Transcript Ms Lerato Matsaunyane (ARC) – A genomic study on the

A genomic study on the unintended
effects of transformation
Lerato B.T. Matsauyane
Promoter:
Co-Promoter:
Prof I.A. Dubery
Dr D Oelofse
Introduction-Fungi
 During pathogenesis
 attach to the plant surface
 germinate on the plant surface and form infection structures
 penetrate the host and colonise the host tissues
 Entry – natural openings or wounds
phytopathogenic fungi secrete a combination of hydrolytic
enzymes to gain entry
 First phytopathogenic enzymes secreted – polygalacturonases
(PGs)
 hydrolyse -1, 4-D linkages randomly
 releases oligogalacturonides-OGs
 OGs activates defence responses
Introduction-Plant
 No circulatory systems and antibodies
 Depend on their own defence mechanisms
 first line of defence – cell wall
 second line of defence – activation of three classes of defence
responses
• 1st class
o hypersensitive response (HR)
• 2nd class
o synthesis of pathogenesis-related (PR)
• 3rd class
o systemic acquired resistance (SAR)
o transcription of a number of plant resistance (R) genes
o expression of polygalacturonase inhibiting proteins
(PGIPs)
Introduction-PGIP
 Extracellular leucine rich repeat (LRR) proteins
 Found in monocotyledonous and dicotyledonous plants
 Recognize PGs
 Form specific, reversible, saturable, high-affinity complexes with PGs
 balance release of OGs and the depolymerisation of the active OGs
into inactive molecules
 increases the release of OGs
 switches on other defence responses
Introduction-PGIP specificity
 Several plants have shown the presence of more than one PGIP
 Phaseolus vulgaris (4 in Bean)
 Psidium guajava (3 in Guava)
 Allium porrum (3 in Leek)
 PGIPs from the SAME plant exhibit differential specificities towards the
same fungal PG
 PvPGIP2 inhibits FvPG and PvPGIP1 does not
 PGIPs from DIFFERENT plant shown differential inhibition of PGs from
the same fungus

PcPGIP inhibits BcPG and SlPGIP does not
Introduction-Potato
 Solanum tuberosum L.
 History of potato
 +/- 6,000-10,000 years ago – first wild type identified
growing in the central Andes of Peru and Bolivia
 1562 – potatoes recorded outside South America in the Canary
Islands
 1563 – rapid growth in Europe and the rest of the world
 Selection and breeding – transformed the wild type potato into
current cultivars
• consistent shapes and colours
• improved agronomical characteristics – e.g. increased yield
Introduction-Potato
 Genetics of potato
 1939 – Bukasov embarked on chromosome counting
• diploid (2n=2x=24)
• triploid (2n=3x=36)
• tetraploid (2n=4x=48)
• pentaploid (2n=5x=60)
 Naming and shaming
 Classification of the modern potato cultivar has been debated
 2002, Huamán and Spooner classified all cultivated potatoes under
S. tuberosum
• lack of relatedness of the cultivars to a single ancestral group
• complications within the taxonomy
• the format of classification
Introduction-Potato
 Fourth most important food crop in the world
 Top three - rice, wheat and barley
 Produced over shorter periods
 Produces large mass of high-value food
 Good source of complex carbohydrates, protein, calcium and
vitamin C
 Important staple crop - Nicknamed “Mother’s Finest”
 Roots and tubers feed over 1 billion people in the developing world
 Provides 40% of the food for half of the people from sub-Saharan
Africa
Introduction-Potato
 Potato is susceptible to several fungal, bacterial, and other pathogens
 Considerable loss in yield and quality products
Producing disease-resistant
cultivars will be an effective and
useful strategy to combat the attack
of pathogens
Introduction-Verticillium Wilt
 Occurs wherever potatoes as grown
 Most severe in irrigated fields
 30 – 50% less yield of potatoes grown in Verticillium infested soils
 The pathogen (V. dahliae Klebahn)
 broad spectrum of host plants
• annual, fibre and food crops, herbaceous and ornamental
crops, and shrubs
 cause the most economic losses and distributed world-wide
 favours warmer environment
 can persist, dormant, for years in the soil or amongst plant rubble
