Michel Caboche, Catherine Golstein, Gilles Pelsy Technologies du Futur Michel Caboche, Catherine Golstein, Gilles Pelsy INRA Paris, 24 Novembre 2008 A L I M E N T A T I O N A G R I C U L T U R E E N V I R O N N E M E N T

Lettre de mission Technologies du Futur Contexte: « (…) l’INRA doit être à même d’anticiper et de susciter les grandes ruptures scientifiques et technologiques susceptibles de survenir dans les décennies à venir et d’avoir un impact fort sur l’agriculture et l’alimentation. » Mission: « (…) compléter les travaux d’Agrimonde* par une étude spécifique sur les nouvelles technologies et leur importance en recherche agronomique, sur un champ suffisamment large pour y inclure la biologie, l’écologie, les sciences agronomiques, et les technologies de transformation et de suivi de la qualité. » Agrimonde*: prospective INRA/CIRAD sur les systèmes agricoles et alimentaires mondiaux à l’horizon 2050.

Objectif Identifier et analyser des technologies émergentes pertinentes pour la recherche agronomique la biologie l’écologie et les sciences agronomiques la transformation et qualité des produits alimentaires et non-alimentaires susceptibles de répondre aux enjeux de l’agriculture de demain Délivrables: Etablissement d’une liste d’une douzaine de technologies émergentes Séminaire de réflexion / validation/élargissement Collection de fiches technologiques Rapport final résumant les conclusions de l’étude Def Larousse - technologie: Étude des outils, des machines, des procédés et des méthodes employés dans les diverses branches de l'industrie. Ensemble des outils et des matériels utilisés dans l'artisanat et dans l'industrie. Ensemble cohérent de savoirs et de pratiques dans un certain domaine technique, fondé sur des principes scientifiques. Théorie générale des techniques. Nouvelles technologies, moyens matériels et organisations structurelles qui mettent en œuvre les découvertes et les applications scientifiques les plus récentes. (On dit aussi haute(s) technologie(s), technologie(s) de pointe, technologie(s) avancée(s).) The Merriam-Webster dictionary: "the practical application of knowledge especially in a particular area" and "a capability given by the practical application of knowledge". The word "technology" can also be used to refer to a collection of techniques. technology refers to tools and machines Confusion technologies / Techniques Rapprochement sciences / technologies

Stratégie Veille scientifique et technologique Littérature scientifique Rapports de projets de recherche Conférences, colloques, workshops et séminaires Sites internets variés Etudes de prospective -> Explorer les sciences et technologies Identifier, analyser, évaluer les technologies émergentes pertinentes Ebauche de fiches technologiques Consultation d’experts des technologies et champs d’application INRA / hors INRA Contacts (collègues, réseau élargi, conférences) Auteurs de publications Recommandations (DS ou CD INRA) -> Enrichir, orienter, corriger, valider les choix

Les nouvelles technologies ainsi que leurs perfectionnements conduisent a de nouvelles découvertes et de nouvelles inventions Leur émergence est un phénomène assez rare, souvent fortuit Rien ne permet de penser que ce processus ralentisse

Sélection de technologies pertinentes pour TF Intérêt prolongé ou renouvelé Technologie nouvelle? non oui Potentiel d’application atteint? Publication scientifique? non oui non oui (PCR) Champ d’application pertinent pour l’agronomie? (Single-Molecule Real-Time Sequencing) (Association d’espèces) (Recombinaison homologue chez la souris) non oui Fiche technologique Veille Fiche technologique

Standard technological worksheet 1- Introduction, background 2- Definition, description How does it work? What makes it a new technology? What does it bring to previous technologies? 3- Current and prospective applications How could it contribute to meet the needs of tomorrow’s agriculture? 4- Current limitations and challenges Potential technical limitations, risk assessments, ethical issues, research funding and management 5- Glossary of scientific terms 6- Key references 7- Consulted experts

Eventail de technologies émergentes et domaines d’application correspondants Omics technologies Genetic engineering Nanotechnologies Imaging technologies Phenotyping technologies Agronomy and agricultural production technologies Bioinformatics and computational tools Research in biology x Plant and animal breeding Plant and animal production systems Environment and industrial ecology Feed and food Non-food and green chemistry (industrial biotech) So far: 19 technologies qui peuvent être divisées en 7 catégories. 19 technologies, 7 catégories

