Resistencia de las plantas a

enfermedades y plagas
(Elicitores y efectores)

Isabel Díaz
CBGP UPM-INIA
ETSI Agrónomos-UPM Master Biotecnología Agroforestal
i.diaz@upm.es (ETSIA-UPM) – Curso 2017/18
Plant defences against pests

STRUCTURAL DEFENCES:
(1) Trichomes, thorns, cuticles, waxes, cell walls,…
DIRECT DEFENCES:
(2) Toxic, deterrents and antimetabolic compounds
INDIRECT DEFENCES:
(3) Induced volatiles to attract parasitoids and
natural enemies
(4) Interplant priming: induced volatiles to prime
defence in neighbouring plants
 Plant inducible defences against pests

Enzymes
.. Volicitins
... .. Caeliferins
Plant Inceptins
Peps
ELICITORS/EFFECTORS perception eggs Bruchins
ELICITORS …

RECEPTORS
plant cell
ROS
RECEPTORS Ca2+

RECEPTORS
EFFECTORS
CMs & MAPKs
CDPKs
NETWORK OF Signal
REACTIONS, transduction
COMPOUNDS, pathway Hormone cross-talk

FACTORS…
MYC
TFs WRKY
…

Inducible
nucleus
Direct and indirect
defences defences

Santamaria et al. (2013) Transgenic Res 22:697

 Plant perception

Plants recognize pest through wounding, saliva and oviposition fluids

- Specific alterations and physical damage produced by the pest (DAMPs: Damage-Associated
Molecular Patterns) are perceived by plants.
- Arthropod fluids contain bioactive compounds known as HAEs (Herbivore-Associated Elicitors or
effectors) or HAMPS (Herbivore-Associated Molecular Patterns) that trigger inducible defence
responses known as HTI (HAMP-Triggered Immunity).

ETI: effector-triggered immunity

WIR: wound-induced resistance
 Plant perception

QUESTIONS:
 Can plants distinguish between pest feeding, oviposition and mechanical damage?
 Can plants differentiate chewing, snipping, tearing and piercing-sucking arthropods?
masticador cortador rasgador perforador chupador
 Can plants discriminate between phloem- and mesophyll cell-sucking arthropods?

YES ¡¡¡
 Plants detect the first insect contact (touch, pressure, landing, walking, wounding,...)
 Plants evaluate the quality and quantity of damage tissue and differentially recognize
compounds in arthropod oral secretions and oviposition fluids
 Plants distinguish special patterns of feeding: place and type of feeding, arthropod
behaviour (many herbivore remove similarly sized pieces of tissues in a highly
choreographed and predictable manner, or repetitive wound before feeding,...), etc.
 Plant perception
Aphids Thrips
Mites
Caterpillars SUCKING SUCKING
Beetles CHEWING (stylets) (mandibles + stylets)
Flies (larvae) (mandibles)

??MPs: Alteration-Associated
Patterns
DAMPs HAMPs
DAMPs: Damage-Associated
PERCEPTION PRRs Molecular Patterns

SHORT-TERM
 Ion imbalance
 Membrane depolarization
HAMPs: Herbivore-Associated
RESPONSES  Ca2+ influx Molecular Patterns
 ROS production

LONG-TERM PRRs: Pattern Recognition

RESPONSES Direct and indirect Receptors
defences
 Plant perception

Graham Calow Plants respond to vibrations caused by phytophagous insect

Feeding → acoustic enegy Can insect vibrations elict chemical defences?

- Arabidopsis thaliana rosettes pre-treated with the vibrations (mimic or

caused by caterpillar feeding) had higher levels of glucosinolate and
anthocyanin defenses when subsequently fed upon by Pieris rapae (L.)
caterpillars than did untreated plants.
-The plants also discriminated between the vibrations caused by chewing
and those caused by wind or insect song.
-Plants thus respond to herbivore-generated vibrations in a selective and
ecologically meaningful way.

Appel and Cocroft (2014) Oecologia 175:1257

 Plant perception

Vibrations produced by Pieris rapae caterpillar

feeding on A. thaliana, recorded using two laser
vibrometers on the fed-upon leaf (pbl) and a
second leaf (sl) on the opposite side of the plant.

