Gene Transfer in Plants

1. Gene Transfer in Plants Assoc. Prof. Dr. Sumontip Bunnag

2. Fundamentals of Gene Transfer in Plants Plant cell and tissue culture - Plants are unique among higher organisms in having the capacity to regenerate whole organisms from a variety of cell types as well as from a variety of cell types as well as from a fertilized ovum. - Genetic transformation or the successful introduction and integration of exogenous DNA into these cells can give rise to transgenic plants.

3. Regeneration Plants Organogenesis Embryogenesis Callus Cell Suspensions Protoplasts Microspores/Anthers Tissue Explants Donor Plants Potential pathways for the growth and differentiation of plant cells and tissue in culture Fig. 1 The regeneration of plantlets in culture may occur by organogenesis or embryogenesis.

4. Potential pathways for the growth and differentiation of plant cells and tissue in culture Organogenesis usually involves the differentiation of shoots followed by roots. Embryogenesis in culture follows stages of maturation similar to zygotic embryos in seeds but originates from different cells. Cells at any of the stages (shown in Fig.1) can be use as recipients for transformation.

5. Gene Transfer Technique To transfer a gene fromoneDNAmolecule to another DNA molecule. Gene Transfer includes isolation of gene, manipulation (target gene insertion ) and reintroduction of DNA into the cells or model organisms. It used to make a crop resistant to a particular herbicide, pest, weedsorintroducing a noveltrait, orproducing a new protein or enzyme.

6. Gene Transfer Technique Two Types of Transformation • Based on vectors, • mainly Agrobacterium tumefaciens • 2. Direct DNA transfer(small plasmid) • The gene transfer results can be transient and stable transduction

7. Transfection Types Transient transfection In transient transfection, the transfected DNA is not integrated into host chromosome. DNA is transferred into a recipient cell in order to obtain a temporary but high levelof expression ofthe target gene.

8. Transfection Types Stable transfection Stable transfectionis also called permanent transfection. By the stable transfection, the transferred DNA is integrated (inserted) into chromosomalDNA and the genetics of recipient cells is permanent changed.

9. Transfection Types Stable transfection Time consuming, labor intensive, influenced by position effects, large production volumes, requires regeneration system, large amounts of glasshouse space, gene expression in all plant tissues.

10. Agrobacterium-mediated gene transfer Bacteria of the genus Agrobacterium are free-living soil bacteria that have evolved the unique capacity to interact genetically with susceptible plants. This interaction results in the stable insertion of part of the genome of the bacterium into the genome of the plant. This natural ability of the bacterium to genetically transform the plant tissue.

11. Introduction to Agrobacterium tumefaciens Agrobacterium tumefaciens is a ubiquitous soil borne pathogen responsible for Crown Gall disease, affecting many higher species of plant.

12. Agrobacterium tumefaciens It’s a gram negative, motile, rod shaped bacterium which is nonsporing, and is closely related to the N-fixing rhizobium bacteria which form root nodules on leguminous plants. The bacterium is surrounded by a small number of peritricious flagella. Virulent bacteria contain one or more large plasmids, one of which carries the genes for tumour induction and is known as the Ti (tumour inducing) plasmid.

13. Agrobacterium tumefaciens The bacteria transfers a T-DNA which located in tumor-inducing plasmid (Ti plasmid) into the nucleus of an infected plant cell The newly introduced T-DNA is incorporated into the plant genome and is consequently transcribed.

14. Agrobacterium tumefaciens • The T-DNA that is integrated into the plant genome contains • cancer-causing oncogenic genes and genes that synthesize • opines which are a food source for A. tumefaciens

15. Agrobacterium tumefaciens Crown Gall Disease A.tumefaciens cells attached to a plant cell.

16. Mechanism of infection of Bacterium in plants

17. Ti plasmid A central role in crown gall formation and it is the portion of Ti plasmid that is integrated into the host genome. Ti plasmid is responsible for the tumorous phenotype. Ti plasmid features - contain T-DNA regions - contain a vir region - contain an origin of replication - contain genes for the catabolism of opines (a class of amino acid conjugates)

18. Ti plasmid Two regions on the Ti plasmid are essential to this transmission, the T-DNA region and another region called the vir(virulence) region.

