319-regulated TCP4[END_REF]. A.

thaliana transgenic plants overexpressing miR319 have increased leaf size, and a similar phenotype is observed in the tcp KO mutants [START_REF] Schommer | Control of jasmonate biosynthesis and senescence by miR319 targets[END_REF] The flowering stage, is regulated by an important number of miRNAs and their targets to synchronize flowering [START_REF] Spanudakis | The role of microRNAs in the control of flowering time[END_REF][START_REF] Teotia | To bloom or not to bloom: Role of micrornas in plant flowering[END_REF][START_REF] Samad | MicroRNA and transcription factor: Key players in plant regulatory network[END_REF]. We can cite for example, miR172/APETALA2-like (AP2) [START_REF] Zhu | Regulation of flowering time and floral patterning by miR172[END_REF][START_REF] Teotia | To bloom or not to bloom: Role of micrornas in plant flowering[END_REF][START_REF] Tripathi | Genome-wide analysis of the SPL/miR156 module and its interaction with the AP2/miR172 unit in barley[END_REF][START_REF] Ó'maoiléidigh | Systematic analyses of the MIR172 family members of Arabidopsis define their distinct roles in regulation of APETALA2 during floral transition[END_REF], miR156/ SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) [START_REF] Chuck | The maize tasselseed4 microRNA controls sex determination and meristem cell fate by targeting Tasselseed6/indeterminate spikelet1[END_REF][START_REF] Efroni | A protracted and dynamic maturation schedule underlies Arabidopsis leaf development[END_REF][START_REF] Yamaguchi | Regulation of reproductive development by non-coding RNA in Arabidopsis: To flower or not to flower[END_REF][START_REF] Hong | Floral induction and flower formation-the role and potential applications of miRNAs[END_REF][START_REF] Teotia | To bloom or not to bloom: Role of micrornas in plant flowering[END_REF][START_REF] Tripathi | Genome-wide analysis of the SPL/miR156 module and its interaction with the AP2/miR172 unit in barley[END_REF] and miR319/TCP [START_REF] Schommer | Control of jasmonate biosynthesis and senescence by miR319 targets[END_REF]. As described above for leaf development, these microRNAs act coordinately to regulate transition from vegetative to flowering phase. miR156 family targets 11 of the 17 SPL genes. Among these genes, SPL3, 4, and 5 promote floral transition [START_REF] Chuck | The maize tasselseed4 microRNA controls sex determination and meristem cell fate by targeting Tasselseed6/indeterminate spikelet1[END_REF][START_REF] Xie | Gradual Increase of miR156 Regulates Temporal Expression Changes of Numerous Genes during Leaf Development in Rice[END_REF][START_REF] Hong | Floral induction and flower formation-the role and potential applications of miRNAs[END_REF][START_REF] Teotia | To bloom or not to bloom: Role of micrornas in plant flowering[END_REF]. Tomato plants overexpressing SlymiR156a showed a delay in flowering and an extended juvenile phase (Zhang et al., 2011b). miR172 repress translation of AP2, a floral repressing transcription factor inducing defect in floral organ identity and mimicking the phenotype of the loss-of-function ap2 mutants (Chen, 2004;[START_REF] Teotia | To bloom or not to bloom: Role of micrornas in plant flowering[END_REF]. Different studies highlighted the complex organization of miR172/AP2 and miR156/SPL module in stabilizing the floral state (Figure 19). In A. thaliana, miR156 and miR172 act together: in vegetative phase, miR156 maintains the juvenile phase by repressing SPL15 which inhibits floral transition. While in the flowering phase, TCP15 activates MIR172 expression that represses AP2 expression that induces the activation of floral transition [START_REF] Teotia | To bloom or not to bloom: Role of micrornas in plant flowering[END_REF][START_REF] Tripathi | Genome-wide analysis of the SPL/miR156 module and its interaction with the AP2/miR172 unit in barley[END_REF][START_REF] Lian | Redundant and specific roles of individual MIR172 genes in plant development[END_REF][START_REF] Ó'maoiléidigh | Systematic analyses of the MIR172 family members of Arabidopsis define their distinct roles in regulation of APETALA2 during floral transition[END_REF]. The root growth is sustained by the root apical meristem (RAM) which contains undifferentiated stem cells able to divide and differentiate to produce the different tissues of the root [START_REF] Petricka | Control of arabidopsis root development[END_REF]. The function of miR160 [START_REF] Mallory | MicroRNA-directed regulation of Arabidopsis Auxin Response Factor17 is essential for proper development and modulates expression of early auxin response genes[END_REF][START_REF] Wang | Control of root cap formation by MicroRNA-targeted auxin response factors in Arabidopsis[END_REF] and miR396 [START_REF] Rodriguez | Control of cell proliferation in Arabidopsis thaliana by microRNA miR396[END_REF]Bazin et al., 2013) has been established in RAM. It is important to mention that most miRNAs that are involved in root development are involved also in auxin and cytokinin hormone pathway. These two hormones are well known modulators of root formation and the cross-talk between them regulates RAM maintenance and lateral root emergence [START_REF] Bishopp | Sending mixed messages: Auxin-cytokinin crosstalk in roots[END_REF][START_REF] Bielach | Genetic approach towards the identification of auxin -cytokinin crosstalk components involved in root development[END_REF]. Thus, miR156 and its target SPL10 control root meristem activity and root-derived de novo shoot regeneration via cytokinin in A. thaliana [START_REF] Barrera-Rojas | MiR156-targeted SPL10 controls Arabidopsis root meristem activity and root-derived de novo shoot regeneration via cytokinin responses[END_REF]. Arabidopsis plants overexpressing miR156a showed a reduced meristem size. The opposite root phenotype was observed in knock down miR156a expressing short tandem target mimic (STTM) that act as molecular sponge to sequester the microRNA and block its action to cleave the targets. Interestingly, CRISPR/Cas9-derived spl10-2 mutant (35-bp deletion in SPL10) displayed shorter meristems whereas rSPL10 lines that resist to miR156a cleavage showed the opposite phenotype suggesting that miR156 contributes to modulation of root meristem activity by targeting SPL10. Cytokinin controls root meristem size by activating the transcription factors of type-B ARABIDOPSIS THALIANA RESPONSE REGULATORS (ARRs) [START_REF] Mason | Type-B response regulators display overlapping expression patterns in Arabidopsis[END_REF]. ARR5 fused to the gene reporter GUS showed a strong expression in root explants of plants overexpressing miR156a. Conversely, weaker GUS staining was observed in rspl10 mutants resistant to cleavage. Moreover, medium supplement with different cytokinin presented an important regenerative capacity for controlling root explants, but it did not improve the low regenerative capacity of plants overexpressing miR156 neither for rspl10. All together, these results indicated that the meristem activity is regulated by miR156-SPL10 module probably through the reduction of cytokinin responses, via the modulation of ARR expression (Barrera-Rojas et al., 2020).

Moreover, miR393 (Figure 20) , miR390 [START_REF] Chen | Regulation of auxin response by miR393-targeted transport inhibitor response protein 1 is involved in normal development in Arabidopsis[END_REF][START_REF] Lu | MiR393 and miR390 synergistically regulate lateral root growth in rice under different conditions[END_REF], miR164 [START_REF] Li | miRNA164-directed cleavage of ZmNAC1 confers lateral root development in maize (Zea maysL.)[END_REF][START_REF] Geng | miR164-targeted TaPSK5 encodes a phytosulfokine precursor that regulates root growth and yield traits in common wheat (Triticum aestivum L.)[END_REF], miR167 [START_REF] Gutierrez | Phenotypic Plasticity of Adventitious Rooting in Arabidopsis Is Controlled by Complex Regulation of AUXIN RESPONSE FACTOR Transcripts and MicroRNA Abundance[END_REF], miR476 (Xu et al., 2021a) or miR847 [START_REF] Wang | Cleavage of INDOLE-3-ACETIC ACID INDUCIBLE28 mRNA by microRNA847 upregulates auxin signaling to modulate cell proliferation and lateral organ growth in arabidopsis[END_REF] have been shown to be involved in the formation of lateral and adventitious roots. Several of these miRNAs regulate lateral root formation via Auxin

Response Factor (ARFs). ARFs are key transcriptional factors regulators of auxin signaling that bind to auxin response elements (AuxREs) included in promoters of numerous auxinresponsive genes (Reviewed in [START_REF] Guilfoyle | Auxin response factors[END_REF][START_REF] Chandler | Auxin response factors[END_REF]Li et al., 2016b). miR390 induces the production of tasiRNAs that silence ARF2, ARF3 and ARF4 removing the repression of lateral root growth (Marin et al., 2010). miR160 is known to repress ARF17 (negative regulators of adventitious rooting) while miR167 repress the two targets ARF6 and ARF8 (positive regulators of adventitious rooting). A complex regulation and interaction between the three ARFs (ARF6, ARF8 and ARF17) and miR160/miR167 was observed using different grf mutant lines [START_REF] Gutierrez | Phenotypic Plasticity of Adventitious Rooting in Arabidopsis Is Controlled by Complex Regulation of AUXIN RESPONSE FACTOR Transcripts and MicroRNA Abundance[END_REF]. Thus, the balance between these two miRNAs control lateral root development [START_REF] Couzigou | Plant microRNAs : key regulators of root architecture and biotic interactions[END_REF].

MicroRNAs in plant response to abiotic stress

Plants are subjected to different environmental challenges such changes in temperature, soil water potential and nutrients. Induced phenotypic and physiological changes help the plant to adapt and survive in response to environmental changes (Figure 21) [START_REF] Singh | Role of abiotic stress responsive miRNAs in Arabidopsis root development[END_REF]. Interestingly, some miRNAs respond to different abiotic stresses such as miR408 in response to cold, drought and copper stresses [START_REF] Ma | miR408 is involved in abiotic stress responses in Arabidopsis[END_REF].

Under drought or salinity stresses, several miRNAs are differentially expressed and can positively or negatively regulate plant tolerance to drought stress [START_REF] Song | miR394 and LCR are involved in Arabidopsis salt and drought stress responses in an abscisic acid-dependent manner[END_REF][START_REF] Fang | Conserved miR164-targeted NAC genes negatively regulate drought resistance in rice[END_REF][START_REF] Ma | miR408 is involved in abiotic stress responses in Arabidopsis[END_REF] or salinity stress [START_REF] Jung | Expression and functional analyses of microRNA417 in Arabidopsis thaliana under stress conditions[END_REF][START_REF] Song | miR394 and LCR are involved in Arabidopsis salt and drought stress responses in an abscisic acid-dependent manner[END_REF][START_REF] Zhou | Constitutive expression of a miR319 gene alters plant development and enhances salt and drought tolerance in transgenic creeping bentgrass[END_REF][START_REF] Iglesias | MiR393 Regulation of Auxin Signaling and Redox-Related Components during Acclimation to Salinity in Arabidopsis[END_REF][START_REF] Kong | System analysis of microRNAs in the development and aluminium stress responses of the maize root system[END_REF]. miR169 is a negative regulator of drought stress. In A. thaliana, plants overexpressing miR169 have an increase in susceptibility towards drought stress. Mutant overexpressing miR169 showed increased in size of the stomatal aperture that mimics the phenotype observed in miR169 target mutant nfya5 (NUCLEAR FACTOR Y SUBUNIT A5) [START_REF] Li | The Arabidopsis NFYA5 transcription factor is regulated transcriptionally and posttranscriptionally to promote drought resistance[END_REF]. miR394 and its target LEAF CURLING RESPONSIVENESS (LCR) are involved in plant responses to both drought and salinity stresses [START_REF] Song | miR394 and LCR are involved in Arabidopsis salt and drought stress responses in an abscisic acid-dependent manner[END_REF].

Using RT-PCR and GUS reporter fusion, miR394a and miR394b were showed to be induced under saline and drought stresses in A. thaliana. Conversely, plants overexpressing miR394b and lcr mutant showed a tolerance to drought stress. On the other hand, treatment with 100 mM NaCl showed a decrease in the germination of plants overexpressing miR394a and lcr seeds compared to wild type. These results suggest that both miR394 and LCR are critical for plant response to salt and drought stresses.

NGS showed that several microRNAs like miR156, miR396 and miR402, are differentially expressed under heat or cold stress suggesting that these microRNAs play important roles in the regulation of heat or cold tolerance in plants [START_REF] Zhou | Identification of cold-inducible microRNAs in plants by transcriptome analysis[END_REF][START_REF] Kim | MicroRNA402 affects seed germination of arabidopsis thaliana under stress conditions via targeting DEMETER-LIKE Protein3 mRNA[END_REF]Wang et al., 2012b;[START_REF] Guan | Heat stress induction of miR398 triggers a regulatory loop that is critical for thermotolerance in Arabidopsis[END_REF][START_REF] Dong | Over-expression of miR397 improves plant tolerance to cold stress in Arabidopsis thaliana[END_REF][START_REF] Li | HEAT-INDUCED TAS1 TARGET1 mediates thermotolerance via heat stress transcription factor A1a-directed pathways in arabidopsis[END_REF][START_REF] Stief | Arabidopsis miR156 regulates tolerance to recurring environmental stress through SPL transcription factors[END_REF]Wang et al., 2014a;[START_REF] Ma | miR408 is involved in abiotic stress responses in Arabidopsis[END_REF]Zhang et al., 2016b;[START_REF] Zhou | Identification of miRNAs and their targets in wild tomato at moderately and acutely elevated temperatures by high-throughput sequencing and degradome analysis[END_REF][START_REF] Ding | microRNAs involved in auxin signalling modulate male sterility under high-temperature stress in cotton (Gossypium hirsutum)[END_REF][START_REF] Mangrauthia | Genome-wide changes in microRNA expression during short and prolonged heat stress and recovery in contrasting rice cultivars[END_REF][START_REF] Song | Response of microRNAs to cold treatment in the young spikes of common wheat[END_REF][START_REF] López-Galiano | Expression of miR159 Is Altered in Tomato Plants Undergoing Drought Stress[END_REF][START_REF] Tiwari | Identification of small RNAs during cold acclimation in Arabidopsis thaliana[END_REF]. A. thaliana plants overexpressing miR397a improves plant tolerance under cold stress. The transcript levels of COLD REGULATED (COR15A), (COR47A) and RESPONSIVE TO DEHYDRATION (RD29A) plants have been shown to be higher in 35S:miR397 than in wild-type plants after cold treatment for over 48 h at 4°C. These results showed that miR397 is involved in the cold signaling pathway and enhanced cold responsive gene expression that may contribute to tolerance to cold stress.

Finally, several miRNAs and their targets have been shown to be differentially expressed in response to nutrients deficiency : Nitrogen, Phosphorus, Sulfur and Copper [START_REF] Yamasaki | Regulation of copper homeostasis by micro-RNA in Arabidopsis[END_REF][START_REF] Gifford | Cell-specific nitrogen responses mediate developmental plasticity[END_REF][START_REF] Hsieh | Uncovering small RNAmediated responses to phosphate deficiency in Arabidopsis by deep sequencing[END_REF][START_REF] Liang | MicroRNA395 mediates regulation of sulfate accumulation and allocation in Arabidopsis thaliana[END_REF][START_REF] Zhao | Involvement of miR169 in the nitrogen-starvation responses in Arabidopsis[END_REF][START_REF] Jagadeeswaran | Redox signaling mediates the expression of a sulfatedeprivation-inducible microRNA395 in Arabidopsis[END_REF]. The role of miRNAs that respond to copper will be discussed later in the results (chapter 2).

MicroRNAs in plant-microorganism interactions

Plant are constantly challenged by various pathogens, like bacteria, viruses, oomycetes, fungi, nematodes but also in interaction with beneficial symbiotic microorganisms, like nitrogenfixing bacteria and arbuscular mycorrhizal fungi. Several studies assessed the role of important components of the RNAi machinery, in particular DCLs and AGOs, in plantmicroorganisms interactions [START_REF] Blevins | Four plant Dicers mediate viral small RNA biogenesis and DNA virus induced silencing[END_REF][START_REF] Zhang | Cucumber mosaic virus-encoded 2b suppressor inhibits Arabidopsis Argonaute1 cleavage activity to counter plant defense[END_REF][START_REF] Navarro | Suppression of the microRNA pathway by bacterial effector proteins[END_REF][START_REF] Li | Identification of microRNAs involved in pathogen-associated molecular pattern-triggered plant innate immunity[END_REF]Medina et al., 2017). These studies demonstrated that miRNAs are involved in plant response to microorganism infection. In addition, several effectors of plant pathogens have been described as targeting the silencing pathway, e.g. in virus, bacterial, fungi infection and RKN [START_REF] Navarro | Suppression of the microRNA pathway by bacterial effector proteins[END_REF][START_REF] Qiao | Oomycete pathogens encode RNA silencing suppressors[END_REF][START_REF] Csorba | Viral silencing suppressors: Tools forged to fine-tune hostpathogen coexistence[END_REF][START_REF] Ye | Filamentous pathogen effectors interfering with small RNA silencing in plant hosts[END_REF][START_REF] Yin | A novel fungal effector from Puccinia graminis suppressing RNA silencing and plant defense responses[END_REF][START_REF] Mejias | The root-knot nematode effector MiEFF18 interacts with the plant core spliceosomal protein SmD1 required for giant cell formation[END_REF].

In symbiotic interactions, small RNA sequencing approach identified microRNAs differentially expressed in nitrogen fixing nodules in several host plants like soybean [START_REF] Subramanian | Novel and nodulation-regulated microRNAs in soybean roots[END_REF][START_REF] Wang | Identification and expression analysis of miRNAs from nitrogen-fixing soybean nodules[END_REF][START_REF] Joshi | Prediction of novel miRNAs and associated target genes in Glycine max[END_REF][START_REF] Turner | Genome organization and characteristics of soybean microRNAs[END_REF][START_REF] Yan | Identification of microRNAs and their mRNA targets during soybean nodule development: Functional analysis of the role of miR393j-3p in soybean nodulation[END_REF][START_REF] Yan | Identification and functional characterization of soybean root hair microRNAs expressed in response to Bradyrhizobium japonicum infection[END_REF], M. trunctula (Lelandais-Brière et al., 2009), Lotus japonicus (De [START_REF] Luis | Two MicroRNAs linked to nodule infection and nitrogen-fixing ability in the legume Lotus japonicus[END_REF][START_REF] Holt | micro RNA 172 (miR172) signals epidermal infection and is expressed in cells primed for bacterial invasion in Lotus japonicus roots and nodules[END_REF] and common bean [START_REF] Formey | Regulation of Small RNAs and Corresponding Targets in Nod Factor-Induced Phaseolus vulgaris Root Hair Cells[END_REF]. The function of several miRNAs in plant response to nitrogen-fixing bacteria was demonstrated in soybean (miR167; Wang et al., 2015), in L. japonicus (miR171 & miR397; De [START_REF] Luis | Two MicroRNAs linked to nodule infection and nitrogen-fixing ability in the legume Lotus japonicus[END_REF], soybean (miR393; [START_REF] Subramanian | Novel and nodulation-regulated microRNAs in soybean roots[END_REF] and M. truncatula (miR169; Lelandais-Brière et al., 2009). Several miRNAs, including miR156, miR167 and miR168, were found up-regulated in tomato roots in response to colonization by the endophyte Pochonia chlamydosporia [START_REF] Pentimone | Identification of tomato miRNAs responsive to root colonization by endophytic Pochonia chlamydosporia[END_REF]. In soybean, a role for the module miR167-ARF6/8 was established in nodule formation. A strong expression is observed for miR167c in mature soybean root nodules [START_REF] Wang | Identification and expression analysis of miRNAs from nitrogen-fixing soybean nodules[END_REF]Wang et al., 2015). Soybean plants overexpressing miR167c showed increase in number and length of lateral roots and an increase in numbers of nodule when infected by Bradyrhizobium japonicum. STTM knockdown of miR167c showed the opposite phenotype of the overexpression of miR167c. RNAi of the two miR167 validated targets, ARF8a and ARF8b (Wang et al., 2015), also produced more nodules in roots while plants overexpressing ARF8a showed less number of nodules than control plants infected by B. japonicum. These results suggest that the regulation of ARF8A and ARF8B by miR167 regulates soybean nodulation and lateral root development. The role of miR172 was also established in nodule formation in common bean infected by Rhizobium etli: miR172 is overexpressed during nodule development and cleaves the target NODULE NUMBER CONTROL1 (NNC1) to regulate nodule formation (Wang et al., 2014b;[START_REF] Íñiguez | The micro-RNA72c-APETALA2-1 node as a key regulator of the common bean-Rhizobium etli nitrogen fixation symbiosis[END_REF] (Figure 22).

Plant viruses cause serious damages in plant and affect crop production [START_REF] Scholthof | Top 10 plant viruses in molecular plant pathology[END_REF]Wang et al., 2012a). The infection with viruses regulates the accumulation of small RNAs in plants which can affect either plant defense or virus pathogenicity [START_REF] Prasad | Recent advances in small RNA mediated plant-virus interactions[END_REF]. Virus dsRNAs are recognized and cleaved by DCLs to produce primary virus-derived small interfering RNAs (vsiRNAs). Plants have developed antiviral defense systems such as RNAi that targets viral RNAs [START_REF] Ruiz-Ferrer | Viral suppression of RNA silencing: 2b wins the Golden Fleece by defeating Argonaute[END_REF]. The components of miRNAs pathways are involved in plant defense towards viruses [START_REF] Blevins | Four plant Dicers mediate viral small RNA biogenesis and DNA virus induced silencing[END_REF][START_REF] Csorba | The p122 Subunit of Tobacco Mosaic Virus Replicase Is a Potent Silencing Suppressor and Compromises both Small Interfering RNA-and MicroRNA-Mediated Pathways[END_REF][START_REF] Havelda | Plant virus infection-induced persistent host gene downregulation in systemically infected leaves[END_REF][START_REF] Prasad | Recent advances in small RNA mediated plant-virus interactions[END_REF]. A recent study, using microarray analysis, revealed 129 miRNAs differentially expressed in N. benthamiana infected by Beet necrotic yellow vein virus (BNYVV) [START_REF] Liu | Genome-Wide microRNA Profiling Using Oligonucleotide Microarray Reveals Regulatory Networks of microRNAs in Nicotiana benthamiana During Beet Necrotic Yellow Vein Virus Infection[END_REF]. Functional analysis showed a role for miR398 induction in the defense response towards BNYVV.

The role of small RNAs in plant defense towards bacterial infection was described precisely in response to Pseudomonas genus, like miR160 [START_REF] Fahlgren | High-Throughput Sequencing of Arabidopsis microRNAs: Evidence for Frequent Birth and Death of MIRNA Genes[END_REF], miR164 [START_REF] Lee | An Arabidopsis NAC transcription factor NAC4 promotes pathogen-induced cell death under negative regulation by microRNA164[END_REF], miR167 [START_REF] Fahlgren | High-Throughput Sequencing of Arabidopsis microRNAs: Evidence for Frequent Birth and Death of MIRNA Genes[END_REF][START_REF] Li | Identification of microRNAs involved in pathogen-associated molecular pattern-triggered plant innate immunity[END_REF], miR393 [START_REF] Fahlgren | High-Throughput Sequencing of Arabidopsis microRNAs: Evidence for Frequent Birth and Death of MIRNA Genes[END_REF]Zhang et al., 2011a), miR398 [START_REF] Jagadeeswaran | Biotic and abiotic stress down-regulate miR398 expression in Arabidopsis[END_REF] and miR482 [START_REF] Shivaprasad | A microRNA superfamily regulates nucleotide binding site-leucine-rich repeats and other mRNAs[END_REF]. A recent study highlighted the role of miR167 and its targets, the two auxin response factors ARF6 and ARF8 and salicylic acid (SA) in defense against Pseudomonas syringae pv. tomato DC3000 in A. thaliana [START_REF] Caruana | Overexpression of Arabidopsis microRNA167 induces salicylic acid-dependent defense against Pseudomonas syringae through the regulation of its targets ARF6 and ARF8[END_REF]. The overexpression of miR393 in A. thaliana restrict the development of Pseudomonas, while the overexpression of one of its target resistant to cleavage, AUXIN SIGNALING F-BOX PROTEIN (AFB1), increase the sensitivity of the plant to bacterial infection (Navarro et al., 2006). A recent study identified the miRNAs and their targets expressed in susceptible and resistant ginger transcriptomes in response to the soil borne bacteria, Ralstonia solanacearum, infection [START_REF] Snigdha | Transcriptomic analysis to reveal the differentially expressed miRNA targets and their miRNAs in response to Ralstonia solanacearum in ginger species[END_REF]. Some studies also showed the involvement of small RNAs during the interaction of plants with

Agrobacterium tumefaciens. In N. tabacum, miR393 is induced by the gall-forming bacteria [START_REF] Pruss | Infiltration with Agrobacterium tumefaciens induces host defense and development-dependent responses in the infiltrated zone[END_REF].

