Plants and insects living together for more than 350 million years Evolutionary between plants and insects resulted in the development of defence system in plants that has the ability to recognize signals from damaged cells Activates the plant immune response against the insects Plants have the ability to distinguish between herbivory and mechanical damage, such as hail and wind, as well as to recognize oviposition. This feature is needed to avoid wasting expensive defence resources, since production and release of defence responses only benefits herbivore challenged plants.

1. Assignment
On
Defense Mechanism in Plants Against Insects
Advanced Insect Physiology
ENT 602
JAYANT YADAV (2017A39D)

2. OUTLINE
•INTRODUCTION
• RECOGNITION OF INSECT HERBIVORE ATTACK
• EARLY EVENTS IN THE PLANT DEFENSE
• REGULATION OF DEFENSE RESPONSES
• TYPES OF DEFENSE RESPONSES

3. INTRODUCTION
• Plants and insects living together for more than 350 million years
• Evolutionary between plants and insects resulted in the development
of defence system in plants that has the ability to recognize signals from
damaged cells
• Activates the plant immune response against the insects
• Plants have the ability to distinguish between herbivory and mechanical
damage, such as hail and wind, as well as to recognize oviposition.
• This feature is needed to avoid wasting expensive defence resources,
since production and release of defence responses only benefits
herbivore challenged plants.

4. Video No. 1: Plant Defence Mechanism (By TED)

6. Recognition of Insect Herbivore Attack
• Conjugation of plant- and herbivore-derived precursors result in the
formation of fatty acid-amino acid conjugates (FACs).
• N-17-hydroxylinolenoyl-L-glutamine (volicitin ): First identified in
Spodoptera exigua (beet armyworm) oral secretions
• Inceptins: Disulfide-bonded peptides formed by proteolytic
fragments of chloroplastic ATP synthase γ-subunit, produced through
the digestion of plant proteins in the gut of Spodoptera frugiperda
(fall armyworm)
Elicitors (Present in saliva of insects)

7. • Caeliferins: Disulfoxy fatty acids, were identified in the oral
secretions of Shistocerca americana (American bird grasshopper) and it
release of volatile terpenoids
• Bruchins: Long-chain α,ω-diols, esterified at one or both oxygen
atoms with 3-hydroxypropanoic acid found in Callosobruchus
maculatus (cowpea weevil)
• β-glucosidase: In the oral secretion of the larvae of Pieris brassicae
elicits the release of volatile organic compounds that attracts the parasitic
wasp Cotesia glomerata
Doss et al 2000

8. Bonaventure et al 2011
Herbivore Associated Elicitors

10. • Insect oviposition fluids can give rise to defense responses
in the plant
• Many female adult herbivorous insects lay eggs directly
into plants, and some species are known to perceive
insects’ oviposition activities and deploy defenses
responses
OVIPOSITION FLUIDS

11. Oviposition induced plant responses acting against the herbivores

12. Hilker andMeiners, 2006
Contd….

13. Early Events in the Plant-Insect Interaction
• Changes in the transmembrane
potential (Vm) appear
immediately upon herbivory
damage
• Changes in the intracellular
Ca2+ concentration and
generation of H2O2
• Kinases and phytohormone
jasmonic acid (JA) are detectable
within minutes
• After 1 h, gene activation
followed by metabolic changes
(Mithöfer and Boland, 2008)

14. Membrane potential changes
• The plant plasma membrane is in direct contact with the
environment, and is therefore able to recognize outer changes and
initiate cascade events leading to apossible defense response.
• Herbivore feeding will lead to an immediate change in the cell
membrane potential (Vm), or modulate the ion flux at the plasma
membrane level.
• The Vm changes induced by herbivory are followed by afast electric
signal (action potential), which travels through the entire plant from
the point where the signal was induced.

15. • Calcium ions function as a second messenger in several
plant signaling pathways.
• In healthy cells, the cytosolic Ca2+ concentration is lowerthan in the
apoplastic fluid andcellularorganelles.
• This creates adriving force for the influx of Ca2+ into the
cytosol, via channel proteins where it acts as a messenger to
induce defense related signals.
Ca2+ Homeostatis

16. Systemic Signaling
• In plants attacked by insect herbivores, the expression of
several defense genes is induced in undamaged leaves
• Several components have been identified that are involved
in the systemic induction of defense responses
• Systemin peptides
• Oligogalacturonides (OGAs)
• Jasmonates

17. Regulation of Defence Responses
 Levels of jasmonic acid rise in response to herbivor
damage.
 This hormone can trigger many types of plant defenses
including bioactive compounds.
 The action of jasmonic acid induces the transcription of
many genes involved in plant defense.
 Jasmonic acid turns on genes for proteinase inhibitor.

