Last time: Obtaining information from a cloned gene

Last time: Obtaining information from a cloned gene Objectives: 1. What is the biochemical role of the gene? 2. Where and when is the gene expressed (transcribed)? 3. Where and when is the protein made? Reference: Westhoff et al. Molecular Plant Development: from gene to plant. Chapter 3:39-65.

Panel 4-6, ECB p.164 Indirect Immunodetection

When and where is the protein made? 2. In situ protein localization (tissue or individual cells) (a) using an antibody raised against protein of interest Expression of DEF and GLO proteins is confined to the epidermis of floral organs

Immunofluorescence Wasteneys lab UBC Anti-tubulin primary antibodies together with fluorescently labeled secondary antibodies were used to detect microtubules in roots

When and where is the protein made? 2. In situ protein localization (tissue or individual cells) (b) using GFP or fluorescently labeled antibody to a protein tag

When and where is the protein made? Epitope tagging Immunolocalization using antibodies to protein tag Detection of GFP-protein fusion

When and where is the protein made? 2. In situ protein localization (tissue or individual cells) (b) using GFP or fluorescently labeled antibody to a protein tag CER5 GFP CER5 NOS CER5::GFP::CER5 CER5 CER5 GFP NOS CER5::CER5::GFP

When and where is the protein made? 2. In situ protein localization (tissue or individual cells) (b) using GFP or fluorescently labeled antibody to a protein tag Plant epidermal cells showing localization of the GFP-tagged surface lipid ABC transporter in the plasma membrane

Green Fluorescent Protein (GFP) http://nobelprize.org/nobel_prizes/chemistry/ absorbs blue light emits green

Dr. Andreas Nebenfuhr, U. Tenn. Fluorescent proteins (GFP, RFP, CFP) to label organelles in live cells (red) Golgi stacks, (green) mitochondria, and (blue) peroxisomes in an onion epidermis cell.

Plant transformation Objectives: 1. What is plant transformation? 2. What is Agrobacterium? How and why does it transform plant cells? 3. How is Agrobacterium used as a tool in molecular genetics? References: Hooykaas and Schilperoort. 1992. Agrobacterium and plant genetic engineering. Plant Molecular Biology 19: 15-38. Westhoff et al. Molecular Plant Development:from gene to plant. Chapter 7, 236-243.

Plant transformation Introduction of exogenous DNA into a plant cell Transient no incorporation of exogenous DNA into the genome Stable incorporation of introduced exogenous DNA into the genome Transformation of multicellular organisms: Cannot directly transform every cell - Transformation involves one cell which then regenerates an entire organism

Agrobacterium tumefaciens: a natural tool for plant transformation Soil gram positive bacterium Martha Hawes Agrobacterium tumefaciens attached to a plant cell

Agrobacterium tumefaciens: a natural tool for plant transformation Causes Crown Gall disease - tumors (galls) form at base of stem in many dicotyledonous plants (dicots) Photographs supplied by Sharon von Broembsen, Oklahoma State University production of tumors is caused by the transfer of bacterial DNA to the plant, which integrates into the plant genome

Agrobacterium tumefaciens: a natural tool for plant transformation Genes involved in crown gall disease are not present on the chromosome of A. tumefaciens but on a large plasmid, called the Ti (tumor-inducing) plasmid. Circular chromosome LB Ti A. tumefaciens vir genes T-DNA Ti plasmid ~ 120 kbp ori RB

A. tumefaciens T-DNA Structure LB RB Shi Shi Roi Nos LB and RB 25 bp direct repeats Nos - nopaline synthase opine biosynthetic gene* Shi - shoot inducing - 2 genes for auxin synthesis* Roi - root inducing - gene for cytokinin synthesis* *have eukaryotic promoters these genes are not expressed in Agrobacterium!!!

T-DNA transfer into plants T-DNA transfer process is activated when Agrobacterium gets in contact with damaged plant tissue T-DNA is nicked at the RB, T-DNA gets replicated to the LB and moved into the plant cell these processes are catalyzed by products of vir genes http://www.plantsci.cam.ac.uk/haseloff/sitegraphics/agrotrans.gif

T-DNA transfer into plants T-DNA is inserted into plant nuclear genome at random sites. Transformed cell starts proliferating upon DNA integration resulting in tumor formation. Why? Transformed cells make opines = N-rich nutrients (amino acid derivatives) for bacterium ( Genetic colonization ) http://www.plantsci.cam.ac.uk/haseloff/sitegraphics/agrotrans.gif

Agrobacterium tumefaciens as a tool for genetic engineering Problem: tumor How can we engineer Agrobacterium to make it useful for genetic engineering? Delete auxin, cytokinin and opine genes Retain vir genes, LB&RB, ori Ti plasmid is huge (~120 kb) need to make it smaller

Agrobacterium tumefaciens as a tool for genetic engineering vir genes and T-DNA can be on separate plasmids only left and right borders (LB & RB) are required for T-DNA to be transferred Cloning site for plant genes LB Selectable marker (Plants) vir genes Ti plasmid Selectable marker (Bacteria) T-DNA Binary vector RB Selectable marker (Bacteria) ori (Agrobacterium) ori (E.coli) ori (Agrobacterium)

Steps in plant transformation 1. Propagate binary vector in E. coli 2. Isolate binary vector from E.coli and engineer (introduce a gene of interest) 3. Re-introduce engineered binary vector into E. coli to amplify 4. Isolate engineered binary vector from E. coli and introduce into Agrobacterium already containing a modified (smaller) Ti plasmid with vir genes 5. Infect plant tissue with engineered Agrobacterium (T-DNA containing the gene of interest gets inserted into a plant cell genome at random sites)

Plant transformation In each cell T-DNA gets integrated at a different site in the genome Each cell is hemizygous for the insertion only one of the homologous chromosomes gets the insertion Consequences of the insertion: - Foreign DNA is inserted - Insertional mutagenesis (does not kill the cell the organism is diploid!)

Plant transformation Solanum chacoense http://en.wikipedia.org/wiki/file:transformation_with_agrobacterium.jpg

Plant transformation Problem: We want to transform the whole organism, not one cell!!! This is done by: Transforming plant cells in culture, selecting transformed cells and regenerating the entire plant from the transformed cell (eg. tobacco) In planta transformation of Arabidopsis - Dip flowering plants into Agrobacterium suspension

Plant transformation plbio.life.ku.dk In planta transformation of Arabidopsis (Floral dip method) Systemic infection in Arabidopsis is accomplished by transformation of female gametes!

Selection of transformants http://krauthammerlab.med.yale.edu/imagefinder/

Selection of transformants DsRed selection using green light excitation https://www.emsdiasum.com/microscopy/technical/datasheet/sfa-2.aspx http://www.isb.vt.edu/articles/jan0803.htm