Using functional genomics to elucidate plant defense networks in poplar

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Using functional genomics to elucidate plant
defense networks in poplar
Steven Ralph, Ph.D. Department of
Biology University of North Dakota
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Ecology of Trees Survival with No Escape in Time
or Space

• During the 150 year lifespan of a poplar tree
  the surrounding world can change dramatically
• Environment (e.g., temperature, drought,
  increased pollution)
• Insect and pathogenic pests (e.g., evolving and
  invasive species)
• Insect pests pose a challenge to the
  sustainability of natural and plantation forests
• Forest insect pest epidemics cannot be addressed
  with short term crop rotation or pesticide
  application, as is possible in agriculture
• Sustainability of our forests can be achieved by
  understanding and utilizing the genetic diversity
  and genome plasticity of our forest trees

Photo credit to Dr. Joerg Bohlmann, University of
British Columbia
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Direct Defense
Plant
Herbivore
Membrane Lipids
PPOs Condensed Tannins Protease Inhibitors
Defense Response
Slide credit to Dr. Joerg Bohlmann, University of
British Columbia
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Indirect Defense
Natural Enemies
Plant
Herbivore
Metabolic Pathways Terpenoid... Oxylipin
... Shikimate...
Volatiles
Membrane Lipids
Direct
Defense Response
Indirect
Slide credit to Dr. Joerg Bohlmann, University of
British Columbia
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Treenomix Genomics Resources
Poplar
Spruce
210,000 ESTs
140,000 ESTs
4,700 FLcDNAs

• Data warehousing analysis
• EST FLcDNA database
• cDNA microarray database

defoliator
stem borer
defoliator
stem borer
White pine weevil
Western spruce budworm
Forest tent caterpillar
Poplar borer
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Ecology of Poplar Trees

• Genus Populus consists of ca. 40 species
• Found in temperature zone
• Fire-following (aspens)
• Riparian (cottonwoods)
• Out-crossing, wind-pollinated and highly
  heterozygous
• Hybridization common in overlapping distribution
  zones
• Short life-span (50-150 years)

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Poplar (Populus spp.) A Model Angiosperm Forest
Tree

• Rapid growth in diverse experimental
  environments
• Relatively short time to reproduction of 7-10
  years
• Facile vegetative propagation
• Facile transformation of many genotypes
• Modest genome size (485 Mb), 4.5x larger than
  Arabidopsis thaliana
• Diploid inheritence
• 7.5x genome sequence coverage complete
• Dense genetic and physical maps

Tuskan, DiFazio, Jansson, Bohlmann, Grigoriev,
Hellsten, Putnam, Ralph et al. (2006) Science
313 1596-1604
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Poplar for Plantation Forestry

• Populus is an ecologically dominant and
  environmentally important species
• Wildlife habitat, stream stabilization,
  filtration of agricultural runoff
• Populus has great economic potential
• Intensively cultivated in plantation forestry
• Low density, short fiber wood used for paper,
  molding and box production
• Future applications include
• Carbon sequestration
• Phytoremediation
• Biofuel (e.g., ethanol) production
• Need for genetic improvement and domestication of
  Populus

10-year-old plantation
3-year-old plntation Photo Credits to William M.
Ciesla, FHMI
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Domestication of Poplar

• Preferred attributes
• Reduced height growth
• Less extensive root system
• Strong apical control narrow, confined crown
  minimal branching
• Non-competitive even at close spacing
• Improved wood chemistry
• Flowering control
• Phytoremediation
• Greater carbon allocation to stem or roots
• Pest resistance

Photo credit to US DOE Genome Program http//genom
ics.energy.gov
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Forest Tent Caterpillar (Malacosoma disstria)

• Forest tent caterpillars are distributed
  throughout the United States and southern Canada,
  hatch in spring, and immediately feed on host
  leaves
• During outbreak periods, which occur every 10-12
  years, they cause extensive defoliation to
  Populus stands, particularly trembling aspen
• By their final instar, larvae grow to over
  1,000x their mass at hatching and consume 15,000x
  their initial body weight in leaf tissue
• During outbreaks trees covering millions of ha.
  are defoliated, with as many as 20,000
  insects/tree
• Although defoliation normally does not kill the
  tree, repeated attacks increase the incidence of
  fungal disease and infestation by other insects

