Transcript Molecular_Nematology..

Nematodes
Nematodes are extremely abundant
and diverse
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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”
Figure 2. The relationships of the Nematoda.
Blaxter M (2011) Nematodes: The Worm and Its Relatives. PLoS Biol 9(4): e1001050. doi:10.1371/journal.pbio.1001050
http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001050
Plant parasitic nematodes
Feeding Strategy
Example Genera
Infective Stage
Resistant Stage
Ectoparasite
Belonolaimus
Xiphenema
Trichodorus
J2-adult
J2-adult
J2-adult
Semi-Endoparasites
Rotylenchulus
Tylenchulus
J4
J2
J4
J2
Cotton, citrus
J4
J2
Migratory
Endoparasites
Pratylenchus
Radopholus
J2-adult
*
Cottton, tobacco,
citrus, corn
*
Sedentary
Endoparasites
Meloidogyne
Heterodera
Naccobus
J2
J2
J2
Egg/cyst
soybean, rice, corn,
potato, cotton,
cereal, pea,
vegetables
Egg/cyst
Stem and Bulb
Nematodes
Bursaphelenchus
Ditylenchus
J4
J4
J3
J4
Coconut, rice
J3
J4
Seed Gall Nematodes
Anguina
J2
J2
Cereal, rice
J2
Foliar Nematodes
Aphelenchoides
J2-adult
Adult
rice
Adult
Important hosts
Resistant Stage
Notes
Citrus, woody plants
Vector viruses
Vector viruses
J4 vectored by
insects
Adaptation for parasitism: Stylet
Sedentary endoparasites
Family Heteroderidae
Cyst Nematodes (Heterodera & Globodera)
Root-Knot Nematodes (Meloidogyne)
Rows of stunted, chlorotic soybean
plants damaged by soybean cyst
nematode
Tomato root
system galled
by root-knot
nematode
Life cycle
Six stages (egg, 4 juvenile stages, and adult)
Cyst filled with
hundreds of
embryonated eggs
Hatch
J-1 occurs in
the egg
Preparasitic J2 Hatches
from the egg
Feeding Sites Formation
Gland Cells
- Extensive endoreduplication
- Increased cytoplasmic density
- Cell wall degradation
- Breakdown of large vacuoles
- Increased numbers of organelles
- High metabolic activity
Feeding Sites
Syncytium
• Fused cells
Giant-Cells
• Discrete and
enlarged cells
• Dense cytoplasm
• Dense cytoplasm
• Cell wall changes
• Cell wall changes
• No nuclear division • Nuclear division
without cytokinesis
• No cell division
• No cell division
Giant-Cells
Giant-Cells
Syncytium
Identifying nematode effectors
Parasitism Genes: Nematode
Effectors
The genetic determinants that enable a nematode to infect
plants
• Parasitome
Parasitism Genes
Parasitism Proteins
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
MNWMHYCLIACFSIYYFNTVESSTINSVTVQVNKIEN
NEKGRQFNLKFTNQVYERVCHVDFRVDLPDTAKLDK
YSKMVPIPDTCGQYALPKSLDLLPGETFDAQLTLLGH
DGKPNVTVLNTNNIPTSKQCKK-
SCN Cellulases
2-Gland-specific expression
in situ hybridization
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. 376–381.
Gao et al. MPMI Vol. 16, No. 8, 2003, pp. 720–726.
Evidences for Secretion
-Enzymes without substrates (cellulase and
pectinase)
-Enzymes without pathway (chorismate mutase,
shikimate pathway) Putative Function
Assignment
-Similarities are with other parasitic nematodes,
bacteria, fungi or plants but not with proteins
from C. elegans
Experimental Approaches for
Functional Characterization of
Nematode Effectors
1-Developmental expression profile
High expression level during parasitic stages
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
3-In Planta Localization of effector Proteins
Cellulase secretion into root tissue around the head of a J2
Wang, et al. 1999; 12:64-67
Secretion of cellulase (green fluorescence) associated with cell
wall degradation along the migratory path of the J2
Wang, et al. 1999; 12:64-67
4 Intracellular localization of the effectors
Plasma
membrane
Cytoplasmic
Nuclear
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 2005;6:187-191.
