Agrobacterium tumefaciens: A natural genetic engineer

Agrobacterium tumefaciens

1. PRESENTED BY- RATNAKAR UPADHYAY M.Sc.- III Sem Agrobacterium tumefaciens : A Natural genetic engineer DEPARTMENT OF BOTANY GUIDED BY - Dr.SHRIRAM KUNJAM GOVT. V.Y.T.PG AUTONOMOUS COLLEGE DURG (C.G.) Session- 2018-19

2. INTERNATIONAL CODE OF NOMENCLATURE OF PROKARYOTES Updated the Scientific name of . Agrobacterium tumefaciens Rhizobium radiobacter Kingdom- Bacteria Phylum- Proteobacteria Class- Alphaproteobacteria Order- Rhizobiales Family- Rhizobiaceae Genus- Rhizobium species- radiobacter

3. INTRODUCTION Agrobacterium tumefaciens only known natural example of inter-kingdom DNA transfer. Established by Sir H.J.Conn Soil Bacterium, Rod shaped, Gram negative, Motile, having Peritrichous flagella(1-6). Broad host range pathogen (Dicots, Monocots, Gymnosperms, Fungi.) e.g. walnuts, grape, nut trees, sugar beats, yeast. It is considered as Natural genetic engineer because it transforms the plant. It has Ti plasmid, responsible for tumor induction (crown gall tumor) in plants. To be virulent, bacterium must contain a “pTi” which contain the T-DNA. Agrobacterium rhizogenes has Ri plasmid, responsible for hair disease in plants. Agrobacterium rubi cause cane gall in Sugarcane plant. Agrobacterium vitis cause gall in grapes.

4. WHY Agrobacterium IS USED TO PRODUCE TRANSGENIC PLANT ? The T-DNA element is defined by its borders but not by the sequence present within it. So Researchers can substitute the T-DNA coding region with any DNA sequence without any effect on its transfer from Agrobacterium to the plant. Many monocot plants can be transformed now, although they do not form crown gall tumors.

5. Crown gall formation

6. MOLECULAR BASIS The process of infection by Agrobacterium tumefaciens culminates in the transfer of a small part of p Ti into the plant cell genome ; this DNA sequence is called T-DNA. The infection process is governed by both chromosomal and plasmid-borne genes of Agrobacterium tumefaciens. Attachment of bacteria to plant cells begins the infection, governed by chromosomal virulence genes ( chv); which are expressed constitutively. The continued presence of virulent bacteria is not needed for tumor maintenance. Bacteria do not penetrate the plant cells that are converted into tumor cells. Only a small part of the Ti plasmid is transferred into the host cell (The T-DNA)

7. Large sized plasmid of 200kbp. Modification of this plasmid is very important in the creation of transgenic plant. The Ti plasmids are classified into different types based on the type of opine, namely, octopine, nopaline, succinamopine and leucinopine produced by their genes. The different Ti plasmids can be grouped into two general categories: octopine type and nopaline type. Ti- PLASMID (TUMOR INDUCING)

8. T-DNA The T-DNA is the transferred DNA of the tumor inducing plasmid of virulent species of bacteria. The size of T-DNA is between 15-30kb. T-DNA contain genes for tumor induction. It has LB & RB (left border & right border). RB play a important role in transfer & integration of T-DNA. Absence of RB will terminate the T-DNA transfer. T-DNA carry genes for Phytohormones (Auxin, Cytokinin) & Opine that are expressed in plant cell. Over production of these hormones at the site of infection is responsible for the proliferation of wounded cell in gall/tumor. These tumor can harbor a plenty of bacteria.

9. All the genes present in T-DNA contain eukaryotic regulatory sequences. As a result, these genes are expressed only in plant cells, and they are not expressed in the Agrobacterium.