Infection of mutants of PTGS pathway demonstrates a role of small RNAs in plant response to fungi and oomycetes [START_REF] Ellendorff | RNA silencing is required for Arabidopsis defence against Verticillium wilt disease[END_REF][START_REF] Weiberg | Fungal Small RNAs Suppress Plant Immunity by Hijacking Host RNA Interference Pathways[END_REF]. In cotton infected by Verticillium dahlia, miR164 and its target NAC100 plays a role in plant resistance to fungal infection (Hu et al., 2020a). miR164 is known to target NAC genes and participate in plant development and defense [START_REF] Mallory | MicroRNA regulation of NAC-domain targets is required for proper formation and separation of adjacent embryonic, vegetative, and floral organs[END_REF][START_REF] Sieber | Redundancy and specialization among plant microRNAs: Role of the MIR164 family in developmental robustness[END_REF]. Indeed, plants overexpressing STTM miR164 showed more severe disease symptom compared to wild type plants, while overexpressing miR164 plants were more resistant to the infection. In response to oomycete infection, overexpression of miR393 in A. thaliana leads to a resistance towards Hyaloperonospora arabidopsidis and Phytophthora capsici [START_REF] Robert-Seilaniantz | The microRNA miR393 re-directs secondary metabolite biosynthesis away from camalexin and towards glucosinolates[END_REF][START_REF] Hou | A Phytophthora Effector Suppresses Trans-Kingdom RNAi to Promote Disease Susceptibility[END_REF]. [START_REF] Wong | Roles of small RNAs in soybean defense against Phytophthora sojae infection[END_REF] identified an overexpression of miR166, miR393, miR1507, miR2109 and miR3522, and a downregulation of miR168, miR319 and miR482 in soybean infected by Phytophthora sojae [START_REF] Wong | Roles of small RNAs in soybean defense against Phytophthora sojae infection[END_REF]. STTM knock down of miR393 in soybean leads to a susceptibility towards Phytophthora infection suggesting that miR393 is a positive regulator of soybean defense towards oomycete infection.

miRNAs have also been described as a key regulator in the plant-nematode interactions. I wrote as a co-first author, a review entitled "MicroRNAs, New Players in the Plant-Nematode Interaction" published in Frontiers in Plant Science [START_REF] Jaubert-Possamai | MicroRNAs, New Players in the Plant-Nematode Interaction[END_REF] This review summarizes the current knowledge about the function of plant miRNAs in plant response to RKN and CN. In this review, we listed the microRNAs that were DE in the two types of feeding sites, galls/giant cells and syncytia, induced by RKN and CN, respectively. Expression profile of most conserved miRNAs (except miR390) in feeding site had different expression profiles according to the type of feeding structures, the kinetic points and/or the plant species. These differences in giant cell and syncytia can be explained by their distinct ontogenesis, but also in the different biological material and sequencing analyzes.

This mini-review was published in 2019. New studies have been published since then describing miRNAs differentially expressed in response to RKN and CN.

Sequencing of small RNAs from control and infected roots of resistant and susceptible soybean cultivars at early stage (1 and 5 dpi) of infection induced by Heterodera glycines identified 14 known and 26 novel microRNAs differentially expressed [START_REF] Lei | Identification of MicroRNAs That Respond to Soybean Cyst Nematode Infection in Early Stages in Resistant and Susceptible Soybean Cultivars[END_REF]. The expression of 19 over 24 miRNAs were confirmed using RT-qPCR.

In cotton-RKN interaction, Cai et al. ( 2021) identified 266 miRNAs including 193 known miRNAs and 73 novel miRNAs expressed in cotton roots 2 months after infection with M.

incognita [START_REF] Cai | Small RNA and degradome deep sequencing reveals important roles of microRNAs in cotton (Gossypium hirsutum L.) response to root-knot nematode Meloidogyne incognita infection[END_REF]. Among them, 50 miRNAs were DE: 28 miRNAs were up-regulated and 22 miRNAs down-regulated in galls. In a first attempt to understand the functions of these miRNAs, the targets of miRNAs were predicted by using "TargetFinder" algorthim and degradome sequencing. Degradome is a biological identification of microRNA targets that consists in the specific sequencing of cleaved mRNA products. This approach is based on the difference between the 5 'end of cleaved mRNA and intact mRNA. RNA adapters specifically binds to the 5 'monophosphate end of cleaved mRNA. The ligation products are then retrotranscribed, the cDNAs are amplified by PCR and then sequenced. 87 gene targets were identified to be targeted by 57 miRNAs. Based on GO (gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis, miRNA targets were categorized in different processes including those associated with organism responses to the environmental stresses.

Interestingly, in galls of rice infected by M. graminicola, 25 long non-coding RNAs (lncRNAs), 3739 siRNAs and 16 miRNAs differentially expressed 3 dpi were recently identified in comparison to control roots [START_REF] Verstraeten | Non-coding RNAs in the interaction between rice and Meloidogyne graminicola[END_REF]. The expression of three miRNAs (miR408-3P, miR3979-3P and miR850.1) was confirmed using RT-qPCR. In addition, miRNA targets were identified in galls using the degradome approach.

All these sequencing data enable to identify microRNAs that are DE in galls and therefore that could regulate gene expression in plant-nematode interactions. However, most of these studies lack functional validation of these miRNAs. So far the function of only four and three microRNAs has been biologically validated in plant response to RKN and CN infection, respectively. These DE microRNAs and the key role of important components of the RNAi machinery (DCLs and AGOs), are a strong elements showing that microRNAs are key regulators of gene expression in galls.

. Plant-parasitic root-knot and cyst nematodes are microscopic worms that cause severe damage to crops and induce major agricultural losses worldwide. These parasites penetrate into host roots and induce the formation of specialized feeding structures, which supply the resources required for nematode development. Root-knot nematodes induce the redifferentiation of five to seven root cells into giant multinucleate feeding cells, whereas cyst nematodes induce the formation of a multinucleate syncytium by targeting a single root cell. Transcriptomic analyses have shown that the induction of these feeding cells by nematodes involves an extensive reprogramming of gene expression within the targeted root cells. MicroRNAs are small noncoding RNAs that act as key regulators of gene expression in eukaryotes by inducing the posttranscriptional silencing of protein coding genes, including many genes encoding transcription factors. A number of microRNAs (miRNAs) displaying changes in expression in root cells in response to nematode infection have recently been identified in various plant species. Modules consisting of miRNAs and the transcription factors they target were recently shown to be required for correct feeding site formation. Examples include miR396 and GRF in soybean syncytia and miR159 and MYB33 in Arabidopsis giant cells. Moreover, some conserved miRNA/target modules seem to have similar functions in feeding site formation in different plant species. These miRNAs may be master regulators of the reprogramming of expression occurring during feeding site formation. This review summarizes current knowledge about the role of these plant miRNAs in plant-nematode interactions.

1

INTRODUCTION

Sedentary endoparasitic nematodes are the most damaging plant-parasitic nematodes (PPNs) that cause massive crop yield losses worldwide (Blok et al., 2008). There are two main groups of PPNs: the root-knot nematodes (RKNs) of the genus Meloidogyne and the cyst nematodes (CNs) of the genera Heterodera and Globodera (Jones et al., 2013). After penetrating the root and migrating to the vascular cylinder, mobile second-stage juvenile (J2) selects one (CNs) or a few (RKNs) initial root cells, into which it injects a cocktail of secretions that transform these cells into hypertrophied multinucleate feeding cells that supply nutrients required for nematode development: the giant cells induced by RKNs (Figure 1A) or the syncytium induced by CNs (Figure 1B).

Common and Specific Processes Involved in Feeding Site Formation

Both hypertrophied and multinucleate feeding cells are highly active metabolically and have a dense cytoplasm, with a large number of organelles and invaginated cell wall (Figure 1A,B) (Grundler et al., 1998;Sobczak and Golinowski, 2011;Favery et al., 2016). They accumulate sugars and amino acids (Hofmann et al., 2010;Baldacci-Cresp et al., 2012). The nuclei and nucleoli of both giant cells and syncytia are larger than normal root cells, due to endoreduplication (de Almeida Engler and Gheysen, 2013). However, these two feeding structures have very different ontogenies. RKN J2 selects five to seven parenchyma cells and induces their dedifferentiation into giant cells through successive mitosis without cytokinesis (Caillaud et al., 2008b). Expansion of giant cells by isotropic growth (Cabrera et al., 2015) together with hyperplasia of the root cells surrounding the giant cells results in a swelling of the root, known as a gall, the characteristic symptom of RKN infection. By contrast, CN J2 targets a single initial root cell. This cell expands within the vascular tissue by progressive cell wall dissolution and incorporation into the syncytium of adjacent cells via cytoplasm fusion (Golinowski et al., 1996;Grundler et al., 1998).

Studies of the feeding site formation have greatly benefited from whole-transcriptome analyses. Such analyses were initially developed in the model host plant Arabidopsis thaliana and were then extended to various crop species (Escobar et al., 2011;Favery et al., 2016;Yamaguchi et al., 2017). All these analyses showed that feeding site formation involves an extensive reprogramming of gene expression within the root The pri-miRNA is then cleaved, in the nucleus, by Dicer-like 1 (DCL1), in association with hyponastic leaves 1 (HYL1) and serrate (SE), to produce a precursor miRNA (pre-miRNA). The pre-miRNA is, in turn, cleaved by DCL1 and its cofactors, thus generating a duplex composed of the mature miRNA and its complementary strand. The HUA ENHANCER 1 protein (HEN1) then adds a methyl group to the OH end of each strand of the miRNA duplex, to protect against degradation. The miRNA duplex is then actively transported from the nucleus to the cytosol through interaction with the hasty (HST) exportin. One of the two strands of the duplex is then loaded onto the argonaute 1 (AGO1) protein, the main constituent of the multiprotein RNA-induced silencing complex (RISC). The AGO1-associated strand guides the RISC to target mRNAs by sequence complementarity, resulting in target cleavage or the inhibition of protein synthesis (reviewed by [START_REF] Yu | The "how" and "where" of plant microRNAs[END_REF].

3

October 2019 | Volume 10 | Article 1180 Frontiers in Plant Science | www.frontiersin.org cells targeted by the nematodes. These analyses suggested that CNs and RKNs establish feeding sites by recruiting and/ or manipulating several plant functions, including plant defense and phytohormone pathways (Gheysen and Mitchum, 2019), cell wall modification (Sobczak and Golinowski, 2011), cytoskeleton (Caillaud et al., 2008a), and the cell cycle (de Almeida Engler and Gheysen, 2013). These analyses also revealed the conservation of some nematode-responsive genes within the plant kingdom (Portillo et al., 2013).

MicroRNAs Are Key Regulators of Gene Expression

Plant miRNAs are 20-to 22-nucleotide-long noncoding RNAs (Bartel, 2004) that regulate gene expression through posttranscriptional gene silencing. Plant miRNA precursors are produced from MIR genes and are processed by several proteins, including Dicer-like 1 (DCL1), to generate a mature miRNA duplex. One strand of the duplex is loaded into the RNA-induced silencing complex (RISC), in which its sequence complementarity directs gene silencing (Figure 1C) [START_REF] Yu | The "how" and "where" of plant microRNAs[END_REF]. Perfect miRNA/mRNA complementarity generally induces cleavage of the mRNA at nucleotide position 10 or 11 (Franco-Zorrilla et al., 2007;[START_REF] Bartel | MicroRNAs: target recognition and regulatory functions[END_REF]. However, in some cases, such as the miR172/ APETALA2 module in Arabidopsis, the miRNA inhibits mRNA translation (Chen, 2004;Zhang and Li, 2013). Interestingly, the miRNA target may activate the expression of its regulator miRNA, e.g. CUC2 and MIR164a [START_REF] Nikovics | The balance between the MIR164A and CUC2 genes controls leaf margin serration in Arabidopsis[END_REF]. Therefore, regulation of genes by miRNA does not always imply a negative correlated expression between mature miRNA and the targeted transcripts. Plant MIR genes are often organized into multigene families in which the sequences of the precursors differ, but the mature sequences are almost identical, suggesting that they share some target mRNAs (Palatnik et al., 2007). Moreover, many MIR families are conserved between evolutionarily distant plant species, either targeting conserved genes or having different targets in different plant species (Jones-Rhoades, 2012). Small regulatory RNAs are major regulators of gene expression in plant development and in responses to various microorganisms such as beneficial mycorrhizal fungi (Bazin et al., 2013) and fungal [START_REF] Park | MicroRNA400-guided cleavage of pentatricopeptide repeat protein mRNAs renders Arabidopsis thaliana more susceptible to pathogenic bacteria and fungi[END_REF] or bacterial pathogens (Navarro et al., 2006). Plant miRNA may regulate the plant defense or the neoformation of specific structures during plant-microbe interactions (Combier et al., 2006;[START_REF] Park | MicroRNA400-guided cleavage of pentatricopeptide repeat protein mRNAs renders Arabidopsis thaliana more susceptible to pathogenic bacteria and fungi[END_REF][START_REF] Lee | MicroRNA844-guided downregulation of cytidinephosphate diacylglycerol synthase3 (CDS3) mRNA affects the response of Arabidopsis thaliana to bacteria and fungi[END_REF]. Plant-parasitic nematodes induce the neoformation of feeding structures within host roots by inducing an extensive reprogramming of gene expression in the targeted root cells. The role of small noncoding RNAs in the plant-nematode interaction was established with the increased resistance to RKN and CN of A. thaliana mutants disrupted for miRNA or siRNA pathway (Hewezi et al., 2008;Medina et al., 2017;[START_REF] Ruiz-Ferrer | Silenced retrotransposons are major rasiRNAs targets in Arabidopsis galls induced by Meloidogyne javanica[END_REF]. The development of sequencing technologies has made it possible to initiate studies of the role of plant miRNAs in this process in various plant species. This review provides an overview of current knowledge about of the conserved and species-specific plant miRNAs involved in responses to RKNs and CNs.

Plant MicroRNAs Responding to RKNs

The identification of novel and differentially expressed (DE) miRNAs involved in plant response to nematodes is based principally on the sequencing of small RNAs (< 35 nt) from infected and uninfected root tissues. If three independent replicates per sample are available, the comparison can be performed directly, by digital expression profiling. Otherwise, sequencing identifies the miRNAs expressed in the samples analyzed, and the levels of these miRNAs are then compared between samples by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). The miRNAs involved in the gall formation induced by RKN have been investigated in Arabidopsis dissected galls and uninfected roots, 3 (Cabrera et al., 2016), 7, and 14 dpi (Medina et al., 2017). This approach identified 62 miRNAs as DE in galls induced by Meloidogyne javanica at 3 dpi, and 24 miRNAs as DE in galls induced by Meloidogyne incognita at 7 and/or 14 dpi. Only two DE miRNAs with the same expression profile were common to these three stages of gall formation: miR390, which is upregulated in galls, and miR319, which is repressed in galls. Using RT-qPCR, identified 17 miRNAs as DE in tomato galls at one or more of the five developmental stages analyzed (Kaur et al., 2017), while Pan et al. (2019) identified 16 miRNAs as DE in whole cotton roots infected by M. incognita at 10 dpi (Table 1). A comparison of susceptible and resistant tomato cultivars identified five RKNresponsive miRNAs in the WT and/or the jasmonic acid-deficient spr2 mutant at 3 dpi (Zhao et al., 2015). Some conserved miRNA families present similar expression profiles in galls from different plant species at similar time points. For example, the evolutionarily conserved miR159 is upregulated in Arabidopsis, tomato, and cotton galls at 10 to 14 dpi, and miR172 is upregulated in A. thaliana and tomato at 3 to 4 dpi (Table 1). The genes targeted by miRNAs have been identified by in silico prediction (Zhao et al., 2015;Cabrera et al., 2016;Pan et al., 2019) or by 5′ RNA ligase-mediated (RLM)-rapid amplification of cDNA ends (RACE) sequencing (Kaur et al., 2017). The expression profiles of genes predicted or known to be targeted by miRNAs were analyzed by transcriptomic analysis or RT-qPCR. A negative correlation between the levels of several DE miRNAs and their targeted transcripts, for miR156/ SPB or miR159/MYB, for example, was observed in galls from Arabidopsis, tomato, and cotton (Zhao et al., 2015;Cabrera et al., 2016;Pan et al., 2019).

Multiple miRNAs have been shown to be DE, but the functions of only four plant miRNAs in plant-RKN interactions have been validated to date. Functional validation involves the characterization of expression profile, often with reporter gene lines or by in situ hybridization, and analyses of the infection status of plants with modified expression or functions for either miRNAs (e.g. overexpression, KO or buffering "target mimicry" lines) or their targets (e.g. overexpression of a miRNA-resistant form, with a mutation in the miRNA target site or knockout lines). For example, miR319 is upregulated in tomato galls at 3 dpi, whereas its target, TCP4 (TEOSINTE BRANCHED 1∕CYCLOIDEA∕PROLIFERATING FACTOR 4), is downregulated (Zhao et al., 2015). Tomato plants overexpressing a miR319-resistant TCP4 have fewer galls and higher levels of endogenous JA, whereas the opposite effect is observed in lines overexpressing Ath-MIR319. These results

4

October 2019 | Volume 10 | Article 1180 Frontiers in Plant Science | www.frontiersin.org suggest that the miR319/TCP4 module is essential in tomato galls by modulating the JA biosynthesis induced by RKN invasion (Zhao et al., 2015). miR159 is a conserved family of miRNAs upregulated in Arabidopsis galls at 14 dpi (Medina et al., 2017). Studies on transgenic GUS lines demonstrated the posttranscriptional regulation of MYB33, the main target of miR159, in Arabidopsis galls at 14 dpi. The mir159abc triple loss-of-function mutant displays enhanced resistance to RKN, with decreased numbers of galls and egg masses, demonstrating the role of the miR159 family in the response of Arabidopsis to M. incognita, probably through the regulation of MYB33. Furthermore, in situ hybridization has shown that miR159 is also expressed in tomato giant cells (Medina et al., 2017) and a conserved upregulation of miR159 associated with a downregulation of MYB transcription factors has also been observed in galls from tomato (3 dpi and 13-15 dpi; Zhao et al., 2015;Kaur et al., 2017) and cotton (10 dpi; Pan et al., 2019). These results suggest that the function of the miR159/ MYB module may be conserved in the galls Arabidopsis, tomato and cotton (Medina et al., 2017). The conserved auxin-responsive miR390 family is overexpressed in A. thaliana galls at 3, 7, and 14 dpi (Cabrera et al., 2016;Medina et al., 2017). In Arabidopsis, the cleavage of TAS3 transcripts by miR390 generates secondary siRNAs (tasiRNAs) that induce post-transcriptional repression of the auxin-responsive transcription factors ARF2, ARF3, and ARF4 (Marin et al., 2010). Cabrera et al. (2016) demonstrated the coexpression of MIR390A and TAS3 in galls and giant cells at 3 dpi and the post-transcriptional regulation of ARF3 by tasiRNAs in galls, in experiments comparing ARF3 sensor lines sensitive or resistant to cleavage by tasiRNAs. Studies of miR390a and tas3 loss-of-function mutants reported the production of fewer galls, suggesting that the miR390/TAS3/ARF3 regulatory module is required for correct gall formation (Cabrera et al., 2016). Finally, a role for the regulatory gene module composed by miR172 and the two transcription factors TOE1 (target of early activation tagged 1) and FT (flowering locus T) has been demonstrated in root galls during the formation of giant cells in Arabidopsis (Díaz-Manzano et al., 2018). The role for the miR172/TOE1/FT module has been first described during Arabidopsis flowering [START_REF] Aukerman | Regulation of flowering time and floral organ identity by a MicroRNA and its APETALA2-like target genes[END_REF]. In Arabidopsis root, the 3′ strand of mature miR172 has been shown to be downregulated in galls at 3 dpi, whereas the pri-miR172 precursor is induced, and its target TOE1 repressed, according to transcriptome data for microdissected A. thaliana giant cells at the same time point (Barcala et al., 2010). Consistent with the negative regulation of FT by TOE1, an induction of FT was observed in galls at 3 dpi. Arabidopsis plants expressing miR172resistant TOE1 or KO for FT were less susceptible to RKNs and had smaller galls and giant cells. Like miR390, miR172 is an auxin responsive microRNA. Auxin is a crucial signal for feeding site formation and parasitism. An enhanced auxin response has been observed in RKN feeding sites (Hutangura et al., 1999) and auxin has been identified in the secretion of RKNS (De Meutter et al., 2005). The function of miR390 and miR172 in the feeding site is probably a part of the auxin response.

Plant Small Noncoding RNAs Responding to CNs

The identification and analysis of miRNAs involved in plant-CN interaction are based on the same approaches that the ones described above. Sequencing identified 30 mature DE miRNAs in Arabidopsis syncytia induced by Heterodera schachtii at 4 and 7 dpi, and qPCR analyses revealed inverse expression profiles for six miRNAs and their targets (Hewezi et al., 2008). A recent analysis of syncytia from tomato plants infected with Globodera rostochiensis, performed at 3, 7, and 10 dpi, identified between 200 and 300 miRNAs at each stage as DE (Koter et al., 2018). Reverse transcriptase-qPCR analyses revealed inversely correlated expression patterns for six miRNAs and their targets (Koter et al., 2018). Moreover, the expression of eight tomato miRNAs regulating defense-related proteins was specifically analyzed by qPCR at 3 and 7 dpi; an inverse correlation between the expression of these miRNAs and their targets in response to CN infection was observed (Święcicka et al., 2017). Finally, several studies have analyzed expression of soybean miRNAs in response to infection with Heterodera glycines by comparing expression levels in resistant and susceptible cultivars [START_REF] Li | miRNA164-directed cleavage of ZmNAC1 confers lateral root development in maize (Zea maysL.)[END_REF]Xu et al., 2014;Tian et al., 2017). Tian et al. (2017) identified 60 miRNAs from 25 miRNA families as DE relative to uninfected roots in susceptible and/or resistant cultivars and validated the expression profiles of most of these miRNAs by qPCR. While most of the miRNAs identified by Tian et al. (2017) are upregulated in resistant lines relative to susceptible lines, the majority of miRNAs were downregulated in the study performed by [START_REF] Li | miRNA164-directed cleavage of ZmNAC1 confers lateral root development in maize (Zea maysL.)[END_REF]. These discrepancies may reflect differences in resistance between these soybean cultivars or a technical bias related to the number of replicates analyzed in these two studies. A comparison of the expression profiles of conserved miRNAs in response to CN infection identified some miRNAs as DE, with the same expression profile, in several plant species. miR396b and the miR167 family were downregulated in Arabidopsis roots infected by H. schachtii at 4 and 7 dpi (Hewezi et al., 2008) and in tomato syncytia induced by G. rostochiensis at 3 and 7 dpi (Święcicka et al., 2017) (Table 1). Three miRNAs DE in syncytia were validated by functional approaches. In Arabidopsis, miR396 was repressed at the onset of syncytium formation in roots infested with H. schachtii and upregulated at later stages, whereas its target transcription factors, the growth-regulating factors (GRF) GRF1, GRF3, and GRF8, displayed the opposite pattern [START_REF] Hewezi | The Arabidopsis microRNA396-GRF1/GRF3 regulatory module acts as a developmental regulator in the reprogramming of root cells during cyst nematode infection[END_REF]. Arabidopsis thaliana mutants overexpressing miR396 have smaller syncytia and greater resistance to CN. These results suggest that the coordinated regulation of miR396 and GRF1 and GRF3 is required for correct syncytium development in Arabidopsis. Interestingly, a repression of the miR396 family associated with an upregulation of soybean GRF genes was observed in soybean syncytia induced by H. glycines at 8 dpi (Noon et al., 2019). A combination of 5′ RLM-RACE and a reporter gene approach demonstrated that the GRF6 and GRF9 genes were targeted by miR396 in syncytia. Transgenic soybean lines overexpressing pre-miR396 and GRF9 RNAi lines displayed similar decreases in the number of H. glycines females per root, reflecting an increase in resistance to CN. These results indicate that the miR396/GRF module is essential for H. glycines infection, and this role is conserved in Arabidopsis and soybean. Furthermore, the use of a reporter gene strategy made it possible to demonstrate an inverse correlation in the expression profiles of the conserved miR827 and its known target NLA (nitrogen limitation adaptation) during syncytium development in Arabidopsis [START_REF] Hewezi | Arabidopsis miR827 mediates post-transcriptional gene silencing of its ubiquitin E3 ligase target gene in the syncytium of the cyst nematode Heterodera schachtii to enhance susceptibility[END_REF]. The overexpression of miR827 increased susceptibility to H. schachtii, whereas the expression of a miR827-resistant NLA decreased plant susceptibility. These results show that miR827 downregulates Arabidopsis immunity to H. schachtii by repressing NLA activity in the syncytium [START_REF] Hewezi | Arabidopsis miR827 mediates post-transcriptional gene silencing of its ubiquitin E3 ligase target gene in the syncytium of the cyst nematode Heterodera schachtii to enhance susceptibility[END_REF]. Finally, a role for the miR858/MYB83 module has been established in Arabidopsis syncytia induced by H. schachtii, in which an inverse correlation of transcript levels was observed between miR858 and its target MYB83 at 7, 10, and 14 dpi [START_REF] Piya | Cooperative regulatory functions of miR858 and MYB83 during cyst nematode parasitism[END_REF]. Modulation of the expression of these genes through gain-and loss-of-function approaches altered the Arabidopsis response to nematode infection, demonstrating a role for this module in syncytium formation.