How does a little bug become such a BIG problem?
Defoliated aspen forest Photo credit to William
M. Ciesla, FHMI, Bugwood.org

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Transcript Profiling in Poplar Forest Tent
Caterpillar

• Clonal Populus trichocarpa x deltoides trees
• Leaves harvested 2hrs, 6hrs and 24hrs after
  initiation of treatment
• Transcript profiling using poplar 15.5K cDNA
  microarrys
• 90 trees and 92 slide hybridizations
• Protein profiling using iTRAQ

Methyl jasmonate
Mechanical damage plus regurgitant
Insect herbivory
Mechanical damage
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Source for Natural Defense Elicitors Leads
for Synthetic Derivatives
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Treenomix Poplar 15.5 k cDNA Microarray

• cDNA libraries of clones
• Phloem (FL) 2,439
• Developing xylem (EST) 1,387
• Developing xylem (N-EST) 1,351
• Developing xylem (FL) 368
• Leaf developmental stages and stems (FL) 1,765
  
• Forest tent caterpillar treated leaves
  (EST) 975
• Forest tent caterpillar treated leaves
  (N-EST) 1,013
• Forest tent caterpillar treated leaves
  (FL) 299
• Weevil treated bark (EST) 1,496
• Weevil treated bark (N-EST) 1,691
• Cell cultures / stress treatments (EST) 414
• Cell cultures / stress treatments (N-EST) 434
• Nitrogen stressed roots (EST) 1,360
• Male terminal vegetative buds (EST) 494
• Clones selected from an assembly of 37,000 ESTs
  (3)
• 4,500 clones selected from normalized libraries
• 7,700 clones selected from stress-treated
  libraries, including 5,500 from herbivore stress

15,496 poplar cDNAs
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cDNA Microarray Hybridization
Slide credit to the University of British
Columbia Advanced Molecular Biology Laboratory
http//www.bioteach.ubc.ca
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Transcriptional Response to Caterpillar Herbivory
5 and 95 DE
min./max. DE

• Contrasting temporal patterns
• MeJA and regurgitant responses are rapid, peak
  _at_2h, and dissipate by 24h
• Mechanical wounding response is somewhat slower,
  peaking _at_2-6h, with elevated differential
  expression after 24h
• Herbivory response is weaker at all time points
  and peaks at 24h, which reflects continuous
  feeding by insects

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Genes by Functional Class Responsive to FTC
Herbivory
Aminopeptidase M Apyrase Leucine-rich repeat
transmembrane protein kinase Phosphorylase family
protein Kunitz protease inhibitor Acid
phosphatase Superoxide dismutase copper
chaperone Choline kinase Thymidylate
kinase Glutathione S-transferase Glycosyl
hydrolase Basic endochitinase Lipoxygenase Thiored
oxin Allene oxide synthase Allene oxide
cyclase Metal transporter Lycopene beta
cyclase Cytochrome P450 Lipase Peroxidase Zinc
finger C3H type transcription factors Isoflavone
reductase Jacalin/lectin family
protein (-)-germacrene D synthase ABC
transporter Polyphenol oxidase Major intrinsic
protein Phytoene synthase DAHP synthase Calreticul
in 4-coumarate CoA ligase Carbonic
anhydrase Laccase/diphenol oxidase AP2, WRKY,
bHLH transcription factors ----------------------
------------------------------- Galactinol
synthase Lipid transfer protein Photosystem II
protein Chlorophyl A-B binding protein Ferredoxin
reductase
General metabolism
Transcriptional regulation and signaling
40x
Response to stress
Detoxification/redox processes
Octadecanoid and ethylene signaling
Transport
20x
Secondary Metabolism
Photosynthesis
10x
5x
no change
-2x
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Protein Profiling in Poplar Forest Tent
Caterpillar
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Proteomic Response to Caterpillar Herbivory
Log2(FC)

