Genomics of plant defense against insects - UNIL
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Genomics of plant defense against insects Dr. Philippe Reymond, MER
Transcript of Genomics of plant defense against insects - UNIL
Genomics of plant defense against insects
Dr. Philippe Reymond, MER
From: Insect-Plant Biology, Schoonhoven, Jermy & van Loon (1998)
Herbivorous insects are abundant on earth and cause severe damage to crops
Proportion of plant and animal species excluding fungi, algae and microbes
Biomass of ants compared to biomass of vertebrates (4 to 1)Insects to vertebrates (9 to 1)
Leaf mining
Piercing/sucking
Gall forming
Chewing
Several types of insect damage
Constitutive defenses against herbivores
Constitutive defenses
Secondary metabolites
SpinesThick cuticle
Mimetism
Hairs
Cyanogenic glycosides
Amygdalin from almond
GlucC
R'
R
CN
O beta-glucosidase
CR'
R
CN
OH lyaseC
R'
R
O + HCN
Toxic degradation products
Glucosinolates(GS)
R comes from Met: aliphatic GSR comes from Trp: indole GSR comes from Phe, Tyr: aromatic GS
Glucoraphanin from cauliflower
Ion intensity map of 4MSOB
Non uniform distribution of glucosinolates in leaves
Shroff et al. (2008) PNAS 105, 6196
Inducible defenses against herbivores
Protease inhibitor
Manduca sexta
WT
acx1
WT acx1
Tomato
Induced defenses
Maize rootworm(Diabrotica virgifera)
Induced indirect defenses
NematodeHeterorhabditis megidis
Cotesia marginiventrislaying eggs on Spodopteralittoralis
Parasitic wasps
Transcriptionalchanges
Global gene expression profiling in plant/insect interactions
Are insects specifically detected by the plant?What are the induced genes and signaling pathways?
?
?
Harvest damaged leavesExtract RNAHybridize to microarrays
Experimental protocol
Arabidopsis thaliana
Plant/Insect Microarray Reference
Arabidopsis/Aphids 500 ESTs Moran et al. (2002) Arch Insect Biochem PhysiolLima bean/Spider mite 2032 ESTs Arimura et al. (2000) Biochem Biophys Res CommNicotiana/Caterpillar 241 cDNAs Hui et al. (2003) Plant PhysiologyNicotiana/Caterpillar 11'000 cDNAs Schmidt et al. (2005) Plant PhysiologyArabidopsis/Caterpillar 150-12'000 ESTs Reymond et al. (2000, 2004) Plant CellArabidopsis/insects Affymetrix ATH01 De Vos et al. (2005) MPMI
Challenge with larvae
Pieris rapae
Spodoptera littoralis
A conserved transcript pattern in response to a specialist and a generalist herbivoreReymond et al. (2004) Plant Cell 16: 3132-3147
Transcriptional responses to different insects are not similar
Comparison of trancript profiles between pathogens and insects
Bacterial pathogen
Fungal pathogen
Lepidopteran herbivore
Thrips
Aphid
Insects
De Vos et al. (2005) Mol Plant-Microbe Interact 18, 923
Comparison of gene expression profilesbetween insect feeding and mechanical wounding
vs
Transcriptionalchanges
?
Transcriptionalchanges
?
CATMA 25K microarray> 4 biological replicates
Gene expression changes after insect herbivory or mechanical wounding in Arabidopsis
No change in gene exprressionSimilar expression in response to insects and woundingDifferent expression in response to insects and wounding(statistically significant)
Activation of defense genes by insect oral secretion
Suppression of defense genes by insect oral secretion
Insect-derived elicitors
Fragments of chloroplastic ATP synthasein oral secretionSchmelz et al. (2006) PNAS 103, 8894
All these compounds induce plant defense responses
In oral secretion of grasshopperAlborn et al. (2007) PNAS 104, 12976
In oral secretion of lepidopteran larvaeHalitschke et al. (2001) Plant Physiology 125, 711
Found in eggs of pea weevilDoss et al. (2000) PNAS 97,6218
Howe and Jander (2008) Annu. Rev. Plant Biol. 59, 41
Signaling of defense responses
e.g.LOX2VSP
e.g.PDF1.2PR1
e.g.HELCHIB1
e.g.PR1 Defense genes
JAJA SA
e.g.PR5
Aggressors (insects/wounding, fungi, bacteria, viruses)
Signals involved in defense
JA: jasmonic acidSA: salicylic acidET: ethylene
ET
0 60 120
2
4
6
8Ja
smon
ic ac
id (
µg/g
)
Time (min)
Jasmonic acid
Jasmonic acid accumulates in response to herbivory
A conserved transcript pattern in response to a specialist and a generalist herbivoreReymond et al. (2004) Plant Cell 16: 3132-3147
Spodoptera littoralis
WT coi1-1 WT coi1-1
Spodoptera littoralis larvae feeding for 10 days on WT or the jasmonate-insensitive coi1-1 mutant
Similar effect of COI1 in other species (tomato, Nicotiana sp.)
A
JA
B C D
Sets of defense genes
Insect-derived elicitors and suppressors
Mechanical wounding+
Salicylic acid (SA) and ethylene (ET) do not play a major role in the induction of insect-responsive genes
sid2-1 : Arabidopsis mutant that lacks SA ein2-1 : Arabidopsis mutant that is insensitive to ET
Role of SA and ET in resistance to Spodoptera littoralis
npr1 mutant does not respond to SA
Abscisic acid (ABA) modulates the expression of some insect-inducible genes
Bodenhausen and Reymond (2007) Mol Plant-Microbe Interact 20, 1406
aba2-1 makes only 10% of abscisic acid
Spodoptera littoralis
Role of ABA
A
JA
B C D
Sets of defense genes
ABA ?