Sensors, remote sensing and spatial analysis A variety of wireless sensors for monitoring biomass, soil and fruit quality, combined with spatial analysis can feed agronomic models in real time and help rapid decision making at the farm level. A high GPS resolution is needed. The Egnos satellite coupled with on site beacons will provide a less than 1 meter resolution European satellite Egnos, GPS Plant vigor, N stress, maturity LAI, NDVI, sugar/acid content Agronomic models Decision support

Sensors, remote sensing and spatial analysis Potential applications: Collecting informations Environment monitoring, Nutritional status and epidemiology Surveillance of illicit crops Identification of management mistakes Agronomic model feed with collected data Improvement of model predictions Crop management decision support Precision farming tracking and automatic guiding systems, variable-rate fertilisation water irrigation control in real time Crop protection treatments Optimal harvest conditions Limitations Cost and complexity Part of the collection of data is still hand-based (ex Spectrophotometer) Present GPS resolution is limiting. Cloudy areas are a problem Consulted experts: Jean-Michel Roger, Bruno Tisseyre, Alexia Gobrecht 

Landscape modelisation Modelisation is a basic tool for many aspects of agronomy sciences. Models have been set up to handle the complexity of the development of a crop, or to describe a cropping system and its management at the field level. The management of water ressources in a river basin can also be modelized. But for the evaluation of the socio-economic and environmental consequences of a decision (ex: Large scale biofuel production) these different levels have to be integrated. Landscape modelisation which aims at integrating these different levels from the plant to the ecosystem is a challenging research domain which has potentially important applications. AMAP modelisation of the consequences of climatic changes on the landscape

Landscape modelisation Potential applications: Predictions on landscape evolution as a consequence of climatic changes Optimization of soil management (farming, natural areas, towns) Management of water ressources and biological invasions Management of the spreading of pathogens and creation of barriers Management of GMO / organic farming Optimization of farming practices as a consequence of market evolution Limitations: Models are still far from integrating the desired informations. They are not ofte interoperable New modelisation tools need to be set up, reducing the size of complex objects and managing the heterogeneity of data Consulted experts: F. Garcia, J. Wery, H. Descamps

High-throughput sequencing technologies Three companies have developped new sequencing technologies (454, Illumina and SOLiD) that open multiple possibilities of applications. What is their respective interest?   ABI 3730XL (Applied Biosystems) (released in 2005) GS FLX System (Roche Diagnostics) (released in October 2005) Illumina Genome Analyzer (Illumina) (released in June 2006) SOLiD DNA Sequencer (released in October 2007) Read length 600-900 bp 400 bp 18, 26, 36 bp 35 bp Data per run 1 Mbp 500 Mbp 1.5 Gbp (3 Gbp for mate pairs) 4 Gbp (8 Gbp for mate pairs) Machine cost €400,000 € 450,000 €500,000 € 550,000 Run cost per base ~€1000/Mbp €20/Mbp 50-fold cheaper than Sanger ~€5/Mbp 200-fold cheaper than Sanger Limitations - Expensive, - low-throughput, - labour intensive, relatively short reads: issues with assembly of repetitive regions Very short reads: issues for assembly or mapping and annotation. - Accuracy limitations? GC Short reads - Adoption of color space in sequence analysis? Applications of choice Method of choice for de novo sequencing of complex genomes Best Sanger competitor for de novo sequencing projects for small and simple genomes and metagenomics projects. Best for “seq-based” method analyses applied to sequenced genomes Best for resequencing projects, ultra deep SNP discovery and transcript. Too few publications at this time to assess the range of SOLiD applications, and to compare its performance with competing technologies.    

