L5-6: Tools for plant gene transfer

promoters

marker genes - reporter genes

- selectable markers
vectors
 Categories of Promoters
• Constitutive:
– Strong constitutive promoters can deliver a high-level
expression of transgenes to almost all tissues and
development stages in plants

• Inducible:
– regulated (induced) in presence of certain abiotic or biotic
factors which may include certain biomolecules

• Tissue-specific:
– drive the transgene expression exclusively in targeted
tissues

2
Promoters
• Most commonly used
• CaMV 35S
• Cauliflower Mosaic Virus 35S promoter
• Strong, constitutive
• Highly effective for dicot plants
• less effective in monocots, especially cereals

3
 CaMV

• infects mostly plants of the Brassicaceae family

• an icosahedron
• contains a circular dsDNA molecule of ~8 kb

https://upload.wikimedia.org/wikipedia/commons/8/8e/Virions-Electron_micrograph_of_CaMV_virions.png 4
http://www.virology.wisc.edu/virusworld/imgency/camCAMV5.jpeg
 CaMV genome & 35S promoter

Svedberg unit
Monir Shababi et al. J. Virol. 2006;80:3811-3822 Sedimentation coefficient 5
Markers usually driven from
CaMV 35S promoter

bar: bialaphos resistance, herbicide resistance

CaMV35S
bar Nos-ter

Selectable Nos-ter: nopaline synthase 3’

flanking sequence,
GUS: β-glucuronidase polyadenylation signal
CaMV35S
GUS Nos-ter

Reporter

6
Plant promoters

• Promoter of plant genes with desirable

regulatory or organ-specific upstream elements

• Direct specific gene expression in target cells

• Wound-induced promoters

• Organ- and tissue-specific promoters

7
 Wound-induced promoter
• AoPR1 (Asparagus officinalis pathogenesis-
related protein 1)
• Strong expression (wound sites)
• Extremely low levels in leaves, roots & seeds
No/ Low background

• Introduction of transgenes into plant cells

• Selectable marker genes are essential in
recognition/ recovery of transformants
• Selection early in plant transformation
• Unnecessary after selection

8
https://4.bp.blogspot.com/_AnLEco_ABfc/S8yRYJqRJ0I/AAAAAAAADKE/coIbr2musTE/s1600/asparagus.jpg
 AoPR1
strong expression at wound sites
Wound sites on CaMV 35S-GUS
AoPR1-GUS

9
Plant Journal 3: 191
 Fruit-specific promoter

• Tomato: Solanum lycopersicum

• Tomato E8 promoter
• Activated at onset of fruit-
ripening
• Target sweet protein (monellin)
in fruit
• No activity in leaf, flower, and
unripe fruit E8 Relative level

10
Kneissl & Deikman 1996 Plant Physiology 112, 537-547. https://i0.wp.com/awaytogarden.com/wp-content/uploads/2011/08/usda-tomato-ripeness-color-chart.gif?ssl=1
Marker genes: Reporter

• Optimize DNA delivery

• Identify and track genetic modification
• Early stages developing transformation
• Readily quantifiable product
• Analysis of gene expression
• Transient assay or stable transformants
• Easily assayed (compared to gene of interest where
an assay is not readily available or developed)
• Do not facilitate survival of transformed cells
under particular conditions
11
Use of reporter genes
• To assay gene expression
• Fusions between regulatory signals (pro) of
gene of interest and reporter (product easily
detected) offers advantages:
• Simplifies analysis
• Can compare different/altered regulatory sequences
• Enhances sensitivity (gene activity determination)

12
What makes a good reporter?
Expression of reporter produce visibly
detectable change in phenotype of
transformed cell
• Enzymatic activity not present in host
• Gene/product well characterized genetically
and biochemically
• Gene product stable under different
physiological conditions - organs/tissues, pH,
light
• Enzymatic activity easily detectable and
quantifiable
13
 Commonly used reporters

