Nematodes

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Nematodes
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Nematodes are extremely abundant and diverse

• Variable size 0.2 mm to over 3 m
• Found in virtually all the ecosystems.
• Over 20,000 species have been described.
• Numerically extremely dominant, over 80 of all
  living animals on earth are nematodes!
• Grouped into a phylum Nematoda

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• Figure 2. The relationships of the Nematoda.

Blaxter M (2011) Nematodes The Worm and Its
Relatives. PLoS Biol 9(4) e1001050.
doi10.1371/journal.pbio.1001050 http//www.plosbi
ology.org/article/infodoi/10.1371/journal.pbio.10
01050
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Plant parasitic nematodes
Feeding Strategy Example Genera Infective Stage Resistant Stage Important hosts Resistant Stage Notes
Ectoparasite BelonolaimusXiphenemaTrichodorus J2-adultJ2-adultJ2-adult   Citrus, woody plants   Vector virusesVector viruses
Semi-Endoparasites RotylenchulusTylenchulus J4J2 J4J2 Cotton, citrus J4J2  
Migratory Endoparasites PratylenchusRadopholus J2-adult Cottton, tobacco, citrus, corn  
Sedentary Endoparasites MeloidogyneHeteroderaNaccobus J2J2J2 Egg/cyst soybean, rice, corn, potato, cotton, cereal, pea, vegetables Egg/cyst  
Stem and Bulb Nematodes BursaphelenchusDitylenchus J4J4 J3J4 Coconut, rice J3J4 J4 vectored by insects
Seed Gall Nematodes Anguina J2 J2 Cereal, rice J2  
Foliar Nematodes Aphelenchoides J2-adult Adult rice Adult  
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Adaptation for parasitism Stylet
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Sedentary endoparasites Family Heteroderidae
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(No Transcript)
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Rows of stunted, chlorotic soybean plants damaged
by soybean cyst nematode
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Tomato root system galled by root-knot nematode
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Life cycle
Six stages (egg, 4 juvenile stages, and adult)
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Cyst filled with hundreds of embryonated eggs
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Hatch
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J-1 occurs in the egg
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Preparasitic J2 Hatches from the egg
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(No Transcript)
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Feeding Sites Formation
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Gland Cells
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(No Transcript)
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- Extensive endoreduplication - Increased
cytoplasmic density - Cell wall degradation -
Breakdown of large vacuoles - Increased numbers
of organelles - High metabolic activity
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Feeding Sites
Syncytium
Giant-Cells

• Fused cells
• Dense cytoplasm
• Cell wall changes
• No nuclear division
• No cell division

• Discrete and enlarged cells
• Dense cytoplasm
• Cell wall changes
• Nuclear division without cytokinesis
• No cell division

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Giant-Cells
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Giant-Cells
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Syncytium
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Identifying nematode effectors
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Parasitism Genes Nematode Effectors The genetic
determinants that enable a nematode to infect
plants

• Parasitome

Parasitism Genes
Parasitism Proteins
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Construction of gland-specific cDNA libraries
Microaspiration of esophageal gland cell
cytoplasm
1-Signal peptide prediction N-terminal sequence
that targets proteins to ER and the secretory
pathway
MNWMHYCLIACFSIYYFNTVESSTINSVTVQVNKIENNEKGRQFNLKFTN
QVYERVCHVDFRVDLPDTAKLDKYSKMVPIPDTCGQYALPKSLDLLPGET
FDAQLTLLGHDGKPNVTVLNTNNIPTSKQCKK-
2-Gland-specific expression
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3-High expression level during parasitic stages
Developmental expression profile of CBP in H.
schachtii
These criteria allowed the identification of more
than 50 putative parasitism proteins Huang et
al. MPMI Vol. 16, No. 5, 2003, pp. 376381. Gao
et al. MPMI Vol. 16, No. 8, 2003, pp. 720726.
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Evidences for Secretion -Enzymes without
substrates (cellulase and pectinase) -Enzymes
without pathway (chorismate mutase, shikimate
pathway) Putative Function Assignment -Similaritie
s are with other parasitic nematodes, bacteria,
fungi or plants but not with proteins from C.
elegans
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Experimental Approaches for Functional
Characterization of Nematode Effectors
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1-Developmental expression profile
High expression level during parasitic stages
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2-in situ hybridization
Detection of 10A7 mRNA in dorsal gland cells
mRNA in situ hybridization of a cellulase probe
to transcripts expressed specifically within the
two subventral esophageal gland cells
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3-In Planta Localization of effector Proteins
Cellulase secretion into root tissue around the
head of a J2
Wang, et al. 1999 1264-67
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Secretion of cellulase (green fluorescence)
associated with cell wall degradation along the
migratory path of the J2
Wang, et al. 1999 1264-67
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4 Intracellular localization of the effectors
Plasma membrane
Cytoplasmic
Nuclear
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5-Plant Expression of Parasitism Genes
Transgenic Arapidopsis expressing a nematode
Clavata3-like gene showing an arrested shoot
apical meristem
Wang et al.,, Molecular Plant Pathology
20056187-191.
C24
CBP
Expression of a nematode parasitism gene in
plant tissues stimulated root growth
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5-Plant Expression of Parasitism Proteins
10A06ox
WT
WT
10A07ox
32E03ox
WT
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6-Mutant Complementation
Chorismate mutase complementation
CLV3 Complementation
minimal medium without supplemental phenylalanine
and tyrosine
A CM deficient E coli strain transformed with a
plasmid containing CM coding region was streaked
on the top half of the petri dish The same
CM-deficient E. coli strain containing only the
plasmid was streaked at the bottom half of the
plate (Vector)
A fully restored clv3-1 mutant expressing
nematode CLV3-like gene
Arabidopsis clv3-1 mutant
Arabidopsis wild-type
Lambert et al. MPMI, 1999 12328336.
Wang et al.,, Molecular Plant Pathology
20056187-191.
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7-Gene Silencing
Plant host-derived RNAi is used to silence the
expression of the parasitism genes
Expression of 16D10 dsRNA in Arabidopsis resulted
in resistance effective against the four major
RKN species Huang et al. (2006)10314302-14306.