High-throughput sequencing technologies Potential applications - GENOME LEVEL  HTP de novo sequencing Facilitated sequencing of related genomes Resequencing -        genetic diversity and evolutionary studies: -        genome-wide discovery of genetic polymorphisms -        Low-cost alternatives to whole-genome resequencing: Environmental sequence analysis Detection of rare somatic mutations (somaclonal variation) - TRANSCRIPTOME LEVEL Transcript discovery and gene expression profiling Accurate gene annotation ( splicing sites) Deep sequencing of small RNAs (miRNA, si RNAs, etc) DNA Methylation profiling (BS-seq, methylC-seq) ChIP sequencing: DNA protein binding sites analysis Epigenome analysis ( nucleosome mapping, analysis of epigenetic processes, etc) Limitations Cost. Danger of simplistic views on research. Needs some thinking Consulted experts : P Wincker, Génoscope

High Throughput genotyping Genotyping and phenotyping are at the root of genetic analysis . Phenotyping can be performed on multiple, unrelated criteria, All genotyping technologies exploit the variations occurring in genomic sequences. The same techniques can be used to study human genetics but also cattle and crop genetics. Among DNA polymorphisms, SNP variations are found in all genomes and can be identified by exploitation of htp genome re-sequencing. A large number of SNP detection/genotyping techniques are available. What’s new? Genome-wide association for LD analysis. Rationale: The genes contributing to a specific trait are difficult to predict. Its more efficient to scan them by detection of associations, using a dense array of SNPs (Ex: an average of 10 SNPs per gene and a total of 1000 000 SNPs per human genome) Two main suppliers: ILLUMINA and AFFY. Limitations: only one or two individuals analysed on one array. Large numbers (1000 genotypes) required for statistical significance. Problems of population structures. Very expensive… Consulted experts: A. Eggen, D. Brunel, A. Charcosset, I. Gut

Applications enabled by HTP genotyping Diagnostics, MAS, disease related genes, Domestication traits, bar coding, industrial protection of genotypes Plant and animal breeding for selected traits 100,000 Genome-Wide Association Studies GWAS validation and candidate gene association 10,000 Candidate region fine mapping Genotyped individuals 1,000 Diagnostics 100 Fingerprinting, Whole genome scans 10 10 100 1,000 10,000 100,000 1,000,000 Genotyped loci

High Throughput genotyping techniques Two main suppliers for GWA: ILLUMINA and AFFYMETRIX GoldenGate assay Infinium BeadChips iselect VeraCode SNPlex, GenPlex TaqMan Openarrays iPLEX Gold Pyroseq SNaP shot Invader BeadChips Illumina AB Sequenom Targeted GeneChips Affymetrix Illumina High-Density 1M-Duo chip Affymetrix Genome-Wide Human SNP Array 6.0 100,000 Genome-Wide Association Studies 10,000 Genotyped individuals 1,000 100 10 10 100 Genotyped loci 1,000 10,000 100,000 1,000,000

high-throughput sequencing technologies Metagenomics Metagenomics (whole-community genomics or environmental genomics) : field of study of the metagenome, defined as all the genomes of a microbe community in a particular environment. Metagenomics helps preforming the inventory of living organisms in a specific biotope Enabled by new high-throughput sequencing technologies from the US National Academy of Sciences website http://dels.nas.edu/metagenomics/overview.shtml#process access to the uncultured (>99% bacteria) access to whole microbe communities in a variety of natural environments (unlike pure cultures in artificial stable laboratory conditions) Revolution in microbiology:

Metagenomics applications ** Microbes ubiquitous and essential to life Fundamental research: microbe diversity and evolution, ecology, biology Environment: Biosensors, bioremediation of contaminated soils, industrial treatment of wastewater Agriculture: Optimisation of natural plant fertilisation, rapid identification of pathogens responsible of emerging diseases Human nutrition and health: Search for new antibiotics, role of human gut microbes (microbiome) in nutrition and obesity Bioindustry: discovery of novel enzymatic activities (ex. enzymes specialised in lignocellulose degradation in termite guts) Consulted experts on Metagenomics: Dusko Ehrlich, Génétique microbienne, INRA Jouy-en-Josas, France Denis Le Paslier, Génoscope, Evry, France Jean Weissenbach, Génoscope, Evry, France Pierre Monsan, INSA, Toulouse, France Michael O’Donohue, INSA, Toulouse, France Renaud Nalin, LibraGen, Toulouse, France

