1. A genomic study on the unintended effects of transformation Lerato B.T. Matsauyane Promoter: Prof I.A. Dubery Co-Promoter:Dr D Oelofse

2. 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

3. 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 1 st class o hypersensitive response (HR) 2 nd class o synthesis of pathogenesis-related (PR) 3 rd class o systemic acquired resistance (SAR) o transcription of a number of plant resistance (R) genes o expression of polygalacturonase inhibiting proteins (PGIPs)

4. 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

5. 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

6. 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

7. 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

8. 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

9. 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

10. 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

11. 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

12. 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

13. 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

14. 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

15. 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

16. 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

17. 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

18. 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

19. 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

20. Molecular screening of transgenics 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 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 genome Lane 1: Lambda PstI Marker Lane 2, 4: Empty Lane 3: PCR of AppBP1 Lane 4: PCR of AppA6 The Mdpgip1 transgene was successfully integrated into the S. tuberosum cv BP1 genome

21. Biochemical screening of transgenics MdPGIP1: VdPG inhibition assay 1: VdPG 2: VdPG + AppA6 PGIP MdPGIP1: VdPG inhibition assay 1: VdPG 2: 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

22. Presence of filler DNA 1 2 3 4 5 6 7 1000 500 300 200 100 bp 300bp 200bp 500bp Fragments extracted and sequenced Promoter TEVMdpgip1 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

23. 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 1.2kb kb  Genome Walking to detect filler DNA

24. 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 7kb AppBP1 AppA6

25. Insertion site of Mdpgip1 Fragment nameAlignmentChromosomes C31B-184-714bp of pCAMBIA2300:appgip1A C31B-2No alignments C31B-3No alignments A31BSolanum tuberosum chloroplast, complete genome Chromosome 02:1:49918294:1 Solanum lycopersicum C32B-11650-1845bp of pCAMBIA2300:appgip1A C32B-2No alignments A32B-1Solanum tuberosum chromosome 6 clone RHPOTKEY030E18 SEQUENCING IN PROGRESS A32B-2PPTIA29TF Solanum tuberosum RHPOTKEY BAC ends Solanum tuberosum genomic clone RHPOTKEY193_F09

26. Insertion site of Mdpgip1

27. NoOC 1 bp * Sample 1Tr15/M70180A 2Tr15/M71205B 3Tr16/M64138A 4Tr16/M64125A 5Tr16/M70280B 6Tr17/M63300B 7Tr17/M64258A 8Tr17/M71430B 9Tr18/M64265B 10Tr18/M70310B 11Tr18/M78340B 12Tr18/M78250B 13Tr17/M65160A 14Tr17/M66255A 15Tr18/M65600A 16Tr18/M65365A 17Tr18/M65190A 18Tr18/M65138A 19Tr18/M66380A 20Tr18/M66280A 21Tr18/M66155A 22Tr18/M68160B Gene expression profiling: cDNA-AFLP 22 differentially expressed fragments isolated cDNA-AFLP gel

28. 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

29. 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

30. kb 1 2 3 kb 14.1 5.1 2.0 1.7 0.3 0.2 0.1 0.25 0.15 kb 1 2 3 kb 14.1 5.1 2.0 1.7 0.3 0.2 0.1 0.35 0.25 Second difference products (DP2) Third difference products (DP3) Gene expression profiling: cDNA-RDA

31. Fragment nameBlast xAccession T1 polygalacturonase-inhibiting protein [Malus x domestica] AAB19212.1 T2aldehyde reductaseAAD53967.1 B1 MADS FLC-like protein 3 [Cichorium intybus] ACL54967.1 B2MADS FLC-like protein 3 [Cichorium intybus] ACL54967.1

32. Future studies  qRT-PCR  Repeat Genome Walking

33. Acknowledgements  Agricultural Research Council  Department of Science and Technology  AgriSETA  Dr Dean Oelofse  Prof Ian Dubery