5Biology of A. tumefaciens Well known to induce crown gall tumorA.tumefaciens lives aroundroot surfaces (in rhizosphere)where it using nutrientsthat leak from the root tissuesinfects only through wound sitesand actively chemotactic to themwww-genvagar.slu.se/teknik/ djup/plasm.htmBacterial T-plasmid producesreceptors for acetosyringonePlant wound producesacetosyringone
6The 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.
7Important genes encoded by Ti plasmid 1. Cytokinins(plant hormone for cell plant division and tumorous growth)2. Enzymes for indoleacetic acid (auxin) synthesisAnother 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 speciesIt provides unique niche for A.tumefaciensNopaline
8Cytokinins are plant hormones that are derivatives of the purine adenine. Zeatin is one of cytokinines which synthesismay be encoded by Ti plamidisolated from corn (Zea mays).Cell specific expression of cytokininin the A.tumefaciens infected cell
9Opines are nutrients that are also for quorum sensing The plant cells start to secretethe opinesfrom transferred bacterial T DNAopine diffusesinto the surrounding cellsand serves as a signal moleculesfor the conjugationof the agrobacterium(Quorum sensing)
10Ti Plasmid T-DNA region DNA between L and R borders is transferred to plantas ssDNA;T-DNA encoded genes can be substituted by target genesTumor-producinggenesOpine catabolismVirulence regionORI
16Agrobacterium tumefaciens 1. Soil bacterium closely related to Rhizobium.2. Causes crown gall disease in plants (dicots).
173. 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.
18Only known natural example of DNA transport between Kingdoms 1. (Virulent) strains of A. tumefaciens contain a 200-kb tumor inducing (Ti) plasmid2. Bacteria transfer a portion of the plasmid DNA into the plant host (T-DNA).T-DNA
19The 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)
20Opine Biosynthesis1. 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-DNA3. 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.
24Bacterial 2-Component Signal Transduction Systems 1. Component 1 : Sensor kinasei) Substrate receptor, signal recognition domain, input domain (periplasmic)ii) Signal transduction domain, membrane spanning regioniii) Autokinase domain, phosphorylation domain (cytoplasmic)a) ATP binding (sub) domainb) phosphorylation-phosphotransfer (sub)-domain
252. Component 2 : Response regulator i) Phosphorylation domainii) DNA binding domainSimplest case: transcriptional activator when phosphorylatedFirst component is typically (auto)-phosphorylated on a His residue and transfers to a Asp group on the response regulator (second component).
26The EnvZ/OmpR System of E. coli Senses changes in extracellular osmolarity
27Agrobacterium tumafaciens senses acetosyringone via a 2-component-like system 3 components: ChvE, VirA, & VirG1. ChvEperiplasmic protein binds to sugars, arabinose, glucosebinds to VirA periplasmic domain amplifies the signal
282. VirA : Receptor kinase1. Membrane protein five functional domains:a) Periplasmic binds ChvE-sugar complex does NOT bind acetosyringoneb) Transmembrane domainc) 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 VirGe) Inhibitory domain in absence of analyte will bleed off the phosphate from the His in the transmitter domain (to an Asp)
293. VirG : Response Regulator a) Receiver domain that is phosphorylated on an Asp residue by the His on the transmitter domain of VirAb) Activates the DNA binding domain to promote transcription from Vir-box continaing promoter sequences (on the Ti plasmid)
32Crown gall tumorsa natural example of genetic engineering.32
33Agrobacterium/plant interactions Agrobacterium at wound site transfers T-DNA to plant cell.opinesAgrobacterium in soil use opines as nutrients.33
34Genes 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-DNA23 kbtrabacterial conjugationpTi~200 kbvir genesfor transfer to the plantopine catabolism34
35Ti 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 (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.35
36H2N CNH(CH2)2CHCO2H HN NH HO2C(CH2)2CHCO2H (Nopaline) 3. Agropine plasmids: carry genes for agropine synthesis and catabolism. Tumors do not differentiate and die out.(Nopaline)CNH(CH2)2CHCO2HNHHO2C(CH2)2CHCO2HH2NHN36
37Ti plasmids and the bacterial chromosome act in concert to transform the plant 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.37
383. 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.38
