e ucalyptus p ine p athogen i nteractions
DESCRIPTION
E ucalyptus P ine P athogen I nteractions. Introduction. Forest trees Long – lived Exposed to array of pathogens Do not posses adaptive immunity Innate immunity Resistant genotypes required. What do we know?. Arabidopsis thaliana. Pieterse et al 2009. P. P. Plant Defence. - PowerPoint PPT PresentationTRANSCRIPT
Eucalyptus Pine Pathogen Interactions
Introduction
• Forest trees– Long – lived– Exposed to array of pathogens– Do not posses adaptive immunity
• Innate immunity
• Resistant genotypes required
What do we know?
Pieterse et al 2009
Arabidopsis thaliana
PATHOGEN
ELICITORS
CROSSLINKING OF CELL WALL
PROTEINS
MAPKCASCADES
TRANSCRITION FACTORS ACTIVATED
HR RESPONSE
PHYTOALEXIN ACCUMULATION
ACCUMULATION OF DEFENCE PROTEINS
ROS
DEFENCE GENE ACTIVATION
ROS
PAPILLAE
KINASESPHOSPHATASES
O2 O2 H2O2
Ca2+H+
Cl- K+
H2O2
P P NADPH OXIDASE
SAETJA
PR 1, 2, & 5LOX, PAL, HEL, PDF 1.2
R genes
Volatiles
Proteinase inhibitorsPPOLOX
2ndry metabolitese.g. alkaloids
HerbivoryCell wall fragmentsSecretions in oviposition fluid NPR1
Insect Pest
Fungal
Bacterial
Oomycete
Plant Defence
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.
PATHOGEN
ELICITORS
CROSSLINKING OF CELL WALL
PROTEINS
MAPKCASCADE
S
TRANSCRITION FACTORS ACTIVATED
HR RESPONSE
PHYTOALEXIN ACCUMULATION
ACCUMULATION OF DEFENCE PROTEINS
ROS
DEFENCE GENE ACTIVATION
ROS
PAPILLAE
KINASESPHOSPHATASES
O2 O2 H2O2
Ca2+H+
Cl- K+
H2O2
P P NADPH OXIDASE
SAETJA
PR 1, 2, & 5LOX, PAL, HEL, PDF 1.2
R genes
Volatiles
Proteinase inhibitorsPPOLOX
2ndry metabolitese.g. alkaloids
HerbivoryCell wall fragmentsSecretions in oviposition fluid NPR1
Insect Pest
Fungal
Bacterial
Oomycete
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
EUCALYPTUS
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.jpghttp://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;
Plant Defence
Model Organism
Arabidopsis thaliana
http://www.dreamstime.com/puzzle-with-missing-pieces-thumb5179822.jpghttp://www-ijpb.versailles.inra.fr/en/sgap/equipes/cyto/images/Planche.gif
Plant Defence
PATHOGEN
ELICITORS
CROSSLINKING OF CELL WALL
PROTEINS
MAPKCASCADE
S
TRANSCRITION FACTORS ACTIVATED
HR RESPONSE
PHYTOALEXIN ACCUMULATION
ACCUMULATION OF DEFENCE PROTEINS
ROS
DEFENCE GENE ACTIVATION
ROS
PAPILLAE
KINASESPHOSPHATASES
O2 O2 H2O2
Ca2+H+
Cl- K+
H2O2
P P NADPH OXIDASE
SAETJA
PR 1, 2, & 5LOX, PAL, HEL, PDF 1.2
R genes
Volatiles
Proteinase inhibitorsPPOLOX
2ndry metabolitese.g. alkaloids
HerbivoryCell wall fragmentsSecretions in oviposition fluid NPR1
Insect Pest
Fungal
Bacterial
Oomycete
Eucalyptus plant
defence
Identify and characterize putative
orthologs for defence marker genes
(SA and JA) in E. grandis
AIM
1. Discovery of marker genes
www.phytozome.net
Gene Discovery of defence marker
genes
Primer Design & PCR
Verification of product
Phylogenetic trees
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
EgrPR5
WAG + I + GI = 0.105G = 1.195
Verification of Genes
Gene Name Signaling Pathway TBLASTX valuePR 1
SA
2e – 15PR 2 7e – 15PR 5 4e - 41
NPR1 6e – 28 PR 3
JA
3e – 37PR 4 7e - 21
LOX 2 1e – 13LOX 3 5e – 31LOX 5 6e – 41
2. Dose Response
Grow E. grandis from tissue culture
Spray with various concentration
Harvest material at 24hrs
RT-qPCR profiling
SA - EgrPR2
Control 25 µM 50 µM 100 µM 250 µM 500 µM 1 mM 5 mM0
2
4
6
8
10
A.U
*
*
* Students T-test P < 0.05
MeJA - EgrPR4
Control
25 µM50 µM
100 µM
250 µM
500 µM1 m
M5 m
M0123456789
1011
A.U
*** Students T-test P < 0.001
***
JA
SAJA
SA
SA and JA antagonism
Does this antagonistic relationship exist in
Eucalyptus?
Specificity of marker genes
Control
25 µM50 µM
100 µM
250 µM
500 µM1 m
M5 m
M0
1
2
3
4
5
EgrPR2
A.U
Control
25 µM50 µM
100 µM
250 µM
500 µM1 m
M5 m
M0123456789
1011
EgrPR3
A.U
MeJA Marker + SA treated
tissue
* **
* *
SA Marker + MeJA treated
tissue
* Students T-test P < 0.05
[MeJA]
[SA]
3. Time Course
Grow E. grandis from tissue culture
Spray with a selected
concentration
Harvest material at various time
points
RT-qPCR profiling
0 6 12 24 480
1
2
3
4
5
6
7
8
A.U
SA - EgrPR2
**
**
**
** Students T-test P < 0.01
0 6 12 24 480
2
4
6
8
10
A.U
MeJA - EgrPR4
******
*
Students T-test * P < 0.05, ** P < 0.01, *** P < 0.001
4. Infection trial
Infect TAG5 and ZG14 with C.
austroafricana
Monitor plants for the development
of lesions
Harvest material at selected time
points
RT-qPCR profiling
ZG14 lesion
TAG 5 lesion
Students T-test * P < 0.05; ** P < 0.01, *** P < 0.001
Infection trial results
48 hrs 2 weeks 6 weeks0
2
4
6
48 hrs 2 weeks 6 weeks0
2
4
6
8
48 hrs 2 weeks 6 weeks0
2
4
6
8
48 hrs 2 weeks 6 weeks0
2
4
6
TAG5 ZG14
EgrPR2
EgrPR4
*****
*
*
*
*
*
How do the pieces fit together?
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
• 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
CONCLUSION
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)
FUTURE VISION
ACKNOWLEDGMENTS
Supervisors and Fellow students
Prof Jolanda Roux
Adri Veale and Grieta Mahlangu
EPPI @
Thank You