The post-translational modification ubiquitination (Ub) plays a central role in biology. Among the several forms of Ub, K63-type polyUb is of particular interest since involved in several proteasome-independent functions. However, the large number of factors involved in Ub processes in higher eukaryotes, the complexity of specific polyUb chain formation associated to the lack of resolution on specific Ub types by biochemical and imaging strategies has greatly hampered our ability to tackle the roles of K63 polyUb. This proposal will use an unprecedented combination of cutting edge approaches to unravel new roles of K63 polyUb, and get further insights into the cellular and biological roles of such linkage in Ub-mediated endocytosis. To this purpose, I will take advantage of the model plant Arabidopsis thaliana to grasp the complexity of K63 polyUb in higher eukaryotes. The numerous resources available including a high-quality sequenced genomes, a close-to-saturated knock-out mutant collection, an extensive ORF library covering over 50% of the genes and suitable for large-scale Ub as well as protein-protein interaction analyses associated to powerful cell biology and forward genetics indeed establish Arabidopsis as an excellent multicellular model organism to deconstruct K63 polyUb. The first part of this proposal will investigate deeper the general mechanisms of K63 polyUb-dependent endocytosis. To this purpose, we generated a generic plasma membrane ubiquitinated cargo that undergoes constitutive Ub-dependent endocytosis and targeting to the vacuole for degradation. We propose to identify factors involved in the recognition and the sorting of Ub cargos along the endocytic route by forward genetics looking for mutant plants unable to correctly address the generic Ub cargo to the vacuole. The second part of the proposal will explore new roles of K63 polyUb. We will notably use several complementary approaches using K63 polyUb-specific Ub sensor-based imaging and proteomics, as well as genomics and genetics to provide brand new information at an unprecedented scale on factors and pathways requiring K63 polyUb. This will open pristine areas of research in biology. Altogether, this project will help deconstruct the complexity of K63 polyUb using plant as model and will certainly reveal new roles of K63 polyUb in other model organisms including humans.

Nitrogen fixing root symbioses of legume plants with soil bacteria collectively called rhizobia and of actinorhizal plants with Frankia have a tremendous ecological and agronomic impact. These plants constitute a major input of combined nitrogen in the biosphere, they are key pioneering plants in ecological successions and legume or actinorhizal crops require no or little nitrogen fertilizer for growth. These endosymbioses require the development of nodules, root organs formed by the host plant to house the nitrogen-fixing bacteria. Recent progress in the genetics and molecular biology of nodule organogenesis in legumes has made the prospect of transferring the symbiosis to cereals realistic in a foreseeable future. The ability of legumes and actinorhizal plants to acquire sufficient nitrogen solely from the symbiosis relies on the intimate contact between thousands of intracellular (endosymbiotic) rhizobia or Frankia bacteria and their host cells in the root nodules. However, the understanding how a eukaryotic cell can maintain such a large bacterial population or how bacteria can chronically resist in a plant cell is lagging far behind the progress that has been made on nodule organogenesis. Thus, successful engineering of nitrogen fixing symbiosis in crops will require first a significant knowledge improvement of this central aspect of the symbiosis. The endosymbiotic rhizobia in the nodule cells, called bacteroids, are differentiated bacteria which have a specific metabolism. In many legumes their formation is accompanied with a morphological transformation which is imposed by the host plant. In Medicago and related legumes, nodule-specific antimicrobial peptides, called NCRs, have been shown to control bacteroid differentiation. However, the mechanisms that control bacteroid differentiation in legumes with other bacteroid morphotypes are unknown. Endosymbiont differentiation is also taking place in the actinorhizal symbiosis by the formation of nitrogen-fixing Frankia vesicles. Like in legumes, also this process is under the control of unknown host plant factors. We will study a model system of Bradyrhizobium strains interacting with Aeschynomene (A. evenia and A. afraspera) and soybean legumes in which bacteroids have three different host-dependent morphotypes. We will determine for these morphotypes parameters such as nitrogen fixation