Vectors Used for Gene Cloning in Plants

These are the vectors used exclusively for plant gene cloning

1. Vectors used for gene cloning in plants Submitted By : Arunodaya Maji CARA-2018-110 Batch - D Submitted To : Dr. G. Uma Devi Ma’am College of Agriculture, Rajendranagar Course No. – PATH – 271 Course Title – Principles of Plant Pathology Course Credits – 2(2+0) Assignment - 3

2. Content What is cloning vector ? Why cloning vector ? History What determines choice of vector Features of a cloning vector Types of cloning vector Agrobacterium Mediated Cloning Vectors Ti Plasmid Ri Plasmid 8. Attempts to use Plant Viruses as Cloning Vectors 9. Conclusion 10. References

3. Cloning Vectors The molecular analysis of DNA has been made possible by the cloning of DNA . The two molecules that are required for cloning are the DNA to be cloned and a cloning vector . A cloning vector is a small piece of DNA taken from a virus , a plasmid or the cell of a higher organism , that can be stably maintained in an organism and into which a foreign DNA fragment can be inserted for cloning purposes. Most vectors are genetically engineered . The cloning vector is chosen according to the size and type of DNA to be cloned. The vector therefore contains features that allow for the convenient insertion or removal of DNA fragment in or out of the vector, for example by treating the vector and the foreign DNA with a restriction enzyme and then ligating the fragments together. After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use.

4. Vector In molecular cloning, a vector is a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed . A vector containing foreign DNA is termed recombinant DNA .

5. Why Cloning Vectors Cloning vector is used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and expressed . It is used to amplify a single molecule of DNA into many copes . Cloning vectors are DNA molecules that are used to "transport " cloned sequences between biological hosts and the test tube . Without Cloning Vector, Molecular Gene Cloning is totally impossible .

6. History Scientists ( Herbert Boyer, Keiichi Itakura and Arthur Riggs ) working in Boyer’s lab (University of California) recognized a general cloning vector with unique restriction sites for cloning in foreign DNA and the expression of antibiotic resistance genes for selection of transformed bacteria. Rodriguez Raymon along with Paco Bolivar constructed the vector “ pBR 322 ” in the year 1977 This vector was small, ~4 kb in size, and had two antibiotic resistance genes for selection. Arthur Riggs Herbert Boyer Rodriguez Raymon

7. Features of A Cloning Vector All commonly used cloning vectors have some essential features: Origin of replication ( ori ): This makes autonomous replication in vector. ori is a specific sequence of nucleotide from where replication starts. When foreign DNA is linked to the sequence along with vector replication, foreign (desirable) DNA also starts replicating within host cell. Cloning Site: Cloning site is a place where the vector DNA can be digested and desired DNA can be inserted by the same restriction enzyme. It is a point of entry or analysis for genetic engineering work. Recently recombinant plasmids contain a multiple cloning site ( MCS ) which have many (up to ~20) restriction sites.

8. Selectable Marker Selectable marker is a gene that confers resistance to particular antibiotics or selective agent that would normally kill the host cell or prevent its growth. A cloning vector contains a selectable marker, which confer on the host cell an ability to survive and proliferate in a selective growth medium containing the particular antibiotics . Reporter Gene or Marker Gene Reporter genes are used in cloning vectors to facilitate the screening of successful clones by using features of these genes that allow successful clone to be easily identified. Such feature present in cloning vectors is used in blue- white selection.

9. R eplicate autonomously . Restriction sites. Self replication, multiple copies. Replication origin site . Small size.( Larger plasmids are more difficult to characterize by restriction mapping and replicate to lower copy numbers ) Low molecular weight . No pathogenicity Easily isolated & purified. Easily isolated into host cell. Control elements – promoter, operator, ribosome binding site.