10. COMPONENTS OF T-DNA Gene Function iaaM (auxL,tins I) Auxin biosynthesis; encodes enzymes Tryptophan-2-mono oxygenase, which converts Tryptophan into Indole-3-acetamide (IAM) iaah (aux2 tms2) Auxin biosynthesis; encodes enzymes Indole-3-acetamide hydrolase which converts IAM into IAA (Indole-3-acetic acid) ipt( tmr,Cyt ) Cytokinin biosynthesis; encodes enzyme Isopentenyl transferase, which catalyses the formation of Isopentenyl adenine. Nos Nopaline biosynthesis; encodes the enzyme Nopaline synthase, which produces Nopaline from arginine & pyruvic acid. 24 bp (left & right border sequences) Site of endonuclease action during transfer of T-DNA, the only sequences of T-DNA essential for its transfer.

11. COMPONENTS OF vir REGION OPERON FUNCTION Vir A Encodes a receptor for acetosyringone that functions as an autokinase, also phosphorylates vir G protein; constitutive expression. VirB Membrane proteins; possibly form a channel for T-DNA transport(conjugal tube formation ); also has ATPase activity. VirC Helicase; binds to the overdrive region just outside the right border, involved in unwinding of T-DNA. VirD VirD1 has Topoisomerase activity it binds to the right border of T-DNA & prevent attack by exonuclease at 5’ end of T-DNA , VirD2 is an Endonuclease (play role in cutting phosphodiester bond), it nicks the right border. VirE/E2 Protect T-DNA against nuclease & target T-DNA to plant cell .It act as a single stranded binding protein. VirG DNA binding protein; probably forms dimer after phosphorylation by Vir A & induces the expression of all Vir operons (constitutive expression) VirD2 & VirE2 Have NLS (nuclear localisation signal) VirB & VirD4 Type IV Secretion system (mating pair formation apparatus), it’s a pore channel formation.

12. Plant transformation

13. Steps of Agrobacterium -Plant cell interaction Plant stress condition. Phenolic production. Signal to Bacteria. Virulence system activation. Generation of T-DNA complex. T-DNA transfer (Nuclear import). T-DNA integration in plant genome.

14. PLANT SIGNALS Wounded plants secrete sap with acidic pH (5.0 to 5.8) and a high content of various phenolic compounds (lignin, flavonoid precursors) serving as chemical attractants to Agrobacterium and stimulants for vir gene expression. Among these phenolic compounds, acetosyringone (AS) is the most effective. Sugars like glucose and galactose also stimulate vir gene expression when AS is limited or absent. These sugars are probably acting through the chvE gene to activate vir genes. Low opine levels further enhance vir gene expression in the presence of AS. These compounds stimulate the autophosphorylation of a transmembrane receptor kinase VirA at its His-474. It in turn transfers its phosphate group to the Asp-52 of the cytoplasmic VirG protein

15. PRODUCTION OF T-strand Every induced Agrobacterium cell produces one T-strand. VirD1 and VirD2 are involved in the initial T-strand processing, acting as site-and strand-specific endonucleases After cleavage, VirD2 covalently attaches to the 5’ end of the T-strand at the right border nick and to the 5’-end of the remaining bottom strand of the Ti plasmid at the left border nick by its tyrosine 29.

16. FORMATION OF T-COMPLEX The T-complex is composed of at least three components: one T-strand DNA molecule, one VirD2 protein, and around 600 VirE2 proteins. If VirE2 associates with the T-strand after intercellular transport, VirE1 is probably involved in preventing VirE2-T-strand binding. Judging from the size of the mature T-complex (13nm in diameter) and the inner dimension of T- pilus (10nm width), the T-strand is probably associated with VirE2 after intercellular transport.

17. INTERCELLULAR TRANSPORT Transport of the T-complex into the host cell most likely occurs through a type IV secretion system. In Agrobacterium, the type IV transporter (called T-pilus) comprises proteins encoded by virD4 and by the 11 open reading frames of the virB operon. Intercellular transport of T-DNA is probably energy dependent, requiring ATPase activities from VirB4 and VirB11. Physical contact between Agrobacterium and the plant cell is required to initiate T-complex export. Without recipient plant cells, T-strands accumulate when vir genes are induced.

18. NUCLEAR IMPORT & CHROMATIN TARGETING OF T-COMPLEX Because of the large size of T-complex (50,000 kD , ~13nm in diameter), the nuclear import of T-complex requires active nuclear import. The T-complex nuclear import is presumably mediated by the T-complex proteins, VirD2 and VirE2. Both of them have nuclear localizing activities. CAK2M and TATA-Box binding protein (TBP) both of which bind to VirD2 and VIP1 binds to VirE2. Which leads in chromatin targeting of T-Complex.

