A genomic study on the unintended effects of transformation

Lerato B.T. Matsauyane

Promoter: Prof I.A. DuberyCo-Promoter: 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 classo hypersensitive response (HR)

• 2nd classo synthesis of pathogenesis-related (PR)

• 3rd classo 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

Tan discoloration of the vascular tissue of the stems

Chlorosis and necrosis of the leaves as well as wilting

Tan discoloration of the vascular tissue of the roots

Introduction-Verticillium Wilt

Pathogenesis cycle

Microsclerotia germinate and infect plants through the roots

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

Microsclerotia germination

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

PCR screening to verify the presence of the nptII gene within the transgenic genomeLane 1: Lambda PstI MarkerLane 2: EmptyLane 3: PCR of AppBP1Lane 4: PCR of AppA6

14.1 2.8

0.8 0.6kb

1 2 3 4

14.1

4.0

1.2

1 2 3 4 5

1.0kb

PCR screening to verify the presence of the Mdpgip1 gene within the transgenic genomeLane 1: Lambda PstI MarkerLane 2, 4: EmptyLane 3: PCR of AppBP1Lane 4: PCR of AppA6

The Mdpgip1 transgene was successfully integrated into the S. tuberosum cv BP1 genome

Biochemical screening of transgenics

MdPGIP1: VdPG inhibition assay1: VdPG2: VdPG + AppA6 PGIP

MdPGIP1: VdPG inhibition assay1: VdPG2: VdPG + AppBP1 PGIP

1 2 1 2

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

1 2 3 4 5 6 7

1000

 

500 300 200 100

bp

300bp

200bp

500bp 500bp

      

 

 

 

Fragments extracted and sequenced

Promoter TEV Mdpgip1 Terminator

300bp fragment contained 35S CaMV promoter and TEV leader sequences

600bp fragments contained 35S CaMV promoter, TEV leader and Mdpgip1 sequences

The Mdpgip1 expression cassette is present in the transgenic AppA6

Genome Walking to detect Mdpgip1 expression cassette

Presence of filler DNA

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

1 2 3 4 5 6 7 8

14.1 5.1 2.5

  1.2  

5.1kb 5.1kb

1.2kb 1.2kb

kb

Genome Walking to detect filler DNA

Insertion site of Mdpgip1 Oligonucleotides designed at the left and right borders

Restriction enzymes used – EcoRV, SacI, SmaI, StuI

14.1 5.1 2.5

  1.2  

1 2 3 4 5 6 7 kb

AppBP1 AppA6

Insertion site of Mdpgip1

Fragment name Alignment Chromosomes

C31B-1 84-714bp of pCAMBIA2300:appgip1A

C31B-2 No alignments

C31B-3 No alignments

A31B Solanum tuberosum chloroplast, complete genome

Chromosome 02:1:49918294:1Solanum lycopersicum

C32B-1 1650-1845bp of pCAMBIA2300:appgip1A

C32B-2 No alignments

A32B-1 Solanum tuberosum chromosome 6 clone RHPOTKEY030E18

SEQUENCING IN PROGRESS

A32B-2 PPTIA29TF Solanum tuberosum RHPOTKEY BAC ends Solanum tuberosum genomic clone RHPOTKEY193_F09

Insertion site of Mdpgip1

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 differentially expressed fragments isolatedcDNA-AFLP gel

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

kb 1 2 3 kb

14.1 5.1 2.0

1.7

0.3

0.2 0.1

0.250.15

kb 1 2 3 kb

14.1 5.1 2.0

1.7

0.3

0.2 0.1

0.350.25

Second difference products (DP2) Third difference products (DP3)

Gene expression profiling: cDNA-RDA

Gene expression profiling: cDNA-RDA

Fragment name Blast x Accession T1 polygalacturonase-inhibiting protein

[Malus x domestica]AAB19212.1

T2 aldehyde reductase AAD53967.1B1 MADS FLC-like protein 3 [Cichorium

intybus]ACL54967.1

B2 MADS FLC-like protein 3 [Cichorium intybus]

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