Lecture 3: Agrobacterium Mediated Transformation PDF

Lecture 3 Agrobacterium mediated Transformation Principles of Biotechnology BIOC 3260 Dr. Angela T. Alleyne 1  Lecture 3 Agrobacterium mediated Transformation Principles of Biotechnology...

Lecture 3 Agrobacterium mediated Transformation Principles of Biotechnology BIOC 3260 Dr. Angela T. Alleyne 1  Lecture 3 Agrobacterium mediated Transformation Principles of Biotechnology BIOC 3260 Dr. Angela T. Alleyne 1 LEARNING OUTCOMES At the end of this lecture you will be able to: 1. Describe the Ti plasmid of Agrobacterium tumefaciens 2. Explain the molecular mechanism of T-DNA transfer 3. Describe the experimental techniques used in Agrobacterium transfer 4. Summarize the differences between T-DNA and the vir region of the Ti plasmid 5. Describe the T-DNA binary vector system 6. Discuss the importance of transformed plants 2 Crown gall disease The first written record of crown gall disease, on grape, dates from 1853 Fridiano Cavara (1897) found that a bacterium causes crown gall in grape Crown gall induces growths at wound sites and severely limits crop yields and growth vigor Edward L. Barnard, Florida Department of Agriculture and Consumer Services, Bugwood.org; Mike Ellis, Ohio State University; University of Georgia Plant Pathology Archive, University of Georgia, Bugwood.org; Wikimedia commons © 2014 American Society of Plant Biologists Compounds called opines are found in many crown galls The type of opine is determined by the bacterium, not the plant Octopine-utilizing strain Octopine Nopaline-utilizing Nopaline strain 1960s – 1970s, numerous studies © 2014 American Society of Plant Biologists Nopaline and Octapine Nopaline strains have one T-DNA approx. 20kb in size Octapine strains- two T- DNA regions T1 and TR- 14 and 17 Kb in size respectively 5 A few days after inoculation, tumours become independent of bacteria There's a lag between infection and tumor growth When the tissue was incubated at room temperature for one day before heat-killing the bacteria, no tumors were formed Periwinkle (Catharanthus roseus) stems were inoculated with Agrobacterium tumefaciens, and When the tissue was then incubated at room temperature incubated at room for various times, followed by 5 days temperature for four days at 47°C to kill the bacteria before heat-killing the bacteria, many tumors were formed Viable bacteria are no longer necessary beyond two days post-inoculation. After this period, tumours become independent of the bacteria, because the bacteria have altered the host cells, by transferring some factors into them. Braun, A.C. (1943) Studies on tumor inception in the crown-gall disease. Am. J. Bot. 30: 674-677 © 2014 American Society of Plant Biologists  Agrobacterium Gene transfer ̶ Agrobacterium tumefaciens ( naturally occurring plant pathogen). A. tumefaciens transfers some of its DNA into the genome of plant cells, reprogramming them to divide and produce chemicals called opines The bacteria use these opines as their primary food source Once the DNA transfer is complete the tumour cells can grow and divide independently of the bacteria. Virulence (vir) loci on the Ti plasmid, including virC and virF , were shown to determine the range of plant species that could be transformed to produce crown gall tumours. 7  Agrobacterium tumefaciens mediated transformation in fungi( ATMT) In 1995, Bundock and co-workers reported the successful transformation of Saccharomyces cerevisiae using ATMT Over 125 different fungal species, including members of the ascomycetes, basidiomycetes The most widely used selection marker system in fungi, is the dominant, E. coli antibiotic resistance gene hygromycin phosphotransferase (hph or hpt), which confers resistance to hygromycin 8 ATMT- broad host range Many important crop species( monocots and dicots) are transformed using this bacterium. A. tumefaciens can transform non-hosts, including fungi, algae, sea urchin embryos. A. tumefaciens can be used for both transient and stable transformation methods in plants. 9 Tumor inducing ( Ti) Plasmid ̶ The Ti plasmid contains the T-DNA region flanked by 25 bp repeat sequences ̶ Ti plasmids are approx. 200 to 800 kbp in size ̶ opine catabolism genes allow bacterium to utilize opines as nutrient. 