Recently, new approaches referred to as Synthetic Biology (SB) have emerged. The work done at the J. C. Venter Institute (JCVI) has significantly contributed to the development of SB by showing: (1) the synthesis of complete bacterial genomes starting from chemically produced oligonucleotides, (2) the genome-scale engineering using yeast as a platform, (3) the transplantation of isolated bacterial genomes to other bacteria or from yeast to bacteria. Mycoplasma capricolum subsp. capricolum (Mcap) and Mycoplasma mycoides subsp. capri (Mmc), which are related species, were used as recipient cell and donor genome, respectively. Mycoplasmas belong to the class Mollicutes, a group of wall-less bacteria phylogenetically related to low G+C% Firmicutes. Mycoplasmas are characterized by the use of UGA as a tryptophan codon and a small genome. Mollicutes include several pathogens affecting humans, animals and plants. One of the limiting factors to better understand the molecular basis of their pathogenicity is the lack of efficient genetic tools. The goal of SYNBIOMOLL is to extend Synthetic Biology key technologies to other Mollicutes species of interest. Because several bacterial genomes have already been cloned in yeast and the synthetic assembly of large DNA fragments is in current use, the major technological limitation to a much wider use of these SB tools is our ability to extend the genome transplantation technology to other microbial species. In task 2 (task 1 is the coordination of the project), we propose to study the mechanisms of genome transplantation by evaluating the degree of relatedness necessary between a donor cell and a recipient cell and also by identifying some of the genetic factors responsible for this compatibility. First, we will transplant in Mcap recipient cell the genomes from species with increasing phylogenetic distance from the recipient cell to identify the transplantation compatibility limit. Then, using a troubleshooting approach, we will determine whether non-compatibility is a problem of replication and/or expression of the donor genome in the recipient cell. Specific genome regions, including the chromosomal replication origin, will be exchanged between Mcap and a non-compatible genome to evaluate the impact on transplantation. As a control, the same exchanges will be performed with a compatible genome. For this sub-task, genomes will be cloned into yeast to be modified accordingly. The expected results should help identifying trans-acting proteins and/or cis-acting sequences required to boot-up heterologous genomes into recipient cells. In task 3, we plan to develop genome transplantation in another phylogenetic group using Acholeplasma laidlawii (AL) as a recipient cell. AL can grow in much simpler culture media than mycoplasmas and is considered as an intermediate species between mycoplasmas and low G+C% firmicutes. Genome transplantations will first be performed with homologous donor genome and then with heterologous donor genomes from two other acholeplasmas. Once the conditions of genome transplantation in AL optimized, specific developments will be pursued to evaluate its capacities to replicate and express the genome of more distant non-cultivable, insect-transmitted plant-pathogenic phytoplasmas. The impossibility to cultivate these bacteria is a major barrier to their study and the development of plant protection strategies. In the SYNBIOMOLL project, we will evaluate AL ability to recognize and initiate replication of phytoplasma chromosomes using phytoplasma oriC plasmids and by replacing the oriC of AL with that of the phytoplasma. We will also use proteomic analyses to evaluate the expression of phytoplasma genes in AL. Succeeding genome transplantation in AL would be important per se but also would serve as a launching pad for the culture and the study of non-cultivable phytoplasmas. At the end of SYNBIOMOLL, Carole Lartigue intends to propose this project for an ERC Consolidator Grants.

Stevia rebaudiana Bertoni, a resilient plant species of the Asteraceae family, is cultivated for its leaves in which steviol glycosides (SG) accumulate, which have 300 times the sweetening power of sugar but are acaloric and acariogenic. This species is becoming the world's leading natural sweetener, compatible with diets for diabetics and hypoglycaemic diets. It also has many other interesting properties for human health and other uses. In order to develop a virtuous French production and processing chain for Stevia rebaudiana under organic farming, respectful of human and environmental health and offering an additional breakthrough crop in the rotations, the SELECTVIA joint laboratory aims to set up and optimise innovative strategies for the early selection and improvement of Stevia rebaudiana in order to provide farmers with adapted, productive and resilient genotypes. The SELECTVIA joint laboratory brings together the UMR Biologie du Fruit et Pathologie (BFP), INRAE Nouvelle Aquitaine, the University of Bordeaux and the company OVIATIS based in Estillac (47). In order to be able to offer competitive varieties to French farmers, it is necessary to optimise and accelerate the selection process. The joint laboratory therefore proposes, within the framework of a long-term collaboration, to accelerate access to innovation for the improvement of S. rebaudiana by (1) developing, within 36 months, a marker-assisted selection strategy for the SG content and Septoria sp. response traits a marker-assisted selection strategy (TRL 7-9), (2) developing tools for predicting metabolic content by NIRS and evaluating the value of the phenomenal selection strategy for SG content and response to fungi as an early selection strategy, at 42 months (TRL 4-6), (3) by developing, in a more exploratory way, haplodiploidisation in this heterozygous allogamous species in order to accelerate the selection process and access to heterosis, at 54 months (TRL 2-4). The SELECTVIA joint laboratory relies on the complementary skills of (1) the OVIATIS company in terms of upstream know-how for field production, phenotyping and extraction on an industrial scale and in terms of downstream know-how for the characterisation of extracts and product development, (2) the UMR BFP teams in terms of genetics, plant improvement, metabolomics and in vitro culture. SELECTVIA aims to produce innovations that will give OVIATIS a major competitive advantage in the selection of high-performance S. rebaudiana genotypes. The joint laboratory will also be the source of high quality publications and also aims to contribute to research training. The perpetuation of the laboratory will be based on a longer-term strategy of developing genotypes for the production of other molecules and/or their use in other fields (biocontrol, biostimulants).

