Lecture 11 Promoters and marker genes Neal Stewart 2016

Discussion questions: promoters What heterologous promoters are used to make transgenic plants, and why? Why are constitutive promoters so popular? What other kinds of promoters (besides constitutive) are needed and why?

Discussion questions: marker genes 1. Why use marker genes for producing transgenic plants? 2. What are some differences between selectable markers and scorable markers? 3. What are the relative merits of and enzymatic marker such as GUS and an in vivo marker such as GFP? 4. What are the advantages, if any, for the use of the manA gene over the nptII gene as a selectable marker for food and feed crops, and would the use of the manA gene overcome public concern over the use of the nptII gene? Conversely, what are the disadvantages?

Figure 7.9

Figure 7.7

What would be the properties of an ideal promoter? Broad range of expression in a number of diverse species Of plant origin Maybe constitutive High expression Tissue specificity Developmental specificity Inducibility Compact—short sequence

Figure 10.1 Figure 10.1 Schemes of a gene with a 5’ and 3’untranslated region (UTR) and a TATA-box promoter (a) and with a synthetic promoter (b). The TATA-box promoter is located upstream of the transcription start site (TSS), and contains a TATA-box in the core region, a few key motifs in the proximal region and many more distant motifs in the distal region. These motifs are the binding sites for various transcription factors, activators or repressors, and can be used together with a core promoter (TATA-box) to engineer a synthetic promoter. The motif selection, motif number, arrangement and space as well as the selection of 5’ and 3’ UTR in synthetic promoters are subject to the expected promoter functions and optimization.

TABLE 10.1 The most widely used tissue-specific promoters in plants. Promoter Type Promoter Name Gene Function Species Reference Green tissue Cab3 Chlorophyll a/b-binding protein Arabidopsis Mitra et al., 1989   rbcS Ribulose bisphosphate carboxylase small subunit De Almeida et al., 1989 PEPC Phosphoenolpyruvate carboxylase Maize Ku et al., 1999 Vascular tissue PP2 Phloem protein 2 Pumpkin Guo et al., 2004 Pfn2 Profilin 2 Christensen et al., 1996 Root EIR1 Ethylene insensitive root1 Luschnig et al., 1998 NAC10 NAM, ATAF1-2, CUC2 Rice Jeong et al., 2000 Pollen Lat52;59 Late anthogenesis Tomato Twell et al., 1991 TA29 Tobacco anther-specific protein TA29 Tobacco Koltunow et al., 1990 Zm13 Pollen specific Hamilton et al., 1998 Seed napA Napin storage protein Brassica napus Rask et al., 1998 GluB-1 Glutelin storage protein Wu et al., 2000

TABLE 10.2 The most widely used inducible promoters in plants. Promoter Inducibility Promoter Name Gene Function Species Reference Pathogen PR1 Pathogenesis-related 1 Arabidopsis Lebel et al., 1998 NPR1 Nonexpressor of PR1 Yu et al., 2001 VSP1 Vegetative storage protein 1 Guerineau et al., 2003 PcPR1-1 Parsley Rushton et al., 2002 PcPAL1 Phenylalanine ammonia-lyase 1 Lois et al., 1989 PR2-d Pathogenesis-related 2-d Tobacco Shah et al., 1996 NtGlnP Glucanase 2   Light CHS Chalcone synthase Weisshaar et al., 1991 LHCP Light-harvesting chlorophyll a/b protein Pea Simpson et al., 1985 Rca Rubisco activase Spinach Orozco & Ogren 1993 Wound MPI Maize proteinase inhibitor Maize Cordero et al., 1994 Pin2 Proteinase inhibitor II Potato Thornburg et al., 1987 Drought ERD1 Early responsive to dehydration stress 1 Tran et al., 2004 Salt RD29A,B Responsive to desiccation 29A, B Yamaguchi-Shinozaki & Shinozaki 1994 Cold Cor15A Cold-regulated 15A Stockinger et al., 1997 CBF2/DREB1C C-repeat/DRE binding factor 1 Zarka et al., 2003 ABA HVA22 ABA-inducible Wheat Shen et al., 1993 Osem Rice homolog of Em Rice Hattori et al., 1995 ABA, RD22 Responsive to desiccation Abe et al., 1997 Em Late embryogenesis Guiltinan et al., 1990 Ethanol AlcA Alcohol-regulated Asperqillu nidulans Caddick et al., 1998

Selectable markers Scorable markers (reporter genes) Typically used to recover transgenic plant cells from a sea of non-transgenic cells Antibiotic resistance markers and herbicide resistance markers are most common Can help visualize transient expression Can help visualize if tissue is stably transgenic Useful for cellular and ecological studies

Figure 10.8

TABLE 10.4 Categories of Marker Genes and Selective Agents Used in Plants Category Marker Genes Source of Genes Selective Agent Selectable marker genes:     Antibiotic resistant nptII, neo, aphII Escherichia coli Tn5 (bacteria) Kanamycin hpt, hph, aphIV E. coli (bacteria) Hygromycin Herbicide resistant bar Streptomyces hygroscopicus (bacteria) Phosphinothricin pat S. viridochromogenes (bacteria) CP4 EPSPS Agrobacterium sp. strain CP4 (bacteria) Glyphosate Nutritional inhibitor related manA Mannose xylA S. rubiginosus; Thermoanaerobacterium Thermosulfurogenes (bacteria) D-xylose   Hormone related ipt Agrobacterium tumefaciens (bacteria) N/Aa   Ablation codA 5-Fluorocytosine Reporter genes: aka scorable marker genes  Enzymatic uidA, gusA MUG, X-gluc Luc  Fluorescent proteins gfp Aequorea victoria (jellyfish) N/A pporRFP Porites porites (hard coral) mOrange Discosoma sp. (soft coral)