19. Ti plasmid Ti plasmids can be classified according to the opines produced 1. Nopaline plasmids: carry gene for synthesizing nopaline in the plant and for utilization (catabolism) in the bacteria. Tumors can differentiate into shooty masses. 2. Octopine plasmids: carry genes to synthesize octopine in the plant and catabolism in the bacteria. Tumour do not differentiate, but remain as callus tissue.

20. Ti plasmid 3. Agropine plasmids: carry genes for agropine synthesis and catabolism. Tumors do not differentiate. 4. Ri plasmids: induce hairy root disease on some plants ; have agropine-type genes and may have segments from both nopaline and octopine plasmids.

21. T-DNA • The T-DNA region of any Ti plasmid is defined by the presence • of the right and left border sequences. • Only parts of the T-DNA that are essential are 25 bp of directly repeated sequences that border the T-DNA region.

22. T-DNA • Any DNA between the border will be transferred into the genome • of the plant. • Vir region has not been found in transformed plant cells. It can • function in the bacterium. • One or both T-DNA border regions provide the sites for the • recognition of gene products from the vir region.

23. T-DNA • The oncogenes: • - Two genes auxA and auxB encode proteins involved in the production of auxin, similarly gene cyt for cytokinin production which are the prime determinants of the tumour phenotype.

24. auxA auxB cyt ocs LB RB T-DNA LB, RB – left and right borders (direct repeat) auxA + auxB – enzymes that produce auxin cyt – enzyme that produces cytokinin ocs – octopine synthase, produces octopine

25. T-DNA • Other genes: • - The tml gene which is in involved in determining tumour • size is found in some species, is also found in the T-DNA. • - Genes responsible for the T-DNA transfer are also • situated on the Ti plasmid

26. Vir region Genes in the vir region must play a major role in T-DNA transmission. Acetosyringone (AS) (a flavonoid) released by wounded plant cells activates vir genes. virA,B,C,D,E,F,G (7 complementation groups, but some have multiple ORFs), span about 30 kb of Ti plasmid.

27. Vir gene Function Vir Genes and their Functions Sense phenolic compounds from wounded plant cells and induce expression of other virulence genes. Vir A and Vir G Produced endonuclease; cut T-DNA at right border to initiate T-strand synthesis Also guides theT-DNA complex through the nuclear pores. . Vir D2

28. กลไกการส่งถ่ายยีนสู่พืชกลไกการส่งถ่ายยีนสู่พืช (Christophe, 2000)

29. Vir Genes and their Functions

30. Vir gene Function Vir Genes and their Functions Vir D1 Topiosomerase; Helps Vir D2 to recognise and cleave within the 25bp border sequence Vir C Binds to the ‘overdrive’ region to promote high efficiency T-strand synthesis Vir E2 Binds to T-strand protecting from nuclease attack, and intercalates withlipids to form channels in the plant membranes through which the T-complex passes.

31. Vir gene Function Vir Genes and their Functions Acts as a chaperone which stabilises vir E2 in the Agrobacterium Vir E1 Vir B Assemble into a secretion system which spans the inner and outer bacterial membranes. Required for export of the T-complex into the plant cell

32. Important genes encoded by Ti plasmid 1. Cytokinins (plant hormone for cell plant division and tumorous growth) 2. Enzymes for indoleacetic acid (auxin) synthesis   (inducing stem and leaf elongation, inducing parthenocarpy and preventing aging)

33. Important genes encoded by Ti plasmid 3. Enzymes for synthesis and release of novel plant metabolites: - the opines (uniques amino acid derivatives) - the agrocinopines (phosphorylated sugar derivatives) • Opines and agrocinopines are NUTRIENTS for A.tumefacies. • They can not be used by other bacterial species. It provides unique niche for A.tumefaciens

34. Ti plasmids and the bacterial chromosome 1. Agrobacterium tumefaciens - chromosomal genes: chvA, chvB, required for initial binding of the bacterium to the plant cell and code for polysaccharide on bacterial cell surface. 2. Virulence region (vir) carried on pTi, but not in the transferred region(T-DNA). Genes code for proteins that prepare the T-DNA and the bacterium for transfer .

