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1 Plant response to insects: Plant response to insects: molecular mechanisms of induced defenses molecular mechanisms of induced defenses Giandomenico Corrado Universita’ di Napoli Federico II : [email protected] IX CORSO DI AGGIORNAMENTO SULLA GENETICA VEGETALE Interactions between plants and other organisms: From molecular and chemical signals to crop improvement Calculation does not include fungi, algae and nematodes Proportion of the number of species Other insects, 31% Protozoa, 2% Vertebrates, 4% Green Plants, 22% Other inverterbrates, 15% Herbivorous insects, 26% Plant response to phytophagy should be effective, equally complex and specialised Response should not be independent from the environment Plant response to phytophagy should be effective, equally complex and specialised Response should not be independent from the environment Insect phytophagy is characterized by: • Highly diverse and specialized mouthparts • A functionally complex gut Insects play a dominant role among heterotrophs that feed on terrestrial plants PLANT INSECT INTERACTIONS PLANT INSECT INTERACTIONS

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Plant response to insects: Plant response to insects: molecular mechanisms of induced defensesmolecular mechanisms of induced defenses

Giandomenico Corrado

Universita’ di Napoli Federico II

: [email protected]

IX CORSO DI AGGIORNAMENTO SULLA GENETICA VEGETALEInteractions between plants and other organisms: From molecular and chemical

signals to crop improvement

Calculation does not include fungi, algae and nematodes

Proportion of the number of species

Other insects, 31%

Protozoa, 2%

Vertebrates, 4%

Green Plants, 22%

Other inverterbrates,

15%

Herbivorous insects, 26%

Plant response to phytophagy should be effective, equally complex and specialised

Response should not be independent from the environment

Plant response to phytophagy should be effective, equally complex and specialised

Response should not be independent from the environment

Insect phytophagy is characterized by:

• Highly diverse and specialized mouthparts• A functionally complex gut

Insects play a dominant role among heterotrophs that feed on terrestrial plants

PLANT INSECT INTERACTIONSPLANT INSECT INTERACTIONS

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Are plants resistant, susceptible or both?Are plants resistant, susceptible or both?

A plant trait is “defensive” if it increases fitness when plants are stressed

In natural ecosystems, any given plant

species is consumed by only a small

fraction of the herbivores in that

environment

The world is green!The world is green!

Theory of coTheory of co--evolution (evolution (ErlichErlich and Raven, 1964)and Raven, 1964)

The central idea is that plant-insect interaction promotes speciation

A major evolutionary force is the competion of sets of co-evolving genes that develop adaptations and counter-adaptations against each other

“Patterns of evolutionary interaction among different organisms where exchange of genetic information among the kinds is assumed to be mininal or absent”

Pairwise co-evolution: evolutionary relationships between two species that are ecologically tightly associated

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• related plants, even when present in different environments, have similar defense mechanisms

• related insect species have related host-plants

The interaction between a plant and an insect species is usually highly

specific, restricted to a limited number of species

Two predictions of the theory of coTwo predictions of the theory of co--evolutionevolution

Plant defense can be distinguished according to the pattern of expression in:

constitutive inducible

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Plant defense is costly and, in absence of frequent and recurring attack, it does not provide an obvious benefit to the plant

How difficult is to be resistant?How difficult is to be resistant?

a) wild-type b) an Arabidopsis plant constitutively expressinga bacterial salicylic acid synthase enzyme (Heil and Baldwin, 2002). The mutant plants show elevated salicylic acid levels and enhanced resistance to the pathogen Peronosporaparasitica

a) Untransformed tomato b) transgenic plants constitutively expressing the prosystemin gene. (Corrado et al, in press). The mutant plants show elevated jasmomic acid levels and enhanced resistance to Manduca sexta larvae

(a) (b)

51das

93das

Plant defense can be distinguished according to the pattern of expression in:

In relation to their mechanisms, plant defenses can be classified as direct or indirect

constitutive inducible

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A plant trait that reduces the performance (growth, development, survival) of a phytophagous pests.

Direct defenses are mainly: - Physical barriers (eg: spines, thorns, trichomes, prickles)- Chemical compounds (eg: secondary metabolites)

Larva of T. ni hangingimmobilized and vulnerable to predators after ingesting the cardenolide-containing latex of A. currassavica.

