Nematodes
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Nematodes. 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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Nematodes

Nematodes


Nematodes are extremely abundant and diverse

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”


Nematodes

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


Nematodes

Plant parasitic nematodes


Adaptation for parasitism stylet

Adaptation for parasitism: Stylet


Nematodes

Cyst Nematodes (Heterodera & Globodera)

Root-Knot Nematodes (Meloidogyne)

Sedentary endoparasites

Family Heteroderidae


Nematodes

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


Hatch

Hatch


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J-1 occurs in the egg


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Preparasitic J2 Hatches from the egg


Feeding sites formation

Feeding Sites Formation


Gland cells

Gland Cells


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- Extensive endoreduplication

- Increased cytoplasmic density

- Cell wall degradation

- Breakdown of large vacuoles

- Increased numbers of organelles

- High metabolic activity


Nematodes

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


Nematodes

Giant-Cells


Nematodes

Giant-Cells


Nematodes

Syncytium


Identifying nematode effectors

Identifying nematode effectors


Nematodes

Parasitism Genes: Nematode Effectors

The genetic determinants that enable a nematode to infect plants

  • Parasitome

ParasitismGenes

Parasitism Proteins


Nematodes

SCN Cellulases

in situ hybridization

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

MNWMHYCLIACFSIYYFNTVESSTINSVTVQVNKIENNEKGRQFNLKFTNQVYERVCHVDFRVDLPDTAKLDKYSKMVPIPDTCGQYALPKSLDLLPGETFDAQLTLLGHDGKPNVTVLNTNNIPTSKQCKK-

2-Gland-specific expression


Nematodes

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.


Nematodes

Evidences for Secretion

-Enzymes without substrates (cellulase and pectinase)

-Enzymes without pathway (chorismatemutase, shikimate pathway) Putative Function Assignment

-Similarities 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


Nematodes

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


Nematodes

Secretion of cellulase (green fluorescence) associated with cell wall degradation along the migratory path of the J2

Wang, et al. 1999; 12:64-67


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4 Intracellularlocalization 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 2005;6:187-191.

C24

CBP

Expression of a nematode parasitism gene in plant tissues stimulated root growth


Nematodes

5-Plant Expression of Parasitism Proteins

WT

10A06ox

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 wild-type

Arabidopsis clv3-1 mutant

Lambert et al. MPMI, 1999; 12:328–336.

Wang et al.,, Molecular Plant Pathology 2005;6:187-191.


Nematodes

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.


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8-Determination of Nematode Susceptibility

Enhanced nematode susceptibility in the transgenic plants expressing nematode effectors


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Prey

Prey

Spermidine Synthase (SPDS1)

Spermidine Synthase (SPDS2)

Bait

10A06

Lamin C

Hs-RFCP Lamin C Vector

Vector

SD/-Leu/-Trp

SD/-Leu/-Trp/-Ade/-His

BiFC assay

Bright Field

Bait

Hs-RFCP

YFP

Lamin C

Vector

SD/-Leu/-Trp

SD/-Leu/-Trp/-Ade/-His

Overlay

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

7 dpi

14 dpi

Pro-PK

Pro-IAA16


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Functions of Nematode Effectors


Nematodes

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


Nematodes

Functions of Nematode Effectors

Nematodes need to change plant metabolism in the infected cells!

2-Metabolic Pathway Enzymes

ChorismateMutase


Chorismate mutase cm

ShikimatePathway

Chorismate

Tryptophan

CM

Tyrosine

Phenylalanine

Prephenate

Functions of Nematode Effectors

Chorismate Mutase (CM)


Nematodes

Functions of Nematode Effectors

Nematodes need to alter plant cell development?

3-Small bioactive peptides

CLAVATA3-like peptide

Unknown peptide < 3KDa


Nematodes

CLV3

CLV3

CLV3

CLV1

CLV1

P

P

P

P

P

P

P

P

P

P

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?


Nematodes

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?


Nematodes

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


Nematodes

Functions of Nematode Effectors

5-Suppression of host defenses

Polyamine biosynthesis

An effector 10AO7 specifically

interacts and induces SPDS2

activity and alters spermidine

level.


Nematodes

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


Nematodes

7- Control of Transcriptional Machinery

A Meloidogyne incognita effector is imported into the nucleus and exhibits transcriptional activation activity in planta

Molecular Plant Pathology30 JUN 2014 DOI: 10.1111/mpp.12160http://onlinelibrary.wiley.com/doi/10.1111/mpp.12160/full#mpp12160-fig-0005


Nematode resistance genes

Nematode Resistance Genes


Nematodes

The Rhg4 locus has a gene encoding serine hydroxymethyl transferase (SHMT)

Functional validation of SHMT

by VIGS, RNAi and complementation.

SM Liu et al.Nature000, 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

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


Nematodes

Broad Resistance of Mi-1 Gene

resistance to the root-knot nematode Meloidogyne incognita

Resistance to the potato aphid Macrosiphum euphorbiae


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