1. 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

2. 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.

3. 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

4. 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

5. 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

6. 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

7. 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

8. 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

9. 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

10. 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 

11. 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

12. 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

13. 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 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.

14. 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

15. 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 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

16. 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

17. 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

18. 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
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:

19. 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

20. 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

21. 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:

22. 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

23. 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

24. 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

25. 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

26. 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.

27. 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

28. 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

29. 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

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

31. 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

32. Merci de votre attention
A L I M E N T A T I O N
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