19. Fig: A simplified scheme for co-evolution between plants and their herbivores.

20. (War et al 2012)

21. Types of Defense Responses

22. • Plants produce physical barriers against insect
herbivores.
• Produce compounds that exert repellent, anti-nutritive or
toxic effects on the herbivores.

23. Physical factors
• Laticifers and Oleoresins
• Waxes and Crystals
• Trichomes
• Spines
• Thicker Leaves

24. Crop Morphological structure Effect on insect
soybean Hairy trichomes Prevent insect eggs from
reaching the epidermis
and the larvae starve
after hatching
Potato
Tomato
Glandular trichomes secrete oils that repel
aphids
Cotton Glandular trichomes Attract pink boll warm
Crucifer Wax bloom Deter feeding by DBM
Onion Round leave Resistant to thrips
Okra Long trichomes
Resistant to Amrasca
biguttula
Sorghum Leaf glossiness Shoot fly
Effect of physical factors on insects

25. Laticifers
• Plants contain networks of channels in vascular tissues called laticifers ducts
• More than 50 latex producing plant families, Asclepias (milkweeds) is the
one most studied
• Latex of Cryptostegia grandiflora (rubber wine) may be transported 70 cm
upwards to the wounding site, where it, upon exposure to air, will coagulate
and there by trap small insect larvae.

26. Oleoresins
• Oleoresins (often termed resin or pitch) mixture of terpenoids and phenolics,
and are stored in high pressurized intercellular spaces .
• Herbivore damage results in upward movement of resin .
• When resin is exposed to air, the highly volatile monoterpenes and
sesquiterpenes evaporate, leaving insects trapped in the solidifying resin
acids.

27. •Epicuticular waxes form films and crystals that cover the cuticle of most
vascular plants
•Oviposition of Pieris brassicae on Arabidopsis thaliana induce
changes in the wax composition, increasing the amount of fatty acid
tetratriacontanoic acid , while decreasing the amount of tetracosanoic
acid . These changes lead to attraction of the egg parasitoid
(Blenn, et al 2012)
Waxes

28. Trichomes
• Trichomes : specialized hairy
structure found on aerial plant
parts
• Trichome density negatively
effect
– Ovipositional behavior
– Feeding
– Larval nutrition of insect pests
– Interfere with the movement of
insect
– Type - Straight, spiral, hooked,
branched, or unbranched and can
be glandular or non glandular

29. Types of Trichome

30. The glandular trichomes of the plant Roridula gorgonias release an extremely adhesive, visco-
elastic, resinous secretion that traps a variety of insects and show mutualistic effect of mirid
bug Pameridea roridulae on its prey
(Voigt and Gorb , 2008)

31. Hooked trichomes and resistance of phaseolus
vulgaris to Empoasca fabae
• Two cultivars of the bean, Phaseolus vulgaris L., differing in
densities of hooked trichomes were examined for resistance
to the potato leafhopper, Empoasca fabae
• Leafhoppers were impaled by these epidermal appendages,
leading to wounding and death
• Nymphal survival on the cultivar, “California Light Red Kidney”
(hooked trichome density: 2000/cm2) was significantly less than
that on “Brasil 343” (hooked trichome density: 400/cm2)
(Pillemer and Tingey, 2011)

32. Direct Chemical Defence Responses
• Bioactive Specialized Compounds
• Hypersensitive Response
• Digestibility Reduction
• Reallocation of Resources

33. A. Bioactive Specialized Compounds
• Alkaloids
• Benzoxazinoides
• Cyanogenic Glucosides
• Terpenoids
• Phenolics
• Proteinase Inhibitor
• Non protein Amino Acid
• Polyphenol Oxidases
• Primary metabolites used for
growth, development and
reproduction
• Secondary metabolites
known as bioactive
specialized compounds, are
used to protect the plant
against insects (herbivores)
(Engelberth et al 2006)