10  Oncogenes There are two general classes of genes in T-DNA: 1. Oncogenes Induce growth ̶ aux A and aux B- produce IAA ̶ alter phytohormone synthesis and sensitivity in the infected cell ̶ Increased levels of these hormones stimulate cell division, thus generating the tumor phenotype 2. Opine-related genes ( unique amino acids). octopine and nopaline - derived from arginine agropine - derived from glutamate 11 The Ti plasmid can be used to introduce any gene into plants T-DNA pTi Gene of interest Selectable marker Tumor-inducing and opine synthesis genes on T-DNA can be replaced by a “gene of interest” and selectable marker Hoekema, A., Hirsch, P.R., Hooykaas, P.J.J. and Schilperoort, R.A. (1983). A binary plant vector strategy based on separation of vir- and © 2014 American Society of Plant Biologists T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature. 303: 179-180. T- DNA 13 1. Signal recognition 2. Attachment 3. Induction of vir genes 4. T- strand production 5. Transfer of T- DNA out of bacteria 6. Transfer to T- DNA and vir proteins into plant cell 14  Transfer tools ̶ T-DNA border sequences, demarcate the DNA segment (T- DNA) to be transferred into the plant genome, ̶ vir genes (virulence genes), are required for transferring the T- DNA region to the plant, but are not themselves transferred, ̶ Modified T-DNA region where the genes that cause crown gall formation are removed and replaced with the genes of interest. 15  Transfer procedure Isolate gene of interest from source organism Develop of a functional Promoters transgenic marker genes construct Insert the transgene into the Ti-plasmid 16 Introduce T-DNA- Transfer procedure containing-plasmid into Agrobacterium Mix Allows transfer of transformed Agrob T-DNA into plant acterium with plant chromosome cells Tissue culture Regenerate Field and lab tests transformed cells for trait into plants- performance of transgenic plant. 17  Summary –plant transformation Inoculate plant with engineered Introduce gene of interest into Agrobacterium T-DNA region, and introduce into Agrobacterium carrying vir genes Agrobacterium tumefaciens T-DNA Regenerate plant from transformed cells Arabidopsis floral dip transformation method © 2014 American Society of Plant Biologists Photo by Peggy Greb, USDA  Agrobacterium plant transformation Taken from: https://www.78stepshealth.us/transposable- elements/images/3301_572_440-transgenic-plants-with-foreign-genes.jpg 19  Molecular tools Agrobacterium tumefaciens virulence genes: Vir A-Vir F ̶ Transfer the T-DNA to plant cell ̶ Activated by Acetosyringone (AS) released by wounded plant cells 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. 20 Perception of host signals induces expression of vir genes Plant-derived small molecules such as acetosyringone induce Agrobacterium vir genes Acetosyringone is likely perceived by the VirA protein encoded on the Ti plasmid VirA and VirG induce other vir genes in response to plant signals Stachel, S.E., Messens, E., Van Montagu, M. and Zambryski, P. (1985). Identification of the signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens. Nature. 318: 624-629; Stachel, S.E., Nester, E.W. and Zambryski, P.C. (1986). A plant cell factor induces Agrobacterium tumefaciens vir gene expression. Proc. Natl. Acad. Sci. USA. 83: 379-383. © 2014 American Society of Plant Biologists  Molecular transfer mechanism virE2 VirD2 NLS may serve as can direct forms a pore a pilot protein in the plant fused to guide the reporter cytoplasmic T-strand to membrane to proteins and and through in vitro- facilitate the the type IV passage of assembled T- export complexes to the T-strand. apparatus the nuclei of contains plant. nuclear localization signal (NLS) Mutations in VirD2 can affect either the efficiency or the “precision” of T-DNA 22 integration.  Vir Genes Vir A Periplam membrane sensor protein senses the presence of plant phenolic compounds such as acetosyringone induced on wounding VirG Cytoplasmic response element used for transcription of other vir genes Autophosphorylated by Vir A VirB Operon of 11 proteins, mating pair formation (Mpf) proteins span the cell envelope- are required for substrate transfer and formation of the T-pilus These proteins get the T-DNA through bacterial membranes 23  - Vir Genes Vir D operon encodes five genes, (virD1- virD5) virD1 and virD2 code for proteins that process the DNA substrate (T-DNA) virD2 - endonuclease/integrase that cuts T-DNA at the borders but only on one strand; attaches to the 5' end of the single strand virD2 & virE2 also have NLS, get T-DNA to the nucleus of