Larva of Erinnyis alope starting to feed after trenching a Caricapapaya leaf.

Intoxication

Avoidance

Exploitation

Larvae of the specialist Tyria jacobaeaeare able to completely defoliate Seneciojacobaea, even though it contains pyrrolizidine alkaloids. The larvae detoxify the alkaloids and sequester them for their own defense against predators.

Direct defenseDirect defense

(Wittstock et al, 2002)

Chemical defenses in plantsChemical defenses in plants

Many investigations have focused on chemical defenses

Plants synthesize a broad range of metabolites that are believed to act as defense

compounds; Main groups are:

• Protease inhibitors (PIs): reduce the quantity of proteins that can be

digested, and also cause hyperproduction of the digestive enzymes which

enhances the loss of sulfur amino acids.

• Polyphenol oxidases (PPOs): antinutritive, reduce food quality.

• Toxic/deterrent compounds: alkaloids, cyanogenic glycosides and

glucosinolates, terpenoids, phenolic compounds, etc.

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Potential antinutritional proteins revealed by microarray and proteomic studies

Zhu-Salzman et al 2008

Winterer J, Bergelson J. 2001. Diamondback moth compensatory consumption of protease inhibitor-transformed plants. Mol. Ecol. 10:1069–74

Plants need more than direct defense….Plants need more than direct defensePlants need more than direct defense……..

ave

rag

e p

lan

t le

af

area

(cm

2)

Average per capita growth rate of moths, ±1 standard error. Bt was either absent from treatment plants (open circles) or present (closed circles).

Average leaf area of whole plants, ± standard deviation after treatment with moths. Bt was either absent from treatment plants (open circles) or present (closed circles)

Compensatory consumptionCompensatory consumption

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Indirect defenseIndirect defense

Attraction of predators and parasitoids of pests

Considered universal in plants

Based on the emission of Volatile Organic Compounds (VOC)

(Maffei, 2010)

Major pathways for the production of Major pathways for the production of VOCsVOCs

(A) The MEP pathways give rise to the formation of monoterpenes and diterpenes. Isoprene is generated from DMAPP. (B) Sesquiterpenoids are generated by FPP derived from the cytosolic MVA pathway. (C) oxylipins generate from fatty acids which are cleaved into GLVs and JA derivatives. (D) the volatile indoles generate from anthranilate. (E) aromatic VOCs such as eugenolderive from phelylpropanoids, whereas MeSA derived from SA generated from benzoic acid. (F) Alternatively, MeSA can beformed by methylation of SA deriving from isochorismate.

(Maffei, 2010)

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Direct and indirect defense are coordinatedDirect and indirect defense are coordinated

Direct defense Indirect defense

Gene activation

Prosystemin overexpressionincrease both direct and indirect defense mechanisms in tomato

Vol

ior

ient

ati

del

para

ssit

oide

Aph

idiu

ser

vi(%

)

WT 35S::PROSYS

Corrado et al 2007

Corrado et al 2007

Chen et al 2004

How do plants How do plants ““noticenotice”” attack by herbivorous arthropods?attack by herbivorous arthropods?

TouchPressureScratchingFootprint (tarsal) secretion

OvipositionPersistent contact with eggsElicitorsSecretions on eggs

Feeding

Plants must be equipped with a sophisticated sensory system to mount a defense response

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Feeding habitsFeeding habits

- Chewing insects (lepidoptera, coleoptera, etc):Cause a significant damage to plant tissue, with removal of extensive leaf areas

- Sucking insects:

Aphids, whiteflies etc: cause modest to barely perceptible damage to epidermal and mesophyll cells. Feed on sap.

Trips, etc.: cause a limited and localized damage to plant tIssue, they feed on cellular content.

Phytophagous insects differ in their feeding habits. This is related to their different mouthparts.

1) Physical stimulus: mechanical wounding of the infested tissue

2) Chemical stimulus: introduction of oral secretions

Feeding of insect species differs in various aspects Feeding of insect species differs in various aspects

It is likely that plants discriminate various biotic stressors according to these two factors

The feeding process combines two different stimuli:

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1) The role of mechanical wounding1) The role of mechanical wounding

T. R. GREEN, C. A. RYAN (1972) Department of Agricultural Chemistry, Washington State University

This work also demonstrated that:

“The wounding of the leaf appeared to be the primary cause

of the induction of inhibitor I accumulation since nearly any

type of crushing would cause the same induction”.