Conclusions and Perspectives

The results presented provide the first insigths into the function of miRNAs in the plant response to nematode infection. Except for miR390, expression profile of most miRNAs in feeding site shows heterogeneity (Table 1), with different expression profiles according to the type of feeding structures, the plant species, and/ or the phase of development. Difference of expression in giant cell and syncytia may be explained by their distinct ontogenesis. Whether these variations of expression of plant miRNAs are directly induced by the nematode or are the results of modification of plant hormonal balance is a question that still needs to be investigated. The identification of the targets of these DE miRNAs and the biological pathways they regulate would improve our understanding of feeding cell development. Moreover, resistance genes of the nucleotide binding site-leucine-rich repeat (NBS-LRR) family genes are known to be targeted by miRNAs and phased siRNAs (reviewed by [START_REF] Fei | Small RNAs add zing to the zig-zag-zig model of plant defenses[END_REF]). An inverse correlation on several tomato NB-LRR transcripts and their miRNA regulators has been evidenced after infection by CN (Święcicka et al., 2017). A better understanding of the role of miRNA in PPN feeding sites may lead to new methods of control for these organisms.

Most studies to date have focused on miRNAs, but few studies investigating the siRNAs expressed in roots infected with PPNs in Arabidopsis [START_REF] Hewezi | Cyst nematode parasitism induces dynamic changes in the root epigenome[END_REF][START_REF] Medina | Characterization of siRNAs clusters in Arabidopsis thaliana galls induced by the root-knot nematode Meloidogyne incognita[END_REF][START_REF] Ruiz-Ferrer | Silenced retrotransposons are major rasiRNAs targets in Arabidopsis galls induced by Meloidogyne javanica[END_REF] have highlighted an overrepresentation in galls of 24 nt siRNAs known to be associated with RNA-directed DNA methylation. Two first studies of changes in DNA methylation have been performed in A. thaliana and soybean plants infected with CN [START_REF] Rambani | The methylome of soybean roots during the compatible interaction with the soybean cyst nematode[END_REF][START_REF] Hewezi | Cyst nematode parasitism induces dynamic changes in the root epigenome[END_REF]. These studies support a role for changes in DNA methylation in plant responses to PPN infection. Future combined studies of small RNAs, methylome and transcriptome should result in an integrative understanding of the epigenetic regulation of feeding site formation. Several intriguing questions remain unanswered: i) How do PPNs modify the expression of small RNA genes in the plant genome? ii) Do the small RNAs produced by nematodes play a role in the plant and vice versa? Genomes of several PPN species are now available [START_REF] Cotton | The genome and life-stage specific transcriptomes of Globodera pallida elucidate key aspects of plant parasitism by a cyst nematode[END_REF][START_REF] Eves-Van Den Akker | The genome of the yellow potato cyst nematode, Globodera rostochiensis, reveals insights into the basis of parasitism and virulence[END_REF]Blanc-Mathieu et al., 2017;[START_REF] Masonbrink | The genome of the soybean cyst nematode (Heterodera glycines) reveals complex patterns of duplications involved in the evolution of parasitism genes[END_REF] and should be used to investigate the small RNAs produced by the nematode during parasitism. Finally, cross-kingdom RNAi (reviewed by Weiberg and Jin, 2015) probably also occurs during interactions between plants and PPNs. Integrative analyses of the small RNAs from both side of the interactions should shed light on this molecular dialog.

Objectives

My thesis aims to understand how microRNAs regulate the reprogramming of gene expression involved in the formation of feeding sites induced in Arabidopsis thaliana and tomato (Solanum lycopersicum) roots by M. incognita. Previous transcriptomic analysis has shown that the formation of RKN feeding sites is the result of a wide reprogramming of gene expression. Until now, we lack knowledge on how these genes are regulated. I choose to focus on microRNAs, as conserved key regulators of gene expression.

NGS approach was previously developed to identify the small RNAs expressed in A. thaliana galls induced by RKN and uninfected roots at two key stages in the kinetics of giant cell formation: 7 days post infection (dpi) which corresponds to the phase of successive mitoses without cytokinesis and 14 dpi, the phase of endoreduplication. Bioinformatics and statistical analysis of the NGS data identified 24 microRNAs from A. thaliana DE in galls at 7 and/or 14 dpi (Medina et al., 2017). A comparable analysis was carried out during my thesis on a plant of agronomic interest, the tomato S. lycopersicum. Illumina sequencing was performed enabling the analysis of mRNA and small RNAs (<200 nt) expressed in galls 7 and 14 dpi and in uninfected tomato roots. Data from small RNA sequencing were used to predict MIR genes in the tomato genome. Statistical analysis identified 174 mature microRNAs DE at 7 and/or 14 dpi in galls. To understand the function of these miRNAs and identify their targets in the galls, I performed a specific sequencing of cleaved mRNA: "degradome". An integration of the results of the transcriptome and degradome analyzes identified the miRNAs DE in galls and their targets with anti-correlated expression profiles and enable the selection of miRNA/targets couples for initiating functional analysis.

During my thesis, I focused on three miRNA/target couples. In the first chapter, I will present the first couple that I characterized on in tomato: miR167 and its targets the auxin-response factors ARF8A and ARF8B. This couple is associated with the auxin signaling knowing to play a major role in the formation of feeding sites.

In the second chapter, I will present two other miRNA/target couples, miR408 and miR398, that are conserved microRNAs, and their targets associated to copper signaling. These two miRNAs families are among the very few conserved miRNAs that present the same expression profiles in A. thaliana and tomato galls.

The work presented in my thesis highlights the key role of three miRNAs families and their targets in the formation of nematode-induced feeding cells: the miR167/ARF8 pair associated with auxin signaling and the SPL7/miR408-UCC2/miR398-CSD1-BCBP module involved in copper signaling pathway.

Results

CHAPTER 1: miR167-ARF8, an auxin responsive module involved in the formation of galls induced by root-knot nematodes in tomato Article 1 :

Yara Noureddine 1 , Martine da Rocha 1 , Clémence Médina 1 , Mohamed Zouine 2 , Joffrey Meijias 1 , Michael Quentin 1 , Pierre Abad 1, Bruno Favery 1 and Stéphanie Jaubert-Possamai 1 . miR167-ARF8, an auxin responsive module involved in the formation of galls induced by root knot nematodes in tomato. In preparation This first part of my work, consisted in identification of small RNAs from tomato Solanum lycopersicum uninfected roots and galls induced by M. incognita at 7 and 14 dpi. Illumina sequencing enabled the analysis of messenger RNAs (mRNA) and small RNAs (<200 nt) expressed in galls and uninfected tomato roots. Statistical analyzes (DSeq2 and EdgeR) identified 1,958 differentially expressed genes (DEGs) at 7 dpi, 3,468 DEGs at 14 dpi and 1,239 DEGs at both 7 and 14 dpi. Data from small RNA sequencing were used to predict MIR genes in the tomato genome (V3.0) by using three prediction algorithms: MirCat, Shortstack and MirDeep plant. DSeq2 statistical analysis identified 174 DE mature microRNAs at 7 and/or 14 dpi. To understand the function of these miRNAs, the degradome was sequenced and analyzed by CleaveLand algorithm to identify the targets of the miRNAs in the galls. Predicted targets were classified into five categories based on the abundance of reads at the predicted cleavage site relative to the overall profile of reads across the entire transcript. We have limited our analysis to categories 0 and 1 which correspond to the most robust predictions. An integration of the results of the transcriptome and degradome sequencing identified 12 robust miRNA/target pairs in tomato galls. miR167/ARF8 pair was selected further for functional analysis. In galls, miR167 is downregulated and targets the auxin-response factors ARF8A and ARF8B. This downregulation is correlated with the upregulation of its targets in galls. These ARFs belong to auxin signaling pathway, a key hormone in plant-RKN interaction. Using tomato lines expressing the two ARF8 promoters fused to GUS reporter gene, we showed a strong activity of both ARF8 promoters in galls at 7 and 14 dpi, confirming the results of the transcriptomic analyzes. Moreover, we analyzed the effect of a CRISPR deletions within ARF8A and ARF8B coding sequences on the infection by M. incognita. arf8a, arf8 and arf8ab mutants showed a significantly decrease of susceptibility to nematode infection correlated with defects in feeding cell formation. Altogether, these result showed that ARF8A and ARF8B expression is required for successful tomato-RKN interaction. In order to identify the genes targeted by ARF8A and ARF8B, mRNA from galls of WT and arf8a and arf8b has been sent for sequencing. Since the sequencing is still in progress, the results of this article will be discussed later in the general discussion of the thesis. Once we will get the sequencing data, the article will be submitted to New Phytologist.

Introduction

Root-knot nematodes (RKNs) are major crop pests causing massive loss of yields worldwide estimated at millions of euros per year (Blok et al., 2008;[START_REF] Abad | Plant Nematode Interaction: A Sophisticated Dialogue[END_REF]. These microscopic worms of genus Meloidogyne have a wide host spectrum, including more than 5,000 plant species, as well as a wide geographical distribution. Upon root infection, these obligatory plant parasites induce the neoformation of a specialized feeding site critical for nematode survival. After penetration and migration into the roots, the second stage RKN juveniles (J2) injects a cocktail of molecules into five to seven root parenchyma cells (Favery et al., 2016). In response to RKN signals, targeted root parenchyma cells dedifferentiate into giant multinucleated and hypermetabolic feeding cells. These "giant cells" formed the feeding site that supplies nutrients required for nematode development [START_REF] Favery | Gall-Inducing Parasites: Convergent and Conserved Strategies of Plant Manipulation by Insects and Nematodes[END_REF]. The dedifferentiation into giant cells involves a first phase of successive mitoses without cytokinesis followed a second phase of endoreduplication (de Almeida Engler and Gheysen, 2013). In parallel to the formation of the feeding cells, the root cells surrounding the feeding site, named neighboring cells start to divide. This whole process results in a root swelling named gall, which is the characteristic symptom of RKN infection. Several biological processes are involved in the formation of the feeding site like the cell cycle (de Almeida- Engler et al., 2011), metabolic reprogramming [START_REF] Marella | The amino acid permeases AAP3 and AAP6 are involved in root-knot nematode parasitism of arabidopsis[END_REF], cytoskeleton organization (Caillaud et al., 2008b), or auxin signaling (Gheysen and Mitchum, 2019). Auxin or indole-3 Acetic Acid (IAA) is a major plant hormone that play a key role in root development by regulating cell division and the establishment/maintenance of root primordia [START_REF] Smet | Auxin-dependent regulation of lateral root positioning in the basal meristem of Arabidopsis[END_REF][START_REF] Weijers | Transcriptional Responses to the Auxin Hormone[END_REF]. The formation of RKN-induced feeding site has been shown to involved auxin peak [START_REF] Karczmarek | Feeding cell development by cyst and rootknot nematodes involves a similar early, local and transient activation of a specific auxininducible promoter element[END_REF]Absmanner et al., 2013a) and gall transcriptome analyses showed that auxin biosynthesis and auxin-responsive genes were upregulated in A. thaliana early galls while the genes coding for repressors of the auxin response genes were repressed (Barcala et al., 2010). Moreover, application of auxin indole acetic acid (IAA) compound to tomato roots induces in a concentration-dependent weight increase of galls induced by M. javanica [START_REF] Glazer | Interactions between auxin and ethylene in rootknot nematode (Meloidogyne javanica) infected tomato roots[END_REF].

Multiple transcriptome analyses were performed on infected roots, galls or specifically on feeding cells, from various plant species firstly by microarrays and more recently by RNA sequencing. Four time points of feeding site formation were investigated by these transcriptome analyses: the early phase of feeding site formation at 3 days post infection (dpi), 7dpi that corresponds to the multiple mitoses without cytokinesis, 14 dpi that corresponds to the endoreduplication phase and finally 21 dpi when the feeding cells are matured and fully functional. All these analyses showed similar massive reprogrammation of plant gene expression in response to nematode infection with approximatively 10% of protein coding genes which expression is modified in response to nematode infection. How this reprogramming occurs and how these genes are regulated is still poorly understood.

MicroRNAs are small non coding RNAs that are major repressors of gene expression in eukaryotes. Within plant genome, microRNAs are encoding in by MIR genes, often organized as multigene families, that are transcribed in a single stranded RNA precursor which folds into a typical hairpin structure. This hairpin precursor is processed to generate a duplex of mature 20-22 nt microRNA. One of these two mature strands is then loaded into the ARGONAUTE-1 protein, and guides the RNA silencing complex (RISC) to target messenger RNAs by miRNA/mRNA sequence complementarity. mRNA targeting by a microRNA induce its degradation or the inhibition of its translation depending on the mRNA/miRNA sequence complementarity. Several recent articles identified the microRNAs expressed in galls [START_REF] Jaubert-Possamai | MicroRNAs, New Players in the Plant-Nematode Interaction[END_REF] induced by RKN in Arabidopsis thaliana (Cabrera et al., 2016;Medina et al., 2017), tomato Solanum lycopersicum (Zhao et al., 2015;Kaur et al., 2017), in cotton Gossypium hirsutum (Pan et al., 2019;[START_REF] Cai | Small RNA and degradome deep sequencing reveals important roles of microRNAs in cotton (Gossypium hirsutum L.) response to root-knot nematode Meloidogyne incognita infection[END_REF] or in Oryza sativa [START_REF] Verstraeten | Non-coding RNAs in the interaction between rice and Meloidogyne graminicola[END_REF]. However, the role of only four microRNAs has been validated by functional analyses: miR390/tasiRNA/ARF3 module (Cabrera et al., 2016), the miR159/MYB33 pair (Medina et al., 2017), or miR172/TOE1/FT module (Díaz-Manzano et al., 2018) in Arabisopsis or the miR319/TCP4 pair in tomato (Zhao et al., 2015) .

In this article, we investigated the gene regulation network of plant response to RKN by integrating transcriptome, microRNome and degradome sequencing of tomato uninfected roots and galls induced by the RKN M. incognita at two key time points of the gall development: 7 and 14 days post inoculation (dpi). We identified twelve miRNA/targeted transcripts couples that are robust candidates to regulate gall formation. Among these candidates, the role in gall formation of the auxin responsive miR167/ARF8 transcripts pair was confirmed by functional analyses.

Materials and methods

Biological Materials, Growth Conditions

For in vitro experiments, seeds of Solanum lycopersicum cv Micro-Tom and of transgenic lines (pARF8A:GUS and pARF8B:GUS) (provided by Mohamed Zouine; ENSAT, Toulouse) were surface-sterilized with chlorine solution (44% active chlorine) and washed three times with 1ml of milli-Q water. 10 to 15 sterile seeds were sown on a Gamborg B5 (Duchefa Biochemie) agar plates (1x Gamborg B5; pH = 6.4; 1% Sucrose; 0,7% Agar), placed at 24ºC for 48 hours for germination, and finally transferred in a growth chamber (8h light; 16h dark, 20ºC). M. incognita strain Moreleos" J2s were sterilized with HgCl2 (0.01%) and streptomycin (0.7%) as described before [START_REF] Caillaud | In Vivo Imaging of Microtubule Organization in Dividing Giant Cell[END_REF]. One to two weeks after germination, roots were inoculated with 1,000 sterile J2s resuspended in phytagel (5%) per petri dishes.

In soil infection assay

Tomato (Solanum lycopersicum cv Micro-Tom) plants of Wild type and CRISPR lines (ARF8A, ARF8B and ARF8AB) (provided by Mohamed Zouine; ENSAT, Toulouse) were sown in pots filled with a mix 2:3 soil and 1:3 of sand, kept at 4ºC for 48 hours, then transferred in a growth chamber (16h light and 8h dark, at 24ºC). Seedlings were individually transferred in pots filled with a mixture of sand 50% and soil 50% and kept in the growth chamber. Two weeks after germination, each plant was inoculated, with 200 J2s resuspended in water .

Infection rate was evaluated six weeks after inoculation. The root system of each plant was collected, rinsed with tap water, weighted and stained for 30 s. in eosin solution (0.5%). Galls and egg masses were counted for each root under the binocular magnifier MZFLIII (Leica).

Mann-Whitney U-tests (α = 2.5%) were performed to determine the significance of the differences in the numbers of egg masses and galls per root observed between mutants and WT.

BABB clearing

For giant cell area measurements, galls were collected 21 days post-infection (dpi), cleared in benzyl alcohol/benzyl benzoate (BABB) as previously described (Cabrera et al., 2018;[START_REF] Mejias | The root-knot nematode effector MiEFF18 interacts with the plant core spliceosomal protein SmD1 required for giant cell formation[END_REF] and examined under an inverted confocal microscope (model LSM 880; Zeiss).

The mean areas of giant cells in each gall, for wild type and CRISPR lines, for two biological replicates, were measured with Zeiss ZEN software. The impact of the mutation on the giant cell surface was analyzed using a Mann & Whitney Test.

De novo microRNA encoding genes were predicted in tomato genome V3.0 by using three algorithms MirCat [START_REF] Paicu | MiRCat2: Accurate prediction of plant and animal microRNAs from next-generation sequencing datasets[END_REF], Shortstack (Axtell, 2013b) and MirDeep plant [START_REF] Yang | miRDeep-P: A computational tool for analyzing the microRNA transcriptome in plants[END_REF] with default parameters. The HTSEQCOUNT package [START_REF] Anders | HTSeq--a Python framework to work with high-throughput sequencing data[END_REF] was used to count reads mapping perfectly onto the predicted S. lycopersicum mature microRNA 5P or 3P sequence. Reads mapping to multiple loci were counted for each of the loci concerned.

The counts for mature miRNAs (5P and 3P) from each replicate were used for differential expression analysis by using DSeq2 statistical analysis (Anders and Huber, 2010). Mature miRNAs with an adjusted p value below 0.05 were considered as differentially expressed.

Transcriptome Analysis

GO analyses of genes differentially expressed in galls were performed by using overrepresentation test from PANTHER analysis tools (Mi et al. NAR 2013) with a Fisher's exact test, a FDR threshold of 0,05 and by selecting "Biological Process » as GO category.

Degradome analysis

Degradome libraries were constructed from total RNAs extracted from galls at 7 and 14 dpi by Vertis Biotechnologie (Freising, Germany) using the parallel analysis of RNA ends (PARE) protocol described by [START_REF] German | Construction of parallel analysis of rna ends (Pare) libraries for the study of cleaved mirna targets and the rna degradome[END_REF]. The PARE libraries were sequenced on an Illumina High Sequencing 2000 platform. The full raw sequencing data were submitted to the GEO database (http://www.ncbi.nlm.nih.gov/geo/). To identify miRNA targets, degradome reads were analyzed and classified by using the CleaveLand 4.0 [START_REF] Addo-Quaye | CleaveLand: A pipeline for using degradome data to find cleaved small RNA targets[END_REF] algorithm with default parameters. All hits are classified into five categories based on the abundance of the diagnostic cleavage tag relative to the overall profile of degradome tags matching the targets.

GUS staining analysis

We localized the promoter activity in tomatoes transgenic lines expressing a reporter gene GUS fused to the promoter of the two tomato genes ARF8A and ARF8B (pARF8A:GUS and pARF8B:GUS). We inoculated 21-day-old seedlings in vitro, as described above. We collected inoculated roots and washed them in water 7 and 14 dpi. GUS staining was performed as previously described [START_REF] Favery | RPE, a plant gene involved in early developmental steps of nematode feeding cells[END_REF], and the roots were observed under a Zeiss Axioplan 2 microscope. Stained galls were dissected, fixed by incubation in 1% glutaraldehyde and 4% formaldehyde in 50 mM sodium phosphate buffer pH 7.2, dehydrated, and embedded in Technovit 7100 (Heraeus Kulzer, Wehrheim, Germany), according to the manufacturer's instructions. Sections were cut and mounted in DPX (VWR International Ltd, Poole, UK), and observed under a Zeiss Axioplan 2 microscope (Zeiss, Jena, Germany).

Results

Gall formation is the result of a massive reprogrammation of gene expression of the root cells

Transcripts level between tomato galls and uninfected roots were compared with two statistical methods: DSeq2 (Anders and Huber, 2010) and EdgeR [START_REF] Robinson | edgeR: A Bioconductor package for differential expression analysis of digital gene expression data[END_REF]. 19,918 genes were considered for DSeq2 and EdgeR statistical analyses. Genes found as differentially expressed by the two methods with an adjusted pvalue below 0.05 were selected as differentially expressed genes (DEG). 1,958 DEGs were found at 7 dpi (Supplemental table S1) and 3,468 DEGs at 14 dpi (Supplemental table S2 and figure 1A). 1,239 DEGs at both 7 and 14 dpi including 625 down regulated and 600 upregulated genes at both time points and 14

DEGs with an anti-correlated expression fold change at 7 and 14 dpi were identified. 719 genes were found differentially expressed specifically in galls at 7 dpi including 327 upregulated and 392 down regulated genes. 2,229 genes were found specifically differentially expressed in galls at 14 dpi including 1006 upregulated and 1223 down regulated genes. Gene Ontology (GO) analysis of the DEGs in galls at 7 and/or 14 dpi showed an over-representation of the genes associated with biological processes described previously as involved in the formation of giant cells (Supplemental table S3) like i) «cell division» including multiple categories linked to cytokinesis and cell wall biogenesis, ii) «response to auxin», iii) «response to endogenous stimulus» (including response to hormone and to cytokinin) and iv) «response to abiotic stress».

As previously shown for various plant species, this analysis confirms that formation of galls and feeding cells is the result of a massive reprogrammation of gene expression of the root cells.

microRNAs regulate gene expression in galls

To understand how is regulated the massive reprogrammation of gene expression observed in tomato galls, we investigated microRNAs expression in galls. First, we identified tomato miRNAs differentially expressed in galls using Illumina sequencing technology. Small RNA libraries of tomato galls and uninfected roots were constructed, from three independent replicates at two points of gall development: 7 and 14 dpi. These libraries were sequenced generating a total of 333,949,327 raw reads (Supplemental table S4). Reads were cleaned and mapped to a virtual genome constructed from the S. lycopersicum (genome V3.0; ITAG3.3) concatened with M. incognita (genome V2.0; Blanc-Mathieu et al., 2017) to reflect the dual composition of root galls. A de novo prediction of microRNAs was performed by integrating the results from three prediction algorithms: MirCat [START_REF] Paicu | MiRCat2: Accurate prediction of plant and animal microRNAs from next-generation sequencing datasets[END_REF], Shortstack (Axtell, 2013b) and MirDeep plant [START_REF] Yang | miRDeep-P: A computational tool for analyzing the microRNA transcriptome in plants[END_REF]. The sequence homology between newly predicted miRNA mature sequences and mature miRNA sequences listed in miRBase 22.1 was analyzed by using SSearch algorithm [START_REF] Kozomara | MiRBase: From microRNA sequences to function[END_REF].

Expression level of microRNAs in galls and in uninfected roots was compared by DSeq2 statistical analyses (Anders and Huber, 2010). 174 mature microRNAs (5P and/or 3P) corresponding to 148 MIR genes were identified as differentially expressed (DE) between uninfected roots and galls at 7 and/or 14 dpi (Supplemental table S5). From the 174 mature microRNAs DE in galls, 129 were identified as specifically DE at 7 dpi, 11 were specifically DE at 14 dpi and 34 mature microRNAs were DE in galls at 7 and 14 dpi (figure 1B). These 148 MIR genes DE in galls include 65 known MIR genes listed in miRbase [START_REF] Kozomara | MiRBase: From microRNA sequences to function[END_REF] and 73 novel MIR genes. The 65 known MIR genes DE in galls are organized in 20 miRNA families. 14 miRNA families are conserved between Tomato and other plants, while six miRNA families are specific to tomato.

Integration of data from transcriptome, small RNAs and degradome sequencing to build a gene-microRNA regulation network in gall

Once the miRNAs expressed in galls have been identified, the transcripts cleaved by the microRNAs in galls were identified by degradome sequencing [START_REF] German | Construction of parallel analysis of rna ends (Pare) libraries for the study of cleaved mirna targets and the rna degradome[END_REF] of mRNA extracted from galls at 7 (G7) and 14 dpi (G14). CleaveLand pipeline [START_REF] Addo-Quaye | CleaveLand: A pipeline for using degradome data to find cleaved small RNA targets[END_REF] was used to analyse degradome sequencing data and predict mRNA cleaved by miRNAs in galls. We restricted our analysis to the highest confidence targets by selecting CleaveLand categories 0 & 1 with a degradome pvalue below 0.05. 153 transcripts were identified as targeted by microRNAs in galls (Supplemental table S6) including 58 targets shared by the two libraries G7 and G14, while 45 targets were identified specifically in the G7 library and 50 were only found in the G14 library. 111 targets were identified for 135 known miRNAs belonging to 39 miRNAs known families. Out of the 298 novel miRNAs identified in galls, 46 were found to target 47 transcripts in galls at 7 and/or 14 dpi.