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8-Determination of Nematode Susceptibility
Enhanced nematode susceptibility in the
transgenic plants expressing nematode effectors
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9-Search for Interacting Proteins

10A06 interacts specifically with Spermidine
Synthase 2
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10-Characterization of the interacting
proteins Promoter lines, Overexpression, Mutant
Lines,
4 dpi
14 dpi
7 dpi
Pro-PK
Pro-IAA16
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Functions of Nematode Effectors
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Functions of Parasitism Proteins
Nematodes need to penetrate and migrate through
the roots !
1- Cell wall-digesting enzymes Cellulase
(Obtained from either bacteria or fungi by
HGT) Pectinase Cellulose-binding
protein Expansins
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Functions of Nematode Effectors
Nematodes need to change plant metabolism in the
infected cells!
2-Metabolic Pathway Enzymes
Chorismate Mutase
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Chorismate Mutase (CM)
Functions of Nematode Effectors
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Functions of Nematode Effectors
Nematodes need to alter plant cell development?
3-Small bioactive peptides
CLAVATA3-like peptide Unknown peptide lt 3KDa
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Functions of Nematode Effectors
Model for CLAVATA3 Action
CLV1
Signal transduction leading to developmental
changes
Does the cyst nematode use ligand mimicry to
alter plant cell development?
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Functions of Nematode Effectors
SCN SYV46 functions as CLAVATA3
clv3-1 mutant
SYV46 in clv3-1
wild-type
Does the cyst nematode use ligand mimicry to
alter plant cell development?
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Functions of Nematode Effectors
4-Auxin signaling
Lee et al., 2011. Plant Physiology
Cyst nematode effector 19C07 interacts with the
Arabidopsis LAX3 auxin influx transporter
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Functions of Nematode Effectors
5-Suppression of host defenses
Polyamine biosynthesis
An effector 10AO7 specifically interacts and
induces SPDS2 activity and alters spermidine
level.
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Functions of Nematode Effectors
Nematodes need to cell cycle activities in
parasitized plant cells
6-RanBPM
Secretory protein with high similarity to
proteins binding to the small G-protein Ran
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A Meloidogyne incognita effector is imported into
the nucleus and exhibits transcriptional
activation activity in?planta
7- Control of Transcriptional Machinery
Molecular Plant Pathology30 JUN 2014 DOI
10.1111/mpp.12160http//onlinelibrary.wiley.com/d
oi/10.1111/mpp.12160/fullmpp12160-fig-0005
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Nematode Resistance Genes
Hs1 pro-1 Sugar beet Sugar beet cyst nematode Heterodera schachtii
Mi-1 Tomato Root-knot nematodes Meloidogyne incognita, M. javanica, M. arenaria Potato aphid White fly
Hero A Tomato Potato cyst nematode Globodera rostochiensis Globodera pallida pathotypes
Rhg1 and Rhg4 Soybean Soybean cyst nematode Heterodera glycines type 0
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The Rhg4 locus has a gene encoding serine
hydroxymethyl transferase (SHMT)
Functional validation of SHMT by VIGS, RNAi and
complementation.
SM Liu et al. Nature 000, 1-5 (2012)
doi10.1038/nature11651
Note This figure is from a near-final version
AOP and may change prior to final publication in
print/online
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Copy Number Variation of Multiple Genes at Rhg1
Mediates Nematode Resistance in Soybean
Cook et al.,Science 30 November 2012vol. 338 no.
6111 1206-1209
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Broad Resistance of Mi-1 Gene
resistance to the root-knot nematode Meloidogyne
incognita
Resistance to the potato aphid Macrosiphum
euphorbiae