IX CORSO DI AGGIORNAMENTO SULLA GENETICA VEGETALE
Interactions between plants and other organisms: From molecular and chemical
signals to crop improvement
Plant response to insects:
molecular mechanisms of induced defenses
Giandomenico Corrado
Universita’ di Napoli Federico II
: [email protected]
PLANT INSECT INTERACTIONS
Insects play a dominant role among
heterotrophs that feed on terrestrial
plants
Insect phytophagy is characterized by:
• Highly diverse and specialized mouthparts
• A functionally complex gut
Proportion of the number of species
Green Plants,
22%
Herbivorous
insects, 26%
Vertebrates,
4%
Protozoa, 2%
Other
inverterbrates,
15%
Other insects,
31%
Plant
Plant response
response to
to phytophagy
phytophagy should
should be
be
effective,
effective, equally
equally complex
complex and
and
specialised
specialised
Response
Response should
should not
not be
be independent
independent from
from
the
the environment
environment
Calculation does not include fungi, algae and nematodes
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A plant trait is “defensive” if it increases fitness when plants are stressed
Are plants resistant, susceptible or both?
The world is green!
In natural ecosystems, any given plant
species is consumed by only a small
fraction of the herbivores in that
environment
Theory of coco-evolution (Erlich
(Erlich and Raven, 1964)
“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
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 counteradaptations against each other
2
Two predictions of the theory of coco-evolution
• 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
Plant defense can be distinguished according to the pattern of expression in:
constitutive
inducible
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How difficult is to be resistant?
Plant defense is costly and, in absence of frequent and recurring attack, it
does not provide an obvious benefit to the plant
(a)
(b)
51
das
93
das
a) wild-type b) an Arabidopsis plant constitutively expressing
a bacterial salicylic acid synthase enzyme (Heil and Baldwin,
2002). The mutant plants show elevated salicylic acid levels
and enhanced resistance to the pathogen Peronospora
parasitica
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
Plant defense can be distinguished according to the pattern of expression in:
constitutive
inducible
In relation to their mechanisms, plant defenses can be classified as direct or
indirect
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Direct defense
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)
Exploitation
Intoxication
Avoidance
Larva of T. ni hanging
immobilized and vulnerable
to predators after ingesting
the cardenolide-containing
latex of A. currassavica.
Larva of Erinnyis alope starting
to feed after trenching a Carica
papaya leaf.
Larvae of the specialist Tyria jacobaeae
are able to completely defoliate Senecio
jacobaea, even though it contains
pyrrolizidine alkaloids. The larvae
detoxify the alkaloids and sequester them
for their own defense against predators.
(Wittstock et al, 2002)
Chemical 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
average plant leaf area (cm2)
Compensatory consumption
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)
Winterer J, Bergelson J. 2001. Diamondback moth compensatory consumption of protease inhibitor-transformed plants. Mol. Ecol. 10:1069–74
Plants
….
defense
Plants need
need more
more than
than direct
direct defense…
defense….
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Indirect 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 VOCs
(Maffei, 2010)
(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 eugenol
derive from phelylpropanoids, whereas MeSA derived from SA generated from benzoic acid. (F) Alternatively, MeSA can be
formed by methylation of SA deriving from isochorismate.
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Direct defense
Chen et al 2004
Voli orientati del
parassitoide Aphidius ervi (%)
Direct and indirect defense are coordinated
Indirect defense
WT
35S::PROSYS
Corrado et al 2007
Gene activation
Prosystemin overexpression
increase both direct and indirect
defense mechanisms in tomato
Corrado et al 2007
How do plants “notice”
notice” attack by herbivorous arthropods?
Plants must be equipped with a sophisticated sensory system to mount a
defense response
Touch
Pressure
Scratching
Footprint (tarsal) secretion
Oviposition
Persistent contact with eggs
Elicitors
Secretions on eggs
Feeding
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Feeding habits
Phytophagous insects differ in their feeding habits. This is related to their different mouthparts.
- 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.
Feeding of insect species differs in various aspects
The feeding process combines two different stimuli:
1) Physical stimulus: mechanical wounding of the infested tissue
2) Chemical stimulus: introduction of oral secretions
It is likely that plants discriminate various biotic stressors according to these two
factors
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1) 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 factors
Oral secretions are delivered from the feeding organism into the wounded tissue
ELICITOR
Believed to be widespread, and not limited to Lepidoptera
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
Elicitors are also formed because of the plant-insect interactions
Apparently, elicitors are not mobile, but able to activate systemic response
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HerbivoreHerbivore-derived elicitors are chemically different
Few classes of chemical compunds that activate HAMP have been identified in OSs
Are there OSOS-elicitor receptors in plants?
