Genomics of plant defense against insects. Dr. Philippe Reymond, MER

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1 Genomics of plant defense against insects Dr. Philippe Reymond, MER

2 Herbivorous insects are abundant on earth and cause severe damage to crops Biomass of ants compared to biomass of vertebrates (4 to 1) Insects to vertebrates (9 to 1) Proportion of plant and animal species excluding fungi, algae and microbes From: Insect-Plant Biology, Schoonhoven, Jermy & van Loon (1998)

3 Gall forming Leaf mining Several types of insect damage Chewing Piercing/ sucking

4 Constitutive defenses against herbivores

5 Spines Constitutive defenses Thick cuticle Hairs Secondary metabolites Mimetism

6 Cyanogenic glycosides Gluc N O C C R R' beta-glucosidase N OH C C R R' lyase O C R R' + HCN Amygdalin from almond Glucosinolates (GS) R comes from Met: aliphatic GS R comes from Trp: indole GS R comes from Phe, Tyr: aromatic GS Glucoraphanin from cauliflower Toxic degradation products

7 Non uniform distribution of glucosinolates in leaves Ion intensity map of 4MSOB Shroff et al. (2008) PNAS 105, 6196

8 Inducible defenses against herbivores

9 Induced defenses Tomato Protease inhibitor WT acx1 WT Manduca sexta acx1

10 Induced indirect defenses Parasitic wasps Cotesia marginiventris laying eggs on Spodoptera littoralis Nematode Heterorhabditis megidis Maize rootworm (Diabrotica virgifera)

11 Global gene expression profiling in plant/insect interactions Are insects specifically detected by the plant? What are the induced genes and signaling pathways??? Transcriptional changes

12 Experimental protocol Pieris rapae Arabidopsis thaliana Challenge with larvae Spodoptera littoralis Harvest damaged leaves Extract RNA Hybridize to microarrays Plant/Insect Microarray Reference Arabidopsis/Aphids 500 ESTs Moran et al. (2002) Arch Insect Biochem Physiol Lima bean/spider mite 2032 ESTs Arimura et al. (2000) Biochem Biophys Res Comm Nicotiana/Caterpillar 241 cdnas Hui et al. (2003) Plant Physiology Nicotiana/Caterpillar 11'000 cdnas Schmidt et al. (2005) Plant Physiology Arabidopsis/Caterpillar '000 ESTs Reymond et al. (2000, 2004) Plant Cell Arabidopsis/insects Affymetrix ATH01 De Vos et al. (2005) MPMI

13 A conserved transcript pattern in response to a specialist and a generalist herbivore Reymond et al. (2004) Plant Cell 16:

14 Transcriptional responses to different insects are not similar

15 Comparison of trancript profiles between pathogens and insects Bacterial pathogen Fungal pathogen Lepidopteran herbivore Thrips Insects Aphid De Vos et al. (2005) Mol Plant-Microbe Interact 18, 923

16 Comparison of gene expression profiles between insect feeding and mechanical wounding? vs? Transcriptional changes Transcriptional changes

17 Gene expression changes after insect herbivory or mechanical wounding in Arabidopsis No change in gene exprression Similar expression in response to insects and wounding Different expression in response to insects and wounding (statistically significant) CATMA 25K microarray > 4 biological replicates

18 Activation of defense genes by insect oral secretion

19 Suppression of defense genes by insect oral secretion

20 Insect-derived elicitors All these compounds induce plant defense responses In oral secretion of lepidopteran larvae Halitschke et al. (2001) Plant Physiology 125, 711 In oral secretion of grasshopper Alborn et al. (2007) PNAS 104, Fragments of chloroplastic ATP synthase in oral secretion Schmelz et al. (2006) PNAS 103, 8894 Found in eggs of pea weevil Doss et al. (2000) PNAS 97,6218 Howe and Jander (2008) Annu. Rev. Plant Biol. 59, 41

21 Signaling of defense responses

22 Signals involved in defense Aggressors (insects/wounding, fungi, bacteria, viruses) JA ET SA JA: jasmonic acid SA: salicylic acid ET: ethylene Defense genes e.g. LOX2 VSP e.g. PDF1.2 PR1 e.g. PR5 e.g. HEL CHIB1 e.g. PR1

23 Jasmonic acid accumulates in response to herbivory Jasmonic acid (µg/g) Jasmonic acid Time (min)

24 A conserved transcript pattern in response to a specialist and a generalist herbivore Reymond et al. (2004) Plant Cell 16:

25 Spodoptera littoralis Spodoptera littoralis larvae feeding for 10 days on WT or the jasmonate-insensitive coi1-1 mutant WT coi1-1 WT coi1-1 Similar effect of COI1 in other species (tomato, Nicotiana sp.)