10. What Determines Choice of Vector I nsert size V ector size R estriction sites C opy number C loning efficiency A bility to screen for inserts Vector Insert size (kb) Plasmid <10 kb Bacteriophage 9 – 15 kb Cosmids 23 – 45 kb BACs ≤ 300 kb PACs 100 – 300 kb YACs 100 – 3000 kb

11. Cloning and Expression Vectors

12. Types Of Cloning Vectors S. No. Vectors Targeted Host 1 Plasmid Bacteria, Streptomyces 2 Ti & Ri Plasmid Transformation of cloned genes in Higher Plants 3 Bacteriophages/ Phagemids /Phasmids Bacteria 4 Cosmid (Hybrid Vector) Bacteria 5 Fosmid E. coli 6 Bacterial Artificial Chromosome (BAC) Bacteria 7 Yeast Artificial Chromosome (YAC) Yeast 8 Human Artificial Chrosome (HAC) Human Cells 9 Mammalian Artificial Chromosome (MAC) Mammalian Cells 10 Shuttle Vectors E. coli, Yeast 11 Retroviral Vectors Human and Animal Cells

13. Agrobacterium M ediated T ransformation The important requirements for Agrobacterium- mediated gene transfer in higher plants are as follows:- The plant explants must produce acetosyringone for activation of Vir genes . The induced Agrobacterium should have access to cells that are competent for transformation. Explants include cotyledon, leaf, thin tissue layer, peduncle, hypocotyls, stem, microspores

14. Crown gall on blackberry cane Crown gall disease

15. Agrobacterium tumifaciens Agrobacterium tumefaciens — nature’s smallest genetic engineer A . tumefaciens causes CROWN GALL DISEASE in many species of dicotyledonous plants. Causes a cancerous proliferation of the stem tissue in the region of the crown . A. tumifaciens is a Gram – ve soil bacterium Infects plants through breaks or wounds . Tumor formation is the result of integration of T-DNA (Transfer DNA) in plant genome.

16. Agrobacterium tumifaciens Gram Negative Soil Borne Rod Shaped Motile Nature’s own Genetic Engineer Crown Gall

17. Infection Process

18. Tzvi Tzfira and Vitaly Citovsky, 2002, Trends in Cell Biol. 12(3), 121-129 Cellular Processes of Agrobacterium Host Infection

19. In general, the transformation procedure is as follows : T he recombinant small replicon is transferred via bacterial conjugation or direct transfer to A. tumefaciens harboring a helper Ti plasmid, the plant cells are co-cultivated with the Agrobacterium , to allow transfer of recombinant T-DNA in to the plant genome, and transformed plant cells are selected under appropriate conditions. B inary vector system involves only the transfer of a binary plasmid to Agrobacterium without any integration. This is in contrast to co-integrate vector system wherein the intermediate vector is transferred and integrated with disarmed Ti plasmid. Due to convenience, binary vectors are more frequently used than co-integrate vectors. Compared with co-integrated vectors, binary vectors present some advantages : No recombination process takes place between the molecules involved. Instead of a very large, recombinant, disarmed Ti plasmid, small vector s are used, which increases transfer efficiency from E. coli to Agrobacterium . This vector system is most widely used nowadays. Different types of binary vectors have been devised to suit different needs in a plant transformation process.

20. Process of Transformation

21. Transformation Protocols Transformation was performed using minor modifications of given protocols , using Leaf disk Scutellum-derived callus, or Floral dip methods, respectively. AGROBACTERIUM-MEDIATED PLANT TRANSFORMATIONS HAVE THE FOLLOWING BASIC PROTOCOL :- Development of Agrobacterium carrying the cointegrate or binary vector with the desired gene . Identification of a suitable explant e.g. cells, protoplasts, tissues, calluses, organs. Co-culture of explants with Agrobacterium. Killing of Agrobacterium with a suitable antibiotic without harming the plant tissue. Selection of transformed plant cells. Regeneration of whole plants.