34. Alkaloids
•Alkaloids (>15,000 different alkaloids found in 20% of all vascular
plants), are prevalently found in the Leguminosae spp.
(legumes), Liliaceae spp. (lilies), Solanaceae spp.
•Alkaline contain nitrogen in a heterocyclic ring e.g., nicotine and
atropine
•Pseudoalkaloids, such as caffeine and solanidine, are alkaline but not
derived from amino acids
•Solanum demissum (nightshade potato) containing the alkaloid
demissine is resistant to Leptinotarsa decemlineata (Colorado beetle)
and Empoasca fabae (potato leafhopper)

35. Benzoxazinoides
•Grammineae Family (maize, rye and wheat) produces the defense-
related bioactive specialized compounds 2,4-dihydroxy-1,4-benzoxazin-
3-one-glucoside (DIBOA-Glc) and dihydroxy-7-methoxy-1,4-
benzoxazin-3-one-glucoside (DIMBOA-Glc, from indole-3-glycerol
phosphate
• Conversion is catalyzed by BX1-BX9, of which BX1 cleaves off the
glycerol phosphate, BX2-BX5 (Cytochrome P450s CYP79C1-4)
catalyze the reactions forming DIBOA, BX8/BX9 add the stabilizing
glucosyl group, and BX6-BX7 assists in the conversion from DIBOA-
Glc to DIMBOA-Glc
• DIMBOA has been shown to confer resistance to Ostrinia
nubilalis (first-brood European corn borer)
(Glauser et al 2011)

36. Cyanogenic Glucosides
• Cyanogenic glucosides (CNglcs ) are amino acid derived glucosides,
originating from aromatic or branched-chain amino acids
• Tyrosine (Dhurrin in Sorghum bicolor)
•Valine and isoleucine in Lotus japonicus
•CNglcs are stored in the vacuole plant tissues.
•When the plant tissue is fragmented, for instance due to feeding, the
CNglcs are exposed to β-glucosidases located in plastids or which leads
to hydrolysis and the formation of a sugar and a cyanohydrin that
spontaneously decomposes into toxic hydrogen cyanide (HCN) and a
ketone or aldehyde.

37. • Phenolics, derived from phenyl alanine (such as caffeic and
ferulic acid), phenylpropanoid lactones (known as coumarins)
and benzoic acid derivatives (such as vanillin and salicylic
acid ).
• The cotton phenolic pigment gossypol has repellent effects
against numerous insects and is toxic to Heliothis
virescens (tobacco bollworm), Heliothis zea (bollworm) and
several other insects
Phenolics

38. • Tannins - naturally occurring plant polyphenols.
Their main characteristic is that they bind and
precipitate proteins and stored in vacuoles of
plants.
Effect on insects
 Bind to salivary proteins and digestive enzymes
including trypsin and chymotrypsin resulting in
protein inactivation
• Lignin heterogeneous polymer composed of
phenolic compounds that gives the cell rigidity.
Lignin is the primary component of wood, and cell
walls that become “lignified” are highly
impermeable to pathogens and difficult for small
insects to chew
Effect on insects
 Insoluble, rigid, and virtually indigestible, lignin
provides an excellent physical barrier

39. Glucosinolates
•Glucosinolates (GSL) are sulphur- and nitrogen-containing compounds
found extensively in Brassicaceae
•The GSL are divided into Three groups based on the amino acid
precursor of the side chain:
• Aliphatic GSL (50%) derived from methionine
•Indole GSL (10%) synthesized from tryptophan
•Aromatic GSL (10%) derived from phenylalanine or tyrosine
•Flee beetle Phyllotreta cruciferae feeds preferably on older
cotyledons of Sinapis alba (white mustard), due to the lower levels of
the GSL
( Bodnaryk , 1991)

40. •Terpenoids are biosynthesized from acetyl-CoA or glycolytic
intermediates.
•They are classified by the number of isoprene units or five-carbon
elements (CH3–CH2–CH–(H3C)2)
10-carbon terpenes are called monoterpenes
15-carbon terpenes are sesquiterpenes
20-carbon terpenes are diterpenes
25-carbon terpenes are sesterterpenes
30-carbon terpenes are triterpenes
40-carbon terpenes are tetraterpenes
terpenes with even more isoprene units polyterpenes
•Many of them play a role in plant defense, both as components in resin
or as volatiles, acting as antifeedants, repellents, toxins or as modifiers of
insect development
(Aharoni et al 2005)
Terpenoids