Pollution of terrestrial and aquatic ecosystems by trace metal elements, also referred to as heavy metals, is a major and ever-growing threat to environmental and human health. A better understanding of the effects of toxic elements on land plants and microalgae is critical to develop approaches for treating contaminated environments using phyto- and phycoremediation processes The identification and characterization of organisms that tolerate and accumulate metals are essential to reach these objectives. Indeed, these organisms evolved sophisticated molecular mechanisms to cope with toxic elements. Deciphering these strategies may indicate how plants/algae might behave in contamination scenario and could provide clues for new biotechnological applications for the capture of metals. We isolated a metal-hypertolerant unicellular photosynthetic microalga from an environment contaminated with uranium, a chemotoxic radionuclide. This green microalga was identified by 18S rDNA sequencing as a Coelastrella species, hereafter designated Cos. Because it is able to live in culture media contaminated with high concentrations of uranium or silver, we assume that Cos has evolved unique molecular mechanisms to survive in environments polluted by toxic elements. In support of our hypothesis, it is known that some metal-tolerant land plants and microalgae have established efficient strategies to cope with metals (e.g. cellular uptake and efflux, compartmentalization, detoxification by chelators). The key genetic loci that explain the unique metal-tolerance and accumulation properties have been identified only in some land plants, for example in the zinc and cadmium hypertolerant Arabidopsis halleri species. These findings were essential to better understand metal homeostasis in both tolerant and non-tolerant species. In addition, they were the basis for new strategies to improve the phytoextraction properties of fast-growing, high-biomass but non-tolerant plant species. In green microalgae, however, the genes involved in metal tolerance and accumulation have never been identified. The objective of the DemoniaCo project is to fill this gap and unravel the molecular mechanisms involved in the tolerance and accumulation of toxic metals in Coelastrella sp. To this aim, we will use a combination of cell physiology and systems-based approaches, including a thorough analysis of the toxicological outcomes of metals on the transcriptome, proteome, ionome, and metabolome of the alga. This unprecedented multiscale and integrative strategy will provide new insights into the fundamental and applied biology of a metal-hypertolerant green microalga. Besides the identification of the gene network enabling Cos to tolerate uranium and silver, the expected results of the project include the characterization of Cos tolerance to a variety of toxic elements, the characterization of metal uptake and subcellular distribution in algal cells, the behaviour of the microalga in natural metal-contaminated waters to estimate its performance for phycoremediation, and the investigation of the potential of Cos as an oleaginous model species for the production of lipids for biofuel applications.

Plant response to heat stress has been extensively investigated for the past two decades. Heat stress (HT) applied to plants to study the effects of climate change often corresponds to T°C > at 40°C for 1h or less. However, global warming induces, among other things, long heat waves (1 week or more at 30 °C average / 24h). In order to identify the genetic determinants able to maintain a good trade-off between HT tolerance and plant crop yield, the Taiwanese (NTU) and French (INRAE) teams have developed collaborative and complementary strategies using tomato as model plant. Thus, NTU has identified several HT response genes using the expression-Quantitative Trait Loci (e-QTL) approach and INRAE has isolated HT-resistant tomato mutants to identify key genes for fruit yield maintenance. Using this unique and original plant material, the HEA2T project aims to characterize the response genes of plants to heat waves and to propose new genetic markers for crop breeding.

Plant health is of paramount importance to ensure food supplies for a growing world population. The Potyvirus genus is the most damaging group to crops. The high mutational frequency of these viruses results in an adaptive potential compromising the sustainability of host resistance. Understanding the molecular basis of their adaptation is of prime importance to derive sustainable resistance strategies. Many biologically active proteins possess intrinsically disordered regions (IDRs), e.g. regions devoid of stable structure and yet exerting biological function(s). Given the strong evolutionary potential of potyviruses on the one hand, and the high IDR content in their proteome on the other, this project aims at assessing the contribution of intrinsic disorder to the mechanisms of adaptation to the host.