1. Eucalyptus Pine Pathogen Interactions

2. Introduction Forest trees – Long – lived – Exposed to array of pathogens – Do not posses adaptive immunity Innate immunity Resistant genotypes required

3. What do we know? Pieterse et al 2009 Arabidopsis thaliana

4. PATHOGEN ELICITORS CROSSLINKING OF CELL WALL PROTEINS MAPK CASCADES TRANSCRITION FACTORS ACTIVATED HR RESPONSE PHYTOALEXIN ACCUMULATION ACCUMULATION OF DEFENCE PROTEINS ROS DEFENCE GENE ACTIVATION ROS PAPILLAE KINASES PHOSPHATASES O2O2 O2O2 H2O2H2O2 Ca 2+ H+H+ Cl - K+K+ H2O2H2O2 P P NADPH OXIDASE SAETJA PR 1, 2, & 5LOX, PAL, HEL, PDF 1.2 R genes Volatiles Proteinase inhibitors PPO LOX 2ndry metabolites e.g. alkaloids Herbivory Cell wall fragments Secretions in oviposition fluid NPR1 Insect Pest Fungal Bacterial Oomycete Plant Defence

5. Strategy Information based on model systems – Arabidopsis thaliana Develop pathosystems Use genomic and transcriptomic resources – Dissect defence responses – Understand host responses Identify key targets to improve resistance.

6. PATHOGEN ELICITORS CROSSLINKING OF CELL WALL PROTEINS MAPK CASCADE S TRANSCRITION FACTORS ACTIVATED HR RESPONSE PHYTOALEXIN ACCUMULATION ACCUMULATION OF DEFENCE PROTEINS ROS DEFENCE GENE ACTIVATION ROS PAPILLAE KINASES PHOSPHATASES O2O2 O2O2 H2O2H2O2 Ca 2+ H+H+ Cl - K+K+ H2O2H2O2 P P NADPH OXIDASE SAETJA PR 1, 2, & 5LOX, PAL, HEL, PDF 1.2 R genes Volatiles Proteinase inhibitors PPO LOX 2ndry metabolites e.g. alkaloids Herbivory Cell wall fragments Secretions in oviposition fluid NPR1 Insect Pest Fungal Bacterial Oomycete

7. A step towards understanding the SA and JA signaling pathways in E. grandis R. Naidoo, Dr. S. Naidoo, Prof AA. Myburg and Prof DK. Berger

8. E UCALYPTUS Paper Production Timber Essential Oils Insect Invasion Eucalyptus rust Eucalyptus Stem Cankers http://scheererbearing.com/uploads/images/feature_pulppaper.jpg; http://topnews.in/health/files/Eucalyptus11.jpg; http://www.scielo.br/img/revistas/gmb/2010nahead/223fig01.jpg http://www.fs.fed.us/r5/spf/fhp/hawaii/eucalyptus%20rust%20on%20ohia.JPG; http://wiki.trin.org.au/pub/Wasps/LeptocybeInvasa/gallformer.jpg; http://www.wood- report.de/bilder/clonal_eucalyptus.jpg; http://2.bp.blogspot.com/_3uQiDL3c53s/Rx5nNpTvf0I/AAAAAAAAAWo/5ubDi6mK9ZQ/s400/Eucalyptus-In-Brazil-Celso-Foelkel-01.jpg;http://2.bp.blogspot.com/_3uQiDL3c53s/Rx5nNpTvf0I/AAAAAAAAAWo/5ubDi6mK9ZQ/s400/Eucalyptus-In-Brazil-Celso-Foelkel-01.jpg

9. Plant Defence  Model Organism  Arabidopsis thaliana http://www.dreamstime.com/puzzle-with-missing-pieces-thumb5179822.jpg http://www-ijpb.versailles.inra.fr/en/sgap/equipes/cyto/images/Planche.gif

10. Plant Defence PATHOGEN ELICITORS CROSSLINKING OF CELL WALL PROTEINS MAPK CASCADE S TRANSCRITION FACTORS ACTIVATED HR RESPONSE PHYTOALEXIN ACCUMULATION ACCUMULATION OF DEFENCE PROTEINS ROS DEFENCE GENE ACTIVATION ROS PAPILLAE KINASES PHOSPHATASES O2O2 O2O2 H2O2H2O2 Ca 2+ H+H+ Cl - K+K+ H2O2H2O2 P P NADPH OXIDASE SAETJA PR 1, 2, & 5LOX, PAL, HEL, PDF 1.2 R genes Volatiles Proteinase inhibitors PPO LOX 2ndry metabolites e.g. alkaloids Herbivory Cell wall fragments Secretions in oviposition fluid NPR1 Insect Pest Fungal Bacterial Oomycete Eucalyptus plant defence

11. Identify and characterize putative orthologs for defence marker genes (SA and JA) in E. grandis AIM

12. 1. Discovery of marker genes www.phytozome.net

13. Bioinformatics Pipeline Selection of Eucalyptus candidates Arabidopsis target genes Eucalyptus genome sequence Gene prediction Prediction software (GenScan and GeneMark) Verification of selected candidates TBLASTX and BLASTP predicted gene against TAIR Phylogenetic analysis Alignment (MAFFT) NJ and ML

14. EgrPR5 WAG + I + G I = 0.105 G = 1.195

15. Verification of Genes

16. 2. Dose Response Grow E. grandis from tissue culture Spray with various concentration Harvest material at 24hrs RT-qPCR profiling

17. SA - EgrPR2 * * * Students T-test P < 0.05

18. MeJA - EgrPR4 *** Students T-test P < 0.001 ***

19. JA SA JA SA SA and JA antagonism Does this antagonistic relationship exist in Eucalyptus?

20. Specificity of marker genes MeJA Marker + SA treated tissue * * * * * SA Marker + MeJA treated tissue * Students T-test P < 0.05 [MeJA] [SA]

21. 3. Time Course Grow E. grandis from tissue culture Spray with a selected concentration Harvest material at various time points RT-qPCR profiling

22. SA - EgrPR2 ** ** Students T-test P < 0.01

23. MeJA - EgrPR4 ** *** * * Students T-test * P < 0.05, ** P < 0.01, *** P < 0.001

24. 4. Infection trial

25. ZG14 lesion TAG 5 lesion Students T-test * P < 0.05; ** P < 0.01, *** P < 0.001 Infection trial results TAG5ZG14 EgrPR2 EgrPR4 *** ** * * * * *

26. How do the pieces fit together?

27. Gene Discovery Dose Response Time course Pathogen infection -Gene ortholog pipeline -Candidate defence genes -Identified specific concentrations -Elucidated window periods for improving defence -Diagnostic marker genes

28. Eucalyptus “puzzle” – Possible diagnostic markers for plant defence – Elucidated the possible role of SA in defence against C. austroafricana – Stepping Stone in understanding Eucalyptus – pathogen interactions http://www.pureskinjunkie.com/sitebuildercontent/sitebuilderpictures/greenLeafPuzzle.jpg C ONCLUSION

29. Gene expression profiling of Eucalyptus challenged with a fungal pathogen (Chrysoporthe austroafricana) Transcript profiling of Eucalyptus challenged with an oomycete pathogen (Phytophthora cinnamomi) Gene expression profiling of defence responses in Eucalyptus during challenge with an insect pest (Leptocybe invasa) F UTURE V ISION

30. A CKNOWLEDGMENTS  Supervisors and Fellow students  Prof Jolanda Roux  Adri Veale and Grieta Mahlangu EPPI @

31. Thank You