19. T-DNA integration

20. REQUIREMENTS ADVANTAGES DISADVANTAGES The plants explant must produce acetosyringone or other related compounds. Natural means of transfer, hence plant friendly Limited host range. Can not infect cereal plants. The induced bacteria should give access to cells that are competent for transformation It is capable of infecting intact plant cells. Sometimes cells in a tissue that are able to regenerate are difficult to transform. Transformation competent cells & tissues should be able to regenerate into whole plants. Capable of transferring large fragments of DNA very efficiently without substantial rearrangements. The stability of gene transferred is excellent. Agrobacterium MEDIATED GENE TRANSFER

21. Wild type Agrobacterium Ti plasmid cannot be use as gene cloning vectors because of :- Large size of Ti plasmid, difficult to handle. Presence of oncogenes or tumor causing genes ( Auxin & Cytokinin). Lack of unique restriction sites & makes sites within T-DNA. There are two genetically engineered Ti plasmid based vectors. They are Cointegrate & Binary vector. Ti plasmid based vectors: Co-integrate & Binary vectors

22. Making of Co-integrate vectors Both the T-DNA with our gene of interest & Vir region are present in the same vector used for transformation. At first, an intermediate vector is made using E.coli plasmid + Vir region + T-DNA borders + Ori + pBR322 sequences. Second vector is a disarmed pTi vector + gene of interest + some markers + pBR322 sequences. Both intermediate vector & disarmed pTi has some sequences in common (pBR322 sequences). Therefore, by homologous recombination, cointegration of two plasmids will takes place within Agrobacterium. Result- A cointegrate vector that has both T-DNA with our gene of interest within the T-DNA borders & Vir regions. This complete vector is used for Transformation eg.pGV2260

23. Binary vector strategy: Two vector strategy Here two vectors are used, based on the knowledge that Vir region need not be in the same plasmid alongwith T-DNA for T-DNA transfer. Binary vector consists of pair of plasmids. 1.A disarmed Ti plasmid: This plasmid has T-DNA with gene of interest + Ori for both E.coli & Agrobacterium. Also called as mini-Ti or micro Ti plasmid e.g- Bin 19. 2.Helper Ti plasmid has virulence region that mediates transfer of T-DNA in micro Ti plasmid to the plant

24. Advantages of Binary vector over cointegrate vector Binary vector do not need in vivo recombination. Binary vectors require only an intact plasmid vector to be introduced into the target bacterium, making the process of gene transfer more efficient & quicker. Confirmation of the transformation event is accomplished very easily.

25. UPDATE Recent work has shown that the plant factor KU80 is involved in the T-DNA integration process, most likely by bridging between double-stranded T-DNAs. In addition, Shaked et al. reported that Over expression of the yeast Rad54 protein led to high frequency gene targeting in transgenic plants. These two reports further support the notion that integration of T-DNA molecules is promoted by host cellular factors and open a new direction for plant gene targeting by genetic manipulation of the host genome.

26. CONCLUSION & FUTURE PROSPECTS With an ever-expanding host range that includes many commercially important crops, flowers, and tree species, Agrobacterium is guaranteed a place of honor in nearly every plant molecular biology laboratory and biotechnology company for a long time to come. Furthermore, its recent application to the genetic transformation of non-plant species, from yeast to cultivated mushrooms, and even human cells, places Agrobacterium at the forefront of future biotechnological applications.

27. REFERENCES Bhojwani, S.S,Razdan, M.K, Plant tissue culture, 1996,ELSEVIER,New York. Snustad, D.Peter, Simmons, Michael J, Principles of Genetics, Sixth Edition, John wiley & sons, USA. Karp, Gerald, Cell & Molecular Biology, Sixth Edition, John Wiley & Sons, USA. Tzvi Tzfira and Vitaly Citovsky, www.Sciencedirect.com, Sharma, Vandana, Meena, Rishikesh, Biotechnologyu & genetic engineering of plants,Vardhman Mahaveer open university kota (Raj.)

28. THANK YOU