Figure 10.2 Sometimes “escapes” occur– for kanamycin resistance markers tissue is red—very stressed

Figure 10.5 Barnase kills tapetum cells (and pollen)—non-conditional selection useful to engineer male-sterility

Common reporter genes Beta glururonidase (GUS) uidA protein from Escherichia coli– needs the substrate X-gluc for blue color Luciferase proteins from bacteria and firefly yields light when substrate luciferin is present. Green fluorescent protein (GFP) from jellyfish is an example of an autofluorescent protein that changes color when excited by certain wavelengths of light. Red and orange fluorescence proteins—RFP and OFP.

Figure 10.9 Figure 10.9 Luminescence detected in transgenic tobacco transformed with the firefly luciferase gene driven by the 35S promoter and watered with a solution of luciferin, the luciferase substrate. [Reprinted with permission from Ow et al. (1986), copyright 1986, AAAS.]

Figure 10.6 GUS positive plants and cells

Figure 10.8

http://www.youtube.com/watch?v=90wpvSp4l_0&feature=related

35S:GFP canola White light UV light in a darkened room

Pollen-tagged GFP—segregating 1:1

GFP-tagged pollen on a bee leg. Hudson et al 2001 Mol Ecol Notes 1:321

Green (and other color) fluorescent proteins FP properties Detection and measurement Anthozoan FPs Why red is better than green Why orange is best of all!

What is fluorescence? x = Brightness Excitation 475 nm Emission 507 nm Stokes shift* x = Brightness Quantum yield % light fluoresced Extinction coefficient Absorption and scattering *Named for Sir George G. Stokes who first described fluorescence in 1852

transformation with GFP Horseweed transformation with GFP Blue Light with GFP Filter White Light

Blue Light with GFP Filter White Light

Transgenic flower cross section Transgenic versus wild-type flowers

In planta fluorescence ex = 395 nm Relative fluorescence Wavelength (nm)

LIFI-laser induced fluorescence imaging—for stand-off detection of GFP and other flourescence

Journal of Fluorescence 15: 697-705

A brief FP history Patterson Nature Biotechnol. (2004) 22: 1524

Anthozoan FPs in transgenics Wenck et al Plant Cell Rep 2003 22: 244 Soybean ZsGreen Cotton AmCyan Wheat leaf DsRed Cotton ZsGreen Rice callus ZsGreen Cotton callus AsRed Corn callus AmCyan DsRed tobacco

Fluorescence x = Brightness Emission 507 nm Excitation 475 nm Stokes shift* x = Brightness Quantum yield % fluoresced Extinction coefficient Absorption and scattering *Named for Sir George G. Stokes who first described fluorescence in 1852

A. victoria GFP “Emerald” 487 (58) 509 (68) Species and FP name Ex max nm (Ext Coef) Em max nm Reference (103 M-1 cm-1) (Quantum yield %) Aequorea victoria GFP 395 (27) 504 (79) Tsien 1998 A. victoria GFP S65T 489 (55) 510 (64) A. victoria EGFP 488 (56) 508 (60) A. victoria GFP “Emerald” 487 (58) 509 (68) A. victoria GFPYFP “Topaz” 514 (94) 527 (60) A. victoria GFPYFP “Venus” 515 (92) 528 (57) Nagai et al. 2002 Zoanthus sp. ZsGreen 497 (36) 506 (63) Matz et al. 1999 Zoanthus sp. ZsYellow 528 (20) 538 (20) Anemonia majano AmCyan 458 (40) 486 (24) Heteractis crispa t-HcRed1 590 (160) 637 (4) Fradkov et al. 2002 Discosoma sp. DsRed 558 (75) 583 (79) Discosoma sp. mRFP1 584 (50) 607 (25) Campbell et al. 2002, Shaner et al. 2004 Discosoma sp. dimer2 552 (69) 579 (29) Discosoma sp. mOrange 548 (71) 562 (69) Shaner et al. 2004 Discosoma sp. dTomato 554 (69) 581 (69) Discosoma sp. tdTomato 554 (138) Discosoma sp. mStrawberry 574 (90) 596 (29) Discosoma sp. mCherry 587 (72) 610 (22)

Excitation scan: Nontransgenic leaf fluorescence—why red fluorescence is better than green

With GFP Why RFP is better– less fluorescence “noise” in the red

More colors in fluorescent proteins discovered (mostly from corals…then improved) http://www.photobiology.info/Zimmer_files/Fig6.png

Orange Fluorescent Protein GFP Jennifer Hinds

Orange Fluorescent Protein (OFP)

An old trick: ER targeting Signal transit peptide 5’ GFP HDEL 3’ Signal peptide directs GFP to endoplasmic reticulum for secretion But HDEL tag sequesters assembled GFP in ER—protected environment allows more accumulation. Haseloff et al 1997 PNAS 94: 2122.

ER retention dramatically improves OFP brightness (monomers) 3x brighter!

Big Orange Fluorescent Proteins Mann et al. 2012

Red foliage as output Arabidopsis MYB transcription factor PAP1 regulates the expression of anthocyanin biosynthesis genes: overexpression of PAP1 results in a red-plant phenotype