• β-glucuronidase (GUS) gene uidA

• Luciferase gene Luc

• Anthocyanin regulatory gene Lc

14
β-glucuronidase (GUS) gene
• E. coli uidA locus
• Plant cells lack intrinsic GUS activity
• GUS hydrolyzes β-glucuronides to glucuronic
acid
Histochemical Colourless, soluble product X
→ oxidative dimerization
+ substrate
X-gluc O2 /oxidation catalyst
Tissue 5-bromo-4-chloro-3-
(potassium ferricyanide)
insoluble
(GUS) indoly1-beta-D- indigo dye
glucuronide

Enzymatic
quantitative fluorometric measurement of 4-methylumbelliferone
(Substrate is non-fluorescent 4-methylumbelliferyl β-D-glucuronide)

15
 β-glucuronidase (GUS) gene
• Advantages
• Stable
• Broad pH 5-7.5
• Thermal stable
• Easily & sensitively assayed:
• Can use tissue extracts or histochemical sections
• Widely used in fusion with plant pro or CaMV 35S pro
• Disadvantages
• Destructive assay
• Substrate weakly permeable to cell membrane:
• need to use mild detergent, fixative & vacuum infiltration
• Bacterial contamination gives FALSE positive

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 Example
CaMV35S
GUS driven by CaMV 35S promoter GUS

0.5 mm

Histochemical assay of GUS activity in transformed maize calli

17
Gordon-Kamm et al. 1990. Plant cell 2, 603-618.
 Example: leaf tissue + X-Gluc

Negative control CaMV 35S-GUS

1 cm

Light micrographs
Magnification x230

Visible GUS activity in many cell types

Various intensity?
18
Gordon-Kamm et al. 1990. Plant cell 2, 603-618.
 Example: Tobacco leaf explant
Tissue + X-Gluc → insoluble indigo dye

AoPR1-GUS Transgenic
Transgenic calli 22d
calli 12d
Wound-induced Less

Strong

Transgenic Transgenic
calli 25d calli 25d

& Less activity

CaMV 35S-GUS
Substrate penetrable in tissue Easy and sensitive Destructive assay
19
Firek et al. 1993. Plant Mol. Biol. 22, 129-142.
Localization of a specific protein using GUS
Example:
Knowing the site of action of Acyl-CoA Binding Protein
O
targeted protein would help R C CoA
S
identify its role & function
Wildtype ACBP2 Transgenic Lines

Control
ACBP2pro-GUS was found
expressed in guard cells

After This suggested that ACBP2

affects stomata function and
drought water loss
treatment
Chye et al., US Provisional Patent
Application No. 61/555, 287
Drought-sensitive Drought-resistant 20
Du et. Al., 2013. Overexpression of Arabidopsis acyl-CoA-binding protein ACBP2 enhances drought tolerance Plant Cell Environ. 36(2):300-14.
 Localization of ACBP3pro-GUS

32-day-old rosette leaf Vascular

stem
bundle of
a leaf

ACBP3pro-GUS is expressed in phloem

VS: vascular system; X: xylem; P: phloem; CA: cambium
Zheng et al (2012). J. Exp Botany 63: 2985-3000. 21
ACBP3 function in plant defense
 Analysis of promoter activity

GUS

5’ deletion
analysis

Check expression of
ACBP3pro-GUS

22
Zheng et al (2012). J. Exp Botany 63: 2985-3000.
 Reporter: Luciferase gene
• Firefly luc: catalyzes ATP/oxygen-dependent
oxidation of luciferin produces light emission
(bioluminescence)
Temperature
dependent
Tissue with + substrate photon/light
Luciferase beetle luciferin emitting cells

Cells can be isolated for regeneration to a transgenic plant

23
 Luciferase gene
• Advantages
• Rapid
• Easy
• No background interference
• Non-invasive

• Disadvantages
• Signal decay rapidly
• Require special instrument: luminometer for
measurement of photon

24
 Example: Luc in orchid
Light Transgenic plant
Left: untransformed Light
control
Right: Transgenic
calli producing Luc

CaMV 35S-luc
Dark
Dark

Superimposition of
images pinpointing
source of photon Demonstration of
emission varying expression in
different plant organs

Low-light video-image analysis of Luc activity in transgenic Dendrobium tissues

and plants. Tissues and plants were immersed in luciferin solution for 45 min
25
Chia et al., 1994 Plant Journal 6: 441-446.
 For detection of transformants

Light Dark Superimposed

Dendrobium cells were transformed with CaMV 35S-luc by bombardment.