Signal perception implies the presence of a specific receptor, but…
Induction by MeJA, BAW (beet
armyworm larvae/S. exigua), and
Mechanical Wounding on [3H]-LVolicitin–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
The interaction of elicitor molecules with receptors (or membranes?) involves
a complex response in which a number of events should be triggered,
resulting, ultimately, in increased transcription of defense genes
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Do Caterpillars Secrete OSs?
OSs?
Visual detection of fluorescent
regurgitant from Helicoverpa zea
after eating diet spiked with Alexa
488. a) Tomato leaves with
fluorescent regurgitant along the
feeding site. b) Leaf which H. zea
fed upon but no regurgitant was
detected. c) Leaf fed upon by H.
zea fed control diet
OS are not released during every caterpillar feeding
Do some herbivores minimize their display of elicitors during feeding?
Peiffer & Felton, 2009
How specific is plant response?
black: no variation
blue: both insects
purple: S. littoralis only
green: P. rapae only
A comparsion between the Arabidopsis transcripts activated upon attack of a
specialist (P. rapae) and a generalist (S. littoralis) phytophagous
Reymond et al, 2004
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CROSSCROSS-TOLERANCE
Plants resistant to one stress are resistant to “another” type of stress
(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 saltconcentrations
C: comparative gene expression of non-stressed
plants
Orsini et al, 2010. Systemin-dependent salinity tolerance in tomato: evidence of specific convergence of abiotic and biotic
stress responses. Physiol. Plant.
A model for the activation of plant defense to insects
(1) Elicitor binding
(2) Ca2+ influx
1
(3) ROS production
2
3
4
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).
5
6
7
Red arrows represent direct phosphorylation; blue arrows represent transcriptional regulation.
Herbivory-induced signalling in plants: perception and action 2009, Plant, Cell & Environment
AOC: allene oxide cyclase
AOS: allene oxide synthase
CDPK: calcium-dependent protein kinase
JAZ: jasmonate ZIM-domain
LOX: lipoxygenase
OPDA: 12-oxo-phytodienoic acid
NO: nitric oxide
NOA: NO-associated protein
NR: nitrate reductase
ROS: reactive oxygen species
SCF: Skp, Cullin, F-box
SIPK: salicylic acid-induced protein kinase
WIPK: wound-induced protein kinase
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Jasmonates
• class of related oxylipin signaling
molecules
JA is the proposed systemic signal
• overlapping role in regulating both stress
response and development
• Stress responses depending on JAs
include:
different insect orders
microbial pathogens
UV radiation
ozone
some abiotic stress
• control hundreds of downstream genes
Undamaged
Damaged
+ 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
PLANTPLANT-APHID INTERACTION IS COMPLEX
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
(a) Symptoms of high population densities of the
potato aphid (M. euphorbiae) on tomato
(b) Feeding by the spotted alfalfa aphid Therioaphis
trifolii 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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MODEL OF APHID FEEDING RECOGNITION
1) gene-per-gene  based on R gene in resistant genotypes
2) tissue damage  local and systemic response
Mi1.2 of tomato is the only cloned R-gene for aphid resistance
It encodes a NB-LRR protein
Smith and Boyko, 2006
Can we apply the same model?
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Elicitors from aphids?
Lapitan et al. 2007
Fractionated Extracts of Russian Wheat Aphid (RWA) Eliciting Defense Responses in Wheat
(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.
Defense signaling mechanisms after aphid infestation
Different combinations seems to give apparently conflicting results.
Common themes:
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 relevant
JA-activation is more limited (cross-talk with SA pathway?)
In tomato:
both JA and SA seem to be important (for M. euphorbiae
infestation)
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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
Digilio et al., 2010
DO
DO APHIDS
APHIDS ANTAGONISE
ANTAGONISE CYTOSOLIC
CYTOSOLIC WOUND-HEALING
WOUND-HEALING EVENTS?
EVENTS?
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+]
Kusnierczyc et al, 2008 Plant, Cell and Environment (2008) 31, 1097–1115
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SUMMARY
- 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
- 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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CONCLUSIONS
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...
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