1. Homologous Recombination
the ideal transformation would be gene replacement by homologous recombination (HR), also « gene targeting »
integration of a DNA fragment involves repairing DS DNA breaks >>
2 mechanisms, Homologous recombination (HR) and Non – homologous end-joining (NHEJ)

2. DS break repair
Non-Homologous
End-Joining (NHEJ, IR)
Homologous Recombination
Consequences different: with NHEJ there is sequence loss, whereas with HR the sequence is conserved
However, HR is much less efficient than IR (NHEJ) in plants (HR is ~10-5 as opposed to 10-2 for mouse)

3. Methods of Gene Targeting
Same principle, difference of scale
As v. inefficient, problem of a sensitive assay

4. Homologous Recombination
very low frequencies of gene targeting observed >lower than NHEJ background> currently impracticable in higher plants
Options?
promotion of DNA repair by mutations or transgenes ?
Exploit the moss model?
Alternatives for excision of unwanted transgenes – HR or otherwise?

5. DS DNA break repair by HR involves many gene products in plants
Two models of HR in plants, one involving simultaneous repair of both template strands, the other
Involving successive ss invasion and synthesis
Results in term of recombination are different, but
In both cases numerous proteins required to create the substrates
Many of these proteins are involved in reducing or eliminating errors – their suppression can increase recombination rates
DS DNA break repair by HR involves many gene products in plants

6. Translational fusion of GFP to the storage protein gene Cruciferin
By recombining with the chromosomoally located cruciferin gene, GFP is brought under the control of the Cruciferin promoter and expressed in developing seeds
Independent observation: recombination between two EXTRA-chromosomal sequences was much more efficient than when a chromosomal locus was involved.
Déduction: Chromatin structure is important in controlling HR

7. Chromatin remodeling is involved in homologous recombination
Figure 1. A model for the functions of chromatin remodeling complexes at DNA double-strand breaks in budding yeast.
1. DNA DS breaks >> the Mre11–Rad50–Xrs2 (MRX) complex + Ku70–Ku80 heterodimer >>the DNA ends.
Tel1 and Mec1 phosphorylate H2A Ser 129 over a 50 kb region. NuA4 HAT complex acetylates H2A and H4 histone tails. RSC is recruited to DSBs by interaction with Mre11.
In the homologous recombination pathway, RSC can remodel chromatin around DSBs to promote loading of cohesin, which holds the sister chromatids together to facilitate strand invasion and Holliday junction formation during homologous recombination..
Current Opinion in Genetics & Development : Volume 17, Issue 2, April 2007, Pages

8. The Chromatin Assembly Factor (CAF-1) Subunit FASCIATA1 Is Involved in Homologous Recombination in Plants
The fas1-4 Mutant
Kirik, A., et al. Plant Cell 2006;18:
Copyright ©2006 American Society of Plant Biologists

9. A model for targeting nucleosome assembly to DNA transactions.
Figure 1. Structure and assembly of nucleosomes. (a) View down the DNA helix axis of the nucleosome core particle from Xenopus at 2.8 Å resolution shows organization of the histones and DNA. The DNA strands (brown and green), are on the outside. The histone octamer forms a helical ramp, around which is wrapped 1.7 turns of a DNA superhelix. Each histone consists of a three-helix domain called the ‘histone fold’, together with two unstructured tails. Pale blue hooks indicate points of DNA–histone contact. (b) Model of chaperone-assisted nucleosome assembly during DNA replication in eukaryotic cell nuclei. CAF-1 binds a histone H3–H4 tetramer (blue and green) and docks with the PCNA clamp on replicating DNA. NAP-1 binds a H2A–H2B dimer (red and brown) and transfers it to the docked histone tetramer.
Trends in Biochemical Sciences
Volume 31, Issue 7, July 2006, Pages

10. HR Is Stimulated in fas1-4
How can we measure homologous recombination in plants?
In the fas1-4 mutant, HR was stimulated ~100-fold
How can we determine if chromatin structure has been altered in fas1-4?
Kirik, A., et al. Plant Cell 2006;18:
Copyright ©2006 American Society of Plant Biologists

11. fas1-4 Causes Loss of Heterochromatin
Staining with DAPI gives an estimate of the % of heterochromatin
Kirik, A., et al. Plant Cell 2006;18:
Copyright ©2006 American Society of Plant Biologists

12. fas1-4 Affects Chromatin Conformation
Total DNA is more susceptible to DNAse1 digestion in fas1-4
3 marker genes on different chromosomes
are all more susceptible to DNAse1 digestion in the fas1-4 line
as is the reporter locus used (Gu-Us)
Kirik, A., et al. Plant Cell 2006;18:
Copyright ©2006 American Society of Plant Biologists

13. In moss (Physcomitrella patens) gene targeting is efficient

14. Top: life cycle of the moss
Bottom: An example of an enhancer trap experiment for this moss, showing different specific expression sites identified in different transformants

15. Towards creation of new varieties using Reverse genetics
non-transgenic eg., TILLING, Fast neutron
transgenic - technology challenges!