Nutrigenomics and nutrigenetics Enabled by new high-throuput ‘omics technologies Nutrigenomics: analyses the effects of nutrients and diets at the molecular and system’s level Ex: analysis of transcriptome/metabolome after Iron deprivation or the providing of phytosterols. Nutrigenetics: analyses the effects of genetic makeup on individual responses to nutrients and diets Ex: Identification of genotypes susceptible to obesity Enabled by Human Genome Project, HapMap Project, and HTP genotyping technologies Association statistics of type 2 diabetes genome-wide association studies. From Frayling, Nat. Rev. Genet., 2007

Nutrigenomics and nutrigenetics applications and limits Fundamental research: human and animal nutrition and health Better understanding of nutrition at the molecular level (mode of action of nutrients) Identification of genetic loci predisposing to diet-related chronic disease Identification of biomarkers associated with diet related chronic disease Feed/food industry: Rational development and validation of claims on new functional feed/food products Towards personalised nutrition: genetic testing-based diet recommendation? Nutrition guidelines and market segmentation (ex Coeliac disease) Challenges Challenges in genome-wide association analysis: large population, replication studies, population stratification, limitation to available SNPs… Phenotyping challenges: For human subjects: uncontrollable heterogeneity of nutrition status and high costs Challenges for genetics-based personalised nutrition Validation of relationship between genetic marker and health status? Genetic tests miss extra sources of variability: the environment, the epigenome,etc Consulted experts on nutrigenomics and nutrigenetics:

Single molecule tracking Single molecule track can be exploited to study the diffusion of macromolecules in the cell. They substitute a « real life » visualisation to a statistical view of processes Single molecules can be visualised by binding a chromophore (Ex GFP). Most chromophores bleach rapidly , preventing kinetics analysis. Their localization is limited by optical diffraction Quantum dots can be substituted to chromophores QD are issued from the chip technology They do not bleach and emit at specific wl They can be localized at a precision of 10-20nm not limited by optical diffraction. Single molecules can also now be manipulated in the test tube to study their mechanical properties (Ex topoisomerases and supercoiled DNA) 1mm D Diffusion of a Glycin receptor tagged by a quantum dot Blue: outside synapse Green: inside synapse

Single molecule track Benefits Meeting of statistical physics, biochemistry and cytology Access to various molecular processes Receptor migration, endocytosis, cytoskeleton dynamics, etc… Limits Sophisticated techniques that need optical engineering Not commercially available QD still sterically big, may create artifacs Tracking the interaction of two different molecules is a challenge Consulted experts: B. Satiat Jeunemaitre, CNRS Gif, A Triller, ENS Ulm

Creation of novel enzymatic activities The diversity of X-rays established enzyme 3D structures seem to reach a plateau. How to create diversity? Goal: Creation of new enzyme specificities by active site random mutagenesis and htp test of these activities on new substrates is a novel avenue Example: DNA shuffling of GAT Iterative rounds (After Johannes and Zhao, 2006) Random/targeted mutagenesis or Gene shuffling Selection or HTP screening Novel substrate specificity or Improved catalytic activity Target gene(s) Goal achieved Library of mutant genes Library of mutant enzymes Functionally improved enzymes 10,000 fold improvement in catalytic efficiency Combination rational design / directed evolution Computational design based on crystal structure or homology modeling, and phylogenetic analysis -> Novel/improved biocatalysts for chemical, food and pharmaceutical industries

Creation of novel enzymatic activities Potential applications Design of novel enzymatic activities and analysis of the basis of substrate specificty. Creation of enzymes working on artificial substrates Potential applications in microbiology/fermentation/ second generation of biofuels /remediation Limitations Enzyme design requires a large set of competences to be operational (3D protein analysis, modelisation, site directed mutagenesis, htp screen for the detection of improved enzyme Consulted experts : Pierre Monsan, INSA, Toulouse, France Michael O’Donohue, INSA, Toulouse, France