Pieris rapeA-Arabidopsis tahliana

Increase in apliphatic glucosinolates in systematic leaves

expressed as porcent change from levels in controls
Appel and Cocroft (2014) Oecologia 175:1257
 Plant perception

Appel and Cocroft (2014) Oecologia 175:1257

 Elicitors and Effectors

ELICITOR: is a molecule able to activate signal a transduction

pathways for the synthesis of metabolites which may reduce
damage and increase resistance to pests.

EFFECTOR: is a molecule that selectively can either trigger or compromise plant inmunity
by binding to other molecules and altering the defense machinery.
watery
Saliva
gelling
 Elicitors and Effectors

Elicitors or effectors?

PLANT SIDE PHYTOPHAGOUS SIDE

- Enzymes: proteases - Enzymes: glucose oxidases
- Peptides: - FACs: Caeliferins
- Systemin - Ca-binding-proteins
- Hidrxyproline-systemin - Unknown proteins: C002, others
- Inceptins - Specific proteins: Mps, Mes
- Peps - Bruchins
- FACs: Voticilins
 Elicitors or Effectors?

- ENZYMES: β-glucosidases, Glucose oxidases, Oxyreductases, Proteases,...

Acevedo et al. (2015) Current Opin Plant Biol 26:80

 Elicitors-Effectors (Oral Secretions and Fluids)

- FACs (Fatty acid-Amonia acid Conjugates) derived from plant membranes conjugated with
amino acids from arthropod.
- Volicitins, N-(17-hydroxylinolenoyl)-L-glutamine)

- Are produced mainly by lepidopteran and dipteran species

- Are essential for nitrogen assimilation by the larvae

- S. exigua-maize voticilin induces the emission of volatiles (VOCs) by attractng parasitic

wapsthat prey on larvae.
- Lepidopetran voticilins activate early (ROS) and late (secondary metabolites) responses in
rice.
 FAC mediates
Chemicalspecific
defence plant
barriers
responses

M. sexta larvae feeding on N. attenuata leaves deposits FACs

Lipooxygenase Expression of transcripts:

- Transcription factors (10%)
- Protein Kinases (7%)
- Protein phosphatases (1%)
- Cell wall metabolism (2%)
- Primary metaolism (20%)
- Others
nucleus

elicitors of
defence responses

12,744 unique transcripts identified, 430 and 117 were differentially up- or down regulated 30 min after
FAC elicitation compared with wounding.
A large percentage (25%) encoded putative regulatory components, including 30 TFs and 22 PK
Bonaventure et al. (2011) Trends Plant Sci 16:1360
 Elicitors-Effectors (Oral Secretions and Fluids)

- CAELIFERINS, disulfoxy fatty acids, analogues of FACs

- Specific of Orthroptera (Caelifera suborder)

- Unknown physiological function in the herbivory. Probably related to digestion

- BRUCHINS, long chain ω,α-diols esterified at one or both oxygens

with 3-hydroxypropanoic acid, found in oviposition fluids. Egg-
derived compounds secreted to overcome host defences

- Specific of Coleoptera (Bruchinae family )

- BENCYL CYANIDE found in oviposition fluids

 Other elicitors/effectors

PEPTIDES
(plant side)

Precursor proteins without an N-

terminal secretion signal

Precursor proteins with an N-

terminal secretion signal (Hydroproline-systemin)

Criptic peptides from proteins

with primary functions

Yamaguchi & Huffaker (2011) Current Opin Plant Biol 14:351

 Other elicitors/effectors

INCEPTINS
- Proteolytic products of plant chloroplastic ATP synthase γ-subunit (cATPC)
- Derived from the regulatory domain of the cATP of legumes
- Ingested cATPC is cleaved in insect midguts, partially digested and form inceptins

SYSTEMINS
- Peptides (18 aas) derived from prosystemin (200 aas)
- Prosystemin accumulates in the cytosol of vascular
phloem of solanaceae
- Wounding/herbivore induced prosystemin processing
 Plant defense responses
Manduca sexta
PEST-PLANT
HAEs/HAMPs (wounding/OS) POTATO &TOMATO

PROSYSTEMIN

+ SYSTEMIN

PPRs
pmb R
Phospholipase NADPH
(MAP Kinases) PGs
A2 oxidase
- Ca Production
2+
LINOLIEIC ACID - CalmodulinH2synthesis
O2 “Late Genes”
- Open of ion channels
(octodecanoid pathway) ↓
JASMONIC ACID Proteinase
Inhibitors