To build a gene-miRNAs regulation network involved in the formation of galls, data from transcriptome, microRNAs and degradome sequencing were integrated. From the 153 transcripts were identified as targeted by a microRNAs expressed in galls, 32 were shown as DE in galls by transcriptome analysis. 19 targeted genes were DE in galls at 7 and 14 dpi including 11 upregulated and eight downregulated genes. Five targeted genes were specifically DE at 7 dpi including three transcripts upregulated and two downregulated in galls. At 14 dpi, only eight transcripts identified as targets were DE including three upregulated and five downregulated. Since most plant miRNAs silence gene expression though the cleavage of targeted transcripts, a negative correlation of expression profiles between the microRNA its targeted gene is usually expected. We identified twelve miRNA/mRNA pairs that show negative correlation of their expression level (Table 1). These twelve miRNA/mRNA pairs are the most robust candidates to be involved in the formation of galls.

ARF8 auxin-related transcription factors are involved in tomato-RKN interactions

Among the twelve negatively correlated microRNAs/mRNA pairs, two of the strongest candidate to be regulated by a microRNA are the AUXIN RESPONSE FACTORS 8A and 8B and their common regulator miR167. These two genes belong to ARF transcription factors that relay auxin signaling at the transcriptional level by regulating the expression of auxinresponsive genes [START_REF] Guilfoyle | Auxin response factors[END_REF]. Gall transcriptomic analyzes showed that ARF8B is overexpressed in tomato galls at 7 and 14 dpi and ARF8A is overexpressed at 14 dpi (Table 1). ARF8A and ARF8B were identified as cleaved by miR167 in galls by degradome sequencing. Four MIR167 genes were identified in tomato genome all sharing the same mature sequence and all are down regulated in galls while both ARF8 genes were found to be upregulated. ARF8A and ARF8B transcripts were previously shown to be cleaved by miR167 in tomato [START_REF] Liu | Down-regulation of AUXIN RESPONSE FACTORS 6 and 8 by microRNA 167 leads to floral development defects and female sterility in tomato[END_REF] and this regulation in conserved in Arabidopsis [START_REF] Wu | Arabidopsis microRNA167 controls patterns of ARF6 and ARF8 expression, and regulates both female and male reproduction[END_REF].

The down regulation of miR167 and the upregulation of ARF8A and ARF8B observed in galls suggest that, by repressing MIR167 expression, RKN infection prevents ARF8 silencing by miR167 that occurs in uninfected root.

The tissue expression of ARF8A and ARF8B in roots infected by RKN was further investigated in vivo, by analyzing the activity of both ARF8A and ARF8B promoters in transgenic tomato lines expressing promoter-GUS fusions. A strong GUS signal was observed in galls 7 and 14 dpi of two pARF8B::GUS and pARF8A::GUS lines [START_REF] Bouzroud | Auxin Response Factors (ARFs) are potential mediators of auxin action in tomato response to biotic and abiotic stress (Solanum lycopersicum)[END_REF] and in root tips from uninfected roots (Figure 2A-F). Histological sections of the galls showed a strong GUS signal within the feeding giant cells and in neighboring cells at 7 and 14 dpi for both ARF8A and ARF8B lines (Figure 3A-D). The strong activity of both promoters ARF8A and ARF8B observed in galls in vivo confirm the results of transcriptomic data.

To investigate the role of both ARF8A and ARF8B in gall development, we analyzed the effect of CRISPR deletions within ARF8 coding sequences on the infection by M. incognita (arf8a CR- 2 (deletion 2nt), arf8b CR-11 (deletion 11nt) and arf8ab CR-2,4 , (double mutant) (all provided by M.

Zouine, ENSAT, Toulouse). All these CRISPR lines did not show any root phenotype compared to wild type (Supplemental figure S2). Infection rate of these CRISPR lines inoculated with M. incognita was quantified by counting the galls and egg masses produced by the adult females at the root surface. A strong and significant decrease, of approximately 50%, in the number of galls and egg masses was observed for the arf8a CR-2 , arf8b CR-11 and arf8ab CR-2,4 lines in comparison to wild type plants. These results showed that disruption of ARF8 leads to resistance and therefore demonstrated that ARF8A and ARF8B genes are involved in the plant-RKN interaction (Figure 4A). To check whether the increased resistance of the arf8a CR-2 , arf8b CR-11 and arf8ab CR-2,4 lines was due to defects of giant cells, the feeding site surface was measured directly with a confocal microscope, after gall BABB clearing. A comparison of the mean surface areas of the giant cells in each gall showed that giant cells from CRISPR lines were approximately 30% smaller than those from control plants (Figure 4B).

Altogether, these results showed that ARF8A and ARF8B expression is required fo the proper formation of feeding site during tomato-RKN interaction. This second part of my work consist in studying the role of two copper-miRNAs, miR408 and miR398, in the formation of giant cells in A. thaliana induced by M. incognita interaction. MiR408 and miR398 families and their targets are known to be involved in the copper signaling pathway. In response to copper starvation, the expression of MIR398 and MIR408 genes is activated by the SPL7 transcription factor, and they repress the expression of genes encoding copper binding proteins non-essential for plant development. These two conserved microRNA families are upregulated in tomato and A. thaliana galls. In this study, I have used Arabidopsis lines expressing transcriptional fusion with GUS reporter gene. I showed that both MIR408 and SPL7 were expressed within nematode induced feeding cells. Moreover, infection assays with mir408 and spl7 KO mutants or lines expressing mutated targets resistant for miR398 cleavage showed a decreased of susceptibility towards nematode infection. Moreover, watering plants with copper sulfate, at concentration below toxic concentrations for the plants or for the nematodes, induced a strong resistance to nematode infection. Altogether, these results demonstrate the role of disruption of copper homeostasis through activation of miR398 and miR408 by SPL7 in the formation of giant feeding cells. This article is in revision in the journal New Phytologist.

CHAPTER 2: Copper microRNAs govern the formation of giant feeding cells induced by the root knot nematode Meloidogyne incognita in Arabidopsis thaliana

Introduction (1124 words)

MicroRNAs are small non-coding RNAs that regulate the expression of protein-coding genes, mostly at the post-transcriptional level, in plants. They are major post-transcriptional regulators of gene expression in various biological processes, including plant development (Li and Zhang, 2016), responses to abiotic stresses [START_REF] Barciszewska-Pacak | Arabidopsis microRNA expression regulation in a wide range of abiotic stress responses[END_REF], hormone signalling [START_REF] Curaba | miRNAs in the crosstalk between phytohormone signalling pathways[END_REF], and responses to pathogens or symbiotic micro-organisms (Weiberg and Jin, 2015;[START_REF] Hoang | The role of microRNAs in the legume-Rhizobium nitrogen-fixing symbiosis[END_REF]. MicroRNAs and short interfering RNAs (siRNAs) were recently shown to play a key role in plant-pathogen crosstalk through trans-kingdom RNAi processes [START_REF] Weiberg | Fungal Small RNAs Suppress Plant Immunity by Hijacking Host RNA Interference Pathways[END_REF]Cai et al., 2018a;[START_REF] Dunker | Oomycete small RNAs bind to the plant RNA-induced silencing complex for virulence[END_REF]. MicroRNAs are produced by the cleavage of long double-stranded RNA precursors by the DICER RNAse, generating 20-22 nucleotides miRNA duplexes composed of a mature (5P) and a complementary (3P) strand.

One of the two strands is then incorporated into the RNA-induced silencing complex (RISC) to guide the major RISC protein, ARGONAUTE1 (AGO1), to the targeted messenger RNA (mRNA) on the basis of sequence complementarity. The hybridization of AGO1-bound miRNAs to their targets induces predominantly targeted mRNA degradation in plants, but it may also lead to an inhibition of mRNA translation (Axtell, 2013a).

The miR408 and miR398 microRNA families are conserved so-called "copper microRNAs", due to their involvement in the plant response to copper deficiency [START_REF] Yamasaki | Regulation of copper homeostasis by micro-RNA in Arabidopsis[END_REF][START_REF] Zhang | Microrna408 is critical for the HY5-SPl7 gene network that mediates the coordinated response to light and copper[END_REF]. Copper is an essential nutrient for plants, due to its function as a cofactor for many proteins. Copper proteins are involved in electron transport chains or function as enzymes in redox reactions. In plants, copper is involved in respiration, photosynthesis, ethylene perception, the metabolism of reactive oxygen species and cell wall remodelling (reviewed in [START_REF] Burkhead | Copper homeostasis[END_REF]. Copper microRNAs accumulate in response to copper deficiency and their synthesis is repressed when copper concentrations are sufficiently high [START_REF] Yamasaki | SQUAMOSA promoter binding protein-like7 is a central regulator for copper homeostasis in Arabidopsis[END_REF]. The underlying mechanism has been described in Arabidopsis thaliana, in which the regulation of MIR408, MIR398B and MIR398C by copper levels was shown to be mediated by the SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE7 (SPL7) transcription factor [START_REF] Yamasaki | SQUAMOSA promoter binding protein-like7 is a central regulator for copper homeostasis in Arabidopsis[END_REF]. The A. thaliana genome contains a single copy of MIR408, and three MIR398 genes: MIR398A, -B and -C. At high copper concentrations, the DNA-binding activity of the SPL7 transcription factor is repressed, preventing the induction of transcription for downstream genes, such as MIR408 or MIR398B and MIR398C, but not MIR398A [START_REF] Yamasaki | SQUAMOSA promoter binding protein-like7 is a central regulator for copper homeostasis in Arabidopsis[END_REF][START_REF] Sommer | The CRR1 Nutritional Copper Sensor in Chlamydomonas Contains Two Distinct Metal-Responsive Domains[END_REF]. In the presence of low concentrations of copper, SPL7 activates the expression of copper-responsive microRNAs that target and repress the expression of genes encoding copper-binding proteins. These proteins are replaced by proteins that do not bind copper, to save copper resources for the functions for which this element is essential, such as photosynthesis (reviewed in [START_REF] Burkhead | Copper homeostasis[END_REF]. For example, the mRNA for the cytosolic COPPER/ZINC (Cu-Zn) SUPEROXIDE DISMUTASE, CSD1, which can be replaced by an iron (Fe)-dependent SOD, is targeted by the copper microRNA miR398. In addition to their regulation as a function of copper levels through the activity of SPL7, MIR408 and the MIR398 family are also regulated by several environmental cues and abiotic stresses, such as light, which regulates MIR408 activity through the ELONGATED HYPOCOTYL5 (HY5) or PHYTOCHROME INTERACTING FACTOR1 (PIF1) transcription factors in A. thaliana [START_REF] Jiang | The PIF1-miR408-PLANTACYANIN repression cascade regulates light-dependent seed germination[END_REF], and salinity, oxidative and cold stresses, which have been shown to induce miR408 in A. thaliana [START_REF] Ma | miR408 is involved in abiotic stress responses in Arabidopsis[END_REF], or cadmium treatment in Brassica napus [START_REF] Fu | MicroRNA-mRNA expression profiles and their potential role in cadmium stress response in Brassica napus[END_REF]. The miR408 and miR398 families have been widely analysed in plant responses to abiotic stresses, but little is known of their role in plant responses to biotic stresses. In sweet potato, MIR408 has been associated with plant defences, as it is repressed by jasmonic acid (JA) and wounding, and miR408-overexpressing plants have attenuated resistance to insect feeding [START_REF] Kuo | MicroR408 regulates defense response upon wounding in sweet potato[END_REF]. Moreover, miR398 has been shown to regulate cell death in response to the causal agent of barley powdery mildew, Blumeria graminis (Xu et al., 2014).

Root-knot nematodes (RKN) of the genus Meloidogyne are obligatory sedentary plant parasites capable of infesting more than 5,000 plant species (Blok et al., 2008;[START_REF] Abad | Plant Nematode Interaction: A Sophisticated Dialogue[END_REF]. The RKN larvae penetrate the roots, in which they induce the de-differentiation of five to seven parenchyma root cells and their reprogramming into the multinucleate, hypertrophied feeding cells that form the feeding site. These metabolically overactive feeding cells provide the nutrients required for RKN development [START_REF] Favery | Gall-Inducing Parasites: Convergent and Conserved Strategies of Plant Manipulation by Insects and Nematodes[END_REF]. During the dedifferentiation of vascular cells and their conversion into 'giant' feeding cells, the cells surrounding the feeding site begin to divide again. The growth of the feeding cells and the division of the surrounding cells lead to a root swelling known as a gall. The feeding cell induction occurs in the first three days after root infection. Feeding cell formation can be split into two phases. Firstly, the cells undergo successive nuclear divisions coupled with cell expansion until ten days post infection (dpi) in A. thaliana (Caillaud et al., 2008). In the second phase, from 10 to 21 dpi, the successive nuclear divisions stop and the nuclei of the feeding cells undergo extensive endoreduplication [START_REF] Wiggers | DNA content and variation in chromosome number in plant cells affected by Meloidogyne incognita and M. arenaria[END_REF]de Almeida Engler and Gheysen, 2013). The dedifferentiation of vascular cells and their conversion into giant cells result from an extensive reprogramming of gene expression in root cells, in response to RKN signals. In A. thaliana, the expression of approximately 10% of protein-coding genes is modified in galls induced by RKN (Cabrera et al., 2014;Yamaguchi et al., 2017;[START_REF] Jammes | Genome-wide expression profiling of the host response to root-knot nematode infection in Arabidopsisa[END_REF]reviewed in Escobar et al., 2011). The sequencing of small RNAs identified 24 mature microRNAs differentially expressed between A. thaliana galls induced by M. incognita and uninfected roots at 7 and 14 dpi (Medina et al., 2017). The miR408 and miR398 families of copper-responsive microRNAs were found to be upregulated in galls at 7 and/or 14 dpi.

In this article, we showed a conserved upregulation of these two microRNA families in Arabidopsis and tomato galls. Moreover, we found that the upregulation of miR408 in response to nematode was required for successful infection. Our findings highlighted a strong activity of MIR408 promoter (pMIR408) in early galls that is i) driven by the modulation of environmental copper levels, ii) colocalised with strong SPL7 expression. Moreover, we also demonstrated the involvement of this transcription factor in giant cell formation. In addition, we showed that the silencing of CSD1 and BLUE COPPER BINDING PROTEIN (BCBP) transcripts by miR398 is involved in gall development. Finally, the watering of Arabidopsis with copper sulphate solutions at concentrations below the toxicity thresholds for nematode and plant development greatly decreased the RKN infection and impaired feeding cell development.

Results

The copper microRNA miR408 is crucial for the Arabidopsis-Meloidogyne interaction Our previous analysis of microRNAs expressed in Arabidopsis galls induced by M. incognita revealed an upregulation of mature miR408 in Arabidopsis galls at 7 and 14 dpi, whereas miR398b/c was specifically upregulated in galls at 14 dpi. Sequencing of small RNAs from uninfected roots and galls of Solanum lycopersicum showed that these two microRNA families were also upregulated in tomato galls at 7 and 14 dpi (Table 1 andsupplemental Table S1).

Therefore, these microRNAs are among the very few conserved microRNAs which expression profile is conserved in Arabidopsis and tomato galls. We investigated the role of miR408 in gall development using two previously described Arabidopsis KO mutant lines: miR408-1 and miR408-2 [START_REF] Maunoury | AGO1 and AGO2 Act Redundantly in miR408-Mediated Plantacyanin Regulation[END_REF]. The KO lines and corresponding wild-type plants were inoculated with M. incognita second stage juveniles (J2s) and their susceptibility was quantified by counting the galls and egg masses produced by adult females at the root surface.

The two KO lines for MIR408 had 40 to 50% fewer galls and egg masses than the wild type (p<0.05; Figure 1 and Supplemental Data Set S1). The roots of these KO lines were of similar weight and global architecture to those of wild-type plants (Supplemental Figure S1 and Supplemental Data Set S1). We then investigated the effect of the MIR408 mutation on feeding site development, by comparing the area of feeding cells within galls collected from KO and wild-type plants (Figure 1A-C and Supplemental Data Set S1). Both KO mutants had a significantly smaller feeding site area than the wild type. Overall, these results demonstrate that MIR408 is involved in feeding cell development in the Arabidopsis-nematode interaction, and that the lower susceptibility of the miR408 KO lines is due to defects of feeding site formation.

We investigated the mechanisms by which miR408 regulates feeding cell formation in galls, by identifying the targets of miR408. The psRNA target algorithm [START_REF] Dai | PsRNATarget: A plant small RNA target analysis server (2017 release)[END_REF]predicted 101 genes as putative targets of miR408 in Arabidopsis (Supplemental Data Set S2A). The expression profiles of these genes in galls at 7 dpi and 14 dpi were obtained from previous transcriptome analyses [START_REF] Jammes | Genome-wide expression profiling of the host response to root-knot nematode infection in Arabidopsisa[END_REF]. Only seven of the 101 putative targets were differentially expressed in galls at 7 and/or 14 dpi, and only two putative targets were repressed: a gene encoding a copper-binding protein, UCLACYANIN2 (UCC2, At2g44790), which is known to be cleaved by miR408 in senescing leaves and siliques [START_REF] Thatcher | Differential expression of miRNAs and their target genes in senescing leaves and siliques: Insights from deep sequencing of small RNAs and cleaved target RNAs[END_REF], and a gene encoding a PHOSPHATASE 2G (PP2CG1, At2g33700) (Supplemental Data Set S2B).

MIR408 induction in galls is driven by modulation of copper level

We investigated the induction of MIR408 in response to nematode infection, by inoculating plants expressing pMIR408::GUS with M. incognita J2s (Zhang and Li, 2013) in vitro in the presence of normal copper levels (0.1 µM CuSO 4 ) or with a high copper concentration (Gamborg B5 plus 5 µM CuSO 4 ). In the presence of normal concentrations of copper, we observed a strong GUS signal in developing galls at 3 and 7 dpi (Figure 2A-B). This signal had decreased in intensity by 14 dpi (Figure 2C) and disappeared completely from fully developed galls at 21 and 28 dpi (Supplemental Figure S2). On gall sections, the GUS signal was localised in the giant feeding cells and neighbouring cells, at the 3 dpi, 7 dpi and 14 dpi time points (Figure 2D-F). By contrast, in plants grown in the presence of high copper concentrations, the GUS signal was much weaker in galls at 3 and 7 dpi (Figure 2G-H), and undetectable in galls at 14 dpi (Figure 2I). The repression of MIR408 expression in galls by high copper concentrations indicates that MIR408 expression within Arabidopsis galls is regulated by modulation of copper levels.

SPL7 is an activator of MIR408 transcription in galls

The regulation of MIR408 by modulation of copper levels has been shown to be mediated by the SPL7 transcription factor [START_REF] Zhang | Microrna408 is critical for the HY5-SPl7 gene network that mediates the coordinated response to light and copper[END_REF][START_REF] Bernal | Transcriptome sequencing identifies SPL7-regulated copper acquisition genes FRO4/FRO5 and the copper dependence of iron homeostasis in Arabidopsis[END_REF]. Transcriptome analyses from Arabidopsis galls showed that SPL7 was expressed in galls at 7, 14 and 21 dpi [START_REF] Jammes | Genome-wide expression profiling of the host response to root-knot nematode infection in Arabidopsisa[END_REF]. We further investigated the expression of SPL7 within galls, by inoculating a pSPL7::GUS Arabidopsis line [START_REF] Araki | SPL7 locally regulates copper-homeostasis-related genes in Arabidopsis[END_REF] with M. incognita in the presence of a normal copper concentration. SPL7 promoter activity was observed within the gall from 3 to 14 dpi, with lower levels at 14 dpi (Figure 3A-C). As observed for pMIR408::GUS, sections of pSPL7::GUS galls revealed a GUS signal in the giant feeding cells and neighbouring cells (Figure 3D). We investigated the putative function of SPL7 in plant responses to RKN, by inoculating the Arabidopsis spl7 KO mutant described by [START_REF] Zhang | Microrna408 is critical for the HY5-SPl7 gene network that mediates the coordinated response to light and copper[END_REF] with M. incognita J2s. SPL7 knockout led to the production of smaller numbers of galls and egg masses per plant than were observed for the wild-type (Figure 3E-F and Supplemental Data Set S3). This knockout had no effect on root weight (Supplemental Figure S3 and Supplemental Data Set S3). Measurements of the area of the feeding site within galls revealed defects of feeding site formation in the spl7 KO mutants, resulting in smaller giant cells than were observed in wildtype plants (Figure 3G and Supplemental Data Set S3). Overall, these results demonstrate the requirement of miR408 and SPL7 for the development of giant cells. The upregulation of mature miR408 observed in galls suggest an induction of MIR408 expression driven by SPL7 due to a decrease in copper availability within the gall.

micro398, a second copper-responsive microRNA family involved in the Arabidopsis-Meloidogyne interaction

MiR408 is not the only copper-responsive microRNA differentially expressed in galls. The expression of MIR398B and MIR398C, from the conserved miR398 family, has also been shown to be induced in response to copper deficiency, via SPL7 activity [START_REF] Araki | SPL7 locally regulates copper-homeostasis-related genes in Arabidopsis[END_REF].

We previously described an induction of the mature miR398b and miR398c in Arabidopsis galls at 14 dpi (Medina et al., 2017). Three targets of the miR398 family have been biologically A-H, The activity of the MIR408 promoter was analysed in galls induced by M. incognita in Arabidopsis expressing the pMIR408::GUS construct grown in the presence of normal concentrations of copper (0.1 µ M CuSO 4 )(A-F) or in the presence of high concentrations of copper (5.0 µM CuSO 4 )(G-L). A-C, a strong GUS signal was observed in galls 3 days post infection (dpi) (A), 7 dpi (B) and 14 dpi (C) in plants grown with 0.1 µ M CuSO 4 . D-F, Section of gall at 3 dpi (D), 7dpi (E) and 14 dpi (F) showing the GUS signal in giant cells and in the cells surrounding the giant cells. G-I, a weaker GUS signal was observed in galls from plants grown with 5.0 µM CuSO 4 analysed at 3 dpi (G) and 7 dpi (H) and no GUS signal was observed in galls at 14 dpi (I). J-L, Section of gall at 3 dpi (J), 7dpi (K) and 14 dpi (L). Galls are indicated with an arrow; N, nematode; (*) giant feeding cells; nc, neighbouring cells. Bars 500 µ m (A-C; G-I) or 50 µ m (D-F; J-L). validated: the At1g08830 and At2g28190 transcripts encoding two copper superoxide dismutases, CSD1 and CSD2, respectively, and the At5g20230 transcript encoding the blue copper binding protein (BCBP), identified as a non-canonical target of miR398 [START_REF] Brousse | A non-canonical plant microRNA target site[END_REF]. We investigated the role of miR398 further, by infecting Arabidopsis lines expressingmodified versions of CSD1 (mcsd1) or BCBP (mbcbp) mRNAs rendered resistant to cleavage by miR398 [START_REF] Beauclair | MicroRNA-directed cleavage and translational repression of the copper chaperone for superoxide dismutase mRNA in Arabidopsis[END_REF][START_REF] Brousse | A non-canonical plant microRNA target site[END_REF]. The target mRNA levels is therefore artificially increased in these plants. The prevention of CSD1 transcript cleavage by miR398 had no effect on root weight (Figure S4), but led to lower levels of nematode infection, with the mutant having less galls and egg masses than the wild type (Figure 4 and Supplemental Data Set S4). The mbcbp line also had fewer egg masses than the wild-type (Figure 4 and Supplemental Data Set S4). No defect of feeding cell formation, such as slower feeding cell growth, was observed in either of these lines (Figure 4 and Supplemental Data Set S4). The specific decrease in egg mass production by females provides evidence for a role for miR398 in the functionality of feeding cells, although the mutations in the mcsd1 and mbcbp lines did not affect feeding site size. These findings demonstrate that the cleavage of CSD1 and BCBP transcripts by miR398 is required for plant-RKN interaction.

Modulation of copper levels is essential for plant-RKN interaction

To study further the effect of copper on nematode infection, we analysed the direct effects of copper on nematode survival and gall development. Free-living M. incognita J2s were incubated in several concentrations of copper sulphate (50 µ M to 2 mM) used in previous studies assessing the effect of copper on plant development [START_REF] Schulten | Do Arabidopsis Squamosa promoter binding Protein-Like genes act together in plant acclimation to copper or zinc deficiency?[END_REF]. As a negative control, J2s were incubated in tap water. Living J2 counts after 24 hours in the copper sulphate solution showed that copper was non-toxic at a concentration of 50 µM (Supplemental Figure S5 and Supplemental Data Set S5). By contrast, toxic effects were observed for all other concentrations tested (0.5 mM, 1 mM and 2 mM). We then analysed the effect of copper on gall formation in Col0 and pMIR408::GUS plants grown in soil watered with 50 µ M CuSO 4 .