26 Insect-derived elicitors and suppressors + Mechanical wounding JA A B C D Sets of defense genes

27 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

28 Role of SA and ET in resistance to Spodoptera littoralis npr1 mutant does not respond to SA

29 Abscisic acid (ABA) modulates the expression of some insect-inducible genes aba2-1 makes only 10% of abscisic acid Bodenhausen and Reymond (2007) Mol Plant-Microbe Interact 20, 1406

30 Role of ABA Spodoptera littoralis

31 Insect-derived elicitors and suppressors SA ET + Mechanical wounding JA ABA?? A B C D Sets of defense genes

32 The specialist P. brassicae is adapted to Arabidopsis defenses Schlaeppi, Bodenhausen et al. (2008)

33 Plant responses to insect eggs

34 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, Meiners and Hilker 2000) Elm leaf beetle (Xanthogaleruca luteola) Egg parasitoid (Oomyzus galleruca) Pine sawfly (Diprion pini ) Egg parasitoid (Chrysonotomia ruferum)

35 Egg mimicry reduces egg laying by butterflies Passiflora sp. Heliconius cydno Resistant to cynogenic glycosides Williams And Gilbert (1981) Science 212, 467

36 Arabidopsis responses to insect eggs? Defense genes?

37 Pieris rapae Pieris brassicae

38 Analysis of expression changes after oviposition 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.

39 Oviposition by pierid butterflies triggers defense responses in Arabidopsis Little et al. (2007) Plant Physiology 143:

40 Oviposition vs herbivory Overlap of expression profiles P. brassicae eggs only 10% P. rapae herbivory

41 Egg composition Egg chorion (proteins, wax, carbohydrates) Terpenes, alkaloids, fatty acids, aldehydes Embryo Egg cement (composition unknown)

42 Activation of PR1::GUS P. brassicae 48 h P. brassicae 72 h P. rapae 72 h Accessory glands

43 Insect eggs contain generic elicitors that activate gene expression Pieris brassicae Egg extract Spodoptera littoralis Drosophila melanogaster Arabidopsis reporter lines

44 Characterization of the egg elicitor Egg glue alone, if the eggs are removed after oviposition, fails to induce PR1::GUS Eggshell after hatching Proteinase K- treated egg extract Lipid extract < 3kDa

45 Detection of microbe-associated molecular patterns (MAMPs) Bacteria Fungi Eggs EF-Tu flagellin chitin? Viruses Caterpillars? EFR FLS2 CERK1???? Defenses Plant cell ~600 receptor-like kinases in Arabidopsis thaliana

46 Oviposition-induced RLK genes P. b. 72 h flg 22 NPP elf 26 AGI ECD Subfamily Gene Symbol Ratio At5g47850 CR4L CR4L At4g23200 DUF26 DUF At4g23220 TKT DUF At4g23190 DUF26 DUF26 RLK At4g23150 DUF26 DUF At3g45860 DUF26 DUF At4g23260 DUF26 DUF At4g23320 DUF26 DUF At4g23210 DUF26 DUF At4g04490 DUF26 DUF At4g04500 DUF26 DUF At4g11890 K DUF At2g37710 LEC L-Lectin LRK At3g53810 LEC L-Lectin At5g01540 LEC L-Lectin At5g60280 LEC L-Lectin 5.72 At2g32800 K L-Lectin At1g66880 LRKL LRK10L At5g15730 K LRR I At1g51890 LRR3 LRR I At5g45800 LRR7 LRR VII 3.63 At3g14840 LRR9 LRR VIII At3g09010 K LRR VIII At1g16670 K LRR VIII At5g48380 LRR4 LRR X At5g42440 K LRR X 2.04 At1g74360 LRR20 LRR X At4g28490 LRR21 LRR XI HAESA 2.47 At1g09970 LRR19 LRR XI 2.86 At5g25930 LRR22 LRR XI At3g47090 LRR22 LRR XII 2.12 At1g35710 LRR27 LRR XII 1.77 At4g39270 LRR10 N.A At5g58540 K RLCK I 3.18 At2g39660 K RLCK VII At1g14370 K RLCK VII APK2a At4g35600 K RLCK VII At5g47070 K RLCK VII At5g51770 SK RLCK XI 1.93 At1g61380 SD SD At4g18250 THN Thaumatin

47 Looking for the receptor of egg-derived elicitors

48 SA accumulates under the eggs Salicylic acid Local Distal

49 No effect of the plant substrate on egg development Hatching time Fertility: 96.6% on Col % on parafilm

50 Eggs induce necrosis Arabidopsis thaliana A et B : Brassica oleracea C: Eruca sativa

51 Role of egg detection for plant defense against herbivores? SA? Defense genes

52 Testing the effect of oviposition on plant defense Egg extract S. littoralis 5 days 8 days Measurement of larval weight 2 days qpcr on insect-responsive genes S. littoralis 5 days 8 days Measurement of larval weight 2 days qpcr on insect-responsive genes

53 Treatment with egg extract suppresses defense gene expression and enhances larval performance Control Egg extract Spodoptera littoralis + Egg extract

54 Manipulation of plant defense signaling patways by the eggs Egg-mediated negative SA/JA cross-talk? SA JA Defense genes Defense genes