Targeted gene modification/inactivation Homologous recombination is used in model organisms to perform targeted gene modification. However it does not work on most species. Four technologies can partially substitute. TILLING is a methodology that allows the screen of mutations affecting a gene of interest in large populations of plants issued from a mutagenic treatment. It’s a well established technology RNAinterference is a process of gene inactivation induced by the recognition by the cell machinery of short (20-23nt) double stranded RNAs. It has a high sequence selectivity. It’s exploited through GM technology TILLING and RNAi are well established technologies but recent developments are promizing Zinc Finger Nucleases (Sangamo biosciences, Ca) are able to recognize and cut a specific DNA sequence. They act as dimers of three zinc finger proteins that recognize a set of 3X3 nt linked to an endonuclease, leading to a specificity of 18 nt (14 in fact). Meganucleases (Cellectis SA) are restriction enzymes with very high sequence selectivity. They can be engineered in heterodimeric endonucleases in which a set of 8 aai recognize seven bases in the DNA target leading to a specificity of 2X7 nt. As for ZFN technology once a double strand break is generated, NHEJ repair generates mutations at the site. Meganucleases can be used for gene exchange by deleting a fragment of up to 8kb and replace it by a new version. In both technique a combinatorial screen for ZFN or Mega nucleases of desired specificity is necessary.

Targeted gene modification Potential applications of TILLING, RNAi, ZFN and Meganucleases Reverse genetics and targeted gene inactivation (Ex creation of recessive resistances to viruses with TILLING) Inactivation by KO of genes with undesirable effects (ex: synthesis of alkaloïds ) Induction of allelic diversity to optimize an agronomic trait RNAi The technique relies on the production of transgenics. No expected toxicity The production of a Si RNA by the host can lead to the inactivation of an essential gene in a pest that feeds on this host, resulting in protection of the plant against the pest. Potential applications of ZFN and Meganucleases More performant than TILLING and RNAi as regards the diversity of target modifications. New therapies against DNA virus infection Claimed targeted introduction of genes (Ex: to cure a disease) Limitations ZNF and Meganuclease technologies are work intensive and expensive The ZFN technology does not work well in different labs. Consulted experts: A. Choulika, Cellectis; B. Dujon, Pasteur

Induced pluripotent stem cells The regeneration of an organism from one of its somatic cells can be achieved in the plant kingdom, but generally not in the animal kingdom, including mammals. This restricts the exploitation of cell therapy approaches as well as gene transfer techniques to embryogenic stem cells which are not easy to handle. iPS (Induced pluripotent stem cells) provide a breakthrough in this field by dedifferentiating somatic cells and reinitiating a pluripotent state. This is achieved by the transfection of several transcription factors that trigger the process of epigenetic reprogramming. Such reprogrammed cells injected in blastocysts lead to viable animals

Induced pluripotent stem cells Potential applications Basic research. Understanding the basis of cell differenciation… Cell therapy. A fibroblast cell issued from an animal carrying a genetic disease can be cured of the mutation by gene transfer, and then converted into an iPS cell that can be differentiated, upon proper stimulation in a specific type of stem cell susceptible to colonize the Diseased animal, in the absence of non self rejection. Numerous applications foreseen to cure human genetic diseases Farm animal engineering. Despite numerous efforts, pluripotent ES cells from farm animals have not been obtained. This is strongly limiting the production of trangenic animals Limitations The technique of transfection of the cocktail of four transcription factors is based on retroviral vectors. This can unpredictably generate tumor formation. This could be alleviated by transient expression of the introduced TF that dont need to be permanently expressed to confer the iPS phenotype Consulted experts: JP Renard, INRA

Ecological intensification Phenotyping technologies Synchrotron beams Mass spectrometry, Proteome, Metabolome Nanotechnologies Synthetic biology

Séminaire de validation Paris, 23 Janvier 2009 Participants sur invitation : DS INRA/CIRAD, chercheurs consultés et additionnels, public 64 invitations/privé 69 invitations, toutes disciplines Objectifs : Evaluer de la pertinence des choix des technologies sélectionnées informer les participants sur les technologies émergentes susciter des décloisonnements de champs d’application susciter des pistes d’applications non-envisagées, Modalités Une session pleinière présentation de l’étude/ analyse par trois experts de ce qui bouge dans leur domaine Trois groups de travail Biologie + Agronomie; Agronomie+ Transfo et Qualité; Transfo et Qualité+ Biologie Travail de synthèse Sur place: conclusions des trois groupes Les participants seront convié a transmettre leurs remarques aux organisateurs

Merci de votre attention A L I M E N T A T I O N A G R I C U L T U R E E N V I R O N N E M E N T