CBP
C24
Expression of a nematode parasitism gene
in plant tissues stimulated root growth
5-Plant Expression of Parasitism
Proteins
WT
10A07ox
WT
WT
32E03ox
10A06ox
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)
Lambert et al. MPMI, 1999; 12:328–336.
Arabidopsis
wild-type
Arabidopsis
clv3-1 mutant
A fully restored clv3-1
mutant expressing
nematode CLV3-like
gene
Wang et al.,, Molecular Plant Pathology 2005;6:187-191.
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)103:14302-14306.
8-Determination of Nematode
Susceptibility
Enhanced nematode susceptibility in the transgenic
plants expressing nematode effectors
9-Search for Interacting Proteins
Prey
Spermidine Synthase (SPDS2)
Bait
Hs-RFCP Lamin C Vector
10A06
Lamin C
Vector
SD/-Leu/-Trp
SD/-Leu/-Trp/-Ade/-His
BiFC assay
Prey
Bright Field
Bait
Spermidine Synthase (SPDS1)
Hs-RFCP
YFP
Lamin C
Vector
SD/-Leu/-Trp
SD/-Leu/-Trp/-Ade/-His
Overlay
10A06 interacts specifically with Spermidine Synthase 2
10-Characterization of the interacting proteins
Promoter lines, Overexpression, Mutant Lines, …
4 dpi
Pro-PK
Pro-IAA16
7 dpi
14 dpi
Functions of Nematode Effectors
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
Functions of Nematode Effectors
Nematodes need to change plant metabolism in
the infected cells!
2-Metabolic Pathway Enzymes
Chorismate Mutase
Functions of Nematode Effectors
Chorismate Mutase (CM)
Shikimate
Pathway
Chorismate
Tryptophan
CM
Prephenate
Tyrosine
Phenylalanine
Functions of Nematode Effectors
Nematodes need to alter plant cell development?
3-Small bioactive peptides
CLAVATA3-like peptide
Unknown peptide < 3KDa
Functions of Nematode Effectors
Model for CLAVATA3 Action
CLV3
CLV3
CLV3
CLV1
P
CLV1
P
P
P
P
P
P
CLV1
P
P
P
Signal transduction leading to
developmental changes
Does the cyst nematode use ‘ligand mimicry’ to alter plant
cell development?
Functions of Nematode Effectors
SCN SYV46 functions as CLAVATA3
wild-type
clv3-1 mutant
SYV46 in clv3-1
Does the cyst nematode use ‘ligand mimicry’ to alter plant cell
development?
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
Functions of Nematode Effectors
5-Suppression of host defenses
Polyamine biosynthesis
An effector 10AO7 specifically
interacts and induces SPDS2
activity and alters spermidine
level.
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
7- Control of Transcriptional Machinery
A Meloidogyne incognita effector is imported into the nucleus
and exhibits transcriptional activation activity in planta
Molecular Plant Pathology
30 JUN 2014 DOI: 10.1111/mpp.12160
http://onlinelibrary.wiley.com/doi/10.1111/mpp.12160/full#mpp12160-fig-0005
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
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) doi:10.1038/nature11651
Note: This figure is from a near-final version AOP and may change prior to final publication in print/online
Copy Number Variation of Multiple Genes at
Rhg1 Mediates Nematode Resistance in
Soybean
Cook et al.,Science 30 November 2012:vol. 338 no. 6111 1206-1209
Broad Resistance of Mi-1 Gene
resistance to the rootknot nematode
Meloidogyne
incognita
Resistance to the
potato aphid
Macrosiphum
euphorbiae