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Lecture 1&2 Agrobacterium mediated plant transformation.

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1 Lecture 1&2 Agrobacterium mediated plant transformation

2 A.Tumefaciens gall is not a tiny thing



5 Biology of A. tumefaciens djup/plasm.htm Well known to induce crown gall tumor A.tumefaciens lives around root surfaces (in rhizosphere) where it using nutrients that leak from the root tissues infects only through wound sites and actively chemotactic to them Plant wound produces acetosyringone Bacterial T-plasmid produces receptors for acetosyringone

6 The basis of Agrobacterium- mediated genetic engineering T-DNA of A. tumefaciens is 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.

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

8 Cytokinins are plant hormones that are derivatives of the purine adenine. Cell specific expression of cytokinin in the A.tumefaciens infected cell Zeatin is one of cytokinines which synthesis may be encoded by Ti plamid isolated from corn (Zea mays).

9 Opines are nutrients that are also for quorum sensing The plant cells start to secrete the opines from transferred bacterial T DNA opine diffuses into the surrounding cells and serves as a signal molecules for the conjugation of the agrobacterium (Quorum sensing)

10 Ti Plasmid Tumor- producing genes Virulence region Opine catabolism ORI T-DNA region DNA between L and R borders is transferred to plant as ssDNA; T-DNA encoded genes can be substituted by target genes





15 Agrobacterium A unique bacterial species Plant-Fungal-Animal Transformation

16 Agrobacterium tumefaciens 1. Soil bacterium closely related to Rhizobium. 2. Causes crown gall disease in plants (dicots).

17 3. Infects at root crown or just below the soil line. 4. Can survive independent of plant host in the soil. 5. Infects plants through breaks or wounds. 6. Common disease of woody shrubs, herbaceous plants, particularly problamatic with many members of the rose family. 7. Galls are spherical wart-like structures similar to tumors.

18 Only known natural example of DNA transport between Kingdoms 1. (Virulent) strains of A. tumefaciens contain a 200-kb tumor inducing (Ti) plasmid 2. Bacteria transfer a portion of the plasmid DNA into the plant host (T-DNA). T-DNA 

19 The T-DNA is transferred from the Bacteria into the Nucleus of the Plant 1. Stably integrates (randomly) into the plant genome. 2. Expression of genes in wild-type T-DNA results in dramatic physiological changes to the plant cell. 3.  Synthesis of plant growth hormones (auxins and cytokinins)  neoplastic growth (tumor formation)

20 Opine Biosynthesis 1. Within tumor tissues, the synthesis of various unusual amino acid-like compounds are directed by genes encoded on the integrated plasmid. 2. The type of opine produced is specified by the bacterial T-DNA 3. Opines are used by the bacteria as a carbon (nutrient) source for growth. 4. Opine catabolism within bacteria is mediated by genes encoded on the Ti plasmid.

21 Overview of the Infection Process

22 How is the signal recognition (acetosyringone and other plant phenolics) converted to gene activation and other cellular responses?


24 Bacterial 2-Component Signal Transduction Systems 1. Component 1 : Sensor kinase i) Substrate receptor, signal recognition domain, input domain (periplasmic) ii) Signal transduction domain, membrane spanning region iii) Autokinase domain, phosphorylation domain (cytoplasmic) a) ATP binding (sub) domain b) phosphorylation-phosphotransfer (sub)-domain

25 2. Component 2 : Response regulator i) Phosphorylation domain ii) DNA binding domain Simplest case: transcriptional activator when phosphorylated First component is typically (auto)-phosphorylated on a His residue and transfers to a Asp group on the response regulator (second component).