35. Ti plasmids and the bacterial chromosome 3. T-DNA encodes genes for opine synthesis and for tumor production. 4. octopine catabolism genes carried on the pTi and allows the bacterium to utilize opines as nutrient.

36. Agrobacteriumchromosomal DNA pscA chvA chvB T-DNA tra bacterial conjugation pTi vir genes opine catabolism oriV inc

37. The basis of Agrobacterium-mediated genetic engineering T-DNA of A. tumefaciensis excised and integrates into the plant genome as part of the natural infection process. Any foreign DNA inserted into the T-DNA will also be integrated.

38. Binary vector system Strategy: 1. Move T-DNA onto a separate, small plasmid. 2. Remove aux and cyt genes. 3. Insert selectable marker (kanamycin resistance) gene in T-DNA. 4. Vir genes are retained on a separate plasmid. 5. Put foreign gene between T-DNA borders. 6. Co-transform Agrobacterium with both plasmids. 7. Infect plant with the transformed bacteria.

40. Ti plasmid vector systems are often working as binary vectors ADVANTAGE: - Smallvectorsareused, which increases transfer efficiencyfromE.colitoAgrobacterium.Nointermolecularrecombinationisneeded. DISADVANTAGE: - Dependingontheorientation, plasmidswithtwo different origins of replication may be unstable in E.coli.

41. Agrobacterium-Mediated Transformation • In order to make a transgenic plants, there are main steps: 1. extracting DNA 2. cloning a gene of interest 3. designing the gene for plant infection 4. transformation 5. finally plant breeding. Overview of how transgenic plants

42. The process of T-DNA transfer and integration Agrobacterium contains a tumour-inducing (Ti) plasmid, which includes virulence (vir) genes and a transferred DNA (T-DNA) region. wounded plant cell produce phenolic compounds, which can trigger the expression of the Agrobacterium vir genes.

43. The process of T-DNA transfer and integration Vir D produced, endonuclease cut T-DNA at right border and left boder to initiate T-stand synthesis. After the bacterium attaches to a plant cell, the T-strand are transferred to the plant through a transport channel. Inside the plant cell, the vir protein interact with the T-strand, forming a T-complex.

44. The process of T-DNA transfer and integration Step 1. Signal recognition by Agrobacterium Step 2. Attachment to plant cells - two step process, attachment via polysaccharide and subsequently of cellulose fibers produced by bacterium Step 3. Induction of vir genes Step 4. T-strand production Step 5. Transfer of T-DNA out of the bacterial cell Step 6. Transfer of T-DNA into the plant cell and nuclear localization.

46. Advantages of Agrobacterium-mediated transformation • High rate of incorporation • Often simple integration pattern • Different from direct gene transfer • (vs. incorporation of multiple copies, with frequent • rearrangements )

47. Genes (or components) used for transfer Marker genes A critical step in the development and evaluation of transformation strategies for plants was the construction of vectors with genes that could act as dominant selectable markers. Under various selection pressures, these genes provide a growth advantage to cells that integrate the vectors and express the marker gene.

48. Genes (or components) used for transfer For example: nptII from the bacterial transposon Tn5 which codes for the enzyme neomycin phosphotransferase.In the presence of kanamycin, transfomed cell can grow and differentiate into plantlets; however, normal cells and tissues of most plant species cannot.

49. Genes (or components) used for transfer A number of selectable marker genes have been developed which provide resistance to a range of antibiotics and chemicals (Table 1). Table 1 Examples of selectable markers Chimeric gene Source Selective agent nos-nptII-nos Tn5 kanamycin ocs-aphIV-nosE.coli hygromycin 35S-ble-nos Tn5 bleomycin 35S-dhfr-nos mouse methotrexate

50. Genes (or components) used for transfer Most of the selectable markers are not of plant origin. To achieve constitutive expression in culture and in the various tissues of the plants, the coding regions of the genes for the marker have been fused to promoters or other regulatory sequences known to function in plants such as those from the nopaline synthase (nos) or octopine synthase (ocs) gene of the Ti plasmids and from the cauliflower mosaic virus (CaMV 35S )