We also minimised J2 exposure to copper in the soil, by beginning to water plants 50 µM CuSO 4 two days after inoculation, after the J2s had already penetrated the roots. Watering with 50 µM CuSO 4 repressed pMIR408 activity, confirming the effects of such treatment in galls (Supplemental Figure S6 and Supplemental Data Set S6). Watering with 50 µM CuSO 4 had no visible effect on root weight and architecture (Supplemental Figure S7 and Supplemental Data Set S7), but it resulted in a strong and significant decrease in the number of galls and egg masses A-D, The activity of the SPL7 promoter (pSPL7) was studied in galls induced by M. incognita from A. thaliana expressing the pSPL7::GUS construct, at 3 days post inoculation (dpi)(A), 7 dpi (B) and 14 dpi (C). D, GUS activity was observed within 5.0 µ mthick gall sections at 7 dpi. E-G, the KO spl7 line (SALK093849c) was infected with M. incognita J2. This line was significantly less susceptible to RKN than Col0, as shown by the smaller mean number of galls (E) and egg masses (F) per plant in two infection assays. (G), the effect of spl7 mutation on the development of feeding cells was further evaluated by measuring the size of the feeding site produced. Galls were collected seven weeks post in vitro infection for measurement of the area (µ m 2 ) covered by the giant cells, by the BABB clearing method (Cabrera et al., 2018). Mann-Whitney tests were performed for statistical analysis in each experiment; significant differences relative to Col-0: *, P < 0. relative to control plants watered with tap water (Figure 5). These results demonstrate that perturbation of plant copper homeostasis governs the formation plant-RKN interaction.

Discussion

RKN induce the formation of similar giant feeding cells in thousands of plant species. The conservation of the ontogeny and phenotype of nematode-induced feeding cells between species, strongly suggests that the plant molecular mechanisms manipulated by RKN are widely conserved across the plant kingdom. Previous transcriptome analyses on various plant species have shown that the development of galls in roots infected by RKN is associated with a massive reprogramming of gene expression (reviewed in Escobar et al., 2011). MicroRNAs are small non-coding RNAs that regulate gene expression at the post-transcriptional level, and some microRNA families, such as the miR156 and miR167 families, are widely conserved in plants (Chavez Montes Nature Communications 2014). The role for microRNAs in controlling gene expression during the formation of galls was recently reported in Arabidopsis (reviewed in [START_REF] Jaubert-Possamai | MicroRNAs, New Players in the Plant-Nematode Interaction[END_REF], for the conserved microRNAs miR390, miR172 and miR159 (Escobar et al., 2015;Medina et al., 2017).

miR408 and miR398: two copper-responsive microRNA families activated in Arabidopsis galls induced by M. incognita

A previous analysis of the levels of mature microRNAs in galls at 7 and or 14 dpi and in uninfected roots showed that mature mi408 and mir398b/c were induced in galls in response to M. incognita (Medina et al., 2017). A combination of in silico predictions of the transcripts targeted by miR408 and previous transcriptional analyses of galls and uninfected roots identified two putative targets downregulated in galls the genes the UCLACYANIN-2 (UCC2) and the PHOSPHATASE (PP2CG). The cleavage of UCC2 transcripts by miR408 has been biologically validated in Arabidopsis and rice [START_REF] Thatcher | Differential expression of miRNAs and their target genes in senescing leaves and siliques: Insights from deep sequencing of small RNAs and cleaved target RNAs[END_REF][START_REF] Zhang | MiR408 Regulates Grain Yield and Photosynthesis via a Phytocyanin Protein[END_REF].

Moreover, several targets of miR398 have been biologically validated, including the cytosolic CSD1 and chloroplastic CSD2, and the non-canonical target BCBP [START_REF] Beauclair | MicroRNA-directed cleavage and translational repression of the copper chaperone for superoxide dismutase mRNA in Arabidopsis[END_REF][START_REF] Brousse | A non-canonical plant microRNA target site[END_REF]. Our analysis, thus, identified several biologically validated and conserved targets that may be considered robust candidates for mediating the functions of miR398b/c and miR408 in galls. The inactivation of miR408 in T-DNA mutant lines, or of miR398b/c function in transgenic plants expressing mutated CSD1 or BCBP resistant to miR398 cleavage, led to decreases in both the parameters used to assess parasitic success (the number of galls and the number of egg masses per root). The smaller number of galls in the mutant lines demonstrates the involvement of the miR398 family and miR408 in the early plant response to RKN. Moreover, the smaller feeding sites observed in the two miR408 KO lines and the spl7 mutant demonstrate that this miR408 and SPL7 are involved in the formation of the giant cells, which are essential for nematode growth and development. Females are unable to develop normally if the feeding cells are too small, as already reported in some Arabidopsis mutants, such as lines with a knockout of PHYTOSULFOKINE RECEPTOR1 (PSKR1) [START_REF] Rodiuc | Evolutionarily distant pathogens require the Arabidopsis phytosulfokine signalling pathway to establish disease[END_REF]. Only a few genes and plant functions have been demonstrated to be essential for the formation of giant feeding cells [START_REF] Favery | Gall-Inducing Parasites: Convergent and Conserved Strategies of Plant Manipulation by Insects and Nematodes[END_REF]. In the absence of changes in giant cell size in the mcsd1 and mbcbp mutants, we hypothesise that the miR398-regulated CSD1 and BCBP genes may play a role in giant cell functioning, potentially in reactive oxygen species (ROS)-related redox regulation and signalling [START_REF] Zhao | The root-knot nematode effector MiPDI1 targets a stress-associated protein (SAP) to establish disease in Solanaceae and Arabidopsis[END_REF]. Further studies will be required to determine their precise roles in the plant-RKN interaction.

SPL7 is a regulator of miR408 and miR398 in galls

In A. thaliana, it has been shown that MIR408, MIR398B and MIR398C are activated by the same transcription factor, SPL7, the activity of which is dependent on copper levels [START_REF] Yamasaki | SQUAMOSA promoter binding protein-like7 is a central regulator for copper homeostasis in Arabidopsis[END_REF][START_REF] Araki | SPL7 locally regulates copper-homeostasis-related genes in Arabidopsis[END_REF]. We confirmed the activity of the SPL7 promoter and MIR408 in feeding cells and neighbouring cells. The co-expression of SPL7 and MIR408 within developing galls, and the similar nematode infection phenotype, with feeding site formation defects, strongly suggest that SPL7 is responsible for activating MIR408 transcription in galls, as already reported in leaves and the root vasculature [START_REF] Yamasaki | SQUAMOSA promoter binding protein-like7 is a central regulator for copper homeostasis in Arabidopsis[END_REF][START_REF] Araki | SPL7 locally regulates copper-homeostasis-related genes in Arabidopsis[END_REF].

We therefore hypothesised that the expression of MIR408 and MIR398B and -C is activated by the SPL7 transcription factor in response to a decrease in copper concentration within galls.

Other transcription factors, such as HY5 and PIF1, which are known to regulate the expression of MIR408 in response to light stress [START_REF] Zhang | Microrna408 is critical for the HY5-SPl7 gene network that mediates the coordinated response to light and copper[END_REF][START_REF] Jiang | The PIF1-miR408-PLANTACYANIN repression cascade regulates light-dependent seed germination[END_REF], are expressed in galls and could also play a role in the regulation of MIR408 expression. However, the strong repression of MIR408 by excess copper observed in galls suggests that MIR408 upregulation in galls is predominantly driven by copper and SPL7.

Modulation of copper levels, a key conserved factor for gall formation

Infection assays with A. thaliana plants watered with a copper sulphate solution at a concentration non-toxic for plants and nematodes, showed a strong decreased RKN infection rates and resulted in defective feeding site formation. Together with the upregulation in galls of two microRNA families known to be induced by copper deficiency, the miR408 and miR398 families, and the regulation of MIR408 expression by copper, this finding suggests that copper content decreases in the galls induced by RKN infection. This hypothesis is supported by the downregulation of the COPT2 gene, encoding a copper importer, in the Arabidopsis gall transcriptome [START_REF] Jammes | Genome-wide expression profiling of the host response to root-knot nematode infection in Arabidopsisa[END_REF]. Assays in RKN-infected susceptible tomato roots have also demonstrated a decrease in copper concentration [START_REF] Lobna | Biochemical and plant nutrient alterations induced by Meloidogyne javanica and Fusarium oxysporum f.Sp.radicis lycopersici co-infection on tomato cultivars with differing level of resistance to M. javanica[END_REF].

The SPL7/MIR408-UCC2/MIR398-CSD1 copper signalling cascade may be a key factor in gall formation, conserved across the plant kingdom. MiR408 and miR398 have been identified in more than 40 plant species (Griffiths- [START_REF] Griffiths-Jones | miRBase: tools for microRNA genomics[END_REF] and SPL7 is widely conserved throughout the plant kingdom [START_REF] Yamasaki | SQUAMOSA promoter binding protein-like7 is a central regulator for copper homeostasis in Arabidopsis[END_REF]. Moreover, the targeting of UCLACYANIN by miR408 and of CSD1 by miR398 is conserved in both dicotyledonous and monocotyledonous plants [START_REF] Zhang | MiR408 Regulates Grain Yield and Photosynthesis via a Phytocyanin Protein[END_REF][START_REF] Thatcher | Differential expression of miRNAs and their target genes in senescing leaves and siliques: Insights from deep sequencing of small RNAs and cleaved target RNAs[END_REF]. A role for UCLACYANINS in the formation of a lignified nanodomain within the Casparian strips known to form an endodermal barrier in Arabidopsis roots has recently been described [START_REF] Reyt | Uclacyanin Proteins Are Required for Lignified Nanodomain Formation within Casparian Strips[END_REF]. Casparian strip defects have been observed in the endodermis bordering the giant cell area within sorghum galls induced by M. naasi [START_REF] Ediz | Life cycle, pathogenicity, histopathology, and host range of race 5 of the barley root-knot nematode[END_REF]. Moreover, Arabidopsis mutants with disrupted Casparian strips are particularly susceptible to RKN [START_REF] Holbein | Root endodermal barrier system contributes to defence against plantparasitic cyst and root-knot nematodes[END_REF]. The infection of plants with nematodes may, therefore, provides a unique model for investigating the role of copper modulation, via miR408 and its UCLACYANIN2 target, in the formation of Casparian strips.

Methods

Biological material, growth conditions and nematode inoculation

Seeds of A. thaliana Col0 and mutants miR408-1(SALK_038860), miR408-2 (SALK_121013.28.25.n), spl7 (SALK_093849), mcsd1 and mbcbp [START_REF] Beauclair | MicroRNA-directed cleavage and translational repression of the copper chaperone for superoxide dismutase mRNA in Arabidopsis[END_REF], pmiR408::GUS (Zhang et al., 2013) and pSPL7::GUS [START_REF] Yamasaki | SQUAMOSA promoter binding protein-like7 is a central regulator for copper homeostasis in Arabidopsis[END_REF] were surfacesterilised and sown on Gamborg B5 medium agar plates (0.5 x Gamborg, 1% sucrose, 0.8% agar, pH 6.4). The plates were incubated at 4°C for two days, then transferred to a growth chamber (20°C with an 8 h light/ 16 h darkness cycle). M. incognita strain "Morelos" was multiplied on tomato plants in a growth chamber (25°C, 16 h light/8 h darkness). For the RKN infection of plants in soil, two-week-old plantlets grown in vitro were transferred to a mixture of 50% sand (Biot B5)/50% soil in a growth chamber (21°C, 8 h light/16 h darkness). For studies of the effect of copper on gall development on plants in vitro, Arabidopsis plantlets were sown and cultured in vitro, as described above, on Gamborg B5 medium supplemented with 50 µM CuSO 4 .

Root knot nematode infection assay

For nematode infections in vitro, J2s were surface-sterilised with HgCl 2 (0.01 %) and streptomycin (0.7 %), as described by [START_REF] Caillaud | In Vivo Imaging of Microtubule Organization in Dividing Giant Cell[END_REF]. We inoculated each 25-dayold seedlings grown individually in vitro with 200 sterilised J2s resuspended in Phytagel (5 %).

Infection assays were performed on Arabidopsis mutants and a wild-type ecotype in soil. We inoculated 20 to 30 two-month-old plantlets with 150 J2s per plant and incubated them in a growth chamber (21°C, 8 h light/16 h darkness). Seven weeks after infection, the roots were collected, washed in tap water and stained with eosin (0.5 %). Stained roots were weighed and galls and egg masses were counted on each root under a binocular microscope. Mann and Whitney tests (2.5 %) were performed to determine the significance of the observed differences in the numbers of egg masses and galls per root.

Small RNA sequencing from galls and uninfected tomato roots

Biological material, RNA extraction, small RNA sequencing, read mapping and statistical analysis are presented as supplemental material.

BABB clearing

Feeding site development was evaluated by the BABB clearing method described by Cabrera et al., (2018). Briefly, the area occupied by the giant cells was measured on galls collected 14 dpi, cleared in benzyl alcohol/benzyl benzoate (BABB) and examined under an inverted confocal microscope (model LSM 880; Zeiss). Zeiss ZEN software was used to measure the area occupied by the giant cells in each gall, on two biological replicates. Data were analysed in Mann and Whitney tests.

Copper treatment

M. incognita eggs were collected as previously described [START_REF] Caillaud | In Vivo Imaging of Microtubule Organization in Dividing Giant Cell[END_REF] and placed on a 10 µ m-mesh sieve for hatching in tap water. Free-living J2s were collected from the water with a 0.5 µm-mesh sieve. We evaluated the toxicity of copper to J2s by incubating freshly hatched J2s in solutions of copper sulphate of various concentrations for 24 hours. The numbers of living or dead J2s were then determined by counting under a binocular microscope.

We investigated the effects of copper on plant-nematode interactions in Arabidopsis Col0 grown in soil. Arabidopsis Col0 plantlets were prepared and inoculated as previously described for in-soil infection. Half the plants were watered with 50 µ M CuSO 4 two days after inoculation with J2s and then once per week for the next seven weeks. Control Col0 plants were watered with tap water in place of copper sulphate solution, at the same frequency. Seven weeks after inoculation, the plants were collected, their roots were washed and weighed, and the numbers of galls and egg masses on the roots were counted, as described above.

Studies of promoter-GUS fusion gene expression

We localised the promoter activity of MIR408 and SPL7 in A. thaliana lines expressing various fusions of the GUS reporter gene to promoters from these genes (ref & Supplemental Table S7). We inoculated 21-day-old seedlings in soil and in vitro, as described above. We collected inoculated roots and washed them in water, 3, 7, 14 and 21 dpi. GUS staining was performed as previously described [START_REF] Favery | RPE, a plant gene involved in early developmental steps of nematode feeding cells[END_REF], and the roots were observed under a Zeiss Axioplan 2 microscope. Stained galls were dissected, fixed by incubation in 1% glutaraldehyde and 4% formaldehyde in 50 mM sodium phosphate buffer pH 7.2, dehydrated, and embedded in Technovit 7100 (Heraeus Kulzer, Wehrheim, Germany), according to the manufacturer's instructions. Sections were cut and mounted in DPX (VWR International Ltd, Poole, UK), and observed under a Zeiss Axioplan 2 microscope (Zeiss, Jena, Germany). Supplemental Figure S4. Root phenotype and area covered by the feeding site in the mcsd1 and mbcbp lines. (A), Weight of infected roots from the mcsd1 and mbcbp lines relative to that for Col0 plants, seven weeks after infection, as assessed by Mann & Whitney statistical tests. No difference in weight was observed between the roots of the wild-type Col0 and those of the two mutant lines. (B), The effect of mCSD1 and mBCBP mutations on the development of giant feeding cells was evaluated further, by measuring the size of the feeding site produced in each KO line and compared it with that in wild-type roots. Galls were collected seven weeks after in vitro infection for measurement of the area (µ m 2 ) covered by the giant cells by the BABB clearing method (Cabrera et al., 2018) predicted in tomato genome V3.0 by using three algorithms MirCat [START_REF] Paicu | MiRCat2: Accurate prediction of plant and animal microRNAs from next-generation sequencing datasets[END_REF], Shortstack (Axtell,2013) and MirDeep plant [START_REF] Yang | miRDeep-P: A computational tool for analyzing the microRNA transcriptome in plants[END_REF] with default parameters. The HTSEQCOUNT package [START_REF] Anders | HTSeq -A Python framework to work with high-throughput sequencing data[END_REF] was used to count reads mapping perfectly onto the predicted S. lycopersicum mature microRNA 5P or 3P sequence. Reads mapping to multiple loci were counted for each of the loci concerned. The counts for mature miRNAs (5P and 3P) from each replicate were used for differential expression analysis by using DSeq2 statistical analysis (Andres & Huber, 2010). Mature miRNAs with an adjusted p value below 0.05 were considered as differentially expressed.

Complementary results

In addition to the results presented in the article submitted to The Plant Cell, additional analyses were performed. The expression profiles of key genes of Casparian Strip (CS) was investigated in available A. thaliana transcriptome and measurement of copper and other micronutrients has been performed by Dr. Sébastien Thomine at I2BC institute, Gif-sur Yvette, France.

1) Expression profile of genes involved in the formation of CS

In order to identify the expression profile for the genes involved in the CS strip formation, we analyzed in the transcriptomic data of A. thaliana galls at 3, 5 and 7 dpi (Yamaguchi et al., 2017) (Table 3). These data showed a global repression of most genes involved in the formation and lignification of CS. Surprisingly, MYB36, the transcription factor that orchestrates the CS formation, is upregulated in galls at 3, 5 and 7 dpi, while most of MYB36 targets are downregulated. These results suggest that RKN induce a defect in CS formation through a disruption of copper homeostasis.

2) Measurement of Copper and other nutrients

The nutrient content (copper, iron, calcium, magnesium, manganese and zinc) in dissected galls, whole infected and uninfected roots of tomato and A. thaliana was performed by Microwave Plasma-Atomic Emission Spectroscopy (MP-AES).

Since sampling tomato galls in vitro is easy, we first measure the level of micronutrients in galls and uninfected roots collected at 14 dpi in vitro (Table 4a). A reduction of copper level in tomato galls compared to uninfected roots was observed in the three independent replicates. This result supports our hypothesis of a decrease of copper level in galls. A reduction of Fe, Mn and Zn was observed in tomato galls compared to the roots. Then the level of nutrients was measured in whole infected (RI) and uninfected roots (RNI) in soil, from 3 independent replicates. A similar reduction of copper level is observed in whole infected roots compared the uninfected roots (Table 4b).

For the measurement in A. thaliana, we didn't measure nutrients level in galls in vitro due to the need of important quantities of material for the experiment. We measured the level of micronutrients in galls and uninfected roots collected at 14 dpi in soil, from three independent replicates (Table 5a). A slightly reduction of copper level was observed in galls compared to uninfected roots in the first and third replicate. Since the expression of MIR408 decreased in galls at 14 dpi (miR408::GUS), this suggested that the copper level decrease at 14 dpi. Therefore, we measured the copper at earlier stage of gall development. Since the experiment required important quantities of material, we have collected whole infected roots at 4 dpi in soil. Surprisingly, we observed an increase of copper level in whole infected roots (RI)

Discussion

Sequencing analysis in tomato

Understanding the transcriptional modifications that result in the formation of giant feeding cells, enables to understand the molecular dialogue between the plant and the nematode. The development of NGS made transcriptomic analysis possible in different non model plant species (reviewed in Favery et al., 2016). All the different transcriptomic analyses presented in the introduction, showed the profound molecular changes observed in forming giant cells: approximatively 15 % of genes are differentially expressed in galls compared to the uninfected roots. Although the biological functions of these genes are starting to be deciphered, little is known about the regulators of gene expression reprogramming. The regulation of gene expression by microRNAs involved in giant cell formation induced by RKN was first evidenced in 2015 (reviewed in [START_REF] Jaubert-Possamai | MicroRNAs, New Players in the Plant-Nematode Interaction[END_REF]. My PhD aimed to characterize the microRNAs involved in the formation of giant feeding cells induced by M. incognita in a plant of agronomic interest: tomato (Solanum lycopersicum). This characterization was performed at two key kinetic points of formation of giant feeding cells, which corresponds to the first phase of successive mitosis without cytokinesis (7 dpi) and a later phase of endoreduplication and cell growth (14 dpi).

Identification of miRNAs differentially expressed in tomato galls

Illumina sequencing was performed enabling the analysis of messenger RNAs (mRNA) and small RNAs (<200 nt) expressed in galls and uninfected tomato roots 7 and 14 dpi. Bioinformatics and statistical analysis identified 174 microRNA differentially expressed between galls and uninfected roots at 7 and/or 14 dpi. A comparable analysis was carried out in 2017 in our lab with A. thaliana (Medina et al., 2017). Surprisingly, we found only two miRNAs (miR408 and miR398) that share the same expression profile in A. thaliana and tomato galls. In the literature, comparison of small RNA sequencing in different plant species [START_REF] Jaubert-Possamai | MicroRNAs, New Players in the Plant-Nematode Interaction[END_REF] identified very few miRNAs with the same profile expression. miR408 and miR827 upregulated in our tomato data, were also upregulated in cotton galls at 10 dpi (Pan et al., 2019). This few similarities of conserved miRNA expression profile seems to rely on the phase of feeding site development and the biological material.

Two genome wide analyses of RKN-responding microRNAs in tomato have been previously published (Zhao et al., 2015;Kaur et al., 2017). [START_REF] Zhao | Suppression of microRNA159 impacts multiple agronomic traits in rice (Oryza sativa L.)[END_REF] identified miRNAs expressed in whole root infected by M. incognita at early stage of gall formation (from 6 hours post infection until 3 dpi), in wild-type plants and in spr2 (suppressor of prosystemin-mediated response 2, JA-deficient) mutant disrupted in the acid jasmonic pathway and resistant to RKN. On the other hand, the work of Kaur et al., (2017) identified miRNAs expressed in whole root infected by M. incognita at five stage of nematode infection with stage 2 (5, 6 and 7dpi) and stage 3 (12, 13, 14 dpi) that overlap with our analyses. The biological material (whole root vs hand dissected galls) and/or the timing of infection kinetic and/or the number of replicates of these studies are different from our analysis. It is therefore difficult to compare the data between these three studies. Despite this, some RKN responsive tomato microRNAs are shared by our analyses and Kaur et al., like the miR164 family which is upregulated in galls at 14 dpi, or miR169 which is upregulated at 7 dpi.

Many analyses of miRNAs involved in plant-CN interaction were also performed in different plant species (Hewezi et al., 2008;Święcicka et al., 2017;Tian et al., 2017;Koter et al., 2018;Noon et al., 2019). CN and RKN are closely related nematode that induce feeding sites within host plant. As RKNs have a large range of hosts, similar giant cells are observed in almost all vascular plants while host range of CN is more restrained. The formation of feeding sites by RKN and CN differ in several points. Following intercellular migration, RKN J2 selects five to seven parenchyma cells and induces their dedifferentiation into giant feeding cells through successive mitosis without cytokinesis. By contrast, CN J2 targets a single initial root cell that expands within the vascular tissue by progressive cell wall dissolution and incorporation into the syncytium of adjacent cells via cytoplasm fusion (Golinowski et al., 1996;Grundler et al., 1998). While endoreduplication occurs in giant cells and syncytia, the successive mitoses without cytokinesis is specific of giant feeding cells [START_REF] Huang | Wall modifications in developing giant cells of Vicia faba and Cucumis sativus induced by root knot nematode, Meloidogyne javanica[END_REF][START_REF] Almeida Engler | Dynamic cytoskeleton rearrangements in giant cells and syncytia of nematode-infected roots[END_REF]. In contrast, cell fusions following partial cell wall dissolution are only observed in syncytia. Although they differ in their ontogeny, syncytium and giant cells have similar phenotype. Both of these feeding sites are hypertrophied and multinucleated, highly active metabolically, have a dense cytoplasm (Sobczak and Golinowski, 2011;Favery et al., 2016) and accumulate sugars and amino acids (Hofmann et al., 2010;Baldacci-Cresp et al., 2012). Cell wall composition of syncytium and giant cells appears similar and its mainly composed of polysaccharides [START_REF] Rodiuc | On the track of transfer cell formation by specialized plant-parasitic nematodes[END_REF]). In addition, both feeding sites develop cell wall ingrowth to facilitate the uptake of solutes [START_REF] Offler | Transfer Cells: Cells Specialized for a Special Purpose[END_REF]. Around both syncytia and giant cells, sieve elements are interconnected by PDs at early stage of feeding site formation. On the contrary of giant cells, symplastic transport enables movement of nutrients into syncytia [START_REF] Hoth | Nematode infection triggers the de novo formation of unloading phloem that allows macromolecular trafficking of green fluorescent protein into syncytia[END_REF][START_REF] Hoth | Differential vascularization of nematode-induced feeding sites[END_REF]. Expression profile of conserved miRNAs DE between giant cell or syncytia and giant cells have different expression profiles. The difference of miRNAs expression profile can be explained by the distinct ontogenesis of these two feeding sites (reviewed in [START_REF] Jaubert-Possamai | MicroRNAs, New Players in the Plant-Nematode Interaction[END_REF]. However, we find two miRNAs families with the same expression profile in giants cells and syncytium in tomato: miR396 and miR167 families that are downregulated in our data and in syncytium induced in tomato infected by Globodera rostochiensis (Święcicka et al., 2017;Koter et al., 2018).