even if no susceptible plants are present for infection
Introduction-Verticillium Wilt
 Symptoms
Chlorosis and necrosis of the leaves as
well as wilting
Tan discoloration of the vascular tissue of
the roots
Tan discoloration of the vascular tissue of
the stems
Introduction-Verticillium Wilt
 Pathogenesis cycle
 Microsclerotia germinate and infect plants through the roots
Microsclerotia germination
 Colonization of the plant
• hyphae moves from the cortex to the xylem vessels
• conidia is produced and moves up the transpiration system
resulting in vascular invasion
Conidia produced in a xylem vessel of an
infected plant
Introduction-Verticillium Wilt
 Current control measures
 crop rotation and chemical fumigation
 limited irrigation, and applying optimal rates of N and P
 Plant biotechnology applications offers a number of sustainable solutions
 Malus domestica polygalacturonase inhibiting protein 1 (Mdpgip1)
(V. dahliae inhibitor)
 Popular potato cultivar AppBP1 (susceptible to V. dahliae)
 Mdpgip1 transgenic potato plant (AppA6) produced for increased
resistance against V. dahliae
Introduction-Genetic modification
 Possible outcomes of production of GM crops
1. GM crop equivalent to traditional counterpart
• No further testing is needed
2. GM crop equivalent to traditional counterpart, except for some well
defined differences
• Safety assessments will target the differences
3. GM crop differs from the traditional counterpart in multiple and
complex ways
• Extensive risk safety assessment required
• Pay attention to potential adverse effects on human and animal
health, and the environment
Introduction-Genetic modification
 Concerns
 Proper regulatory process to address consumer concerns
 Current approaches to compare GM crops to their traditional
counterparts are biased
 Unintended, unexpected effects resulting directly or indirectly from
the genetic modification are not always considered
 Solution
 New molecular techniques available, such as DNA fingerprinting, ….
 Utilize these to address concerns
Aim
 Concerns
 Proper regulatory process to address consumer concerns
 Current approaches to compare GM crops to their traditional
counterparts are biased
 Unintended, unexpected effects resulting directly or indirectly from
the genetic modification are not always considered
 Solution
 Find more comprehensive techniques available to address concerns
Thus
The aim of this project is to study the unintended
effects of transformation of potato with the
Mdpgip1 gene using genomic-based
technologies
Objectives
 Study the unintended effects of plant transformation, if any, in the
transgenic plants through gene expression analysis
 Gene expression profiling to determine whether the insertion of the
transgene into the potato genome results in differential gene expression
between the transgenic and untransformed potato plants
 Establish this technology within the ARC
Importance of Project
 Gene discovery is being implemented within the ARC
 GM crops will continue to be produced, thus the technology will assist in
the characterisation of these plants
Research Objectives
 Molecular analysis of transgenics
 Screening for the presence of the transgene
 Screening for the presence of the marker gene
 Biochemical analysis of transgenics
 MdPGIP: PG inhibition studies
 Number of copies of the transgene in the transgenic
 Gene expression profiling
 cDNA-AFLP
 cDNA-RDA
 qRT-PCR
 Presence of filler DNA
 Genome Walking
 Insertion site of the transgene
 Genome Walking
Molecular screening of transgenics
1
2 3
4
5
1
2
3
4
14.1
14.1
2.8
4.0
1.2
1.0kb
PCR screening to verify the presence of
the Mdpgip1 gene within the transgenic
genome
Lane 1: Lambda PstI Marker
Lane 2, 4: Empty
Lane 3: PCR of AppBP1
Lane 4: PCR of AppA6
0.8
0.6kb
PCR screening to verify the presence of
the nptII gene within the transgenic
genome
Lane 1: Lambda PstI Marker
Lane 2: Empty
Lane 3: PCR of AppBP1
Lane 4: PCR of AppA6
The Mdpgip1 transgene was successfully integrated into the
S. tuberosum cv BP1 genome
Biochemical screening of transgenics
1
2
MdPGIP1: VdPG inhibition assay
1: VdPG
