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?
Arabidopsis thaliana
Pieterse et al 2009
Plant Defence
CROSSLINKING
OF CELL WALL
PROTEINS
Cl-
K+
O2
O2
H2O2
Insect Pest
Oomycete
Volatiles
Bacterial
P
P
NADPH
OXIDASE
PATHOGEN
Fungal
ELICITORS
PAPILLAE
Ca2+
KINASES
PHOSPHATASES
H+
MAPK
CASCADES
R genes
Herbivory
Cell wall fragments
Secretions in oviposition fluid
ROS
JA
H2O2
ET
ROS
SA
HR RESPONSE
NPR1
TRANSCRITION FACTORS ACTIVATED
DEFENCE GENE
ACTIVATION
Proteinase inhibitors
PPO
LOX
PHYTOALEXIN
ACCUMULATION
LOX, PAL, HEL, PDF 1.2
2ndry
metabolites
e.g. alkaloids
ACCUMULATION OF
DEFENCE PROTEINS
PR 1, 2, & 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.
CROSSLINKING
OF CELL WALL
PROTEINS
Cl-
K+
O2
O2
H2O2
Insect Pest
Oomycete
Bacterial
Volatiles
P
P
NADPH
OXIDASE
PATHOGEN
Fungal ELICITORS
PAPILLAE
Ca2+
KINASES
PHOSPHATASES
H+
MAPK
CASCADE
S
R genes
Herbivory
Cell wall fragments
Secretions in oviposition fluid
ROS
JA
H2O2
ET
ROS
SA
HR RESPONSE
NPR1
TRANSCRITION FACTORS ACTIVATED
DEFENCE GENE
ACTIVATION
Proteinase inhibitors
PPO
LOX
PHYTOALEXIN
ACCUMULATION
LOX, PAL, HEL, PDF 1.2
2ndry
metabolites
e.g. alkaloids
ACCUMULATION OF
DEFENCE PROTEINS
PR 1, 2, & 5
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
E UCALYPTUS
Eucalyptus rust
Paper
Production
Essential Oils
Insect
Invasion
Timber
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.woodreport.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.jpg
http://www-ijpb.versailles.inra.fr/en/sgap/equipes/cyto/images/Planche.gif
Plant Defence
CROSSLINKING
OF CELL WALL
PROTEINS
Cl-
K+
O2
O2
H2O2
Insect Pest
Oomycete
Volatiles
Bacterial
P
P
NADPH
OXIDASE
PATHOGEN
Fungal
ELICITORS
PAPILLAE
Ca2+
KINASES
PHOSPHATASES
H+
MAPK
CASCADE
S
R genes
Herbivory
Cell wall fragments
Secretions in oviposition fluid
ROS
JA
H2O2
ET
ROS
SA
HR RESPONSE
NPR1
TRANSCRITION FACTORS ACTIVATED
DEFENCE GENE
ACTIVATION
Proteinase inhibitors
PPO
LOX
PHYTOALEXIN
ACCUMULATION
LOX, PAL, HEL, PDF 1.2
2ndry
metabolites
e.g. alkaloids
ACCUMULATION OF
DEFENCE PROTEINS
PR 1, 2, & 5
AIM
Identify and characterize putative
orthologs for defence marker genes
(SA and JA) in E. grandis
1. Discovery of marker genes
www.phytozome.net
Bioinformatics Pipeline
Selection of
Eucalyptus
candidates
Gene
prediction
• Arabidopsis target genes
• Eucalyptus genome sequence
• 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 + G
I = 0.105
G = 1.195
Verification of Genes
Gene Name
Signaling Pathway
TBLASTX value
PR 1
2e – 15
PR 2
7e – 15
PR 5
SA
4e - 41
NPR1
6e – 28
PR 3
3e – 37
PR 4
7e - 21
LOX 2
JA
1e – 13
LOX 3
5e – 31
LOX 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
*
10
8
A.U
6
4
*
2
0
Control
25 µM
50 µM
100 µM 250 µM 500 µM
1 mM
5 mM
* Students T-test P < 0.05
MeJA - EgrPR4
3
***
A.U
2
1
0
Control
25 µM
50 µM
100 µM 250 µM 500 µM
1 mM
5 mM
*** Students T-test P < 0.001
SA and JA antagonism
JA
SA
JA
SA
Does this antagonistic
relationship exist in
Eucalyptus?
Specificity of marker genes
EgrPR2
5
SA Marker +
MeJA treated
tissue
A.U
4
3
2
*
1
*
*
0
Control
25 µM
50 µM
100 µM 250 µM 500 µM
1 mM
5 mM
[MeJA]
EgrPR3
5
MeJA Marker +
SA treated
tissue
A.U
4
3
2
*
1
*
0
Control
25 µM
50 µM
100 µM 250 µM 500 µM
[SA]
* Students T-test P < 0.05
1 mM
5 mM
3. Time Course
Grow E. grandis
from tissue culture
Spray with a
selected
concentration
Harvest material
at various time
points
RT-qPCR profiling
SA - EgrPR2
10
**
9
8
7
A.U
6
5
**
4
3
2
**
1
0
0
6
12
24
48
** Students T-test P < 0.01
MeJA - EgrPR4
10
8
6
A.U
*
4
***
2
*
**
0
0
6
12
24
48
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
Infection trial results
TAG 5
lesion
TAG5
8
ZG14
**
EgrPR2
***
6
8
6
4
4
2
2
0
0
48 hrs
8
2 weeks
ZG14
lesion
6 weeks
*
48 hrs
2 weeks
*
8
EgrPR4
6
6 weeks
6
*
4
*
2
*
4
2
0
0
48 hrs
2 weeks
6 weeks
Students T-test * P < 0.05; ** P < 0.01, *** P < 0.001
48 hrs
2 weeks
6 weeks
How do the pieces fit together?
Gene
Discovery
-
Gene ortholog pipeline
-
Candidate defence genes
-
Identified specific
concentrations
-
Elucidated window periods for
improving defence
-
Diagnostic marker genes
Dose Response
Time course
Pathogen
infection
C ONCLUSION
• 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
F UTURE V ISION
Gene expression
profiling of Eucalyptus
challenged with a fungal
pathogen (Chrysoporthe
austroafricana)
Gene expression
profiling of defence
responses in
Eucalyptus during
challenge with an
insect pest
(Leptocybe invasa)
Transcript profiling
of Eucalyptus
challenged with an
oomycete pathogen
(Phytophthora
cinnamomi)
A CKNOWLEDGMENTS

Supervisors and Fellow students

Prof Jolanda Roux

Adri Veale and Grieta Mahlangu
EPPI @
Thank You