Degradome analysis

A degradome sequencing was performed from gall RNAs to understand the function of the miRNAs DE in tomato galls. From 153 transcripts identified as targeted by microRNAs in galls only 12 were selected as robust candidates. However, this is a very stringent selection and expression profile of microRNAs and their biologically validated targets are not always negatively correlated. Firstly, some transcripts can be targeted by several microRNAs with different expression pattern. MiR159 and miR319 are closely related microRNAs that share some targets like MYB and TCP (Palatnik et al., 2007). Gall degradome data showed that these two microRNA families shared four targets in galls 7 and 14 dpi but displayed different expression profile in galls. On the other hand, one miRNA can target multiple genes. For example, the genes of miR396 family that are downregulated in galls, targets different genes, GRF and basic helix-loop-helix (bHLH), that are also repressed. Moreover, a feedback regulation of a miRNA by its own target is common in plant like the negative feedback loop involving miR172 that is positively regulated by the transcription factors TARGET OF EARLY ACTIVATION TAGGED (TOE1 and TOE2) it targets [START_REF] Wu | The Sequential Action of miR156 and miR172 Regulates Developmental Timing in Arabidopsis[END_REF]. Therefore, additional DE miRNAs/mRNA target pairs identified by degradome analyses may be involved in plant RKN interaction despite the absence of negatively correlated expression profile.

From these 12 miRNA/target pairs, three families of microRNAs were selected for functional analysis: miR167, miR408 and miR398. MiR408 and miR398 were selected due to the conservation of their expression profiles in A. thaliana and in tomato while and miR167 was selected based on its role in auxin signaling, a main process for the formation of giant cells.

miR167/ARF8: an auxin responsive module involved in gall formation

Auxin and ARFs

Among the various phytohormones, auxin plays a role in the formation of giant feeding cells (reviewed in Gheysen and Mitchum, 2019). As presented in introduction auxin peaks have been showed in formation of RKN induced feeding site. Likewise, auxin-mimicking compounds have been found in nematode secretions [START_REF] De Meutter | Identification of cytokinins produced by the plant parasitic nematodes Heterodera schachtii and Meloidogyne incognita[END_REF]De Meutter et al., 2005). This phytohormone regulates several mechanisms in plants such as cell division, organ differentiation, embryogenesis or lateral root initiation [START_REF] Quint | Auxin signaling Marcel[END_REF][START_REF] Majda | The Role of Auxin in Cell Wall Expansion[END_REF].

Auxin regulates a large number of genes that are involved in plant growth and development processes in many plant species [START_REF] Zouine | Characterization of the Tomato ARF Gene Family Uncovers a Multi-Levels Post-Transcriptional Regulation Including Alternative Splicing[END_REF][START_REF] Guilfoyle | The PB1 domain in auxin response factor and aux/IAA proteins: A versatile protein interaction module in the auxin response[END_REF]. Auxin signaling pathway is a complex mechanism involving multiple gene families. A. thaliana contains 6 auxin receptors of the TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING 123 F-BOX PROTEIN (TIR1/AFB) family, 29 AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) repressors, and 23 ARFs transcription factors [START_REF] Remington | Contrasting modes of diversification in the Aux/IAA and ARF gene families[END_REF] The distribution of auxin is achieved via the auxin transporters such as PINFORMED1 (PIN) and AUXIN1/LIKE AUX1 (AUX/LAX) family [START_REF] Bainbridge | Auxin influx carriers stabilize phyllotactic patterning[END_REF][START_REF] Adamowski | PIN-dependent auxin transport: Action, regulation, and evolution[END_REF] (Figure 23 A). ARFs mediate auxin signaling by directly transmitting auxin response through the activation or repression of auxin-induced genes [START_REF] Liscum | Genetics of Aux/IAA and ARF action in plant growth and development[END_REF]. ARFs are a large multigene family conserved across plant kingdom that is well described in various plant species such as A. thaliana (23 genes) [START_REF] Hagen | Auxin-responsive gene expression: Genes, promoters and regulatory factors[END_REF], S. lycopersicum (22 genes) [START_REF] Zouine | Characterization of the Tomato ARF Gene Family Uncovers a Multi-Levels Post-Transcriptional Regulation Including Alternative Splicing[END_REF], Oryza sativa (25 genes) [START_REF] Wang | Genome-wide analysis of the auxin response factors (ARF) gene family in rice (Oryza sativa)[END_REF] or Glycine max (51 genes) [START_REF] Van Ha | The auxin response factor transcription factor family in soybean: Genome-wide identification and expression analyses during development and water stress[END_REF].

In high level of auxin, ARF8 together with ARF5 ARF6, ARF7, and ARF19 (class II of ARF) activate transcription of auxin-responsive genes [START_REF] Tiwari | The roles of auxin response factor domains in auxinresponsive transcription[END_REF] (Figure 23B,23C). In A. thaliana roots, the role of ARF8 have been described in the formation of lateral roots [START_REF] Gifford | Cell-specific nitrogen responses mediate developmental plasticity[END_REF] and adventitious roots [START_REF] Gutierrez | Phenotypic Plasticity of Adventitious Rooting in Arabidopsis Is Controlled by Complex Regulation of AUXIN RESPONSE FACTOR Transcripts and MicroRNA Abundance[END_REF]. Recently, the role of ARF6 and ARF8 was attributed to cambium establishment and maintenance [START_REF] Ben-Targem | Auxin and gibberellin signaling cross-talk promotes hypocotyl xylem expansion and cambium homeostasis[END_REF]. arf6arf8 double mutant displayed decrease in xylem occupancy and absence of fiber accumulation until very late stages of plant growth. This regulation is via a crosstalk between GA and auxin.

Regulation of ARF8 in tomato galls

Gall transcriptomic analyzes showed that ARF8B is overexpressed in tomato galls at 7 and 14 dpi while ARF8A is overexpressed at 14 dpi. Using tomato transgenic lines expressing the promoter of ARF8A and ARF8B fused to GUS, we confirmed RNAseq results and located the expression of both genes in giant cells and neighboring cells at 7 and 14 dpi. Degradome analysis identified ARF8A and ARF8B as cleaved by miR167 in galls. In tomato, four MIR167 genes sharing the same mature sequence were identified. They are all down regulated in galls at 7 and 14 dpi. In A. thaliana and in tomato, ARF6 and ARF8 have been shown to be targeted by miR167 [START_REF] Wu | Arabidopsis microRNA167 controls patterns of ARF6 and ARF8 expression, and regulates both female and male reproduction[END_REF][START_REF] Liu | Down-regulation of AUXIN RESPONSE FACTORS 6 and 8 by microRNA 167 leads to floral development defects and female sterility in tomato[END_REF]. In tomato, the two ARF8 and two ARF6 genes are cleaved by miR167 [START_REF] Liu | Down-regulation of AUXIN RESPONSE FACTORS 6 and 8 by microRNA 167 leads to floral development defects and female sterility in tomato[END_REF]. The overexpression of miR167 in tomato, leads to a downregulation of ARF8 and ARF6 genes which caused defect in flower maturation and fertility. ARF6 and ARF8 are closely related putative ARFs with both independent and cooperative functions in plant development [START_REF] Nagpal | Auxin response factors ARF6 and ARF8 promote jasmonic acid production and flower maturation[END_REF][START_REF] Gutierrez | Phenotypic Plasticity of Adventitious Rooting in Arabidopsis Is Controlled by Complex Regulation of AUXIN RESPONSE FACTOR Transcripts and MicroRNA Abundance[END_REF][START_REF] Gutierrez | Auxin Controls Arabidopsis Adventitious Root Initiation by Regulating Jasmonic Acid Homeostasis[END_REF][START_REF] Liu | Down-regulation of AUXIN RESPONSE FACTORS 6 and 8 by microRNA 167 leads to floral development defects and female sterility in tomato[END_REF]Wang et al., 2015a). However, they are expressed differentially throughout plant development [START_REF] Rademacher | A cellular expression map of the Arabidopsis AUXIN RESPONSE FACTOR gene family[END_REF][START_REF] Vernoux | The auxin signalling network translates dynamic input into robust patterning at the shoot apex[END_REF].

qRT-PCR analysis showed that ARF6B displayed a low expression in all tomato tissues, including the root [START_REF] Zouine | Characterization of the Tomato ARF Gene Family Uncovers a Multi-Levels Post-Transcriptional Regulation Including Alternative Splicing[END_REF]. In tomato roots, the level expression of ARF6A, ARF8A and ARF8B is very low, with a higher expression of ARF6A, probably due to the cleavage by miR167 [START_REF] Liu | Down-regulation of AUXIN RESPONSE FACTORS 6 and 8 by microRNA 167 leads to floral development defects and female sterility in tomato[END_REF][START_REF] Zouine | Characterization of the Tomato ARF Gene Family Uncovers a Multi-Levels Post-Transcriptional Regulation Including Alternative Splicing[END_REF]. However, neither ARF6A nor ARF6B have been identified as targeted by miR167 in degradome. Therefore, I propose a model of ARF8 regulation in tomato roots and galls (Figure 24). In roots, ARF8 transcripts are cleaved by miR167. During RKN infection, the inhibition of miR167 in galls prevents the cleavage of ARF8, together with auxin peak, its allows an overexpression of ARF8.

Although the repression of miR167 prevents post-transcriptional silencing of ARF8A and ARF8B in galls, how the expression of these transcription factors is activated in galls needs to be further investigated. Recently the transcriptional regulation of ARF8 by a complex network of multiple activating and repressing transcriptional factors has been shown in A. thaliana figure [START_REF] Truskina | A network of transcriptional repressors modulates auxin responses[END_REF]. This work showed that most TFs regulating ARF8 expression are involved in plant development such as WUSCHEL but many ARF8 regulators are associated with biotic and abiotic stress. The authors proposed that ARF8 may act as an environmental hub mediating auxin responsiveness. The formation of feeding sites by RKN interferes with plant developmental processes, suggesting that nematode may hijack these mechanisms. In A. thaliana, miR167/ARF8 controls the balance between initiating and emerging lateral roots in relation to nitrogen availability [START_REF] Gifford | Cell-specific nitrogen responses mediate developmental plasticity[END_REF]. Indeed, several genes involved in lateral root formation were shown to be required for the formation of giant cell. The transcription factor, LBD16 that is activated by auxin is a perfect example on the role of auxin gene in lateral root development and also in gall formation (Cabrera et al., 2014b).

ARF8 is involved in plant response to microorganisms

In order to understand if ARF8 plays a role in nematode parasitism, we used tomato lines with CRISPR deletion within ARF8A, ARF8B and ARF8AB coding sequence (provided by M. Zouine). arf8a, arf8b and arf8ab lines showed a resistance towards nematode infection. Moreover, phenotyping of giant cells in cleared galls showed a reduced size of giant cells within the two crisper lines. These defects associated to CRISPR lines confirmed that expression of ARF8A and ARF8B genes play a key role in the tomato-RKN interaction and are necessary for a proper formation of giant cell.

During biotic stresses, different ARFs, including ARF8, have shown to be regulated in leaves under different biotic stress such as Flagelline and Pseudomonas syringae [START_REF] Bouzroud | Auxin Response Factors (ARFs) are potential mediators of auxin action in tomato response to biotic and abiotic stress (Solanum lycopersicum)[END_REF]. A recent study highlighted the role of miR167 and its two targets ARF6 and ARF8 against P. syringae pv. tomato DC3000 in A. thaliana [START_REF] Caruana | Overexpression of Arabidopsis microRNA167 induces salicylic acid-dependent defense against Pseudomonas syringae through the regulation of its targets ARF6 and ARF8[END_REF]. In this study, the authors showed that A. thaliana plants overexpressing miR167a had smaller stomatal apertures with less symptoms of infection by P. syringae. arf6 arf8 double mutant were also resistant to P. syringae, with the same phenotypes as plants overexpressing miR167, suggesting that miR167 modulates defense through these two targets. These results suggest that the resistance observed in plants overexpressing miR167 is due to the suppression of auxin responses. In soybean, a role for the module miR167-ARF6/8 was established in nodule formation. The regulation of ARF8A and ARF8B by miR167 regulates soybean nodulation and lateral root development in soybean infected by Bradyrhizobium japonicum [START_REF] Wang | Identification and expression analysis of miRNAs from nitrogen-fixing soybean nodules[END_REF]Wang et al., 2015). These different studies suggest that the regulation miR167/ARF8 it is key actor in the response to the environmental stresses but also in response to pathogen infection, therefore a role in defense in giant cells. We can suggest that the induction of ARF8 in tomato galls, is in favor for nematode parasitism to bypass plant defense.

miRNAs responsive to auxin in plant-RKN interaction

In previous studies, two other auxin-responsive microRNAs, miR390 and miR172, have been shown to be involved in the formation of feeding site induced by RKN (Cabrera et al., 2016;Díaz-Manzano et al., 2018). miR390 is overexpressed in A. thaliana galls at 3, 7 and 14 dpi. miR390 induces the cleavage of the lncRNA encoded by the TAS3 genes resulting in secondary siRNAs production (tasiRNA). TasiRNAs induce the cleavage of mRNAs of ARF2, ARF3 and ARF4 (Marin et al., 2010). KO of either MIR390 or TAS3 gene, leads to the reduction of the number of galls, suggesting that the miR390/TAS3/ARF2-3-4 module is also required for the formation of the gall induced by M. javanica (Cabrera et al., 2016). Moreover, the role of miR172 and the two transcription factors TOE1 (TARGET OF EARLY ACTIVATION TAGGED 1) and FT (FLOWERING LOCUS T) has been demonstrated in the formation of giant cells in A. thaliana. In roots, the 3′ strand of mature miR172 has been shown to be downregulated i n galls at 3 dpi, whereas the pri-miR172 precursor is induced, and its target TOE1 is repressed (Barcala et al., 2010). Consistent with the negative regulation of FT by TOE1, an induction of FT was observed in galls at 3 dpi. A. thaliana plants expressing miR172-resistant TOE1 or KO for FT were less susceptible to RKNs and had smaller galls and giant cells (Díaz-Manzano et al., 2018). Since miR167 is not the only miRNA that responds to auxin, we can suggest that the other miRNAs can interfere in auxin response in galls.

Copper microRNAs

The two copper microRNA families upregulated in galls, miR398 and miR408, are the only conserved miRNAs that that have the same profile expression in our data (tomato and A. thaliana) galls. This study highlighted the role of copper in the plant response to nematode.

Copper in plants

Copper is an essential micronutrient necessary for human, animals and for plant growth, and it is required for different physiological and biochemical processes [START_REF] Yruela | Copper in plants[END_REF][START_REF] Garcia | Mitochondria and copper homeostasis in plants[END_REF]. It has been shown that animals use copper as an antimicrobial weapon. Microbes have also developed mechanisms to counteract the toxic effects of copper [START_REF] Samanovic | Copper in microbial pathogenesis: Meddling with the metal[END_REF]. In plants, copper plays a critical role in mitochondrial respiration, cell wall metabolism and remodeling, hormone signaling and in response to oxidative stress [START_REF] Pilon | Copper cofactor delivery in plant cells[END_REF]. Copper is a cofactor of different enzymes such as laccase, cytochrome c oxidase and superoxide dismutase (SODs). However, the deficiency or the excess of copper is very toxic for the plants. Symptoms of copper deficiency can be spotted by the whitening and curling of leaves, reduction in plant growth, decrease in cell wall formation and lignification in several tissues [START_REF] Tiffin | Mineral Nutrition of Plants: Principles and Perspectives[END_REF][START_REF] Martin | The Mineral Nutrition of Higher Plants[END_REF]. Excess of copper can also interfere with plant growth and development and causes reduction in plant biomass, leaf chlorosis, necrosis and also inhibited root growth [START_REF] Martin | The Mineral Nutrition of Higher Plants[END_REF][START_REF] Prasad | Impact of Heavy Metals on Photosynthesis[END_REF][START_REF] Navari-Izzo | Copper excess triggers phospholipase D activity in wheat roots[END_REF]. The excess of copper causes the generation of reactive oxygen species (ROS) a signaling molecule involved in the regulation of various physiological and developmental processes and in defense against pathogen [START_REF] Hänsch | Physiological functions of mineral micronutrients (Cu, Zn, Mn, Fe, Ni, Mo, B, Cl)[END_REF][START_REF] Karuppanapandian | Reactive oxygen species in plants: Their generation, signal transduction, and scavenging mechanisms[END_REF]. Therefore, it is crucial for plants to maintain copper homeostasis.

Copper homeostasis is mainly regulated by the copper-responsive transcription factor SPL7 [START_REF] Yamasaki | SQUAMOSA promoter binding protein-like7 is a central regulator for copper homeostasis in Arabidopsis[END_REF]. In low copper condition, SPL7 binds to DNA and activates the transcription of copper-responsive microRNAs: miR397, miR398, miR408, and miR857 [START_REF] Yamasaki | Regulation of copper homeostasis by micro-RNA in Arabidopsis[END_REF]. These microRNAs repress the expression of genes encoding nonessential copper-binding proteins such as SODs and laccases; therefore, their expression is repressed to preserve copper for the copper binding proteins involved in most vital functions for the plant like photosynthesis. In contrast, when the plant is in excess of copper, it prevents the binding of SPL7 to DNA and the expression of copper-responding miRNAs enabling expression of genes coding copper binding proteins [START_REF] Yamasaki | SQUAMOSA promoter binding protein-like7 is a central regulator for copper homeostasis in Arabidopsis[END_REF]Zhang and Li, 2013;[START_REF] Shahbaz | Conserved Cu-MicroRNAs in Arabidopsis thaliana Function in Copper Economy under Deficiency[END_REF].

Conservation of copper module

Our sequencing results support a conserved role for modulation of copper homeostasis in plant response to root-knot nematodes in A. thaliana and tomato. Sequencing analyzes show that miR408 and miR398 families are overexpressed in A. thaliana but also in tomato galls. The identification of the targets of these two microRNAs in the degradome analyses of tomato galls and in silico (psRNA target) in A. thaliana, associated with the integration of transcriptomic data in these two plants identified conserved targets for these microRNAs.

COPPER SUPEROXIDE DISMUTASE1 (CSD1) and UCLACYANIN 2 (UCC2), targets of miR398 and miR408 respectively, are both repressed in galls of both plant species. In a collaboration with Dr. Sébastien Thomine (I2BC institute, Gif-sur Yvette, France), we have measured copper level in tomato galls versus uninfected roots. A decrease of copper level has been observed in galls collected at 14 dpi in vitro and in whole infected roots in soil in comparison to uninfected roots (Table 4). Another study in 2017 showed a reduction of copper in tomatoes in whole roots infected by M. javanica in comparison to uninfected roots [START_REF] Lobna | Biochemical and plant nutrient alterations induced by Meloidogyne javanica and Fusarium oxysporum f.Sp.radicis lycopersici co-infection on tomato cultivars with differing level of resistance to M. javanica[END_REF]. Resistance to RKN was correlated with a higher root copper content in resistant tomatoes cultivars in comparison to the susceptible one [START_REF] Lobna | Biochemical and plant nutrient alterations induced by Meloidogyne javanica and Fusarium oxysporum f.Sp.radicis lycopersici co-infection on tomato cultivars with differing level of resistance to M. javanica[END_REF]. These results support our hypothesis that the induction of miR408 and miR398 in tomato galls is due to a reduction of copper level in galls in comparison to uninfected roots during susceptible response.

Copper in biocontrol

Treatment with high level of copper is used to protect the plants against pathogen infection. Copper-based bactericides and fungicides such as Bordeaux mixture, are extensively used in agriculture. In plant response to pathogen, pepper plants treated with copper (50 µM) showed a resistance to Verticillium dahlia fungi [START_REF] Chmielowska | Cross-protection of pepper plants stressed by copper against a vascular pathogen is accompanied by the induction of a defence response[END_REF]. Maize plants treated with copper (10 -80 µM) were more resistant towards Spodoptera frugiperda infection [START_REF] Winter | Heavy metal stress can prime for herbivoreinduced plant volatile emission[END_REF]. The concentrations of copper used in these studies are ten times higher than the concentration (5 µM) we used to water plants during infection test.

Copper treatment is known to enhance ROS level and to increase peroxidase enzyme and laccase activity increasing lignin content [START_REF] Kuc | Fungal Regulation of Disease Resistance Mechanisms in Plants[END_REF][START_REF] Díaz | Induction of shikimate dehydrogenase and peroxidase in pepper (Capsicum annuum L.) seedlings in response to copper stress and its relation to lignification[END_REF]. Lignin is a cross-linked phenolic polymer material that forms key structural materials in the supporting tissues of plants such as vascular plants [START_REF] Printz | Copper Trafficking in Plants and Its Implication on Cell Wall Dynamics[END_REF]. The complex structure of lignin provides the mechanical support, water transmission, as well as blocking the growth of pathogen and infection. The polymerization of lignin is catalyzed by peroxidase in the presence of H 2 O 2 and by laccases in the presence of O 2 [START_REF] Sterjiades | Laccase from sycamore maple (Acer pseudoplatanus) polymerizes monolignols[END_REF]. Soybean plants cultured in various concentrations of CuSO4 showed a decrease in H 2 O 2 level with increase in POX activity and lignin content after 72 hours on 5 µM copper medium [START_REF] Lin | Rapid effect of copper on lignin biosynthesis in soybean roots[END_REF]. Same results have been observed in Raphanus sativus plants cultured on 4 µM of copper medium [START_REF] Chen | Effect of copper on peroxidase activity and lignin content in Raphanus sativus[END_REF]. In these studies, the treatment with copper affected plant growth and photosynthesis, while in our cases plant treated with copper did not show any difference in root development compared to the plants treated with water . The plants were treated two days after infection to let the nematode penetrate to the roots and plants were treated one time per week, in contrast to these studies plants were grown on medium containing different concentrations of copper.

miR398 and ROS

miR398b/c is a copper-miRNA which expression is regulated by SPL7 and it is overexpressed in A. thaliana galls. CSD1 and 2, BLUE COPPER BINDING PROTEIN (BCBP) and COPPER CHAPERO FOR SUPEROXIDE DISMUTASE (CCS) are biologically validated targets of miR398 [START_REF] Jones-Rhoades | Computational identification of plant MicroRNAs and their targets, including a stress-induced miRNA[END_REF][START_REF] Beauclair | MicroRNA-directed cleavage and translational repression of the copper chaperone for superoxide dismutase mRNA in Arabidopsis[END_REF][START_REF] Brousse | A non-canonical plant microRNA target site[END_REF]. The role of BCBP is still unknown, CCS, CSD1 and CSD2 are related to the redox process in the activation of primary defense response of plants. CCS1 delivers the copper to CSD1 and CSD2. CSD1 and CSD2 are superoxide dismutase (SODs) enzymes expressed in response to stresses that generate ROS [START_REF] Se | AtCCS is a functional homolog of the yeast copper chaperone Ccs1/Lys7[END_REF][START_REF] Sunkar | Posttranscriptional induction of two Cu/Zn superoxide dismutase genes in Arabidopsis is mediated by downregulation of miR398 and important for oxidative stress tolerance[END_REF]. In low copper conditions, miR398 is induced and it is involved in the degradation of CSD1 and CSD2 mRNA to restrict copper for plastocyanin [START_REF] Yamasaki | Regulation of copper homeostasis by micro-RNA in Arabidopsis[END_REF]. The expression of CSD1 and BCBP is repressed in A. thaliana galls, while CSD2 is not differentially expressed. We showed that A. thaliana expressing gene with a mutation at the cleavage site, mcsd1 and mbcbp, had fewer egg masses than the wild-type. These findings demonstrate that the cleavage of CSD1 and BCBP transcripts by miR398 is required for plant-RKN interaction.

ROS act as signal molecules to activate plant immunity. Plants produce ROS, such as hydrogen peroxide (H 2 O 2 ), to defend itself from pathogens [START_REF] Forman | Signaling functions of reactive oxygen species[END_REF]. However, plants need to maintain redox homeostasis by balancing ROS production and removal while by ROSscavenging enzymes like SOD [START_REF] Eves-Van Den Akker | The feeding tube of cyst nematodes: Characterisation of protein exclusion[END_REF][START_REF] Camejo | Reactive oxygen species, essential molecules, during plant-pathogen interactions[END_REF]. SODs aids the scavenging of ROS by converting O 2-into H 2 O 2 . miR398 has been mainly studied in the plant response to abiotic stress. In response to oxidative stress, MIR398 is transcriptionally repressed to prevent the cleavage of CSD1 and CSD2 transcripts leading to an accumulation of CSD1 and CSD2 mRNAs that scavenge superoxide radicals [START_REF] Sunkar | Posttranscriptional induction of two Cu/Zn superoxide dismutase genes in Arabidopsis is mediated by downregulation of miR398 and important for oxidative stress tolerance[END_REF][START_REF] Jagadeeswaran | Biotic and abiotic stress down-regulate miR398 expression in Arabidopsis[END_REF]. Recently, miR398 was shown to be induced in Nicotiana benthamiana infected by Beet necrotic yellow vein virus (BNYVV) [START_REF] Liu | Genome-Wide microRNA Profiling Using Oligonucleotide Microarray Reveals Regulatory Networks of microRNAs in Nicotiana benthamiana During Beet Necrotic Yellow Vein Virus Infection[END_REF]. TRV-induced gene silencing (VIGS) of miR398 target UMECYANIN (Cu-superoxide dismutase in A. thaliana), showed a higher concentration of O -2 in silenced plants that in control plants and a lower accumulation of BNYVV in the systemic leaves, suggesting that the induction of miR398 during BNYVV infection is favorable for the activation of plant defense and it inhibits the O 2-scavenging activity of plants. In contrast, in our results the induction of miR398 and the cleavage of its target it's in favor of RKN parasitism.

