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