26 The EnvZ/OmpR System of E. coli Senses changes in extracellular osmolarity

27 Agrobacterium tumafaciens senses acetosyringone via a 2-component-like system 3 components: ChvE, VirA, & VirG 1. ChvE periplasmic protein binds to sugars, arabinose, glucose  binds to VirA periplasmic domain  amplifies the signal

28 2. VirA : Receptor kinase 1. Membrane protein five functional domains: a) Periplasmic binds ChvE-sugar complex does NOT bind acetosyringone b) Transmembrane domain c) Linker region BINDS acetosyringone NOTE this is on the cytoplasmic side! d) Transmitter domain (His) auto- phosphorylates and then transfers to the response regulator protein VirG e) Inhibitory domain  in absence of analyte will bleed off the phosphate from the His in the transmitter domain (to an Asp)

29 3. VirG : Response Regulator a) Receiver domain that is phosphorylated on an Asp residue by the His on the transmitter domain of VirA b) Activates the DNA binding domain to promote transcription from Vir-box continaing promoter sequences (on the Ti plasmid)

30 Periplasmic domain acetosyringone ChvE VirA VirG sugars Transmitter Inhibitory domain receiver DNA- binding


32 Crown gall tumors a natural example of genetic engineering. a natural example of genetic engineering.

33 Agrobacterium/plant interactions opines Agrobacterium at wound site transfers T-DNA to plant cell. Agrobacterium in soil use opines as nutrients.

34 Genes required to breakdown opines for use as a nutrient source are harbored on the Ti plasmid in addition to vir genes essential for the excision and transport of the T-DNA to the wounded plant cell. T-DNA vir genes opine catabolism pTi ~200 kb tra for transfer to the plant bacterial conjugation 23 kb

35 1. Nopaline plasmids: carry gene for synthesizing nopaline in the plant and for utilization (catabolism) in the bacteria. Tumors can differentiate into shooty masses (teratomas). 2. Octopine plasmids: carry genes(3 required) to synthesize octopine in the plant and catabolism in the bacteria. Tumors do not differentiate, but remain as callus tissue. Ti plasmids can be classified according to the opines produced

36 3. Agropine plasmids: carry genes for agropine synthesis and catabolism. Tumors do not differentiate and die out. CNH(CH 2 ) 2 CHCO 2 H NH HO 2 C(CH 2 ) 2 CHCO 2 H H2NH2N HN (Nopaline)

37 1. Agrobacterium tumefaciens chromosomal genes: chvA, chvB, pscA 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. Ti plasmids and the bacterial chromosome act in concert to transform the plant

38 3. T-DNA encodes genes for opine synthesis and for tumor production. 4. occ (opine catabolism) genes carried on the pTi and allows the bacterium to utilize opines as nutrient.

39 vir genes opine catabolism pTi tra for transfer to the plant bacterial conjugation Agrobacterium chromosomal DNA chvA chvB pscA oriV T-DNA- inserts into plant genome

40 Generation of the T-strand overdrive Right Border Left Border T-DNA virD/virC VirD nicks the lower strand (T-strand) at the right border sequence and binds to the 5’ end. 5’

41 Generation of the T-strand Right border Left border D virD/virC gap filled in T-strand T-DNA virE 1. Helicases unwind the T-strand which is then coated by the virE protein. 2. ~one T-strand produced per cell.

42 1. Transfer to plant cell. 2. Second strand synthesis 3. Integration into plant chromosome Right border Left border D T-strand coated with virE T-DNA virD nicks at Left Border sequence

43 The vir region is responsible for the transfer of T-DNA to the wounded plant cell. receptor for acetyl- syringone positive regulator for other vir genes virA constitutive virG virA is the sensor. membrane activated virG Note: activated virG causes its own promoter to have a new start point with increased activity.

44 virA is the sensor. bacterial membrane Acetylsyringone is produced by wounded plant cells (phenolic compound). triggers auto- phosphorylation of virA 1 2 P 3 virG virA virG activates transcription from other vir promoters. VirA phosphorylates virG which causes virG to become activated. virG is the effector. Asg P

45 The vir region is responsible for the transfer of T-DNA to the wounded plant cell. ssDNA binding protein. Binds T- strand. virA virG virB virC virD virE sensor effector endo- nucleas e nicks T- DNA Binds overdrive DNA. membrane protein; ATP-binding Note: The virA-virG system is related to the EnzZ- OmpR system that responds to osmolarity in other bacteria.

