Chapter 05: Transgene expression in plants

Selectable marker genes Reporter genes Protein translocation Herbicide resistance

Reporter and selectable marker genes

Reporter- and selectable marker genes are often of bacterial origin and need to be adjusted for expression in plant cells: appropriate start and stop signals must be added for transcription. Constitutive expression signals are normally used for selectable marker genes, for reporter genes this depends on the aim of the experiment (inducible, tissue-specific). Examples of constitutive expression signals: 35S-promoter (from CaMV plant virus) and 3nos (polyadenylation signal from nopaline synthase gene of A. tumefaciens T-DNA)

Selectable marker genes

Most commonly used antibiotic- and herbicide resistance genes: nptII, kanamycin resistance hpt, hygromycin resistance bar or pat, bialaphos resistance or phosphinothricin acetyltransferase glyphosate tolerance (5-enolpyruvyl-shikimaat3-phosphate synthase or EPSPS)
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Reporter genes
GUS (-glucuronidase): bacterial enzyme that can convert a colourless substrate (X-gluc) into a coloured (blue) product. GFP (green fluorescent protein from jellyfish): no substrate needed. Luciferase enzyme producing light with luciferin as substrate (e.g. firefly luciferase in plant, see picture).

Reporter GFP
Green fluorescent protein (under UV light) Gene comes from jellyfish Aequoria victoria No substrate needed In vivo observations Used a lot also in translational fusions

GFP-fusions
Transcriptional GFP fusion: - to check transformation (constitutive promoter) - to follow an organism (idem, e.g. bacteria) - to analyze promoter activity in different tissues and conditions: when the promoter is active, GFP is seen. GFP
GFP

Translational GFP-fusion - to follow a protein GFPin the cell

GFP
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wrky transcription factor

wrky-gfp fusion goes to the nucleus because of signal in wrky

Translocation of proteins
Proteins have signals for their cellular location in their primary sequence Proteins that are not destined to stay in the cytoplasm are generally produced as preproteins Transport through ER-membrane occurs cotranslationally Hydrophobic AAstretches block proteins in membranes

Film protein sorting/ membrane/protein secretion bcs.whfreeman.com/lodish6e/pages/bcsmain.asp?v=category&s=00010&n=04000&i=04010.02&o=|00510|00520|00530|00540|00550|00560|00570|00580|00590|00600|00610|00010|00020|000 30|00040|00050|01000|02000|03000|04000|05000|06000|07000|08000|09000|10000|11000|12000|13000|14000|15000|16000|17000|18000|19000|2000 0|21000|22000|23000|24000|25000|99000|&ns=198

Protein transport

SSSSS

S = specific
signal

signal

S = in the absence of a S
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Protein transport to mitochondria

Signal-peptide (= transit peptide or TP) at NH2-end leads protein to mitochondrial membrane, signal is cut off during transport through membranes. Transport into chloroplasts very similar.

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Recent herbicides
Novel herbicides with large advantages: Rapid biological degradation Much less toxic for man and animal Reason: specific plant process is inhibited (photosynthesis, amino acid synthesis) Very active therefore low dose is sufficient Examples: Roundup Monsanto (glyphosate) Liberty / Basta Bayer (Phosphinothricin) Bromoxynil Rhone-Poulenc

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Recent herbicides
Problem: total herbicides, toxic for all plants Strategy: make crop resistant to specific herbicide via genetic engineering (expression of an insensitive enzyme or degradation of herbicide): novel market for the agrochemical industry! Two main strategies: - Express extra target protein that is not susceptible to herbicide - Express enzyme that inactivates herbicide

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Herbicides
(RR) Soybean without RR Soybean after herbicide treatment herbicide treatment

Source: Monsanto

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Glyphosate resistant plants

Glyphosate blocks EPSPS (5-enolpyruvylshikimate-3phosphate synthase) = enzym for biosynthesis of aromatic amino acids EPSP synthase of Agrobacterium CP4 is relatively insensitive to glyphosate EPSPS is an enzyme that is active in the chloroplasts of plants Transformation with chimaeric EPSPS of CP4: P35S TP EPSPS Petunia -EPSPS CP4 -3nos (transit peptide added to bing bacterial enzyme to the chloroplast)

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Glyphosate resistant plants

Plants that express this bacterial EPSPS besides their own enzyme are resistant to glyphosate

Recombinant DNA, Watson et al., Freeman and Company, Scientific American Books 15