39for transfer to the plant Agrobacterium chromosomal DNApscAchvAchvBT-DNA-inserts into plant genometrabacterial conjugationfor transfer to the plantpTivir genesopine catabolismoriV39
40Generation of the T-strand Left BorderRight BorderT-DNAoverdrive5’virD/virCVirD nicks the lower strand (T-strand) at the right border sequence and binds to the 5’ end.40
41Generation of the T-strand Left borderRight borderT-DNAgap filled invirET-strandDvirD/virC1. Helicases unwind the T-strand which is then coated by the virE protein.2. ~one T-strand produced per cell.41
42T-strand coated with virE Left borderRight borderT-DNADT-strand coated with virEvirD nicks at Left Border sequence1. Transfer to plant cell.2. Second strand synthesis3. Integration into plant chromosome42
43The vir region is responsible for the transfer of T-DNA to the wounded plant cell. virAconstitutivevirGvirA is the sensor.membraneactivated virGNote: activated virG causes its own promoter to have a new start point with increased activity.positive regulator for other vir genesreceptor for acetyl-syringone43
44virG activates transcription from other vir promoters. virA is the sensor.Asg1P2virAAsgtriggers auto-phosphorylation of virAbacterialmembraneAcetylsyringone is produced by wounded plant cells (phenolic compound).3virG activates transcription from other vir promoters.PVirA phosphorylates virG which causes virG to become activated.virGvirG is the effector.44
45The vir region is responsible for the transfer of T-DNA to the wounded plant cell. effectorsensorvirGvirEvirAvirDvirBvirCssDNA binding protein. Binds T-strand.membrane protein; ATP-bindingendo-nuclease nicks T-DNABinds overdrive DNA.Note: The virA-virG system is related to the EnzZ-OmpR system that responds to osmolarity in other bacteria.45
46Generation of the T-strand Left BorderRight BorderT-DNAoverdrive5’virD/virCVirD nicks the lower strand (T-strand) at the right border sequence and binds to the 5’ end.46
47Generation of the T-strand Left borderRight borderT-DNAgap filled invirET-strandDvirD/virC1. Helicases unwind the T-strand which is then coated by the virE protein.2. ~one T-strand produced per cell.47
48T-strand coated with virE Left borderRight borderT-DNADT-strand coated with virEvirD nicks at Left Border sequence1. Transfer to plant cell.2. Second strand synthesis3. Integration into plant chromosome48
50Assembly of the Agrobacterium T-Complex Transport Apparatus VirB1 may have local lytic activity that allows assembly of the transporter at specific sites in the cell envelope.The processed VirB1* peptide is secreted through the outer membrane by an unknown mechanism.The structural components of the pilus are VirB2 and VirB5.Complexes of VirB7/9, formed by disulfide bridges, may initiate assembly of the VirB channel.The exact role of VirB3, 4, 6, 8, 10 and 11, and VirD4 in the transporter apparatus is unknown.50
51SP, signal peptide; SPI, signal peptidase I. 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.51
53Model for contact-dependent activation of the T-complex transport apparatus (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.53
55Locations of the Vir Protein Components of the T-DNA transfer system 1. The VirB and VirD4 proteins are grouped according to probable functions:exocellular proteins mediating attachment (VirB1*, VirB2 pilin and VirB5)channel proteins (VirB3, VirB6, VirB7, VirB8, VirB9 and VirB10)ATPases (VirB4, VirB11 and VirD4).55
56Agrobacterium can be used to transfer DNA into plants 56
58pTi-based vectors for plant transformation: 1. Shuttle vector is a small E. coli plasmid using for cloning the foreign gene and transferring to Agrobacterium.2. Early shuttle vectors integrated into the T-DNA; still produced tumors.pTiShuttle plasmidconjugationE. coliAgrobacterium58
59Transformed sunflower seedlings Several hundred tumors containing foreign gene can be grown for experimental purposes.Transformed sunflower seedlings59
61Transformation 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.
62Transformation 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
63Agrobacteria attach to plant cell surfaces at wound sites. SummaryAgrobacteria are biological vectors for introduction of genes into plants.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.63
64Activated 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 cellSingle 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.64
65The 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.65