46 Generation of the T-strand overdrive Right Border Left Border T-DNA virD/virC VirD nicks the lower strand (T-strand) at the right border sequence and binds to the 5’ end. 5’

47 Generation of the T-strand Right border Left border D virD/virC gap filled in T-strand T-DNA virE 1. Helicases unwind the T-strand which is then coated by the virE protein. 2. ~one T-strand produced per cell.

48 1. Transfer to plant cell. 2. Second strand synthesis 3. Integration into plant chromosome Right border Left border D T-strand coated with virE T-DNA virD nicks at Left Border sequence


50 1.VirB1 may have local lytic activity that allows assembly of the transporter at specific sites in the cell envelope. 2.The processed VirB1 * peptide is secreted through the outer membrane by an unknown mechanism. 3.The structural components of the pilus are VirB2 and VirB5. 4.Complexes of VirB7/9, formed by disulfide bridges, may initiate assembly of the VirB channel. 5.The exact role of VirB3, 4, 6, 8, 10 and 11, and VirD4 in the transporter apparatus is unknown. Assembly of the Agrobacterium T-Complex Transport Apparatus

51 6. VirD4, VirB4 and VirB11 have nucleotide-binding motifs that are essential for their activity. 7. The T-complex, consisting of a ss copy of T-DNA bound to VirD2 and coated with VirE2, is exported through the transport apparatus. SP, signal peptide; SPI, signal peptidase I.


53 (a) The pilus has not contacted the surface of the recipient plant cell and the apparatus is unable to transport T-complex. (b) The pilus has contacted a receptor (?) on the surface of the recipient plant cell. This induces the VirB transporter, perhaps via a change in conformation, so that it is now competent to transfer the T-complex to the plant cell cytoplasm. OM, outer membrane; IM, inner membrane; CW, plant cell wall; PM, plasma membrane. Model for contact-dependent activation of the T- complex transport apparatus


55 1. The VirB and VirD4 proteins are grouped according to probable functions: a)exocellular proteins mediating attachment (VirB1*, VirB2 pilin and VirB5) b)channel proteins (VirB3, VirB6, VirB7, VirB8, VirB9 and VirB10) c)ATPases (VirB4, VirB11 and VirD4). Locations of the Vir Protein Components of the T-DNA transfer system

56 Agrobacterium can be used to transfer DNA into plants


58 pTi-based vectors for plant transformation: 2. Early shuttle vectors integrated into the T- DNA; still produced tumors. 1. Shuttle vector is a small E. coli plasmid using for cloning the foreign gene and transferring to Agrobacterium. E. coli Agrobacterium pTi Shuttle plasmid conjugation

59 Several hundred tumors containing foreign gene can be grown for experimental purposes. Transformed sunflower seedlings

60 Harvest time! 3 weeks after inoculation

61 Transformation of Arabidopsis plants Dip floral buds in 1 ml of Agrobacterium culture for 5 to 15 min. Detergent added to allow bacteria to infiltrate the floral meristem.

62 Transformation of Arabidopsis plants 700 to 900 seeds per plant. Germinate on kanamycin plates to select transformants. 10 to 20 transformed plants per plant. 10 day old seedlings

63 Agrobacteria are biological vectors for introduction of genes into plants.vectors Agrobacteria attach to plant cell surfaces at wound sites. The plant releases wound signal compounds, such as acetosyringone. The signal binds to virA on the Agrobacterium membrane. VirA with signal bound activates virG. Summary

64 Activated virG turns on other vir genes, including vir D and E. vir D cuts at a specific site in the Ti plasmid (tumor-inducing), the left border. The left border and a similar sequence, the right border, delineate the T-DNA, the DNA that will be transferred from the bacterium to the plant cell Single stranded T-DNA is bound by vir E product as the DNA unwinds from the vir D cut site. Binding and unwinding stop at the right border.

65 The T-DNA is transferred to the plant cell, where it integrates in nuclear DNA. T-DNA codes for proteins that produce hormones and opines. Hormones encourage growth of the transformed plant tissue. Opines feed bacteria a carbon and nitrogen source.

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