plant cell Vir E virE1 - chaperone for virE2 virE2 - binds to the T-DNA and can form channels in artificial membranes Vir F VirF is thought to degrade host cell factors during infection. In the host nucleus, VirF, together with the host proteasome, mediate degradation of the T-DNA complex, causing the release of T-DNA and its subsequent chromosomal integration 24 SUMMARY. Plant cell Agrobacterium Transfer E3 E2 D2 E2 D5 T-DNA processing T4SS F E2 E3 F VirB/D4 Integration D2 LB RB D2 of T-DNA T-DNA vir nucleus genes Ti Plasmid vir genes induction Expression of VirG p T-DNA: auxin, VirA cytokinin, opine VirG p Phenolics Signaling in rhizosphere 25  Vir regions T-strand, and not a double-stranded T-DNA molecule, that is transferred to the plant cell !! VirA auto phosphorylates and subsequently transphosphorylases the VirG protein in coordination with the monosaccharide transporter ChvE, and in the presence of the other phenolic and sugar molecules. VirA and VirG proteins regulate the activation of other vir genes Most VirB proteins either form the membrane channel or serve as ATPases to provide energy for channel assembly or export processes The Vir region is approx. 40Kb in size 26  Transfer proteins- VIP proteins and Actin VIP1 protein shows homology to a class of basic leucine zipper (bZIP) proteins, and is known to localize to the cell nucleus VIP1 interacts with VirE2–ssDNA complexes in vitro and importin to form a ternary complex in some cells both VirD2 and VirE2 proteins interact with filamentous actin in the cytoskeleton in Agrobacterium-mediated transformation b To facilitate movement 27  Chromosomal or chv genes pscA/exoC, chvA, and chvB synthesis, processing, and export of cyclic β-1,2-glucan and other sugars Exo-polysaccharide production, modification, and secretion att genes bacterial attachment to plant cells. Unipolar polysaccharides (UPP) include suagrs N-acetylglucosamine and N-acetylgalactosamine. chvE sugar transporters involved in co-induction of vir genes. Interacts with VirA 28 Overview of the Infection Process 29 Challenges for Biotechnology 1. The oncogenes from the wild-type T-DNA need to be removed to eliminate the pathogenicity of the bacterium, strain becomes nonpathogenic (disarmed) without affecting its ability to transfer T- DNA. 2. The gene of interest and selection markers for transgenic plants need to be introduced into the T-DNA, 3. Molecular biology tools are needed for DNA cloning in vitro. 4. The Ti plasmid size is large and usually low-copy number, which makes isolation and cloning of the Ti plasmid quite challenging. 30 Strategy: Binary vector system 1. Move T-DNA onto a separate, small plasmid ( e.g. cloning into E. coli). 2. Remove aux and cyt genes. 3. Insert selectable marker (e.g. kanamycin resistance) into the 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 bacterium. 31  T- DNA Binary vector A two-component vector system: 1. consisting of a Ti plasmid lacking a T-DNA region (vir helper plasmid/disarmed Ti-plasmid: e.g. pLBA4404 or EHA105) and, a small shuttle vector containing a T-DNA region (binary vector: pPZP). 2. The T-DNA of the binary vector contains only a multiple cloning site (MCS) surrounded by RB and LB. 3. The small, MCS-containing shuttle vector allows for easier manipulation of the T-DNA region using standard molecular biology techniques in Escherichia coli and the finished vector could subsequently be transformed into and replicated in Agrobacterium tumefaciens. 4. Once both of these replicons are located within the same Agrobacterium cell, proteins encoded by vir genes could act upon T- DNA in trans to mediate its processing and export to the plant 32  T-DNA binary vector systems. Genes of interest are maintained within the T-DNA region of a binary vector. Vir proteins encoded by genes are on a separate replicon (vir helper) mediate T-DNA processing from the binary vector and T- DNA transfer from the bacterium to the host cell. The selection marker is used to indicate successful plant transformation. ori, Origin of replication; Abr, antibiotic-resistance gene used to select for the presence of the T-DNA binary vector in E. coli, or in Agrobacterium. Taken from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2245830/figure/fig1/ 33 Components of a T DNA binary vector 1. the T-DNA right and left border repeat sequences that define the T-DNA region; 2. selectable marker genes to function in bacteria (E. coli and A. tumefaciens) and in plants; 3. restriction endonuclease sites within the T-DNA to allow insertion of one or more gene(s) of interest; 4. origin(s) of replication to allow plasmids to replicate in bacteria. 