21d cells were soaked in 1 mM luciferin before imaging.

26
Chia et al., 1994 Plant Journal 6: 441-446.
 Reporter: Maize Lc gene

• Encode regulatory protein controlling anthocyanin

pigmentation
• For visualization of transformed maize cells after
bombardment of various tissues with Lc DNA-
coated gold particles
• Lc: Leaf colour

27
 CaMV 35S-Lc
Embryogenic-derived
protoplast

Transformants
expressing Mesophyll-derived
anthocyanin protoplast
pigment

28
Lusardi et al. 1994. Plant Journal 5: 571-582
 CaMV 35S-Lc
Somatic cells Red-
before (c) and brown
after (d) injection sectors in
leaf

: Cells
producing
anthocyanin

29
Lusardi et al. 1994. Plant Journal 5: 571-582
 Anthocyanin regulatory gene Lc
• Advantages
• Pigment detected in vivo
• Highly sensitive (single transformed cell detected)
• No histological preparation required
• Cells not sacrificed
• Can be detected 48 h after bombardment
• Activation of endogenous pathway – no background
from Agrobacterium
• Can recover transformants without selection

• Disadvantage
• Only applicable to maize/cereal transformation
30
Lusardi et al. 1994. Plant Journal 5: 571-582
 Selectable marker genes
• For stable transformation
• Enable transformed cells/tissues/whole plants
to grow
• Confer dominant phenotype (new trait) on the
transformed
• Confer cell viability in the presence of selective
agent
• Confer distinguishable characteristics like that
of Luc, Lc.

31
 Selectable marker genes

• Essential for introduction of agronomically

important genes into crops
• Fused to gene of interest in vector
• Enable cells with the marker to survive
• Plants regenerated from surviving cells contain
the selectable marker and gene of interest

32
 Selectable marker genes
• Encoding antibiotic-resistant gene
• nptII: for kanamycin resistant
• Encoding herbicide-resistant gene
• bar: bialaphos resistant
• Selection criteria
• Selective agent readily available
• Soluble in plant tissue culture medium
• Inexpensive
• Non-toxic
• Untransformed cells should not grow in media with
selective agent

33
 nptII: neomycin
phosphotransferase II gene
• Most common
• Derived from E. coli Tn5
• Encodes aminoglycoside-3-phospho-transferase II
• Inactivates aminoglycoside antibiotics (Kanamycin,
neomycin) by phosphorylation
• Select transformants on media containing
kanamycin

34
 bar: bialaphos resistant gene
• Derived from Streptomyces hydroscopicus
• Encodes a Phosphinothricin Acetyl Transferase
(PAT) enzyme that detoxify bialaphos
• Bialaphos, PPT
• Phosphinothricin-tripeptide, Bilanafos, Basta
• Nonselective natural herbicide produced by many
Streptomyces
• Consists of a glutamic acid analogue moiety
• Inhibitor of glutamine synthetase of nitrogen
assimilation pathway

35
 Phosphinothricin
Ala Ala PPT

peptidase

glufosinate
Bialaphos

Glutamine
+ NH3 + ATP synthetase + ADP + Pi

glutamate glutamine

36
 Phosphinothricin
Ala Ala PPT

peptidase

glufosinate
Bialaphos

Glutamine
+ NH3 + ATP synthetase + ADP + Pi

glutamate glutamine

No inhibition of
PAT glutamine synthetase
NH Cell resistant to PPT
glufosinate Acetyl-CoA Ac
Acetyl Phosphinothricin

37
 Use of bar gene
• CaMV 35S-bar
• Transform embryonic maize suspension culture
by bombardment
• Select callus on Basta-containing (1 mg/L) media

1 mm

Bialaphos-resistant colonies Embryonic Bialaphos-resistant callus

7 weeks 38
Gordon-Kamm et al., 1990. Plant Cell 2:603-618
 Basta-resistant maize

CaMV 35S-bar CaMV 35S-bar

Wildtype Wildtype
transformed transformed
Effect of Bialaphos on wildtype and CaMV 35S-bar transformed maize.
Photos were taken 6d after application of the herbicide.