41. • Phytoecdysones are plant
steroids that have the same
basic structure as insect
molting hormones and thus
interfere with molting. These
compounds sometimes cause
death of the insect herbivore.
• Phytoecdysteroids are
classed as triterpenoids
PHYTOECDYSONES

42. (a) Nicotine, a true alkaloid derived
from aspartate and ornithine
(b) DIMBOA, a benzoxazinoide
derived from indole-3-glycerol
phosphate
(c) Dhurrin, a cyanogenic glucoside
derived from tyrosine
(d) Sinalbin, a glucosinolate derived
from tyrosine
(e) Canavanine, a nonprotein amino
acid derived from L-homoserine
(f) Salicylic acid, a benzoic acid
derived phenol
(g) limonene, a terpenoid derived
from geranyl pyrophosphate
(Caspi et al 2008)
Structures of plant bioactives

43. Ibanezet al 2012
Examples of Plant Bioactive Specialized Compounds With Insecticidal Activity

44. Contd…..

45. B. Hypersensitive response
• Plant’s response to herbivore results in the formation of
necrotic plant tissue and neoplamal growth that isolates the
invader from plant
• Plants can cast eggs off their leaves
• When an insect deposits its eggs onto a plant, the plant may
respond with growth of neoplastic tissue and formation of
necrotic tissues that results in detachment of eggs.

46. (a) Eggof smallcabbagewhite butterfly, inducinghypersensitive response–like
necrosisin Brassica nigra.
(b) Eggof Heliothissubflexainducingneoplasticgrowth in Physalis angulata leaf.
Hilker andFatouros, 2014

47. C. Digestibility Reduction
•Plants produce a number of defense proteins that reduce insect
herbivores ability to digest the plant
•Anti-digestive proteins limit the rate of enzymatic conversion of
ingested food
•Anti-nutritive proteins limit the utilization of food by altering physical
availability or chemical identity
•Five major classes of defense proteins are
•Protein inhibitors
• α-amylase inhibitors
• Lectins
•Chitinases
• Polyphenol oxidases
(Falco et al 2001)

48. •Endopeptidases and Exopeptidases found in midgut region , used by
insect herbivores to cleave peptide bonds
Serine proteases (trypsin and chymotrypsin )
Cysteine
Aspartic acid proteases
Metalloproteinases
•Plants have inhibitors for all four classes of proteinases
Delay larval development without directly causing mortality
 Proteolytic activity of midgut enzymes and decrease the
availability of amino acid
In sugarcane, trypsin inhibitors detected in leaves, lateral buds and
seed tissue, which effect sugarcane borer larval devlopment
(Falco et al 2001)
Protease Inhibitors

49. •The lectin-like α-amylase inhibitors (α-AI) are found in cereal seeds,
such as Triticum spp.(wheat) and Hordeum vulgare (barley), and in
monocots, such as S. bicolor and Z. Mays
•The activities of these inhibitors are directed against α-amylases from
insects , used for starch breakdown
•Transgenic Wheat α-AIs can inhibit Tenebrio
obscurus (mealworm), Tribolium spp. (flour beetles), Sitophilus spp.
(wheat weevils) and Oryzaephilus spp. (grain beetles)
(Falco et al 2001)
α- Amylase Inhibitors

50. •Polyphenol oxidase (PPO) enzymes cause the typical browning of plant
extracts, mainly fruits, and damaged tissues
•PPOs appear frequently upon wounding, and are therefore suggested to
play a defensive role
•Polyphenol oxidase (PPO) enzymes, over expression genes decreased
the growth rate 2.5-fold in S. litura, and increased the mortality up to
3.3-fold
•PPOs can also be combined with specific phenolic substrates in
glandular trichomes to produce a kind of “super glue” to trap smaller
insects
Polyphenol Oxidases

51. • Lectins are sugar-binding proteins produced by plants as a
defense response
• When lectins come into contact with the glycoproteins lining
the intestinal area of insect herbivores, they are assumed to
inhibit the absorption of nutrients
Lectins

52. QuantificationofNICTABAaccumulationintobaccoleavesafterfeeding bydifferentherbivores
Vandenborreet al 2011

54. Spottedknapweedknapweed
moth
Newingham et al 2007
Fig: Allocating nitrogen away from aherbivore
D. Reallocation of Resources
To protect valuable resources, they might be reallocated by the plant upon attack