34 Marker genes and reporter systems in plants Markers neomycin phosphotransferase (NPTII) gene for kanamycin resistance and hygromycin phosphotransferase (HPT), Reporters cused for expression ß-D-glucuronidase (GUS), both firefly and bacterial luciferases (LUC), green fluorescent protein (GFP) and other fluorescent proteins, can be detected in transformed plant tissues. 35 36  T-DNA Binary vector characteristics 1. T-DNA left and right border repeat sequences ( ensures only T-DNA is inserted). 2. Plant-active selectable marker gene (usually for antibiotic or herbicide resistance). a)The most commonly used are antibiotics such as kanamycin or hygromycin, b)herbicides e.g. phosphinothricin/gluphosinate, 3. Restriction endonuclease, rare-cutting, or homing endonuclease sites within T-DNA into which goi can be inserted. 4. Origin(s) of replication to allow maintenance in E.coli and Agrobacterium. a)This can be important if several plasmids need to co-exist in the Agrobacterium b)As such, these plasmids must belong to different incompatibility groups. c) Antibiotic-resistance genes within the chromosome and within backbone sequences for selection of the binary vector in E. coli and Agrobacterium. a)Many laboratory Agrobacterium strains are resistant to rifampicin due to a chromosomal mutation b)Most commonly used E. coli K12 laboratory strains cannot use Sucrose as a carbon source. Thus, growth on minimal medium containing rifampicin and 37 Sucrose generally will eliminate E. coli from Agrobacterium cultures Transient transformation Non-integrated T-DNA copies remain transiently present in the nucleus that can transcribe and translate, leading to T-DNA transient gene somatic cells may express the transgenes under the control of the constitutive CaMV-35S promoter within 3–5 days after agroinfiltration The Target gene is expressed within 12 hours and high expression could be attained within 2~4 days in some plant cells such as lettuce and tobacco. Transient gene expression can affect plant signal transduction that could be used for studying various regulatory processes and defense mechanisms in the plant system 38  Selectable markers, Promoters and terminators 35S ̶ Widely used to drive expression of plant genes ̶ Expressed in all tissue of ̶ transgenic plants Nos ̶ Widely used terminator ̶ sequence in ATMT ̶ Stops transcription 39  Genetically-modified (GM) plants Transgenic plants Bacillus expressing insecticidal thuringiensis Bt gene expressing Bt toxin Plant cell expressing Bt toxin Agrobacterium tumefaciens allows gene transfer into many crop plants, particularly dicots like soybean and peanut Wild-type peanut Peanut plant expressing the plant Bt gene Photo credits: Herb Pilcher, Scott Bauer © 2014 American Society of Plant Biologists GM modification methods in plants Indirect Agrobacterium-mediated floral dip, - a stable transformation that targets the germ cells agroinfiltration, - target somatic cells, transient and not transferred to the next generation. co-cultivation method- tissue culture of transformed plants Direct gene transfer methods polyethylene glycol method (PEG)-mediated method, particle bombardment (biolistics), microinjection, electroporation 41 Agrobacterium-mediated transformation - other uses T-DNA Insertional mutagenesis Mutated, tagged gene Transient expression studies: Short-term expression of T-DNA genes gives results faster than generating transgenic plants GFP expression in tobacco epidermal cells Alonso, J.M. et al., and Ecker, J.R. (2003). Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science. 301: 653-657; Reprinted by permission from Macmillan Publishers Ltd Sparkes, I.A., Runions, J., Kearns, A. and Hawes, C. (2006). Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat. Protocols. 1: 2019-2025. © 2014 American Society of Plant Biologists 43  PDF Resources: Pritschze, A and Hirt, H. (2010) New insights into an old story: Agrobacterium- induced tumour formation in plants by plant transformation. The EMBO Journal 29, 1021–1032. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC150518/pdf/0003.pdf https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2245830/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6501860/ 44