The role of ROS towards nematode infection have been described in resistant and susceptible cultivars. Mi-resistant tomato cultivar infected by M. incognita produced a higher level of ROS [START_REF] Melillo | Modulation of reactive oxygen species activities and H2O 2 accumulation during compatible and incompatible tomato-root-knot nematode interactions[END_REF]. [START_REF] Siddique | Host-pathogen interactions: Parasitic worms stimulate host NADPH oxidases to produce reactive oxygen species that limit plant cell death and promote infection[END_REF] have showed that CN infection causes a localized ROS burst in A. thaliana. In a recent paper, Chopra et al., have used two mutants upon CN and RKN infection: RESPIRATORY BRUST OXYDATIVE HOMOLOGUES mutants (RbohD/F), that generate ROS in response to pathogens and that interacts with WALLS ARE THIN1 (wat1) mutant, an auxin transporter. Interestingly, these two mutants lead to a decrease in susceptibility to CN but not to RKN. Authors have suggested that RbohD/F-mediated ROS production and activated WAT1 is needed for a syncytium establishment and suggesting that CN use the host's ROS for their own benefit [START_REF] Chopra | Plant parasitic cyst nematodes redirect host indole metabolism via NADPH oxidase-mediated ROS to promote infection[END_REF]. Since ROS are involved in numerous processes throughout the plant life cycle such as germination root, shoot and flower development (reviewed in [START_REF] Mittler | ROS Are Good[END_REF][START_REF] Mhamdi | Reactive oxygen species in plant development[END_REF] we can suggest that RKN also take advantage of ROS for gall potentially via miR398 induction.

The family of miR398 includes three genes, MIR398A, MIR398B and MIR398C. The sequences of the precursor of MIR398A doesn't contain the GTAC motif necessary for SPL7 binding in copper deficiency. Only MIR398B and MIR398C respond to copper deficiency while the level of MIR398A doesn't change in all copper conditions [START_REF] Yamasaki | SQUAMOSA promoter binding protein-like7 is a central regulator for copper homeostasis in Arabidopsis[END_REF]. So the induction of MIR398A observed in A. thaliana galls is not due to copper response and should involve different pathway, that needs to be further investigated.

miR408/Uclacyanin/Casparian Strip

Sequencing of small RNAs showed an upregulation of miR408 in A. thaliana galls at 7 and 14 dpi. The localization of miR408 expression during the A. thaliana -M. incognita interaction using promoter GUS fusion confirmed the strong expression in galls observed in the sequencing analyzes. Infection tests using Arabidopsis KO mutant lines: miR408-1 and miR408-2 [START_REF] Maunoury | AGO1 and AGO2 Act Redundantly in miR408-Mediated Plantacyanin Regulation[END_REF] showed a decrease in susceptibility of these two KO lines associated with a defect of feeding site formation. These results demonstrated the key role of miR408 in the development of the feeding cells during Arabidopsis-nematode interaction.

Using psRNA target, an algorithm that identifies plant small RNA targets based on sequence complementary [START_REF] Dai | psRNATarget: a plant small RNA target analysis server[END_REF], we predicted more than 100 targets of miR408 with only two targets downregulated in A. thaliana galls: UCC2 and PHOSPHATASE 2C (PP2C). The cleavage of UCC2 transcripts by miR408 has been biologically confirmed in A. thaliana and rice [START_REF] Zhou | Degradome sequencing reveals endogenous small RNA targets in rice (Oryza sativa L. ssp. indica)[END_REF][START_REF] Thatcher | Differential expression of miRNAs and their target genes in senescing leaves and siliques: Insights from deep sequencing of small RNAs and cleaved target RNAs[END_REF]. UCC are a sub-family of phytocyanins family, a plantspecific blue copper protein. This family is characterized by a copper binding site and is associated with lignified tissues [START_REF] Drew | Isolation and characterization of a pea pod cDNA encoding a putative blue copper protein correlated with lignin deposition[END_REF][START_REF] Nersissian | Uclacyanins, stellacyanins, and plantacyanins are distinct subfamilies of phytocyanins: Plantspecific mononuclear blue copper proteins[END_REF]. UCC are anchored to the cell surface and associate with the cell wall. In rice, role of UCC8 was described in the fertility, pollen tube formation and growth [START_REF] Zhang | Rice UCL8, a plantacyanin gene targeted by miR408, regulates fertility by controlling pollen tube germination and growth[END_REF] and also in regulating rice photosynthesis and grain yield [START_REF] Zhang | MiR408 Regulates Grain Yield and Photosynthesis via a Phytocyanin Protein[END_REF]. Recently, a study highlighted a role for UCC1 and UCC2 in the formation of lignified nanodomain within Casparian strips (CS) in A. thaliana [START_REF] Reyt | Uclacyanin Proteins Are Required for Lignified Nanodomain Formation within Casparian Strips[END_REF]. CS are belt-like lignin structures surrounding endodermal cells, that seals the apoplastic way in and out of the endodermis to control the uptake of water and solutes [START_REF] Alassimone | A developmental framework for endodermal differentiation and polarity[END_REF][START_REF] Naseer | Casparian strip diffusion barrier in Arabidopsis is made of a lignin polymer without suberin[END_REF][START_REF] Doblas | The endodermis, a tightly controlled barrier for nutrients[END_REF]. In Reyt's article, the authors generated CRISPR CAS9 double mutant of the two genes of uclacyanin, ucc1ucc2. Interestingly, this double mutant showed a strong increase of permeability using Apoplastic tracer and the loss of function of UCC1 and UCC2 reduced lignification in central of CS, suggesting a role of these two genes in the lignification of CS. In 2015, a study has showed that UCC1 is downregulated in the transcriptomic data in A. thaliana of myb36 mutants compared to the control plants [START_REF] Kamiya | The MYB36 transcription factor orchestrates Casparian strip formation[END_REF]. The transcription factor MYELOBLASTOSIS PROTO-ONCO GENE 36 (MYB36) is the main regulator of several genes necessary in the position of CS in the endodermis. The formation of CS required CASPARIAN STRIP MEMBRANE DOMAIN PROTEINS (CASPs). There are five genes of this family in A. thaliana and the double mutant casp1casp3 leads to a defect in CS formation [START_REF] Roppolo | A novel protein family mediates Casparian strip formation in the endodermis[END_REF]. The formation of CS required also an association with CASPs and lignin deposition. The deposition of lignin in CS involved many signaling pathway like the kinase SCHENGEN (SGN1 &3) and their ligands CASPARIAN STRIP INTEGRITY FACTOR 1& 2 (CIFs) [START_REF] Nakayama | A peptide hormone required for Casparian strip diffusion barrier formation in Arabidopsis roots[END_REF]. This deposition required the polymerization of lignin thanks to the peroxidase PER64 and the enhanced suberin 1 (ESB1) [START_REF] Hosmani | Dirigent domain-containing protein is part of the machinery required for formation of the lignin-based Casparian strip in the root[END_REF][START_REF] Lee | A mechanism for localized lignin deposition in the endodermis[END_REF] (Figure 25). esb1 and casp1casp3 mutants shows a strong increase of permeability PI (Apoplastic Tracer), the same result was observed in ucc1.ucc2 mutant [START_REF] Reyt | Uclacyanin Proteins Are Required for Lignified Nanodomain Formation within Casparian Strips[END_REF]. LOTR1 (lord of the rings) is a member of an uncharacterized family and LOTR2 is a subunit of the exocyst complex: LOTR2/ EXO70A1 transiently accumulates on CS membrane domain and guide CASP localization [START_REF] Kalmbach | Transient cell-specific EXO70A1 activity in the CASP domain and Casparian strip localization[END_REF]. The analysis of the expression profile in A. thaliana galls of the different genes involved in the formation of CS showed that MYB36 is upregulated in galls at 3, 5 and 7 dpi while most of MYB36 targets were are downregulated (CASPs, SGN3, SGN1). Earlier studies in plant-nematode interaction have shown that the endodermis bordering the giant-cell in barley infected with Meloidogyne naasi, lacked a CS [START_REF] Ediz | Life cycle, pathogenicity, histopathology, and host range of race 5 of the barley root-knot nematode[END_REF]. More recently, transgenic A. thaliana mutants, with defects in CS strip and lignin deposition (e.g. cap1-1casp3-1), displayed increased in susceptibility towards M. incognita infection [START_REF] Holbein | Root endodermal barrier system contributes to defence against plantparasitic cyst and root-knot nematodes[END_REF]. Finally, a recent article has shown that the root of the rice cultivar resistant towards M. graminicola infection presented a thicker CS lining compared to the susceptible rice cultivar [START_REF] Singh | Suberin Biopolymer in Rice Root Exodermis Reinforces Preformed Barrier Against Meloidogyne graminicola Infection[END_REF]. These different studies showed that CS formation is altered in galls induced by nematode infection. Moreover, in another pathogen infection, Plasmodiophora brassicaei, that also induces galls, transcriptome analysis showed several genes involved in CS formation (e.g. CASP1, CASP3, UCC1, UCC2, MYB36, PER64) were downregulated in A. thaliana galls [START_REF] Liégard | Quantitative resistance to clubroot infection mediated by transgenerational epigenetic variation in Arabidopsis[END_REF].

Moreover, using psRNA target, we found that UCC1 is targeted by miR2934. UCC1 is downregulated in A. thaliana galls at 7 and 14 dpi, while miR2934 is upregulated at 14 dpi. The role of this miRNA is still unknown [START_REF] Borges | MicroRNA activity in the Arabidopsis male germline[END_REF]. Additionally, we have found that CASP1 that is downregulated in A. thaliana galls at 7 dpi is also predicted to be cleaved by miR390b that is upregulated in galls at 7 & 14 dpi. These results suggest that genes involved in CS formation and lignification, could be posttranscriptional repressed by miRNAs, like the cleavage of UCC2 transcripts and its downregulation by miR408 in galls.

The next question that needs to be answered is how nematode can use the CS defect for its own benefit? We can think that since giant cells remain symplasmically isolated [START_REF] Hoth | Differential vascularization of nematode-induced feeding sites[END_REF] and since solutes are unloaded from sieve elements into the apoplast from which the nutrients are further transported into the symplasmically isolated giant cells, defects in CS formation would lead to an outflow of solutes, which may leads to a better nematode development [START_REF] Bartlem | Vascularization and nutrient delivery at root-knot nematode feeding sites in host roots[END_REF][START_REF] Holbein | Root endodermal barrier system contributes to defence against plantparasitic cyst and root-knot nematodes[END_REF]. So far the role of copper in CS regulation is not well known. Interestingly, pharmacological treatments using copper chelator, leads to a defect in CS with a disorganization of lignin deposition (Zhuang et al., 2020). This can suggest the role of copper in CS formation and lignification.

A recent article has shown that two auxin, auxin phenylacetic acid (PAA) and indole-3-acetic acid (IAA), plays a role in the formation of CS [START_REF] Cook | Regulation of suberin biosynthesis and Casparian strip development in the root endodermis by two plant auxins[END_REF]. PAA and IAA treatment induces a decrease in the expression level of the CASP and ESB gene families but didn't affect MYB36 level expression. Moreover, a reduction in auxin level has been observed in the CS mutants esb1 and casp1casp3 but not in myb36 mutant suggesting that a functional MYB36 is necessary for the repression of auxin biosynthesis in CS deficient plants. The authors proposed a model on the role of auxin in regulation CS genes (Figure 26). The contradictory in the expression profile of MYB36 (upregulation) and CASP & ESB (downregulation) observed in A. thaliana galls, could be therefore explained by a role of auxin.

How does the nematode induce copper decrease in galls?

All the results obtained on copper module points a reduction of copper level in galls. The question of how nematode induce this decrease of copper level in galls, needs to be further investigated. One hypothesis could be that nematode secretions/effectors would manipulate copper homeostasis of feeding cells. Indeed, some nematode effectors have a copper-binding domain. A M. graminicola effector, Mg-MO289, has been recently identified to interact with rice copper metallochaperone heavy metal-associated plant protein (OsHPP0 4 ) [START_REF] Song | The Meloidogyne graminicola effector MgMO289 targets a novel copper metallochaperone to suppress immunity in rice[END_REF]. Mg-OsHPP0 4 is involved in copper binding as Cu metallochaperones. HPP0 4 transport Cu to Cu binding enzymes such as COPPER/ZINC-SUPEROXIDE DISMUTASE 2 (Cu/Zn-SOD) that plays an important role in scavenging superoxide radical (O 2-) produced by ROS [START_REF] Pilon | Copper cofactor delivery in plant cells[END_REF]. The treatment of plants with copper for 24 hours showed less accumulation of O 2-in HPP04 and Mg-MO289-transgenic lines than the plants treated with water, suggesting that OsHPP04 and Mg-MO289 can promote O 2-decrease by boosting the rice Cu/Zn-SOD activity. The authors suggest a role of this effector by eliminating O 2 -and suppressing plant immunity. Nematode secretions can therefore play a role in the manipulation of copper in galls induced by RKN.

I propose a model on copper regulation in galls, integratory auxin, key regulator of plant development (Figure 27). RKN secretion induce a decrease in copper level in galls, that increase the activity of SPL7 and therefore induce the expression of MIR398 and MIR408. The genes involved in CS formation, repressed by auxin, all along with the cleavage of UCC2 by miR408, leads probably to a defect in CS formation and lignification. The cleavage of miR398 targets, CSD1 and BCBP, needs to be further investigated.

Perspectives

During my PhD, I performed the functional analysis of two miRNAs and/or their targets modules. I first deciphered the role of miR167/ARF8 in the formation of feeding cells induced in tomato and secondly the role of copper module, copper/SPL7/MIR408-UCC2/MIR398-CSD1-BCBP module, in the formation of nematode-induced feeding cells in A. thaliana.

MiR167/ARF8 module

Functional analysis performed with the two ARF8 in tomatoes galls induced by M. incognita, showed clearly that these transcription factor are involved in giant cell formation. The mechanism of action of ARF8 in tomato galls is still unclear. ARF8 has been described as an activator of genes responding to auxin. In order to decipher further the role of ARF8A and ARF8B in response to nematode infection, expression profile of genes which transcription is regulated by ARF8 should analyzed in galls. We are sequencing mRNA from arf8a and arf8b and wild type galls in order to identify the targets of ARF8 in tomato galls. The sequencing is still in progress (BGI sequencing company, China). The results of the sequencing of arf8a and arf8b tomato galls will enable to identify downstream genes and the biological processes they are involved in. On the other hand, during my PhD I focused on the functional analysis of ARF8 but the role of miR167 in this regulation should be further investigated. Since miR167 is downregulated in tomato galls, I have transformed tomato to overexpress miR167 using promoter 35S. One tomato transformed plant (microtom) that overexpress miR167, has the same phenotype as the double mutant CRISPR arf8ab: smaller plant with smaller and seedless fruits. The sterility of tomato overexpressing miR167 [START_REF] Liu | Down-regulation of AUXIN RESPONSE FACTORS 6 and 8 by microRNA 167 leads to floral development defects and female sterility in tomato[END_REF], required to backcross this plant with wild type. Infecting these lines with RKN infection will complete our understanding and test the role of miR167 in tomato giant cells.

Functional analysis of copper module in tomato galls

As showed before, our sequencing analysis identified a conserved copper module in tomato and A. thaliana galls with the two pairs miR408/UCC2 and miR398/CSD1 conserved in both plants. Functional analyses were initiated in A. thaliana for which the necessary biological material was already available. Functional analyzes in tomato should be performed in the future in order to investigate the conserved role of miR408/UCC2 and miR398/CSD1 in galls. We have started the functional analysis of miR408 in tomato. I performed stable transformation of tomatoes (WVA106) with the precursor of miR408 fused to reporter gene GUS. Since we generated the seeds of F1 that express miR408:GUS, the expression profile of this miRNA should be investigated at early and late stage of gall development, with and without copper. In the future, mutant lines of KO miR408 or UCC2 resistant to cleavage by miR408 should be generated.

Characterization of Casparian strip and lignification in galls

Our results obtained in copper module and the role of UCC in CS formation suggest that RKN induce a reduction of copper level via SPL7/miR408/UCC, and this module may play a role in the formation of CS through lignification. To test hypothesis, this barrier should be characterized in the presence or the absence of copper in A. thaliana and tomato galls and uninfected roots. I will stain lignin in galls and roots in order to compare lignin deposition in CS. A protocol has been set up in order to stain lignin in A. thaliana and its compatible with chemical dyes [START_REF] Ursache | A protocol for combining fluorescent proteins with histological stains for diverse cell wall components[END_REF].

Characterization of ROS in galls

As we saw also most of the components of copper module (peroxidase, the lignification, SODs) are linked to ROS, a more detailed study will be needed to investigate the changes in ROS production during galls formation. We can measure H 2 O 2 in galls and uninfected roots. We propose this hypothesis that the downregulation of UCC expression in A. thaliana galls, leads to a decrease in lignification in important target structures (CS) which may help the nematode infection.

Auxin and copper

In order to identify if copper affected auxin disruption in galls, we can proc eed to the functional analysis of plants expressing auxin transporters (e.g. pin1; aux1) fused to gene reporter cultivated in medium with and without copper. Indeed, a study has showed that Cu excess affected root auxin distribution which affected the mitotic activity of the meristem.

Copper induced an auxin redistribution that involves the efflux carrier of the PINFORMED1 (PIN1), which is responsible for root auxin transport [START_REF] Yuan | Copper regulates primary root elongation through PIN1mediated auxin redistribution[END_REF]. Moreover, auxin have been showed to play a role in the formation of CS [START_REF] Cook | Regulation of suberin biosynthesis and Casparian strip development in the root endodermis by two plant auxins[END_REF]. To identify the role of auxin in CS formation in galls, galls of auxin transporters mutants could be used to characterize CS by lignin staining; treatment with auxin using mutant genes, such as casp1-1casp3-1; esb1; sgn3, could be also a track to explore the effect of auxin in CS formation.

New miRNAs/targets in tomato-RKN interaction

This work has also characterized the miRNAs and mRNAs differentially expressed in the galls during tomato -M. incognita interaction at 7 and 14 dpi. As mid-term perspectives, I propose to identify new miRNAs/targets in tomato-RKN interaction. A list of twelve pairs of miRNA/targets were identified with an anti-correlation of expression. Beyond miR167/ARF8 pair, other pairs would be interesting candidates to investigate their roles in the formation of giant cells in tomato. For example, the patatin family the target of miR7981 is upregulated while miR7981 is downregulated. The patatin genes family is well known in facilitation of pathogens host colonization in different plants [START_REF] Rydel | The crystal structure, mutagenesis, and activity studies reveal that patatin is a lipid acyl hydrolase with a Ser-Asp catalytic dyad[END_REF]. miR164 family are characterized as auxin-responsive miRNA [START_REF] Guo | MicroRNA directs mRNA cleavage of the transcription factor NAC1 to downregulate auxin signals for Arabidopsis lateral root development[END_REF]. miR164 and its target NO APICAL MERISTEM (NAC) are also promising candidates to play a role in the regulation of gall development. A negative correlation between miR164/NAC was observed in our data at 7 and 14 dpi and this regulation was observed and confirmed by qPCR in Kaur et al., at 14 dpi (Kaur et al., 2017). This highly conserved family regulate boundaries in shoot and lateral root development through the negative regulation of NAC family transcription factors with CUP-SHAPED COTYLEDON 2 (CUC2) as main targets [START_REF] Peaucelle | Plants expressing a miR164-resistant CUC2 gene reveal the importance of post-meristematic maintenance of phyllotaxy in Arabidopsis[END_REF][START_REF] Sieber | Redundancy and specialization among plant microRNAs: Role of the MIR164 family in developmental robustness[END_REF]. Since tomato CRISPR lines are available, functional analysis should be performed.

Trans kingdom RNAi

All the previous functional analysis studies and the work done during my thesis showed that plant miRNAs are involved in plant nematode interaction and in the giant cell formation. However, only few studies have investigated RKN microRNAs and no functional validation have been done so far (Wang et al., 2015b;[START_REF] Liu | Small RNA Sequencing Reveals Regulatory Roles of MicroRNAs in the Development of Meloidogyne incognita[END_REF]. Sequencing RKN miRNAs from parasitic stage is in progress in the lab in order to identify RKN miRNAs differentially expressed that may control RKN development by regulating genes in pathways associated with RKN development and/or pathogenicity.

The last 10 years pathogens were shown to manipulate the plant machinery of small RNAs by secreting small RNAs into host plant [START_REF] Weiberg | Fungal Small RNAs Suppress Plant Immunity by Hijacking Host RNA Interference Pathways[END_REF][START_REF] Westwood | RNA mobility in parasitic plant-host interactions[END_REF]Cai et al., 2018b). This mechanism is known as trans kingdom RNAi and vesicles exchanges is bidirectional and it was shown that pathogen too can export sRNAs to silence gene [START_REF] Dunker | Oomycete small RNAs bind to the plant RNA-induced silencing complex for virulence[END_REF]. Botrytis cinerea secrets fungal miRNAs which are loaded into the ARGONAUTE 1 of the plant and induce post-transcriptional repression of the genes involved in the defense of the plant host [START_REF] Weiberg | Fungal Small RNAs Suppress Plant Immunity by Hijacking Host RNA Interference Pathways[END_REF]. In cottonfungal interaction, cotton increase production of miR166 and miR159 and exports both to the fungal hyphae for specific silencing of fungal genes (Zhang et al., 2016a). Exosomes are extracellular vesicles (EV) that are involved in intracellular communication and interaction with others organisms, by transporting proteins, lipids and RNAs [START_REF] Colombo | Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles[END_REF]. Exosomes from A. thaliana was shown to transfer small RNAs in order to silence pathogen genes B. cinerea (Cai et al., 2018a). We can think that similar manipulations in plant host could take place in response to RKN infection. Functional analysis of exosome markers in plant-nematode infection should be investigated.

Finally, the identification of miRNAs and their targets could be a new track to find new strategies to control nematode infection. Interestingly, new studies aim to spray small RNAs on plant surfaces as an efficient way to crop protection [START_REF] Wang | Spray-Induced Gene Silencing: a Powerful Innovative Strategy for Crop Protection[END_REF][START_REF] Sang | Advanced strategies to control plant pathogenic fungi by host-induced gene silencing (HIGS) and spray-induced gene silencing (SIGS)[END_REF][START_REF] Werner | RNA-Spray-Mediated Silencing of Fusarium graminearum AGO and DCL Genes Improve Barley Disease Resistance[END_REF][START_REF] Qiao | Spray-induced gene silencing for disease control is dependent on the efficiency of pathogen RNA uptake[END_REF]. Moreover, the microRNA targets essential for giant cells and nematode development represent loss of susceptibility genes whose inactivation could lead to more resistant plant [START_REF] Favery | Gall-Inducing Parasites: Convergent and Conserved Strategies of Plant Manipulation by Insects and Nematodes[END_REF].

Figure 15 .

 15 Figure 15. Hierarchical classification system for endogenous plant small RNAs. Thick black lines indicate hierarchical relationships. dsRNA, double-stranded RNA; hpRNA, hairpin RNA; miRNA, microRNA; NAT-siRNA, natural antisense transcript small interfering RNA; siRNA, small interfering RNA.(Axtell, 2013a)

Figure 16 .

 16 Figure16. Simplified biogenesis and mechanisms of action of microRNAs in plants. The MIR genes are transcribed by RNA polymerase II (RNA Pol II) to generate single-stranded hairpin-containing primary transcripts (pri-miRNA). The pri-miRNA is then cleaved, in the nucleus, by Dicer-like 1, in association with hyponastic leaves 1 and serrate, to produce a precursor miRNA (pre-miRNA). The pre-miRNA is, in turn, cleaved by DCL1 and its cofactors, thus generating a duplex composed of the mature miRNA and its complementary strand. The HUA ENHANCER 1 protein then adds a methyl group to the OH end of each strand of the miRNA duplex, to protect against degradation. The miRNA duplex is then actively transported from the nucleus to the cytosol through interaction with the HASTY (HST) exportin. One of the two strands of the duplex is then loaded onto the ARGONAUTE-1 protein, the main constituent of the multiprotein RNA-induced silencing complex (RISC). The AGO1-associated strand guides the RISC to target mRNAs by sequence complementarity, resulting in target cleavage or the inhibition of protein synthesis. DCL1, Dicer-like 1; HYL1, hyponastic leaves 1; SE, serrate; HEN1, HUA ENHANCER 1 protein; HST, hasty; AGO1, argonaute 1.[START_REF] Jaubert-Possamai | MicroRNAs, New Players in the Plant-Nematode Interaction[END_REF]

Figure 18 .