55. Indirect Defence Response
The term “Indirect Defence ” is used when plants
attract, nourish or house natural enemy.

56. Two mechanisms are involved in Indirect Defence Response
1. Herbivore-induced Plant Volatiles (HIPV)
2. Extra-floral Nectar

57. Video No. 2: Plant Defence Mediated By HIPV Production

58. HIPV:
HIPV can mediate indirect defences by attracting foraging carnivores predators and
parasitoids

59. 1. Volatiles
•More than 1000 volatile organic compounds (VOCs), primarily
consisting of 6-carbon aldehydes, alcohols, esters and various
terpenoids are released from plant flowers, vegetative parts or roots
• VOCs are used to attract pollinators and predators or repel herbivores
•Green-leaf volatiles (GLVs) are isomers of hexanol, hexenal or hexenyl
acetate
•Roots VOCs: Z. mays roots attacked by Western corn rootworm
Insect larvae release the sesquiterpene (E)-β-caryophyllene as well as
small amounts of α-humulene and caryophyllene oxide which attract its
natural enemy
(Rasmann et al 2005)

60. •Insect oviposition fluids give rise to defence responses in the plant as
well, making the plant attract egg-eating predators or strengthen its
defense in case of a potential future insect herbivore attack
(Hilker and Meiners, 2006)
•Oviposition of P. brassicae on leaves of Brassica oleracea (Brussels
sprouts) changes the leaf surface chemicals leading to attraction of the
egg parasitoid Trichogramma brassicae
(Fatouros et al 2005)
2. Ovipositional Fluids

62. 3. Extrafloral Nectar
• Extrafloral nectar (EFN) appear in more
than 70 plant species spanning
angiosperms, gymnosperms and ferns,
indicating that it is evolutionary more
ancient than floral nectar
• In contrast to floral nectar, used to attract
pollinators, EFN is secreted on leaves
and shoots to attract predators and
parasitoids

63. Extrafloral nectar as an herbivore-induced defense trait
Family Species Herbivore Trait enhanced
Bignoniaceae Catalpabignonioides
(Indian bean)
Ceratomiacatalpae
(CatalpaSphinx)
Sugarcontent inEFN
Euphorbiaceae Ricinuscommunis
(Castor)
Spodopteralittoralis EFNvolume
Euphorbiaceae Triadicasebifera
(Chinese tallow tree)
Gadirthainexacta,
and
Grammodesgeometrica
Secretionof total
solids
Malvaceae Gossypiumherbaceum
(Cotton)
Spodopteralittoralis EFNvolume
Martin, 2015

64. Ant visitation to extrafloral nectaries decreases herbivory and
increases fruit set in Chamaecrista debilis
AlvesandClaro, 2010

65. CONCLUSION
 Insects and plants co-evolved and not only developed morphological defense
mechanisms active against insect herbivory, but also genetic transformations allowing
the production of volatile chemicals.
 Attacked plants use these volatile chemicals as arsenals and signals against attacking
pests. These chemicals not only repell herbivores but also control their population, by
signalling invitations sent to their predators or parasites.
 The interaction between herbivores and biotic environment is thus largely based on
plant mediated mechanisms, including constitutive traits like modifications in plants
anatomy and physiology, or herbivore induced changes in host biochemistry.
 Plants also evolved direct strategies to repel herbivores, through induced and
constitutive defence mechanism. The trichomes constitute a defense feature against a
variety of insects.
 Due to co-evolution of synthesis of herbivore repelling volatiles in plants and their
modification by herbivores, cyclic population changes, concerning both the herbivores
and primary producers, can be also affected.

66. Video No. 3: Plant Defence Mechanism Briefing

67. FUTURE OUTLOOK
 Although induced resistance has attained a considerable momentum recently, and has
attracted the attention of scientists in evolutionary ecology, entomology, plant
physiology and biotechnology, much of the underlying mechanism have still remained
unanswered.
 There is a need to understand the herbivore- specific signal molecules, their
identification, mode of action and further signal transduction.
 An understanding of induced resistance in plants can be utilized for interpreting the
ecological interactions between plants and herbivores and for exploiting in pest
management in crops.
 The future challenge is to exploit the elicitors of induced defense in plants for pest
management, and identify the genes encoding proteins that are up and/or down
regulated during plant response to the herbivore attack, which can be deployed for
conferring resistance to the herbivores through genetic transformation.