39
Gordon-Kamm et al., 1990. Plant Cell 2:603-618
Tools for plant gene transfer
Promoters
Marker genes
Vectors
A. Viral vectors
B. Agrobacterium vectors *

Techniques in plant gene transfer

i) Agrobacterium-mediated transformation
ii) protoplasts
iii) microprojectile bombardment
iv) microinjection
v) plant viruses

i-iv: stable transformation, integration into plant genome

v : no integration
 Plant Viral Vectors
• Plant viral vectors
– Alternative to Agrobacterium-mediated
transformation
– Delivery of target gene into plant for introducing
novel trait
– Use reverse transcriptase of RNA virus to yield
cDNA clones that are infective
– Use desirable properties of viruses as gene
vectors

• Transient expression of protein for large-scale

production: antibodies and vaccine antigens 41
 Desirable properties of plant viruses

• Viruses adsorb to and infect cells of intact plants

• Cloned viral DNA is infectious when rubbed onto
healthy leaves
• Some viruses spread systematically
• Viral nucleic acid is transported from infection site to
virtually every living cell of the host
• Small genome (e.g. Geminiviruses: 2.7-3 kb) for
easy in vitro manipulation
• Region non-essential for infection can be deleted or
replaced by gene of interest

42
 Desirable properties of plant viruses

• Exist at high copy number

• Virus replicate to several thousand/cell (103-106/cell)
• Viral genes driven by strong plant-constitutive
promoters are strongly expressed
• Have extremely broad host range
• Geminiviruses infect both monocots & dicots
• Every plant infected by at least one RNA virus
• Can accumulate large numbers of viruses
• Consequently, large amounts of viral NA/proteins in
infected hosts
43
Can accumulate large numbers of viruses
Consequently, large amounts of viral NA/proteins in infected hosts

44
 Example on the use of viral vector:
Production of -trichosanthin
• -trichosanthin
• an eukaryotic ribosome inactivating protein
from root of Trichosanthes kirilowii
• Inhibits the replication of HIV in vitro
• Transfect Nicotiana benthamiana plants
(close relative of tobacco ) with in vitro
transcripts of plasmid pBGC152 i.e., RNA
• Transfection was confirmed by isolation
of virions from plant leaves
• Accumulate to >2% total soluble protein
in 2-weeks after inoculation
Kumagai M.H. et al. 1993, PNAS 90(2): 427-430 45
https://en.wikipedia.org/wiki/Trichosanthes_kirilowii#/media/File:Trichosanthes_kirilowii.jpg
https://ae01.alicdn.com/kf/HTB1iE9qHpXXXXaEXFXXq6xXFXXXP/202692953/HTB1iE9qHpXXXXaEXFXXq6xXFXXXP.jpg
alpha-trichosanthin gene under control of a tobamovirus subgenomic promoter

Transcriptional
The plasmid was prepared
start point and purified from E. coli
ATG:
translation The plasmid was then
start
Signal peptide:
linearized by cutting with
targeting to KpnI and used as template
extracellular
space of odontoglossum
for in vitro transcription
transfected ringspot virus using SP6 DNA-dependent
plant ORSV coat protein gene
RNA polymerase
Tobacco
Mosaic
Virus In vitro produced RNAs were
TMV then used to mechanically
ORFs inoculate N. benthamiana
plant

The amount of trichosanthin

was then determined after
two weeks
The structural and biological
properties of recombinant
protein (-trichosanthin)
confirmed
Kumagai M.H. et al. 1993, PNAS 90(2): 427-430
46
 Limitations of plant viruses