 18 Figure 18. Interplay of evolutionary conserved microRNA-transcription factor networks during leaf development. The functions associated to each miRNA and their targets network are indicated as well as silhouettes of leaves representing the phenotypes observed in Arabidopsis thaliana after modifying each network. The pink triangle refers to the protein-protein interactions between the miRNA targets of the different networks. GRF, GROWTH REGULATING FACTORS; CUC, CUP-SHAPED COTYLEDON2; TCO, TEOSINTE BRANCHED1, CYCLOIDEA and PROLIFERATING CELL NUCLEAR ANTIGEN BINDING FACTOR; SPL, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE[START_REF] Rodriguez | Control of cell proliferation by microRNAs in plants[END_REF].

Figure 19 .

 19 Figure 19. Schematic representation of the interactions between miR172 and miR156 and their targets during flower transition; Crosstalk between miR156 and miR172 modules are shown along with the regulatory networks and feedback regulation of the target genes. miR156 is regulated by positive and negative feedback loop of SPL9 and SPL15, respectively; and positively regulated by AP2 and AGL15. miR172 is regulated by the positive feedback loop of TOE1/2 and negatively by AP2 through LUG and SEU. TOE1/2 repress the expression of SPL3/4/5 genes. SPL3 positively regulates the expression of TOE3. AP2 and SMZ repress their own expression and also of other miR172 target genes. AGL15, AGAMOUS LIKE15; AP2, APETALA2; LUG, LEUNIG; SEU, SEUSS; SMZ, SCHLAFMU TZE; SNZ, SCHNARCHZAPFEN; SPL, SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE; TOE1-3, TARGETOF EAT1-3.[START_REF] Teotia | To bloom or not to bloom: Role of micrornas in plant flowering[END_REF]

Figure 20 .

 20 Figure 20. Regulation of miR393 and its target in root development. A. thaliana lateral roots (A and E) Col-0 compared with mutants (B) tir1-1 and mutants overexpressing (C) miR393a, (D) miR393b, (F) TIR1 and (G) miR393-resistant form of TIR1 (mTIR1). Overexpressing (F) TIR1 and (G) mTIR1 displayed shorter primary roots, and more lateral roots while (B) tir1 and (C and D) miR393a/b overexpression displayed slightly longer primary root and fewer lateral roots, compared to wild type (A and E) Col0. TIR1, TRANSPORT INHIBITOR RESPONSE1. Bars = 10 mm[START_REF] Chen | Regulation of auxin response by miR393-targeted transport inhibitor response protein 1 is involved in normal development in Arabidopsis[END_REF]

Figure 21 .

 21 Figure 21. Functions of miRNAs and their targets under different abiotic stresses (a) nutrients stresses, (b) drought and salinity stresses, (c) temperature stress.[START_REF] Li | The functions of plant small RNAs in development and in stress responses[END_REF]

FIGURE 1 |

 1 FIGURE 1 | Multinucleate and hypertrophied feeding cells induced by RKN and CN. (A) Confocal section of a gall induced by M. incognita in Nicotiana benthamiana. Galls were fixed and cleared with the BABB method described by Cabrera et al. (2018). Giant cells are colored in blue and marked with an asterisk to differentiate them from surrounding cells of normal size. Bar = 100 μm. (B) Longitudinal section of a syncytium induced by the CN H. schachtii in Arabidopsis roots, 10 days after inoculation. The syncytium is colored in blue. Bar = 20 µm. (C) Simplified biogenesis and mechanism of action of miRNAs in plants. The MIR genes are transcribed by RNA polymerase II (RNA Pol II) to generate single-stranded hairpin-containing primary transcripts (pri-miRNA).The pri-miRNA is then cleaved, in the nucleus, by Dicer-like 1 (DCL1), in association with hyponastic leaves 1 (HYL1) and serrate (SE), to produce a precursor miRNA (pre-miRNA). The pre-miRNA is, in turn, cleaved by DCL1 and its cofactors, thus generating a duplex composed of the mature miRNA and its complementary strand. The HUA ENHANCER 1 protein (HEN1) then adds a methyl group to the OH end of each strand of the miRNA duplex, to protect against degradation. The miRNA duplex is then actively transported from the nucleus to the cytosol through interaction with the hasty (HST) exportin. One of the two strands of the duplex is then loaded onto the argonaute 1 (AGO1) protein, the main constituent of the multiprotein RNA-induced silencing complex (RISC). The AGO1-associated strand guides the RISC to target mRNAs by sequence complementarity, resulting in target cleavage or the inhibition of protein synthesis (reviewed by[START_REF] Yu | The "how" and "where" of plant microRNAs[END_REF].

6

  October 2019 | Volume 10 | Article 1180 Frontiers in Plant Science | www.frontiersin.org

  ., (2021) in revision in New Phytologist Yara Noureddine 1 , Martine da Rocha 1 , Sébastien Thomine 2 , Michaël Quentin 1 , Pierre Abad 1 , Bruno Favery 1 , and Stéphanie Jaubert-Possamai 1* . Copper microRNAs govern the formation of giant feeding cells induced by the root knot nematode Meloidogyne incognita in Arabidopsis thaliana.

Table 1 :

 1 Expression profile of miRNAs of the miR408 and miR398 families in Arabidopsis and tomato galls. Expression level of mature sequence of the miR408 and miR398 families upregulated in A. thaliana and tomato (S. lycopersum) galls were identified by small RNA Illumina (for tomato samples) or SOLID (for Arabidopsis samples) sequencing, followed by DESeq 2 statistical analysis. MicroRNAs significantly upregulated in galls in comparison to uninfected roots (adjusted pvalue <0.05) are indicated in red.

Figure 1 :

 1 Figure 1: The miR408 KO lines were significantly less susceptible to M. incognita than the wild type. A-B, The susceptibility of the two miR408 KO lines, miR408-1 (A) & miR408-2 (B), and Col0 wild type to M. incognita was evaluated by counting the number of galls and egg masses per plant in two independent infection assays in soil. The effect of miR408 mutation on the development of giant feeding cells was further evaluated by measuring the size of the feeding site produced in each KO line and comparing it to that in Col0. (C) Galls were collected seven weeks post in vitro infection to measure the area (µ m 2 ) covered by the giant cells by the BABB clearing method (Cabrera et al., 2018). The impact of plant genotype was analysed in Mann and Whitney tests.*, P < 0.05. Open squares, minimum values; open circles, maximum values; red lines, median values; blue diamond, first quartile; purple star, third quartile. Bars 50µm.

Figure 2 .

 2 Figure 2. Copper modulates MIR408 promoter activity in galls. A-H, The activity of the MIR408 promoter was analysed in galls induced by M. incognita in Arabidopsis expressing the pMIR408::GUS construct grown in the presence of normal concentrations of copper (0.1 µ M CuSO 4 )(A-F) or in the presence of high concentrations of copper (5.0 µM CuSO 4 )(G-L). A-C, a strong GUS signal was observed in galls 3 days post infection (dpi) (A), 7 dpi (B) and 14 dpi (C) in plants grown with 0.1 µ M CuSO 4 . D-F, Section of gall at 3 dpi (D), 7dpi (E) and 14 dpi (F) showing the GUS signal in giant cells and in the cells surrounding the giant cells. G-I, a weaker GUS signal was observed in galls from plants grown with 5.0 µM CuSO 4 analysed at 3 dpi (G) and 7 dpi (H) and no GUS signal was observed in galls at 14 dpi (I). J-L, Section of gall at 3 dpi (J), 7dpi (K) and 14 dpi (L). Galls are indicated with an arrow; N, nematode; (*) giant feeding cells; nc, neighbouring cells. Bars 500 µ m (A-C; G-I) or 50 µ m (D-F; J-L).

Figure 3 .

 3 Figure 3. SPL7 is induced and required for M. incognita infection and giant cell formation in Arabidopsis.A-D, The activity of the SPL7 promoter (pSPL7) was studied in galls induced by M. incognita from A. thaliana expressing the pSPL7::GUS construct, at 3 days post inoculation (dpi)(A), 7 dpi (B) and 14 dpi (C). D, GUS activity was observed within 5.0 µ mthick gall sections at 7 dpi. E-G, the KO spl7 line (SALK093849c) was infected with M. incognita J2. This line was significantly less susceptible to RKN than Col0, as shown by the smaller mean number of galls (E) and egg masses (F) per plant in two infection assays. (G), the effect of spl7 mutation on the development of feeding cells was further evaluated by measuring the size of the feeding site produced. Galls were collected seven weeks post in vitro infection for measurement of the area (µ m 2 ) covered by the giant cells, by the BABB clearing method(Cabrera et al., 2018). Mann-Whitney tests were performed for statistical analysis in each experiment; significant differences relative to Col-0: *, P < 0.05; Open squares, minimum values; open circles, maximum values; red lines, median values; blue diamond, first quartile; purple star, third quartile. Galls are indicated with an arrow; (*) giant feeding cells; nc, neighbouring cells. Bars 50 µ m (A-C) or 500 µ m (D).

  Figure 3. SPL7 is induced and required for M. incognita infection and giant cell formation in Arabidopsis.A-D, The activity of the SPL7 promoter (pSPL7) was studied in galls induced by M. incognita from A. thaliana expressing the pSPL7::GUS construct, at 3 days post inoculation (dpi)(A), 7 dpi (B) and 14 dpi (C). D, GUS activity was observed within 5.0 µ mthick gall sections at 7 dpi. E-G, the KO spl7 line (SALK093849c) was infected with M. incognita J2. This line was significantly less susceptible to RKN than Col0, as shown by the smaller mean number of galls (E) and egg masses (F) per plant in two infection assays. (G), the effect of spl7 mutation on the development of feeding cells was further evaluated by measuring the size of the feeding site produced. Galls were collected seven weeks post in vitro infection for measurement of the area (µ m 2 ) covered by the giant cells, by the BABB clearing method(Cabrera et al., 2018). Mann-Whitney tests were performed for statistical analysis in each experiment; significant differences relative to Col-0: *, P < 0.05; Open squares, minimum values; open circles, maximum values; red lines, median values; blue diamond, first quartile; purple star, third quartile. Galls are indicated with an arrow; (*) giant feeding cells; nc, neighbouring cells. Bars 50 µ m (A-C) or 500 µ m (D).

Figure 4 .

 4 Figure 4. The miR398-resistant mcsd1 and mbcbp mutant lines had smaller numbers of egg masses. The susceptibility of the mcsd1 (A) and mbcbp (B) lines and of wild-type Col0 plants was evaluated by counting the number of galls and egg masses per plant in two independent infection assays in soil. The impact of the plant genotype on the number of galls and egg masses relative to Col0 was analysed in Mann and Whitney statistical tests.*, P < 0.05. Open squares, minimum values; open circles, maximum values; red lines, median values; blue diamond, first quartile; purple star, third quartile.

Figure 5 .

 5 Figure 5. Plants watered with copper sulphate solution were significantly less susceptible to root knot nematodes. The effect of physiological concentrations of copper on M. incognita infection was evaluated by counting galls and egg masses in Col0 plants watered with a copper sulphate solution at a non-toxic concentration (50 µ M) and comparing the results to those for Col0 watered with tap water. The impact of plant genotype on the numbers of galls and egg masses relative to Col0 was analysed in Mann and Whitney statistical tests.*, P < 0.05. Open squares, minimum values; open circles, maximum values; red lines, median values; blue diamond, first quartile; purple star, third quartile.

  . The impact of plant genotype on the area of giant cells was analysed in Mann and Whitney tests.*, P < 0.05. Open squares, minimum values; open circles, maximum values; red lines, median values; blue diamond, first quartile; purple star, third quartile.

  . Significant changes in TCP4 transcripts levels affect organ curvature. TCP4 directly activates the promoters of miR396 and miR164. miR164 targets CUC transcription factors whose activities contribute positively to the generation of leaf serrations. miR156 targets SPL transcription factors which promote the phase change from the juvenile to the adult phases of vegetative development and also to reproductive development; but they also influence leaf growth itself. The miR156, miR319 and miR164 networks are further interconnected by the protein-protein interactions of their targets. During the development of the younger leaves miR156 levels are high and therefore SPL levels are low, and TCP proteins form dimers with CUC proteins which in turn leads to smooth margins. Later on in development, when miR156 level goes down, SPL level increases and the TCP-CUC dimers are replaced by TCP-SPL dimers. The released CUC proteins dimerize and leaf serrations are formed.

TABLE 1 |

 1 List of functionally validated miRNAs differentially expressed in response to RKN and/or CN.

	miRNA	Host plant	Infected	Nematode			miRNA regulation b	References
			material	species a
					3 or 4	7	10	14	27-30
	miR159	Arabidopsis	Galls	M. javanica				Cabrera et al., 2016
			Galls	M. incognita				Medina et al., 2017
		Tomato	Roots					Zhao et al., 2015
			Roots					Kaur et al., 2017
			Roots	G. rostochiensis				Koter et al., 2018; Święcicka et al.,
								2017
		Cotton	Roots	M. incognita				Pan et al., 2019
	miR172	Arabidopsis	Galls	M. javanica				Díaz-Manzano et al., 2018 (pre-miRNA)
			Galls	M. javanica				Cabrera et al., 2016 (mature)
			Roots	H. schachtii	172c	172c		Hewezi et al., 2008
			Roots	H. schachtii		172a		Hewezi et al., 2008
		Tomato	Galls	M. javanica				Díaz-Manzano et al., 2018
			Galls	M. incognita				Kaur et al., 2017
			Roots	G. rostochiensis				Koter et al., 2018
		Pea	Galls	M. javanica				Díaz-Manzano et al., 2018
	miR319	Arabidopsis	Galls	M. javanica				Cabrera et al., 2016
			Galls	M. incognita				Medina et al., 2017
		Tomato	Roots	M. incognita				Zhao et al., 2015
			Roots	G. rostochiensis				Koter et al., 2018
		Cotton	Roots	M. incognita				Pan et al., 2019
	miR390	Arabidopsis	Galls	M. javanica				Cabrera et al., 2016
			Galls	M. incognita				Cabrera et al., 2016
		Cotton	Roots	M. incognita				Pan et al., 2019
		Tomato and	Galls	M. incognita				Díaz-Manzano et al., 2018
		pea
	miR396	Arabidopsis	Roots	H. schachtii	396a	396a		Hewezi et al., 2008; Hewezi et al., 2012
			Roots	H. schachtii	396b	396b		Hewezi et al., 2008; Hewezi et al., 2012
		Tomato	Roots	M. incognita				Zhao et al., 2015; Kaur et al., 2017
			Roots	G. rostochiensis				Święcicka et al., 2017
		Cotton	Roots	M. incognita				Pan et al., 2019
		Soybean		H. glycines				Noon et al., 2019
	miR827	Arabidopsis	Roots	H. schachtii				Hewezi et al., 2016
		Cotton	Roots	M. incognita				Pan et al., 2019
	miR858	Arabidopsis	Galls	H. schachtii				Piya et al., 2017

a nematodes species: RKN in yellow, CN in pink. b expression pattern between 3 and 27-30 dpi; up-regulated in infected material in red; down-regulated in infected material in green. October 2019 | Volume 10 | Article 1180 Frontiers in Plant Science | www.frontiersin.org

ACKNOWLEDGMENTS

We thank Dr. Miroslaw Sobczak (Warsaw University of Life Sciences-SGGW, Poland) for the syncytium picture.

Acknowledgements

The microscopy work was performed at the SPIBOC imaging facility of Institut Sophia Agrobiotech. We thank Dr Olivier Pierre and the entire team of the platform for assistance with microscopy. This work was funded by the INRA SPE department and the French Government (National Research Agency, ANR) through the 'Investments for the Future' LabEx SIGNALIFE: programme reference #ANR-11-LABX-0028-01 and IDEX UCAJedi ANR-15-IDEX-0, and by the French-Japanese bilateral collaboration programme PHC SAKURA 2019 #43006VJ. Y.N. was supported by a doctoral fellowship from Lebanon (Municipal Council of Aazzée).

Acknowledgements

We would like to thank Dr. Nicolas Bouché (INRAE Versailles, France) for helpful discussions and for providing the mbcbp and mcsd1 Arabidopsis lines. We would like to thank Dr. Lei Li (Beijing University, China) for providing the pMIR408:GUS Arabidopsis line. We would also like to thank Dr. Toshiharu Shikanai (Kyoto University, Japan) for providing the pSPL7:GUS and ko spl7 Arabidopsis line. The microscopy work was performed at the SPIBOC imaging facility of Institut Sophia Agrobiotech. We thank Dr Olivier Pierre and the entire team of the platform for assistance with microscopy. This work was funded by the INRA SPE department and the French Government (National Research Agency, ANR) through the 'Investments for the Future' LabEx SIGNALIFE: programme reference #ANR-11-LABX-0028-01 and IDEX UCAJedi ANR-15-IDEX-0, and by the French-Japanese bilateral collaboration programme PHC SAKURA 2019 #43006VJ. Y.N. was supported by a doctoral fellowship from Lebanon (Municipal Council of Aazzée).

FUNDING YN was supported by a Lebanese fellowship from Aazzée city. BF and SJ-P were supported by INRA and the French Government (National Research Agency, ANR) through the "Investments for the Future" LabEx SIGNALIFE: program reference #ANR-11-LABX-0028-01 and by the Plant-KBBE program NESTOR (ANR-13-KBBE-0003-06).

ARF8A and ARF8B regulated genes in galls

In order to identify the targets of ARF8A and ARF8B, mRNA from galls of WT and arf8a and arf8b has been sent for sequencing. Still the sequencing is still in progress. The results of this article will be discussed later in the general discussion of the thesis.

Bioinformatic analysis

We used psRNA target with default parameters for the prediction of miR408 targets [START_REF] Dai | PsRNATarget: A plant small RNA target analysis server (2017 release)[END_REF].

Abbreviations

AUTHOR CONTRIBUTIONS

YN, BF, and SJ-P organized the study and wrote this manuscript. All authors have read and approved the manuscript.

Conflict of Interest:

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Jaubert-Possamai, Noureddine and Favery. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

RNA extraction

Total RNAs, including small RNAs (< 200 nt), were isolated from in vitro galls or uninfected roots at 7 and 14 dpi. Approximately 40 galls or uninfected roots devoid meristems were independently frozen into powder by using a tissue lyser (Retsch; MM301) at 30 Hertz frequency for 30 seconds with 4 mm tungsten balls (Retsch; MM301). Total RNAs were extracted from these samples with the miRNeasy Mini Kit (Qiagen), according to the manufacturer's instructions, with three additional washes in RPE buffer.

RNA sequencing

Small RNA libraries were generated by ligation, reverse transcription and amplification (11 cycles) from total RNAs (1 µ g), with the reagents of the NEBNext Small RNA Library Prep Set for Illumina. Libraries were then quantified with the Bioanalyzer High Sensitivity DNA Kit (Agilent) and sequenced at the Nice-Sophia Antipolis functional genomics platform (France Géenomique, IPMC, Sophia Antipolis, France). The full raw sequencing data were submitted to the GEO database (http://www.ncbi.nlm.nih.gov/geo/).

PolyA-RNA libraries were generated from 500 ng of total RNA using Truseq Stranded mRNA kit (Illumina). Libraries were then quantified with Qubit dsDNA High Sensitivity Assay Kit (Invitrogen) and pooled. 4nM of this pool were loaded on a Nextseq 500 High output Flowcell and sequenced on a NextSeq 500 platform (Illumina) with 2 × 75bp paired-end chemistry.

miRNAs Analysis

For each library, adapters were trimmed and reads matching ribosomal RNA, mitochondrial RNA and repeat sequences were removed by performing Blast analyses with the sequences listed in the Rfam database (Nawrocki et al., 2015). The STAR 2.5 aligner (: --twopassMode Basic --alignEndsType EndToEnd) was then used to align the trimmed reads [START_REF] Dobin | STAR: ultrafast universal RNA-seq aligner[END_REF] on a virtual concatenated genome generated from the S. lycopersicum genome (V3.01, annotation V3.2) and the M. incognita genome (Blanc-Mathieu et al., 2017). Each read was attributed to the S. lycopersicum and/or M. incognita genome on the basis of the best alignment obtained. Low-quality mapped reads were removed. The htseq-count package version 0.9.1 [START_REF] Anders | HTSeq -A Python framework to work with high-throughput sequencing data[END_REF] was used to count reads mapping perfectly onto the S. lycopersicum genome. The counts for protein coding genes from each replicate were used for differential expression analysis with the R package. EdgeR version 3.4.1 [START_REF] Robinson | edgeR: A Bioconductor package for differential expression analysis of digital gene expression data[END_REF] miR408 and miR398 are two conserved microRNAs which expression is activated by the SPL7 transcription factor in response to copper starvation. We identified these two microRNAs families as upregulated in Arabidopsis thaliana and Solanum lycopersicum roots infected by root-knot nematodes. These endoparasites induce the dedifferentiation of a few root cells and the reprogramming of their gene expression to generate giant feeding cells. By combining functional approaches, we deciphered the signaling cascade involving these microRNAs, their regulator and their targets. MIR408 expression was located within nematode-induced feeding cells in which it co-localised with SPL7 expression and was regulated by copper. Moreover, infection assays with mir408 and spl7 KO mutants or lines expressing targets rendered resistant to cleavage by miR398 demonstrated the essential role of the SPL7/MIR408/MIR398 module in the formation of giant feeding cells. Our findings reveals how perturbation of plant copper homeostasis, via the SPL7/MIR408/MIR398 module, governs the formation of nematodeinduced feeding cells.

Author contributions

Y.N., B.F and S.J.P. designed the study and performed the experimental work. All authors analysed and discussed the data. S.J.P and B.F. designed the study and wrote the manuscript. M.Q. and P.A. participated in the writing of the manuscript. S.T. participated in the copper studies. MdR analysed NGS data. The author responsible for distributing materials integral to the findings presented in this article, in accordance with the policy of the journal (https://academic.oup.com/plcell) is: Stephanie Jaubert Possamai (Stephanie.jaubert@inrae.fr).

Supplemental Data Set

Supplemental Data Set 1: Infection assays of miR408-1 and miR408-2 ko lines infected with M. incognita.

Supplemental Data

Supplemental material

Biological Materials, Growth Conditions

Seeds of Solanum lycopersicum cv St Pierre were surface-sterilized with chlorine solution (44% active chlorine) and washed three times with 1ml of milli-Q water. 10 to 15 sterile seeds were sown on a Gamborg B5 medium agar plates (0.5 x Gamborg, 1% sucrose, 0.8% agar, pH 6.4), placed at 24ºC for 48 hours for germination, and finally transferred in a growth chamber (8h light; 16h dark, 20ºC). M. incognita strain Morelos" J2s were sterilized with HgCl2 (0.01%) and streptomycin (0.7%) as described before [START_REF] Caillaud | In Vivo Imaging of Microtubule Organization in Dividing Giant Cell[END_REF]. One to two weeks after germination, roots were inoculated with 1,000 sterile J2s resuspended in phytagel (5%) per petri dishes.

RNA extraction

Total RNAs, including small RNAs (< 200 nt), were isolated from in vitro galls or uninfected roots at 7 and 14 dpi. Approximately 40 galls or uninfected roots devoid meristems were independently frozen into powder by using a tissue lyser (Retsch; MM301) at 30 Hertz frequency for 30 seconds with 4mm tungsten balls (Retsch; MM301). Total RNAs were extracted from these samples with the miRNeasy Mini Kit (Qiagen), according to the manufacturer's instructions, with three additional washes in RPE buffer.

RNA sequencing

Small RNA libraries were generated by ligation, reverse transcription and amplification (11 cycles) from total RNAs (1 µ g), with the reagents of the NEBNext Small RNA Library Prep Set for Illumina. Libraries were then quantified with the Bioanalyzer High Sensitivity DNA Kit (Agilent) and sequenced at the Nice-Sophia Antipolis functional genomics platform (France Génomique, IPMC, Sophia Antipolis, France).

miRNAs Analysis

For each small RNA library, adapters were trimmed and reads matching ribosomal RNAs, mitochondrial RNAs and repeat sequences were removed by performing Blast analyses with the sequences listed in the Rfam database (Nawrocki et al., 2015). The Bowtie aligner was then used to align the trimmed reads (Langmead et al., 2009) on a virtual concatenated genome generated from the Solanum lycopersicum genome (V3.0) and the M. incognita genome (V2.0; Blanc-Mathieu et al., 2017). Each read was attributed to the S. lycopersicum and/or M. incognita genome on the basis of the best alignment obtained. Reads with identical best alignment hits for the two genomes were attributed to both the nematode and the plant. If one read aligned to multiple genomic locations, a single read was attributed to each locus. Lowquality mapped reads were removed and reads corresponding to molecules of between 20 and 24 nt in size were retained for further analysis. De novo microRNA encoding genes were