16. Towards creation of new varieties using Reverse genetics
Problems associated with T-DNA transformants:
Certain selectable markers (herbicide resistance, drug resistance)
Site of transgene integration and stability, copy number
gene silencing
risk of secondary effects on phenotype
Silencing - transgene integration can provoke a number of epigenetic effects
Can check with transcriptome chips if gene expression has been disrupted

17. Towards creation of new varieties using Reverse genetics
Problems:
transgene transcription and translation
low level transcription
little product accumulation
Particularly a problem with heterologous genes. Why ?
Can adjust codon usage to correspond if a synthetic sequence is used

18. Towards creation of new varieties using Reverse genetics
Possible solutions:
Novel plant vectors (environmentally friendly selection systems)
Co-transform with marker and segregate out
use of excisable inserts
Zinc finger endonucleases
use of novel non-drug selectable markers

19. Co-transformation procedure to generate marker-free transgenic plants
« Low-tech » solution
Inefficient, as often multiple integrations and rearrangements

20. use of excisable inserts:
The Cre-Lox system for excising T-DNA fragments in planta
Lox repeat substrate
Substrate after excision
Could also be used to integrate DNA at a LOX site
Which reporter genes are available for plant biotech?
Cre recombinase (expressed in trans)
Lox target sites 23 bp (invert repeat + spacer)
>> can be used to eliminate an unwanted marker gene after transformation
Cre-lox recombination: Creative tools for plant biotechnology
 Gilbertson L ,  TRENDS IN BIOTECH 21 (12): DEC 2003

21. Mode of action of a Zn-finger nuclease
Principle: construction and employment of a nuclease recognizing a defined (and rare) site
Principle: construction of a nuclease recognizing a defined (and rare) site
How to maximize the specificity of the enzyme-target site interaction?
« we observe more than one homologous
recombination event per 10 illegitimate recombination
events, a 104- to 106-fold enhancement over frequencies of
unassisted homologous recombination. »
Trends in Plant Science 11, 159
Uses:
- Promote homologous recombination at a specific site when cleaved (~100 x )
- site-specific excision

22. use of novel non-drug selectable markers
The dsdA gene from Escherichia coli provides a novel selectable marker for plant transformation
Oskar Erikson, Magnus Hertzberg and Torgny Näsholm
Plants are sensitive to D-serine, but expression of the dsdA gene, encoding D-serine ammonia lyase from Escherichia coli, can alleviate this toxicity.
Selection on D-Ser
Selection on Kanamycin
Plant Molecular Biology :  Volume 57, Number 3 Date:  February 2005 Pages: 

23. Towards creation of new varieties using Reverse genetics
Ideally, homologous recombination in plants ?
advantages:
gene replacement/gene targeting
precise positioning of insert
Problems remaining:
How to increase efficiency without deleterious consequences for the plant
High background of NHEJ
No ‘universal’ or ‘one-step’ method

25. Objectif du TP TILLING: identifier mutations dans pools d’ADN de plantes mutées
PCR produits fournis
Digestion avec CelI
Filtration sur Sephadex G-50
Concentrer par évaporation
Déposer sur la Gel
Séparation, analyse
démonstrations:
préparation d’un gel
analyse informatique des séquences
production des plantes

26. References:
Krysan PJ, Young JC, Sussman MR. T-DNA as an insertional
mutagen in Arabidopsis. Plant Cell Dec;11(12):
2. Li X, Song Y, Century K, Straight S, Ronald P, Dong X, Lassner M,
Zhang Y. A fast neutron deletion mutagenesis-based reverse genetics
system for plants. Plant J Aug;27(3):
3. Till BJ, Reynolds SH, Greene EA, Codomo CA, Enns LC, Johnson JE,
Burtner C, Odden AR, Young K, Taylor NE, Henikoff JG, Comai L,
Henikoff S. Large-scale discovery of induced point mutations with
high-throughput TILLING. Genome Res Mar;13(3):
Helpful links for getting the full text:
PubMed:
UBC ejournal:
useful sites

27. Anand, A; Krichevsky, A; Schomack, S; Lahaye, T; Tzfira, T; Tang, YH; Citovsky, V; Mysore, KS Arabidopsis VIRE2 INTERACTING PROTEIN2 is required for Agrobacterium T-DNA integration in plants PLANT CELL
Terada, R; Johzuka-Hisatomi, Y; Saitoh, M; Asao, H; Iida, S Gene targeting by homologous recombination as a biotechnological tool for rice functional genomics PLANT PHYSIOLOGY
Li, J; Hsia, AP; Schnable, PS Recent advances in plant recombination CURRENT OPINION IN PLANT BIOLOGY
Smith, J; Grizot, S; Arnould, S; Duclert, A; Epinat, JC; Chames, P; Prieto, J; Redondo, P; Blanco, FJ; Bravo, J; Montoya, G; Paques, F; Duchateau, P A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences NUCLEIC ACIDS RESEARCH e149
Endo, M; Ishikawa, Y; Osakabe, K; Nakayama, S; Kaya, H; Araki, T; Shibahara, KI; Abe, K; Ichikawa, H; Valentine, L; Hohn, B; Toki, S Increased frequency of homologous recombination and T-DNA integration in Arabidopsis CAF-1 mutants EMBO JOURNAL
Kamisugi, Y; Schlink, K; Rensing, SA; Schween, G; von Stackelberg, M; Cuming, AC; Reski, R; Cove, DJ The mechanism of gene targeting in Physcomitrella patens: homologous recombination, concatenation and multiple integration NUCLEIC ACIDS RESEARCH
Kirik, A; Pecinka, A; Wendeler, E; Reiss, B The chromatin assembly factor subunit FASCIATA1 is involved in homologous recombination in plants PLANT CELL