Phosphinothricin resistant plants

Glufosinate = phosphinothricin = PPT = Basta = Liberty Phosphinothricin acetyltransferase gene (pat) from Streptomyces viridochromogenes or bar (bialaphos resistance) from Streptomyces hygroscopicus

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Phosphinothricin resistant plants

Phosphinothricin is a potent inhibitor of glutamine synthetase (GS) Inhibition of GS in plants leads to accumulation of ammonia and rapid dead Figure: ammonia accumulation in plant after BASTA treatment

NH4+

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Phosphinothricin resistant plants

Transformation of plant with chimaeric pat- gene (35S-bar-3g7) De Block et al., 1987 Plant with pat behaves after treatment with ppt as the untreated control, phenotypically as well as biochemically = resistant to the herbicide phosphinothricin

NH4+

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Phosphinothricin resistant tobacco

Source: PGS

First tested in model plant tobacco

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Phosphinothricin resistant potato

Source: PGS

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Phosphinothricin resistant sugar beet

Source : PGS

Also in oilseed rape, soybean, corn, sugar beet

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Herbicides
(RR) Soybean without RR Soybean after herbicide treatment herbicide treatment

Source: Monsanto

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Roundup Ready soya (Monsanto)

Historical invasion of Europe through the port of Antwerp Greenpeace took this opportunity to start anti-GMO campaign, they tried to stop the boat from unloading Allergenic soybean on the shelves = not true Roundup Ready soybean seed is more expensive = true Roundup Ready soybean is more fragile = not true Roundup Ready soybean enhances herbicide use = not true Roundup Ready soybean yields less = not true anymore But Roundup Ready e. a. herbicide resistant soybean

Herbicide resistant soybean in world

US: soybean 2 % in 1996; 77 % in 2002; >90% in 2007 Globally: 72% of the 65 M ha in 2008 Argentina: since 2002 nearly 100% of 13 Mha soybean = herbicide resistant
http://www.gmo-compass.org/eng/ agri_biotechnology/gmo_planting
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Herbicide resistant soybean in US

US ca. 23 mill. ha soybean in 2007: 90% GM (herbicide resistant) Why? Janet Carpenter and Leonard Gianessi 1999 www.agbioforum.org/v2n2/v2n2a02-carpenter.pdf Better weed control easier management less tillage Plants, Genes and Crop
Biotechnology, Chrispeels & Sadava

Another advantage: less nocious and less persistent herbicide Disadvantage: no real herbicide decrease (change in herbicide use to less toxic one), expensive seed, initial cultivars lower yield

Herbicide resistant oilseed rape in Canada

Ca. 5 M ha oilseed rape in 2007 of which 85% GM (herbicide resistant) Arguments of Canadian farmers : Better weed control easier management less tillage higher yield (+10%) less herbicide (- 6.000 ton) less fuel (- 31 mill. liter) Higher income Disadvantage: herbicide resistance crosses out

Superweed?
Introduction of the Rambo gene of total resistance maybe was not such a good ideabut total resistance is only to one herbicide, the plant is still susceptible to all other herbicides, it is not at all indestructible!

Deze soja blijft even (on)gevoelig voor andere herbiciden als niettransgene soja

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Herbicide resistant plants: environmental risks

Important concerning discussion environmental consequences herbicide resistant plants: Compare potential pro and contra arguments case by case (cfr. higher or lower or equal herbicide use or yield!?) Imagined risks: crossing out to unrelated plant species???? crossing out of herbicide resistance threatens biodiversity???? Discussion was started on GM- herbicide resistant plants, non-GM-herbicide resistant plants (mutants) have the same consequences, but these plants are not questioned/attacked.

Transgenic soybean
Herbicide resistant gene can cross out and result in herbicide resistant weed biodegradable Roundup, low toxicity tested in detail before commercialisation modification known Monsanto

Non-transgenic soybean
Herbicide resistant gene can cross out and result in herbicide resistant weed more persistent Synchrony, low toxicity no procedure before commercialisation modification unknown Dupont

Pictures of Jonathan Gressel, Weissman Institute, Isral

Striga hermonthica

Developing world???
Sorghum in Africa

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Herbicide resistant crops useful in developing countries?

Striga infection on corn burn the field!!

 Striga and Orobanche are parasitairy weeds that suck out the nutrition from the host and therefore result in lower yield Striga is a plant and thus sensitive to herbicides Herbicide resistant mutant corn, seed is treated before sowing
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