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

Leaf miners

Fungus gnatGall forming insects

Induced plant responses to herbivory

Rootworm

Volatile signals

Parasitoidwasps

Entomopathogenicnematodes

Transcriptionalchanges

Global gene expression profiling in plant/insect interactions

Are insects specifically detected by the plant?What are the induced genes and signaling pathways?

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3-5 hours withbig larvae

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

6-8 days withneonate larvae

Pieris rapae

Spodoptera littoralis

Defense proteinsLectinsCysteine proteinasePhenolics and lignin synthesisProtease inhibitorsStrictosidine synthaseTerpene synthaseIndole glucosinolates synthesisVSPs

Reallocation of ressourcesBeta-fructosidasesBeta-amylaseHexose transporterGalactinol synthase

Detoxification, redox processesGlutathione-S-transferasesDehydroascorbate reductasesSerine acetyl transferase (cysteine biosynthesis)Germin (SOD)ThioredoxinOxidoreductases

Unknownca 20% (half of them are plant specific genes)

SignalingJasmonate synthesisCalmodulinCalcium-binding proteinsCDPKPhosphatasesKinases

Transcription factorsERF/AP2sbHLHsC2H2 zinc finger proteinsMYBs NAM-like protein, NAC-like proeinWRKYsHSFs

Abiotic stressAquaporinsTonoplast integral proteinDehydrins (e.g. ERD10)Ribonuclease (RNS1)Imbibition proteinHSP70

Insect-induced genes (2-5 % ot total Arabidopsis transcriptome)

TransportABC transportersMATE transporters

ca. 500-1500 genes

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

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Transcriptionalchanges

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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

Linolenic acid

13-HPOTRE

OPDA

OPC-8:0

OPC-6:0

OPC-4:0

Jasmonic acid

LOX2

OPR3

AOSAOC

ACX1

JA responses

COI1

JA-biosynthesis genes and jasmonates accumulate during herbivory

P. rapaeCTL

nmol

/g F

Wcoi1-1 mutant is insensitive to JA

Gene Identification and Putative Function AGI Code Wild Type P Value coi1-1 P Value