• Requires ‘full virus’: many viral genes

• Usually as a fusion to coat protein gene
• Viruses do NOT integrate into host genome,
even if present in high copy numbers
• Excluded from meristems and germline
• NOT transmitted to offspring
• Introduced as infectious nucleic acid or viral
particle
• Spread systemically: requires time

47
 Limitations of plant viruses

• Unstable: lost of inserted sequence

• A negative correlation between insert size and
vector stability was observed
• Relatively few geminiviruses can be re-
introduced into plants by mechanical
inoculation

48
 Second Generation Viral Vectors
• “Deconstructed virus”:
only the viral elements required for efficient expression left
• Allowing easy chemical conjugation of (separately produced)
proteins to viral surface
• Use of Agrobacterium for delivery of viral vectors to whole plant
• Expression time much shorter: 6-10 days
• Magnifection, combines advantages of three biological systems:
– vector efficiency and efficient systemic DNA delivery
of Agrobacterium
– speed and expression level/yield of a plant RNA virus
– posttranslational capabilities and low production costs of a plant

49
His-tag, FLAG-tag to aid purification
 Infection and spread of replicons
1st generation
Use of infectious NA/virus
Infect localized area and allow to
spread systemically
Relative inefficient processing and
accumulation of viral particles

2nd generation
Use of Agrobacterium as vector for
delivery of viral vector
Transfect whole plant and allow to spread
Efficient processing into active replicons

50
Gleba et al. 2007. Curr Opinion in Biotechnology 18:134-141
 Recombinant Protein Production in
Plants using Magnifection
• Process:
– First step: introduce a gene of interest into Agrobacterium
tumefaciens
– Strain grown in a liquid culture and the resulting bacteria are
washed and suspended into a suitable buffer solution
– Agroinfiltration:
• Syringe infiltration
• Vacuum infiltration
– Once inside the leaf the Agrobacterium remains in the
intercellular space and transfers the gene of interest as part of the
Ti plasmid-derived T-DNA in high copy numbers into the plant
cells
• Produce 0.5–5 g recombinant protein/kg leaf biomass.
– Green biomass yield of 100-300 ton/hectare/year
–  50–600 kg recombinant protein/hectare/year
• Expression time: 6-10 days 51
 Plant Magnifection
transformation
Floral dip

Agrobacterium
Transient expression

Standard, existing industrial processes

Requires special equipment & biological containment 52
Gleba et al. 2007. Curr Opinion in Biotechnology 18:134-141
 Discussion

Possible questions for 2h written examination

• Compare and contrast the use of Lc and luc genes in plant

gene transfer.

• Discuss the advantages and limitations in the use of β-

glucuronidase gene.

• Describe the advantages and limitations in the use of uidA

gene in plant biotechnology.

53
 Possible questions for 2h written examination
• Name 2 specific promoters that have been commonly used in plant
transformation. What are the significant features of each promoter?
Give an example in the use of each promoter for transformation.

• What promoters are used for the expression of foreign genes in

transgenic plants? Discuss the qualities of these promoters.

• Discuss the use of the following:

a) Wound-inducible promoters
b) Cauliflower Mosaic Virus 35S promoter

• Compare and contrast the use of the Cauliflower Mosaic Virus 35S
promoter and plant promoters in plant genetic engineering.
What are the uses of reporter genes in studying plant transformation.
Give a specific example and discuss its advantages and disadvantages.

Why are selectable markers used in plant transformation? Give a specific

example of a selectable marker and discuss some of its advantages and
limitations.

Distinguish between selectable marker genes and reporter genes. Why are
they important in plant genetic engineering? Give a specific example of each
and discuss the advantages and limitations in its use.

Compare and contrast:

a) reporter gene and selectable marker gene
Compare and contrast: nptII and GUS

Selectable markers are common in plant gene transfer.

a) Why are they necessary?
b) Where is the selectable marker located on a plant transformation
vector? Give reasons for your answer.
c) State an example of a commonly-used selectable marker and give
reasons for its popularity.

Compare and contrast the terms, “transfected plant cells” and “transgenic
plant cells”.
Using a specific example for each, describe how they can be used to
produce proteins for biomedical applications.