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 value

PR 1

SA

2e – 15

PR 2 7e – 15

PR 5 4e - 41

NPR1 6e – 28

PR 3

JA

3e – 37

PR 4 7e - 21

LOX 2 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

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

M0

1

2

3

4

5

6

7

8

9

10

11

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