2) Chemical factors2) Chemical factors

Oral secretions are delivered from the feeding organism into the wounded tissue

Believed to be widespread, and not limited to Lepidoptera

Elicitors are also formed because of the plant-insect interactions

Apparently, elicitors are not mobile, but able to activate systemic response

A macromolecule,originating either from the host plant (endogenous elicitors) or from the plant stressor (exogenous elicitors), which is able to induce structural and/or biochemical responses associated with plant resistance

ELICITORELICITOR

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HerbivoreHerbivore--derived elicitors are chemically differentderived elicitors are chemically different

Few classes of chemical compunds that activate HAMP have been identified in OSs

Are there OSAre there OS--elicitor receptors in plants?elicitor receptors in plants?

Signal perception implies the presence of a specific receptor, but…

The interaction of elicitor molecules with receptors (or membranes?) involvesa complex response in which a number of events should be triggered, resulting, ultimately, in increased transcription of defense genes

Induction by MeJA, BAW (beet

armyworm larvae/S. exigua), and

Mechanical Wounding on [3H]-L-

Volicitin–Plasma Membrane Binding.

Saturating levels of [3H]-L-volicitin (10

nM) were used to determine the total

level of binding at the indicated times

after treatment with MeJA,

BAW, or razor blade. The total binding

is shown in fmol/mg.Truitt et al, 2004

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OS are not released during every caterpillar feeding

Do some herbivores minimize their display of elicitors during feeding?

Do Caterpillars Secrete Do Caterpillars Secrete OSsOSs??

Peiffer & Felton, 2009

Visual detection of fluorescent regurgitant from Helicoverpa zeaafter eating diet spiked with Alexa488. a) Tomato leaves with fluorescent regurgitant along the feeding site. b) Leaf which H. zeafed upon but no regurgitant was detected. c) Leaf fed upon by H. zea fed control diet

A comparsion between the Arabidopsis transcripts activated upon attack of a specialist (P. rapae) and a generalist (S. littoralis) phytophagous

How specific is plant response? How specific is plant response?

black: no variationblue: both insectspurple: S. littoralis onlygreen: P. rapae only

Reymond et al, 2004

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CROSSCROSS--TOLERANCETOLERANCE

Plants resistant to one stress are resistant to “another” type of stress

Orsini et al, 2010. Systemin-dependent salinity tolerance in tomato: evidence of specific convergence of abiotic and biotic stress responses. Physiol. Plant.

(A)

(B)

(C)

A: phenotype of control (BB) and transgenic plants (BBS) under salt-stress condition (40 mM NaCl)B: Leaf Proline Content at different salt-concentrationsC: comparative gene expression of non-stressed plants

A model for the activation of plant defense to insectsA model for the activation of plant defense to insects

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Herbivory-induced signalling in plants: perception and action 2009, Plant, Cell & Environment

AOC: allene oxide cyclaseAOS: allene oxide synthaseCDPK: calcium-dependent protein kinaseJAZ: jasmonate ZIM-domainLOX: lipoxygenaseOPDA: 12-oxo-phytodienoic acid NO: nitric oxideNOA: NO-associated proteinNR: nitrate reductaseROS: reactive oxygen speciesSCF: Skp, Cullin, F-boxSIPK: salicylic acid-induced protein kinaseWIPK: wound-induced protein kinase

Red arrows represent direct phosphorylation; blue arrows represent transcriptional regulation.

2 3

4

5 6

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(1) Elicitor binding

(2) Ca2+ influx

(3) ROS production

Activation of various metabolic pathways,which increase the production of NO (4), Ethylene (5) and JA (6), ultimately resulting in the transcriptional

activation of defense genes (7).

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• class of related oxylipin signaling molecules

• overlapping role in regulating both stress response and development

• Stress responses depending on JAsinclude:different insect ordersmicrobial pathogensUV radiation ozonesome abiotic stress

• control hundreds of downstream genes

JasmonatesJasmonates

JA is the proposed systemic signal

Damaged

Undamaged

+ PIs+ VOCs

+ PIs+ VOCs

The jasmonate signaling pathway is an evolutionarily conserved mechanism to regulate the expression of direct and indirect defenses.