• 24h time point analyzed
• 3 biological replicates per treatment
• 651 identified proteins
• 71 up-regulated 2-fold over control
• 56 down-regulated 2-fold below control
• protein induction most common in mechanical
  wounding
• protein suppression most common in regurgitant

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Proteomic Response to Caterpillar Herbivory
MW Reg. FTC

• Protein profiles less consistent across
  treatments in comparison to transcript profiles
• 1/3 of proteinstranscripts positively
  correlated, 1/3 of proteinstranscripts
  negatively correlated, 1/3 of proteinstranscripts
  not correlated

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Refined Expression Analysis of Selected Kunitz PI
Genes
Relative expression level
2
6
2
6
2
6
2
6
2
6
2
6
24
24
24
24
24
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Time (hrs)
Untreated Control
Oral Secretions Mechanical Wounding
FTC Herbivory
Tween Control
Mechanical Wounding
Methyl Jasmonate (MeJA)
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Constitutive Expression Analysis of Selected
Kunitz PI Genes
Relative expression level (ln scale)
Tissue Source
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KPI-67?
KPI-44?
KPI-49?
KPI-69?
KPI-74?

KPI-75?

A
KPI-10?
KPI-42?

KPI-43?

KPI-82?

KPI-12??
KPI-68?
KPI-11?

KPI-14?
KPI-13???

KPI-73?
KPI-48?
KPI-16?

KPI-62?

KPI-41?


KPI-39??
KPI-40?

KPI-59?


B
KPI-56?
KPI-45?

KPI-31?

KPI-15?

KPI-46?

KPI-38?

KPI-47?

KPI-51?
Phylogenetic Relationships Among Populus Kunitz
Protease Inhibitors
KPI-37?
KPI-71?

KPI-18?
KPI-63?

KPI-64?
KPI-55?
KPI-54??

KPI-52?

KPI-32?
C
KPI-53?

KPI-78?


KPI-72?
KPI-60?
KPI-50?


KPI-34?
KPI-33?
KPI-17?
KPI-65?

0.1 substitutions per site
KPI-20?
KPI-57?
KPI-2?

KPI-79?

KPI-61?

KPI-35??

KPI-77?
KPI-4??


D
KPI-70??
KPI-3??
KPI-25?


KPI-30?

KPI-27?

KPI-26?

KPI-76?
KPI-5?

KPI-58?

E
KPI-36?
KPI-66?

KPI-19?

KPI-21?

KPI-24?

KPI-22?

KPI-6?

KPI-9?

KPI-80?
F
KPI-28?

KPI-81??
KPI-23?

KPI-29?


KPI-8?
KPI-7?
KPI-1?
CAA45777 G. max
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fuzzy Data Illustrating Insect Resistance

• For petri dish development assays using 1st
  instar larvae
• Larvae on WT disks (black bar) developed through
  4th to 5th instar and gained on average 20.9 mg
• Larvae on fuzzy disks (white bar) developed
  through 3rd to 4th instar and gained on average
  5.6 mg (P lt 0.05 N 8 trees with 3 replicate
  bioassays/tree)
• For caged on tree development assays using 2nd
  instar larvae
• Larvae on WT trees developed through 4th to 5th
  instar and gained 20.6 mg
• Larvae on fuzzy trees developed through 3rd to
  4th instar and gained 13.3 mg (P lt 0.05 N 6
  trees with 3 replicate bioassays/tree)

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fuzzy Data Illustrating Insect Resistance

• For petri dish feeding choice assays using 3rd
  instar larvae, tussock moth larvae forage
  significantly less on fuzzy than WT leaf disks (P
  lt 0.05 N 5 trees with 6 replicate
  bioassays/tree)
• Data from these 3 assays combined demonstrate
• Tussock moth larvae prefer consuming WT leaves
  over fuzzy leaves
• Tussock moth larvae feeding on fuzzy trees
  display substantial developmental retardation

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www.und.edu/instruct/sralph/