2: VdPG + AppBP1 PGIP
1
2
MdPGIP1: VdPG inhibition assay
1: VdPG
2: VdPG + AppA6 PGIP
The transgenic AppA6 successfully expresses an active MdPGIP1
The MdPGIP1 is successful in inhibiting the PGs from V. dahliae in vitro
Presence of filler DNA
 Genome Walking to detect Mdpgip1 expression cassette
bp
1
2
3
4
5
6
7
 300bp fragment contained 35S CaMV
promoter and TEV leader sequences
 600bp fragments contained 35S CaMV
promoter, TEV leader and Mdpgip1
sequences
1000
500bp
500bp
300bp
500
300
200
100
 The Mdpgip1 expression cassette is
present in the transgenic AppA6
200bp
Fragments extracted and sequenced
Promoter
TEV
Mdpgip1
Terminator
Presence of filler DNA
 Genome Walking to detect filler DNA
kb
14.1
5.1
2.5
1
2
3
4
5
6
5.1kb
5.1kb
1.2kb
1.2kb
7
8
1.2
 No amplification products obtained from the transgenic AppA6
 5.1kb and 1.2kb fragments amplified from pCAMBIA2300
 No filler DNA present in the transgenic AppA6
Insertion site of Mdpgip1
 Oligonucleotides designed at the left and right borders
 Restriction enzymes used – EcoRV, SacI, SmaI, StuI
kb
1
2
3
4
5
14.1
5.1
2.5
1.2
AppBP1
AppA6
6
7
Insertion site of Mdpgip1
Fragment name
Alignment
C31B-1
84-714bp of pCAMBIA2300:appgip1A
C31B-2
No alignments
C31B-3
No alignments
A31B
Solanum tuberosum chloroplast,
complete genome
C32B-1
1650-1845bp of
pCAMBIA2300:appgip1A
C32B-2
No alignments
A32B-1
Solanum tuberosum chromosome 6
clone RHPOTKEY030E18
A32B-2
PPTIA29TF Solanum tuberosum
RHPOTKEY BAC ends Solanum
tuberosum genomic clone
RHPOTKEY193_F09
Chromosomes
Chromosome
02:1:49918294:1
Solanum lycopersicum
SEQUENCING IN
PROGRESS
Insertion site of Mdpgip1
Gene expression profiling: cDNA-AFLP
cDNA-AFLP gel
22 differentially expressed fragments isolated
No
OC1
bp*
Sample
1
Tr15/M70
180
A
2
Tr15/M71
205
B
3
Tr16/M64
138
A
4
Tr16/M64
125
A
5
Tr16/M70
280
B
6
Tr17/M63
300
B
7
Tr17/M64
258
A
8
Tr17/M71
430
B
9
Tr18/M64
265
B
10
Tr18/M70
310
B
11
Tr18/M78
340
B
12
Tr18/M78
250
B
13
Tr17/M65
160
A
14
Tr17/M66
255
A
15
Tr18/M65
600
A
16
Tr18/M65
365
A
17
Tr18/M65
190
A
18
Tr18/M65
138
A
19
Tr18/M66
380
A
20
Tr18/M66
280
A
21
Tr18/M66
155
A
22
Tr18/M68
160
B
Gene expression profiling: cDNA-AFLP
 22 TDFs analysed as putatively differently expressed genes between the
untransformed and transgenic tobacco plants
 Protein grouping
 Abiotic stress expression
• Aromatic-ring hydroxylase (Flavoprotein monooxygenase)
• C2 calcium/lipid-binding region-containing protein
• S-phase kinase-associated protein 1 (SKP1)-like protein 1A
• Aminotransferase, class v; protein
• Ripening regulated protein DDTFR10-like
• Elongation factor Tu C-terminal domain containing protein
• Putative receptor kinase-like protein, identical
 Biotic stress expression
• Ripening regulated protein DDTFR10-like
• Putative receptor kinase-like protein, identical
• Cystathionine beta-synthase (CBS) protein
Gene expression profiling: cDNA-AFLP
 Protein grouping
 Plant Organ and development expression
• Aromatic-ring hydroxylase (Flavoprotein monooxygenase)
• C2 calcium/lipid-binding region-containing protein
• SKP1-like protein 1A
• Ripening regulated protein DDTFR10-like
• Elongation factor Tu C-terminal domain containing protein
• Non-specific lipid-transfer protein
• Glycoside hydrolase family 47 protein
• Cystine Binding (CBS) protein
 Tissue culture (callus) expression
• Tryptophan/tyrosine permease family protein
• Ankyrin repeat domain-containing protein 2
Gene expression profiling: cDNA-RDA
kb
1
2
3
kb
kb
14.1
5.1
14.1
5.1
2.0
2.0
1.7
1.7
0.3
0.2
0.1
0.3
0.25
0.15
Second difference products (DP2)
0.2
0.1
1
2
3
kb
0.35
0.25
Third difference products (DP3)
Gene expression profiling: cDNA-RDA
Fragment name
T1
T2
B1
B2
Blast x
polygalacturonase-inhibiting protein
[Malus x domestica]
aldehyde reductase
MADS FLC-like protein 3 [Cichorium
intybus]
MADS FLC-like protein 3 [Cichorium
intybus]
Accession
AAB19212.1
AAD53967.1
ACL54967.1
ACL54967.1
Future studies
 qRT-PCR
 Repeat Genome Walking
Acknowledgements
 Agricultural Research Council
 Department of Science and Technology
 AgriSETA
 Dr Dean Oelofse
 Prof Ian Dubery