22. Example-scutellum-derived callus method Transformation Protocol for Rice – Abbreviated Seed plating on 2N6 – dark ↓ 4 weeks Subculture onto 2N6 – dark ↓ 4 – 10 days Co cultivation onto 2N6-AS – dark ↓ 3-7 days Selection on 2N6-TCH – dark ↓ 4 weeks, subculture onto 2N6-TCH every 2 weeks Transfer proliferating calli onto 2N6-TCH-dark ↓ 2 weeks Regeneration onto RGH6-dark ↓ 7 days Transfer to light ↓ 4-6 weeks Plantlets onto ½ MSH - light ↓ Transfer plants to the glasshouse

23. Seed Material : Oryza sativa L. ssp japonica cvs. Millin or Nipponbare . Steps- Callus Induction Callus Subculture Bacteria Preparation Transformation Callus washing Selection Regeneration Plantlet Formation To ensure that the gene transfer did not result from contamination with Agrobacterium cells, controls including species specific PCR, selective plating, and use of a tagged binary vector were implemented. Thus , diverse plant associated bacteria, when harbouring a disarmed Ti plasmid and binary vector(or presumably a cointegrate or whole Ti plasmid), are readily able to transfer TDNA to plants. The Ti plasmid is self transmissable, perhaps in dicating the existence of a ubiquitous natural mechanism effecting horizontal gene transfer from bacteria to plants.

24. Procedure for P lant T ransformation IMAGE: Mol bio of the cell by Albert (pg no:599)

25. Regeneration, Selection And Detection Regeneration : for shoot organogenesis, cytokinin (lower amounts of auxin) are required Selection : two antibiotics are required An antibiotic to kill the Agrobacterium, while not affecting the plant's cell growth and division a second antibiotic allows growth of transformed shoots (w/selectable marker) but inhibits growth of untransformed plant cells . Detection of the "trait" gene PCR methods can detect the presence of the "trait" DNA protein detection methods are used where a gene product is produced that defines the trait verification of the incorporation of the trait gene into the plant's chromosome: by Southern hybridization by demonstrating transfer of the trait to the original transformant's progeny.

26. Scientists can insert any gene they want into the plasmid in place of the tumor causing genes and subsequently into the plant cell genome. By varying experimental materials, culture conditions, bacterial strains, etc. scientists have successfully used A. tumefaciens Gene Transfer to produce BT Corn. This method of gene transfer enables large DNA strands to be transferred into the plant cell without risk of rearrangement whereas other methods like the Gene Gun have trouble doing this . The vast majority of approved genetically engineered agriculture has been transformed by means of Agrobacterium tumefaciens Mediated Gene Transfer. Original problems existed in that Agrobacterium tumefaciens only affects dicotyledonous plants. Monocotyledon plants are not very susceptible to the bacterial infection. Benefits and Problems with Agrobacteria

27. Ti Plasmid The Ti plasmid ( approx.size 200 kb each) exist as independent replicating circular DNA molecules within the Agrobacterium cells. The T-DNA is variable in length in the range of 12 to 24 kb. The Ti plasmid is lost when Agrobacterium is grown above 28 ° C. The plasmid has 196 genes that code for 195 proteins. There is one structural RNA. The plasmid is 206,479 nucleotides long, the GC content is 56% and 81% of the material is coding genes. There are no pseudogenes. The modification of this plasmid is very important in the creation of transgenic plants. Genes in the virulence region are grouped into the operons virABCDEFG , which code for the enzymes responsible for mediating conjugative transfer of T-DNA to plant cells.

28. Though Ti plasmids are effective natural vectors they had certain limitations . The phytohormone produced by transformed cells growing in culture prevents their regeneration into mature plants. Hence auxins and cytokinin genes must be removed from the Ti –plasmid derived cloning vector. The opine synthesis gene must be removed as it m ay divert plant resources into opine production in transgenic plant. Generally, Ti- plasmids are large in size (200-800kb) For effective cloning, large segments of DNA that are not essential for cloning has to be removed. As Ti plasmid does not replicate in E.coli Ti-plasmid based vectors require an ori that can be used in E.coli.

29. Binary vector Plasmid DNA VIR genes Bacterial Ch r omos ome Bacterial ORI t-DNA Ampici llin r esista n ce Construction of vector with disired genes

30. To overcome these constraints, Ti plasmid based vectors were organized with the following components : A selectable marker gene that confers resistance to transformed pla nt cells . As these marker genes are prokaryotic origin, it is necessary to p ut them under the eukaryotic control (plant) of post transcriptional regul ation signals, including promoter and a termination- poly adenylation sequence, to ensure that it is efficiently expressed in transformed plant cells. An origin of replication that allows the plasmid to replicate in E.coli . The right border sequence of the T-DNA which is necessary for T- DNA integration into plant cell DNA. A polylinker (MCS) to facilitate the insertion of cloned gene into th e region between T-DNA border sequences.

