Download - Genomics of plant defense against insects - UNIL Accueil · Nitrilase (NIT3) At3g44320 2.15
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)
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
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?
?
?
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
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)
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
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.)
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
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
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
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
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
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