Defense protein

Lectin At3g16400 9.14 <0.001 1.15 0.615

Cysteine proteinase At4g11320 4.35 <0.001 1.07 0.780

Vegetative storage protein (VSP2) At5g24770 6.36 <0.001 0.94 0.639

Indole glucosinolate metabolism

Anthranilate synthase At5g05730 2.62 <0.001 1.08 0.113

Tryptophan synthase alpha subunit At3g54640 2.11 <0.001 1.10 0.477

Cytochrome P450 (CYP79B2) At4g39950 3.37 <0.001 1.07 0.285

Cytochrome P450 (CYP83B1) At4g31500 3.71 <0.001 1.55 0.210

Myrosinase-associated protein, putative At1g54010 2.36 <0.001 1.05 0.644

Phenolic metabolism

Chorismate mutase At5g22630 2.23 <0.001 1.37 0.095

Prephenate dehydratase At3g44720 2.02 0.003 1.65 0.069

Phenylalanine ammonia lyase (PAL1) At2g37040 2.92 0.002 1.41 0.772

Oxylipin metabolism

Lipoxygenase (LOX2) At3g45140 3.21 <0.001 1.01 0.560

Allene oxide synthase (AOS) At5g42650 5.99 <0.001 1.05 0.334

12-oxophytodieonate reductase (OPR3) At2g06050 4.31 <0.001 1.23 0.482

Hydroperoxide lyase (HPL) At4g15440 2.30 0.004 1.01 0.443

Hormone biosynthesis

Nitrilase (NIT3) At3g44320 2.15 <0.001 1.01 0.932

IAA-Ala hydrolase (IAR3) At1g51760 6.97 <0.001 1.13 0.283

Detoxification, redox processes

Glutathione S-transferase (GST5) At2g29450 5.70 <0.001 1.13 0.345

GSH-dependent dehydroascorbate reductase At1g19570 6.01 <0.001 1.20 0.369

Thioredoxin At1g45145 2.17 0.004 1.23 0.234

Abiotic Stress

Aquaporin At2g37180 2.81 0.001 1.31 0.147

Tonoplast integral potein At3g16240 2.83 <0.001 1.20 0.425

Reallocation of resources

Hexose transporter At5g26340 2.32 0.005 1.64 0.085

Galactinol synthase At2g47180 2.81 <0.001 0.94 0.605

Signal transduction

Transducin (WD40 repeat protein) At1g04140 2.05 0.001 1.16 0.389

Transcription factors

bHLH protein (AtMYC2) At1g32640 3.59 <0.001 1.54 0.009

MYB-related protein At5g67300 2.84 0.001 1.70 0.015

Jasmonic acid (JA) is a major signal in plant-insect interactions

Fold change Fold change

In Arabidopsis 60 to 70% of insect-regulated genes depend on the JA pathway

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+

Role of inducible secondary metabolitesGlucosinolates (GS) in Brassicaceae

Indole GS Aliphatic GS

Spodoptera littoralis caterpillars feeding on Arabidopsis plants

Arabidopsis mutants lacking GS (cyp79B2/B3, gsm1-1)or mutant not able to respond to insect attack (coi1-1)

Indole GS

Aliphatic GS

CTL

Spodoptera littoralis

Role of glutathione (GSH)

Schlaeppi et al. (2008) Plant Journal 55, 774

pad2-1 has 20% of WT GSH

GSH1 = PAD2

GSH2

GSSG

GSH

pad2-1 is not affected in its transcriptional response to S. littoralis

CTL

Spodoptera littoralis

pad2-1 accumulates less glucosinolates in response to S. littoralis

GSH as a sulfur donor ?

Plant responses to insect eggs

Trancriptional changes

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?

Detection of oviposition by plants

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

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

Little et al. (2007) Plant Physiology 143, 784

Table I. Functional classification of genes regulated by P. brassicae oviposition using GO categories

Number of genesBiological process Up-regulated Down-regulated

Response to abiotic or biotic stimulus 125 48Response to stress 86 31Protein metabolism 113 65Transport 67 18Transcription 45 18

Developmental processes 29 21Electron transport or energy pathways 37 19Cell organization and biogenesis 33 29Signal transduction 34 24DNA or RNA metabolism 2 1Other processes 318 162Biological process unknown 201 145

24 h 48 h 72 h

303 416 671

53 123 426

Hours after oviposition

Nb of genes up-regulated

Nb of genes down-regulated

7810

688442

33028

P. brassicae eggs766452

P. rapae herbivory40838Up-regulated genes

Down-regulated genes

Oviposition vs herbivory

Selected egg-induced genes

Trypan blue (cell death)

DAB (H2O2)Aniline blue (callose)

Oviposition causes cytological changes associated with cell death

Oviposition on Brassica oleracea and Eruca sativa plants triggers necrosis

Bruessow and Reymond (2007) Plant Signaling & Behavior 2, 165

Egg chorion(proteins, wax, carbohydrates)

Terpenes, alkaloids, fatty acids, aldehydes

Egg cement(composition unknown)

Egg composition

Accessory glandsEgg massEgg extract

P. rapae 72 hP. brassicae 48 h P. brassicae 72 h

Egg SN

PR1::GUS activation

Chicken egg white

Chicken yolk

P. brassicaeegg SN

Extract of 24h-oldP. brassicae larvae

Empty eggshells

P. brassicaeegg SN

Spodoptera littoralisegg SN

P. brassicaeegg SN

Drosophila melanogasteregg SN

P. brassicaeegg SN

ca 109 cfu per spot

P. b. egg SN

Colony 1

Colony 2

Colony 3

Egg-associated bacteria?

Specificity of PR1::GUS activation

A

JA

B C D

Sets of defense genes

ABA ?

SAET

Insect-derived elicitors and suppressors

Mechanical wounding+

eggs

Cell death genes

?