31. Ti Plasmid Structure

32. 1. T-DNA Region :This region has the genes for the Biosynthesis of Auxin (aux), Cytokinin (cyt), and Opine (ocs) T-DNA Border : A set of 24 bp sequence present on either side of T-DNA 2. Virulence Region : The genes responsible for the transfer of T- DNA into the host plant are located outside the T-DNA ant the region referred to as vir or virulence region 3. Opine Catabolism region : Uptake and Metabolism of Opine Organization of Ti-Plas mi d A B C D E G

33. Structure of T-DNA

34. virA - Transports acetosyringone into bacterium, activates virG post- translationally (by phosphorylation) virG - Promotes transcription of other vir genes virD 2 - Endonuclease/integrase that cuts T-DNA at the borders but only on one strand. virE2 - Can form channels in membranes virE1 - Chaperone for virE2 virD2 & virE2 also have NLSs, gets T-DNA to the nucleus of plant cell virB - Operon of 11 proteins, gets T-DNA through bacterial membranes Function of Vir genes

35. Opines Derivatives of amino acids synthesized by T-DNA. Ti plasmids can be classified according to the opines produced : Nopaline plasmids Octopine plasmids Agropine plasmids Nopaline plasmids : carry gene for synthesizing nopaline in the plant and for utilization (catabolism ) in the bacteria. Octopine plasmids : carry genes to synthesize octopine in the plant and catabolism in the bacteria. Agropine plasmids : carry genes for agropine synthesis and catabolism.

36. Mechanism of T-DNA Transfer and I ntegration

37. 1. Signal Induction : Wounded Plant cells release certain phenolic compounds which are recognized as signals by agrobacterium. Wound Phenolic compounds Signal 2. Attachment : The Agrobacterium attaches to palnt cells through polysaccharides, particularly via cellulose fibers Plant Cell Bacteria Vir G VirB,C,D,E 3. Production of Virulance Proteins : Signal Vir A Vir A

38. 4. Production of T-DNA Strand : The right and left border of T-DNA are recognized by D1/D2 proteins and these proteins involved in the production of ss-DNA. 5. Transfer of T-DNA out of Agrobacterium : Vir B form transport apparatus and ss-T- DNA in association with vir D2 exported from the bacterial cell . 6. Transfer of T-DNA into plant cells and its Integration : In plant cell ss T-DNA get covered with vir E2 for its protection. Vir D2 and E2 interact with variety of plants proteins which influences T-DNA transport and its integration into plant genome. ( illegitimate Recombination ) Transcription Translation Crown Gall Integration

39. B B A G D 2 D 2 D1/D2 D1/D2 B C D E E2 SS-T DNA Wounded Plant cell E2 Ti 1 2 3 4 5 6 Transcription Translation Production of auxin, cytokinin and opine Autostimulation of Cell division Bacteria

40. Making of Co-Integrate Vectors In this strategy, both the T-DNA with our gene of interest and 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 + origin of replication+pBR 322 sequences . Second vector is a disarmed pTi vector = gene of interest+ some markers+pBR322 sequences. Both intermediate vector and disarmed pTi has some sequences in common (pBR322 sequences ). Therefore by homologous recombination, co-integration of two plasmids will take place within Agrobacterium. Now we have a co-integrate vector that has both T-DNA with our gene of interest with in the T-DNA borders and vir region. This complete vector is used for transformation eg:pGV2260. There are two types of Ti plasmid vectors are used for genetic transformation of plant they are cointegrate vector and binary vector.

41. PGV · s S6 1 n · , {Con! in T T Homologous Co-ilntcgr V °'(P 11 La

42. Advantages of Co-integrate Vector Target genes can be easily cloned. The plasmid is relatively small with a number of restriction sites. Intermediate plasmid is convenientiy cloned in E.coli and transferred to Agrobacterium .