SAET
?
Insect-derived elicitors and suppressors
Mechanical wounding+
The specialist P. brassicae is adapted to Arabidopsis defenses
Schlaeppi, Bodenhausen et al. (2008)
Plant responses to insect eggs
Plant responses to oviposition
- Weevil eggs induce neoplasm growth on pea pods (Doss et al. 2000)
- Eggs induce the release of plant volatiles that attract egg parasitoids (Hilker et al. 2002, Meinersand Hilker 2000)
Pine sawfly (Diprion pini )Egg parasitoid (Chrysonotomia ruferum)
Elm leaf beetle (Xanthogaleruca luteola) Egg parasitoid(Oomyzus galleruca)
Williams And Gilbert (1981) Science 212, 467
Heliconius cydno
Resistant to cynogenic glycosides
Passiflora sp.
Egg mimicry reduces egg laying by butterflies
Defense genes ?
Arabidopsis responses to insect eggs
?
Pieris rapae
Pieris brassicae
Arabidopsis leaf discs were collected 24 h, 48 h, and 72 h after oviposition by Pieris rapae or P. brassicae. RNA was extracted, amplified, and hybridized to CATMA arrays.
Analysis of expression changes after oviposition
Oviposition by pierid butterflies triggers defense responses in ArabidopsisLittle et al. (2007) Plant Physiology 143: 784-800
P. brassicae eggs P. rapae herbivory
Oviposition vs herbivory
Overlap of expression profiles
only 10%
Egg chorion(proteins, wax, carbohydrates)
Terpenes, alkaloids, fatty acids, aldehydesEmbryo
Egg cement(composition unknown)
Egg composition
Accessory glands
P. rapae 72 hP. brassicae 48 h P. brassicae 72 h
Activation of PR1::GUS
Insect eggs contain generic elicitors that activate gene expression
Pieris brassicae
Spodoptera littoralis
Drosophila melanogaster
Egg extract
Arabidopsis reporter lines
Egg glue alone, if the eggs are removed afteroviposition, fails to induce PR1::GUS
Eggshell afterhatching
Proteinase K-treatedegg extract Lipid extract < 3kDa
Characterization of the egg elicitor
EFR FLS2 CERK1 ?
BacteriaFungi
Eggs
Viruses
??
?
Caterpillars
??
Defenses
~600 receptor-like kinases in Arabidopsis thaliana
EF-Tuflagellin
chitin
Plant cell
Detection of microbe-associated molecular patterns (MAMPs)
P. b. 72 h flg 22 elf 26 AGI ECD Subfamily Gene Symbol Ratio
At5g47850 CR4L CR4L 2.68 + +At4g23200 DUF26 DUF26 2.53At4g23220 TKT DUF26 2.21At4g23190 DUF26 DUF26 RLK3 2.67 + +At4g23150 DUF26 DUF26 6.19At3g45860 DUF26 DUF26 2.25At4g23260 DUF26 DUF26 2.42At4g23320 DUF26 DUF26 2.13 +At4g23210 DUF26 DUF26 2.64 + +At4g04490 DUF26 DUF26 5.35At4g04500 DUF26 DUF26 4.85At4g11890 K DUF26 3.98 +At2g37710 LEC L-Lectin LRK1 2.92At3g53810 LEC L-Lectin 2.24 + +At5g01540 LEC L-Lectin 5.80 + +At5g60280 LEC L-Lectin 5.72At2g32800 K L-Lectin 1.82 + +At1g66880 LRKL LRK10L-1 3.95 + +At5g15730 K LRR I 2.26 + +At1g51890 LRR3 LRR I 3.93 + +At5g45800 LRR7 LRR VII 3.63At3g14840 LRR9 LRR VIII-2 2.03At3g09010 K LRR VIII-2 2.72 +At1g16670 K LRR VIII-2 2.02 + +At5g48380 LRR4 LRR X 2.47 + +At5g42440 K LRR X 2.04At1g74360 LRR20 LRR X 2.43 + +At4g28490 LRR21 LRR XI HAESA 2.47At1g09970 LRR19 LRR XI 2.86At5g25930 LRR22 LRR XI 3.32 + +At3g47090 LRR22 LRR XII 2.12At1g35710 LRR27 LRR XII 1.77At4g39270 LRR10 N.A. 2.89At5g58540 K RLCK I 3.18At2g39660 K RLCK VII 2.06 + +At1g14370 K RLCK VII APK2a 2.74 + +At4g35600 K RLCK VII 2.12 + +At5g47070 K RLCK VII 1.72 + +At5g51770 SK RLCK XI 1.93At1g61380 SD SD-1 2.11 +
At4g18250 THN Thaumatin 4.20 + +
Oviposition-induced RLK genes
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
NPP
Looking for the receptor of egg-derived elicitors
SA accumulates under the eggs
Local
Distal
Salicylic acid
Fertility: 96.6% on Col-096.5% on parafilm
No effect of the plant substrate on egg development
Hatching time
Eggs induce necrosis
A et B : Brassica oleraceaC: Eruca sativa
Arabidopsis thaliana
Defense genes
?
Role of egg detection for plant defense against herbivores
SA
?
Egg extract
5 days
S. littoralis
8 daysMeasurementof larval weight
5 days 8 daysMeasurementof larval weight
S. littoralis
2 days
2 days
qPCR on insect-responsive genes
qPCR on insect-responsive genes
Testing the effect of oviposition on plant defense
Treatment with egg extract suppresses defense gene expressionand enhances larval performance
ControlEgg extract
Spodoptera littoralis
+ Egg extract
JA
Defense genes
Defense genes
?SA
Egg-mediated negative SA/JA cross-talk
Manipulation of plant defense signaling patways by the eggs