PLANT Proteinase Other products

“Early Genes” → Inhibitors
DEFENSES
Other products
(parenchyma phloem) (células de mesófilo)
 Crosstalk-Plant defense responses

PEST-PLANT Manduca sexta

PIN I and PIN II
(wounding/OS) POTATO/TOMATO
PROSYSTEMIN

SYSTEMIN

mp R

PGs

LINOLEIC ACID H2O2 “Late Genes”

JASMONIC ACID

“Early Genes”
(Parenchyma phloem) (Mesophyll cells)
 Elicitor/Effector examples

Bonaventure (2011) Plant Sign Behav 6:2060

 Elicitors/effectors in different aphid species

Comparative analyses of salivary

proteins from 3 aphid species

A. pisum

M. viciae

M. persicae

Vardermorten et al. (2014) Insect Mol Biol 23:67

 Elicitor/Effector examples

Acevedeo et al. (2015) Current Opin Plant Biol 26:80

 Elicitor/Effector in eggs

Eggs
↓
suppress defences

Hilker & Fatouros (2016) Current Opi Plant Biol 32:9

 How to find and identify elicitors/effectors & defence genes?

In silico analyses

Microarrays/RNAseq or Proteomic/Metabolomics and other -omics

T. urticae T. evansi
How to find and identify elicitors/effectors?

Salivary poroteins of T. urticae/T. evansi

Villaroel et al. (2016) Plant J 86:119

 Salivary secreted effectors in mites

Candidates

Control

Candidate effectors causes chlorosis

in Nicotiana benthamiana
(agroinfiltration)

Villarroel et al. (2016) Plant J 86:119

 How to find and identify elicitors/effectors?

Tetranychus urticae

N. benthamiana

Villarroel et al. (2016) Plant J 86:119

 How to find and identify elicitors/effectors?

The salivary protein repertoire of the spider mite T. urticae: a quest for effectors

Jonckheere et al. (2017) Mol Cell Poteomics 15:3594

 How to find and identify elicitors/effectors & defnce genes?

In silico analyses

Microarrays/RNAseq or Proteomic/Metabolomics and other -omics

T. urticae T. evansi
 How to find and identify elicitors/effectors & defnce genes?

Microarrays/RNAseq or Proteomic/Metabolomics or other omics

T. urticae T. evansi
Scan whole plant post-infection control
4 days post-infection Damage quantification

Arabidopsis-T. urticae
Foliar damage (mm2)

Zhurov et al. (2014) Plant Physiol

 How to find and identify elicitors/effectors?

Microarrays/RNAseq or Proteomic/Metabolomics or other omics

Arabidopsis-T. urticae
ARRAYS
Bla-2 vs Kondara

T. urticae
Foliar damage (mm2)

Tomato-T. evansi
ARRAYS
Tomato Heinz

T. urticae vs T. evansi
 How to find and identify elicitors/effectors & defnce genes?

Microarrays/RNAseq or Proteomic/Metabolomics or other omics

Tetranychus
urticae

Short-term responses (one day) Long-term responses (1-5 days)

10 mites/plant 10 mites/plant
Bla-2 and Kondara plant accessions Bla-2 and Kondara plant accessions
6 plants/genotype/time point/treatment 6 plants/genotype/time point/treatment
 How to find and identify elicitors/effectors & defnce genes?

Microarrays/RNAseq
Repartition of genes in biological process
Short term Long term
4

3
∼36%

Enrichment %
635
2

307 842

1
Short and Long experiments are comparable

KON BLA 0
88 65 12 60 162 148 18 103

Response to JA: 6 genes

Defense response: 34 genes
Response to wounding: 11 genes
∼53% Response to others stress: 54 genes Response to stress
Response to abiotic or biotic stimulus
502
Electron transport or energy pathways
Other biological processes
281 159
Other metabolic processes
Other cellular processes
942 genes differentially expressed during short term Transcription
Unknown biological processes
Spider mite bodies and faeces

PROTEASE ACTIVITIES
&
PROTEOMICS
 Spider mite bodies and faeces

Gel free nanoLC–nanoESI-QTOF MS/MS

proteomic analysis of mite faeces
resulted in the identification of
- 4 cathepsins L-like (Cys-Prot)
- 1 aspartyl protease

Tetarnychus urticae genome:

- 29 cathepsins Llike
- 27cathepsins B-like
- 19 legumains and
- 2 aspartyl proteases