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CRISPR-Cas9 has proven to be highly valuable for genome editing in plants, including the model plant Physcomitrium patens. However, the fact that most of the editing events produced using the native Cas9 nuclease correspond to small insertions and deletions is a limitation., CRISPR-Cas9 base editors enable targeted mutation of single nucleotides in eukaryotic genomes and therefore overcome this limitation. Here, we report two programmable base-editing systems to induce precise cytosine or adenine conversions in P. patens., Using cytosine or adenine base editors, site-specific single-base mutations can be achieved with an efficiency up to 55%, without off-target mutations. Using the APT gene as a reporter of editing, we could show that both base editors can be used in simplex or multiplex, allowing for the production of protein variants with multiple amino-acid changes. Finally, we set up a co-editing selection system, named selecting modification of APRT to report gene targeting (SMART), allowing up to 90% efficiency site-specific base editing in P. patens., These two base editors will facilitate gene functional analysis in P. patens, allowing for site-specific editing of a given base through single sgRNA base editing or for in planta evolution of a given gene through the production of randomly mutagenised variants using multiple sgRNA base editing., The work, including study design, data collection, analysis and interpretation and manuscript writing, was supported by the French National Research Agency (ANR11-BTBR-0001-GENIUS). The IJPB benefits from the support of the LabEx Saclay Plant Sciences-SPS (ANR-10-LABX-0040-SPS).

The Tomato spotted wilt virus (TSWV) is the second most destructive plant virus, particularly in Solanaceae. Because the resistance genes to TSWV available in tomato (Solanum lycopersicum) and pepper (Capsicum annuum) are overcome by resistance breaking isolates, there is an urgent need to develop new durable genetic resistance. Resistance by loss-of-susceptibility is one of the great opportunities to provide such resistances. The aim of this present work is first to characterize a TSWV isolate and then identify tomato host proteins that interact with the viral proteins, aiming at characterizing new resistances. - Firstly, Tomato spotted wilt virus RNA-dependent RNA polymerase adaptive evolution and most constrained domains was analyzed. The TSWV-LYE51 genome was sequenced. We decided to focus on the RNA-dependent RNA polymerase which is the key protein for cycle life by being responsible for both the transcription and replication of the viral genome. A 3D model of this RdRp protein was built by homology modeling and phylogenetic and selection analyses were used to characterize hot-spot of adaptive mutation as well as the most constrained domains. Those results allow to pinpoint RdRP regions of interest toward which efficient resistances can be targeted. - Based on those data, candidates genes implicated in susceptibility to TSWV were sought after. Viruses have to hijack host cellular protein –i.e. named susceptibility factors- in order to accomplish their cycle and infect their host. Those factors can be turned into genetic resistance through their absence or modification. To characterize such susceptibility factors in tomato, yeast-two hybrid screenings were carried out using the identified RdRP constrained domain as a bait. We identify several candidates’ genes currently being validated. This work, by obtaining new information about TSWV infectious mechanism and its adaptation to plan, may bring new perspectives in order to develop loss-of-susceptibility resistances to TSWV in crops by using natural or induced variability.; Tomato spotted wilt virus (TSWV, un tospovirus) est un pathogène majeur de la tomate et du piment. Il y a un besoin urgent de développer de nouvelles et durables résistances génétiques pour assurer leur culture. Dans le cadre de ce doctorat, nous voulons caractériser des protéines de la plante hôte (tomate) qui sont essentielles pour le cycle infectieux TSWV. Ceci grâce à la recherche d'interactions directes aux protéines de TSWV par la méthodes du double hybride. Nous allons surtout essayer de trouver des protéines de tomates qui interagissent avec la protéine TSWV virale impliquée dans la réplication du virus et de la traduction: le grand ARN polymérase dépendante de l'ARN codé par l'ARN L et la protéine N. Dans une deuxième étape, nous allons voir si des allèles modifiés de ces gènes candidats de tomates peuvent être utilisés comme gène de résistance. Des variants de gènes seront caractérisées par deux façons : 1 / par l'exploration des séquences génomiques et transcriptomiques disponibles à la recherche de la variation naturelle entre les candidats 2 / par criblage de la population TILLING pour les mutations affectant les gènes candidats. Les lignes sélectionnées (accession ou lignée TILLING) seront ensuite testés pour la résistance à une gamme de TSWV. Les variantes sélectionnées seront également combinées avec le gène de résistance dominant (Sw5 pour la tomate) pour voir si cela peut augmenter la résistance durable au TSWV.

National audience

Tospovirus is the only member of the Bunyavirus family which infects plants (the other members infecting animals and humans). Tomato spotted wilt virus (TSWV), the most famous tospovirus, is a major pathogen for tomato and pepper cultures. Because there is no chemical treatment against viruses, the only way to fight them is to develop genetic resistance. Genetic resistances to tospoviruses are available, but are largely overcome. Therefore, there is an urgent need to characterize new sources of resistance. The aim of my PhD is to find new genetic resistances based on susceptibility factors, which are proteins required by the virus to accomplish its cycle and infect the plant. As a first step, I will develop a 3D model of the TSWV RNA-dependent RNA polymerase (RdRp), a key viral protein for its infectious cycle. Based on this model, a part of the RdRp protein will be selected as bait in order to identify plant protein interactors by yeast-two hybrid screening. Finally, I will search for new tomato alleles encoding variant forms of those tomato susceptibility factors. If those variants impair the interaction between the plant factor and the viral protein, those alleles are very likely to be associated with the crop resistance to the pathogen.