As relatively nonspecic sentinels of cellular injury, jasmonates promote resistance to a wide variety of biotic aggressors

Aphids have the ability to manipulate host plant physiology and response

The nature and extent of symptoms vary widely depending upon the aphid and plant

PLANTPLANT--APHID INTERACTION IS COMPLEXAPHID INTERACTION IS COMPLEX

(a) Symptoms of high population densities of the potato aphid (M. euphorbiae) on tomato

(b) Feeding by the spotted alfalfa aphid Therioaphistrifolii on Medicago sativa

(c) Russian wheat aphids (Diuraphis noxia) on wheat (Triticum aestivum) cause leaf rolling and longitudinal streaks

(d) Pemphigus betae induces foliar galls on its overwintering host, the narrowleaf cottonwood (Populus angustifolia).

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1) gene-per-gene based on R gene in resistant genotypes

2) tissue damage local and systemic response

MODEL OF APHID FEEDING RECOGNITIONMODEL OF APHID FEEDING RECOGNITION

Smith and Boyko, 2006

Mi1.2 of tomato is the only cloned R-gene for aphid resistance

It encodes a NB-LRR protein

Can we apply the same model?Can we apply the same model?

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Elicitors from aphids?Elicitors from aphids?

(A) Gamtoos injected with RWA whole extract(arrow shows leaf rolling). (B) Gamtoos injected with protein extract from RWA (arrows show leaf rolling and chlorosis). (C and D) Same as B, showing close-up view of leaves with leaf rolling and chlorosis (C) and only chlorosis (D). (E) Gamtoos injected with protein extract from RWA showing a trapped head. (F) Gamtoos injected with metabolite showing normal leaf morphology. (G) Gamtoos injected with buffer showing normal leaf morphology.

Fractionated Extracts of Russian Wheat Aphid (RWA) Eliciting Defense Responses in Wheat

Lapitan et al. 2007

Defense signaling mechanisms after aphid infestationDefense signaling mechanisms after aphid infestation

Different combinations seems to give apparently conflicting results.

1) Aphids (and other phloem-feeders) cause rapid increase in SA levels/PR gene transcriptions

2) Indirect defenses are also activated

3) In Arabidopsis: JA-regulated defenses appear to be relevantJA-activation is more limited (cross-talk with SA pathway?)

In tomato: both JA and SA seem to be important (for M. euphorbiaeinfestation)

Common themes:

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Digilio et al., 2010

JA and SA levels are related to aphid resistance in tomato? JA and SA levels are related to aphid resistance in tomato?

Relative quatification of the expression levels of major genes involved in insect response pathway after aphid infestation

A comparison of the constitutuve express level in aphid susceptible (‘M82’, white columns) and aphid-resistant (‘AN5’, grey cloumns; ‘AN7’, black colums) tomato genotypes

DO APHIDS ANTAGONISE CYTOSOLIC WOUND-HEALING EVENTS?DO APHIDS ANTAGONISE CYTOSOLIC WOUND-HEALING EVENTS?

Kusnierczyc et al, 2008 Plant, Cell and Environment (2008) 31, 1097–1115

1) some products must be transported in transcriptionally active cells (i.e. from SE to companion cells)

2) [Ca2+] increase is associated to mechanisms of sieve block

Plants increase [Ca2+] Aphids lower [Ca2+]

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- In natural ecosystems, any given plant species is consumed by only a small fraction of the herbivores in that environment

- Terrestrial plants use a combination of constitutive and inducible defensive traits to resist challenge by herbivorous insects

- Plant defensive metabolites and proteins limit herbivory by exerting direct repellent, antifeedant, and toxic effects on the insect. Synergistic interactions between these compounds strengthen the host defense response

SUMMARYSUMMARY

- Herbivore-induced plant volatiles serve various important functions in plant immunity to insect herbivores, including the attraction of insect predators and priming of defense responses

- Defense responses to insect attack are elicited by compounds in insect oral secretions.

-For plant interactions with some hemipterans, there is evidence for the involvement of R genes in the control of host plant resistance.

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A plant response to herbivore attack is generally so complex that establishing the relevance of a particular trait for the interaction is often difficult

Omics data integration is probably necessary to identify genes and molecules that are “important” for plant resistance

Few gene products have been shown to play a direct role in plant resistance, so...

CONCLUSIONSCONCLUSIONS