43. Binary vector strategy: Two vector strategy Here two vectors are used. This vector was devised based on the knowledge that vir region need not be in the same plasmid along with T-DNA for T DNA transfer. Binary vector consists of a pair of plasmids A disarmed Ti plsmid : This plasmid has T-DNA with gene of interest + ori for both E.coli and Agrobacterium. Also called as mini-Ti or micro Ti plasmid eg: Bin 19 Helper Ti plasmid has virulence region that mediates transfer of T-DNA in micro Ti plasmid to the plant.

44. The binary vector system consist of an Agrobacterium strain along with a disarmed Ti plasmid called vir helper plasmid (the entire T-DNA region including borders deleted while vir gene is retained). It may be noted that both of them are not physically linked (or integrated). A binary vector with T-DNA can replicate in E.coli and Agrobacterium . The binary vector has following components- : Left and right borders that delimit the T-DNA region. A plant transformation marker (PTM) e.g. npt2 that confers kanamycin resistance in plant transformed cells. A multiple cloning site (MCS) for introducing target/foreign genes. A bacterial resistance marker e.g. tetracycline resistance gene for selecting binary vector colonies in E.coli and Agrobacterium. Ori T sequence for conjugal mobilization of the binary vector from E.coli to Agrobacterium . A broad host- range origin of replication such as RK2 that allows the replication of binary vector in Agrobacterium. The target (foreign) gene of interest is inserted into the multiple cloning site of the binary vector. In this way, the target gene is placed between the right and left border repeats and cloned in E.coli. By a mating process, the binary vector is mobilised from E.coli to Agrobacterium. Now , the virulence gene proteins of T-DNA of the vector into plant cells. The binary vector system involves only the transfer of a binary plasmid to Agrobacterium without any integration. This is in contrast to cointegrate vector system wherein the intermediate vector is transferred and integrated with disarmed Ti plasmid. Due to convenience, binary vectors are more frequently used than cointegrate vectors.

45. Limitations of Ti – Plasmid LARGE SIZE. TUMOR INDUCTION PROPERTY. ABSENCE OF UNIQUE RESTRICTION SITES.

46. Ri Plasmid The virulence plasmid of A. rhizogenes is commonly known as Ri - Plasmid ( pRi ). Agrobacterium rhizogene is a soil borne , gram negative bacterium . All strains of A . rhizogenes are known to produce agrocinopine . Causes hairy root disease , i.e ., massive proliferation of a highly branched root system. This is used for obtaining large amounts of protein from genes cloned in plants Ri plasmids are large (200 to greater than 800 kb) . Contain one or two regions of T-DNA and a vir ( virulence) region , all of which are necessary for hairy root formation. The Ri -plasmids are grouped into two main classes according to the opines synthesized by hairy roots . First , agropine -type strains induce roots to synthesise agropine , mannopine and the related acids. Second , mannopine -type strains induce roots to produce mannopine and the corresponding acids.

47. How Ri – Plasmids Cause disease

48. D D irect gene transfer by precipitation of NA onto the surfaces of protoplasts. Direct gene transfer.

49. Limitations of cloning with Agrobacterium plasmids Extensively used in dicots, but much more difficult to obtain the same results with monocots. But yet eventually artificial techniques for achieving T-DNA transfer in monocots were devised. Ease of recovery of plants varies with species. Hence gene gun technique is being used

50. ATTEMPTS TO USE PLANT VIRUSES AS CLONI NG VECTORS The potential of plant viruses as cloning vectors has been explored for several years, but without great success. One problem here is that the vast majority of plant viruses have genomes not of DNA but of RNA. RNA viruses are less useful as potential cloning vectors because manipulations with RNA are more difficult to carry out. Only two classes of DNA virus are known to infect higher plants – the caulimoviruses and geminiviruses – and neither is ideally suited to gene cloning. Caulimovirus vectors Although one of the first successful plant genetic engineering experiments, performed back in 1984, used a caulimovirus vector to clone a new gene into turnip plants, two general difficulties with these viruses have limited their usefulness.