efficiency, and the bacteroid metabolome, proteome and transcriptome. These data will permit to identify morphotype-specific or –enhanced pathways and indicate whether particular cell morphotypes represent an advantage to the host plant as it is suggested by a few preliminary studies. A second objective is the identification of bacterial determinants required for bacteroid differentiation. For this, genetic and transcriptomic approaches will be used and candidate functions will be characterized in detail. Third, we will identify the plant effectors that affect bacteroid differentiation. Bioassays based on the change of bacterial morphology or the activation of genes specifically up-regulated during bacteroid morphogenesis will be developed. This global approach will be associated with the evaluation of candidate effectors (nodule-specific antimicrobial peptides) through biochemical and cell biology assays. Fourth, we will explore whether mechanisms for endosymbiont control operating in legumes are conserved in the evolutionary distant actinorhizal symbiosis. Nodule-specific antimicrobial peptides have been identified in the actinorhizal plants Alnus glutinosa and Casuarina glauca and the role of these peptides in vesicle formation will be studied by in vivo and in vitro approaches. This project will enhance our knowledge on the requirements for the intracellular accommodation of bacteria during the legume and actinorhizal symbioses and therefore constitutes an essential step towards transfer of nitrogen-fixing symbiosis to crop plants lacking nitrogen-fixation capability.

Nickel (Ni) is a heavy metal widely used in the industry to produce stainless steel and rechargeable batteries that are used in everyday life. However, the Ni mining industry leads to environmental pollution and has a direct impact on biodiversity. In the context of a sustainable development, it is crucial to limit the negative effects of Ni production on the environment. Phytoremediation and phytomining are promising technologies that use plants to remove Ni from polluted soil and to extract Ni for commercial purpose. Today, the development of these eco-friendly strategies is still limited by our succinct knowledge on the mechanisms of Ni accumulation in plants. The goal of the EvoMetoNicks project is to improve our basic knowledge about molecular mechanisms involved in Ni resistance and hyperaccumulation in plants. Ni is an essential element but becomes toxic at high concentration for most living organisms. Surprisingly, 400 plant species found on serpentine (ultramafic) soils rich in Ni in Europe, New Caledonia and Cuba, are able to accumulate tremendous amount (>0.1%) of Ni in leaves. Sixty of these Ni hyperaccumulators are endemic to the ultramafic soils of New Caledonia that is a biodiversity hotspot but also one of the most important producers of Ni in the world. Ni hyperaccumulators are receiving an increasing interest because of their potential use in phytoremediation and phytomining technologies. In this project, we will take advantage of the important diversity found in Ni hyperaccumulators worldwide to obtain a novel and broad vision on the fundamental mechanisms involved in Ni accumulation and adaptation in plants. We have selected evolutionary distant Ni hyperaccumulators including Noccaea caerulescens, a species of the Brassicaceae family developed as a model plant to study metal accumulation, and two hyperaccumulators endemic to New Caledonia, Psychotria gabriellae (Rubiaceae) and Geissois pruinosa (Cunoniaceae) that will be better characterized at the physiological and molecular levels in this project. Using Next Generation Sequencing strategies, we will compare the transcriptomes of these species with those of closely related non-accumulating species to identify molecular mechanisms linked to Ni accumulation and adaptation that were conserved during evolution of higher plants or on the contrary that are more divergent in plant species. Among candidate genes, we will focus our functional studies on genes and pathways that are involved in transport, chelation, detoxification and sequestration of Ni. We think this project will identify target genes and molecules important for Ni accumulation in plants and therefore will be valuable for the development of phytoremediation and phytomining technologies. Also, according to the Nagoya Protocol for the access to genetic resources and the fair and equitable sharing of benefits, the EvoMetoNicks project will conform to local and international environmental laws for the protection of plant species and we will share the knowledge and experience generated by this project with students and a more general audience through lectures and conferences in New Caledonia.