51. CMV

52. The first problem is that the total size of a caulimovirus genome is, like that of λ, constrained by the need to package it into its protein coat. Even after the deletion of non-essential sections of the virus genome the capacity for carrying inserted DN A is still very limited. Recent studies have shown that it might be possible to circumvent this problem by adopting a helper virus strategy , similar to that used with phagemids . In this strategy, the cloning vector is a cauliflower mosaic virus (CaMV) genome that lacks several of the essential genes, which means that it can carry a large DNA insert but cannot, by itself, direct infection.

53. Plants are inoculated with the vector DNA along with a normal CaMV genome. The normal viral genome then provides the genes needed for the cloning vector to be packaged into virus proteins and spread through the plant. Although this approach has considerable potential it does not solve the second problem, which is the extremely narrow host range of caulimoviruses. This restricts cloning experiments to just a few plants, mainly brassicas such as turnips, cabbages and cauliflowers. Caulimoviruses have, however, been important in genetic engineering as the source of highly active promoters that function in all plants and that are used to obtain expression of genes introduced by Ti plasmid cloning or direct gene transfer.

54. Geminivirus V ect ors The geminiviruses are particularly interesting because their natural hosts include plants such as maize and wheat , and they could therefore serve as potential vectors for these and other monocots. But geminiviruses have presented their own set of difficulties, one problem being that during the infection cycle the genomes of some geminiviruses undergo rearrangements and deletions, which would scramble up any additional DNA that has been inserted – an obvious disadvantage for a cloning vector. I n v e s t i g a tions per f or m ed o v er t h e y ea r s h a v e ad d r es s e d t hes e p r oble m s and ge m ini v ir u s e s a r e n o w beginning to find some specialist applications in plant gene cloning. One such application is virus-induced gene silencing (VIGS) , a technique used to investigate the functions of individual plant genes.

55. The use of a geminivirus vector to silence a plant gene via virus-induced gene silencing. Thi s m e thod e xplo i ts on e o f the n a tu r al d e f ence mechanisms that plants use to protect themselves against viral attack. Thi s m e tho d , t er m ed RN A s i lenc i n g , r esu l t s i n the degradation of viral mRNAs. If one of the viral RNAs is transcribed from a cloned gene contained within a geminivirus genome, then not only are the viral transcripts degraded but also the cellular mRNAs derived from the plant’s copy of the gene . Consequently, the plant gene becomes silenced and the effect of its inactivation on the phenotype of the plant can be studied.

56. Genome maps of (a) African cassava mosaic virus (ACMV) and (b) maize streak virus (MSV), representative of the majority of dicot- and monocot-infecting geminiviruses, respectively. Coding regions are referred to as virion-sense (V) and complementary-sense (C) depending on their orientation relative to the virion ssDNA. ACMV AVI and MSV V2 encode coat proteins. ACMV AC1 and the MSV C1-C2 fusion protein are essential for viral-DNA replication, while ACMV BVl and BCI, and MSV Vl participate in virus spread. ACMV AC2 trans-activates coat-protein expression and AC3 is required for efficient viral-DNA replication. The functions of ACMV AV2 and AC4 are unknown. Intergenic sequences conserved between ACMV DNA A and DNA B (dark shading) contain c/s-acting elements that participate in DNA replication and the control of bidirectional gene expression. Positions of the large intergenic region (LIR) and small intergenic region (SIR) of MSV are indicated. Sequences that are dispensible for viral-DNA replication are light-shaded; the ACMV sequences are also dispensible for systemic infection of Nicotiana benthamiana.

57. References www.asgct.org › General Public › Educational Resources www.link.springer.com www.bios.net Wikipedia http:// nptel.ac.in/courses/102103016/module3/lec23/2.html Plant biotechnology – the genetic manipulations of plants 2nd edition by Adrian Slater, Nigel W.Scott & Mark R. Fowler Principles of gene Manipulations & Genomics – Primrose & Twyman 7th edition Joseph Sambrook , David Russell. "Chapter 1". Molecular Cloning – A Laboratory Manual 1 (3rd ed .) https://www.ndsu.edu/pubweb/~mcclean/plsc431/cloning/clone3.htm http:// www.chemistrylearning.com/cloning-vector/