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”

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.1001050http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001050

http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001050

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

Plant parasitic nematodes

Adaptation for parasitism: Stylet

Sedentary endoparasitesFamily Heteroderidae

Root-Knot Nematodes (Meloidogyne)

Cyst Nematodes (Heterodera & Globodera)

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

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 SitesSyncytium

• Fused cells

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

Giant-Cells

• Discrete and enlarged cells

• Dense cytoplasm• Cell wall changes• Nuclear division

without cytokinesis

• No cell division

Giant-Cells

Syncytium

Identifying nematode effectors

Parasitism Genes: Nematode EffectorsThe genetic determinants that enable a nematode to infect plants

Parasitism Proteins

Parasitism Genes

• Parasitome

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-

in situ hybridization

SCN Cellulases

2-Gland-specific expression

Developmental expression profile of CBP in H. schachtii

3-High expression level during parasitic stages

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

mRNA in situ hybridization of a cellulase probe to transcripts expressed specifically within the two subventral esophageal gland cells

2-in situ hybridization

Detection of 10A7 mRNA in dorsal gland cells

3-In Planta Localization of effector Proteins

Cellulase secretion into root tissue around the head of a J2Wang, 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

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

CBP C24

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

5-Plant Expression of Parasitism Proteins

WT 10A07ox 10A06oxWT

WT 32E03ox

6-Mutant Complementation

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)

Chorismate mutase complementation

Arabidopsis clv3-1 mutant

Arabidopsis wild-type

A fully restored clv3-1 mutant expressing nematode CLV3-like gene

minimal medium without supplemental phenylalanine and tyrosine

CLV3 Complementation

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

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

7-Gene Silencing

Expression of 16D10 dsRNA in Arabidopsis resulted in resistance effective against the four major RKN species

Huang et al. (2006)103:14302-14306.

Plant host-derived RNAi is used to silence the expression of the

parasitism genes

8-Determination of Nematode Susceptibility

Enhanced nematode susceptibility in the transgenic plants expressing nematode effectors

10A06 interacts specifically with Spermidine Synthase 2

Bright Field

YFP

Overlay

BiFC assay

Hs-R

FCP L

amin C

Vector

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

Bait

Vector

Lamin C

10A06

PreySpermidine Synthase (SPDS2)

PreySpermidine Synthase (SPDS1)Bait

Vector

Lamin C

Hs-RFCP

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

9-Search for Interacting Proteins

Pro-PK

Pro-IAA16

4 dpi 14 dpi7 dpi

10-Characterization of the interacting proteins

Promoter lines, Overexpression, Mutant Lines, …

Functions of Nematode Effectors

1- Cell wall-digesting enzymes

Cellulase (Obtained from either bacteria or fungi by HGT)

Pectinase

Cellulose-binding protein

Expansins

Functions of Parasitism Proteins

Nematodes need to penetrate and migrate through the roots !

Nematodes need to change plant metabolism in the infected cells!

2-Metabolic Pathway Enzymes

Chorismate Mutase


Chorismate Mutase (CM)Functions of Nematode Effectors

Nematodes need to alter plant cell development?

CLAVATA3-like peptide

Unknown peptide < 3KDa

3-Small bioactive peptides


CLV1

CLV3

P

P

P

P

PSignal transduction leading to developmental changes

CLV1

CLV3

P

P

P

P

P

Model for CLAVATA3 Action

CLV1

CLV3

Does the cyst nematode use ‘ligand mimicry’ to alter plant cell development?


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?


Cyst nematode effector 19C07 interacts with the Arabidopsis LAX3 auxin influx transporter

4-Auxin signaling

Lee et al., 2011. Plant Physiology


5-Suppression of host defensesFunctions of Nematode Effectors

Polyamine biosynthesis

An effector 10AO7 specifically interacts and induces SPDS2 activity and alters spermidine level.

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


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.12160

http://onlinelibrary.wiley.com/doi/10.1111/mpp.12160/full#mpp12160-fig-0005

7- Control of Transcriptional Machinery

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

SM Liu et al. Nature 000, 1-5 (2012) doi:10.1038/nature11651

Functional validation of SHMT by VIGS, RNAi and complementation.

Note: This figure is from a near-final version AOP and may change prior to final publication in print/online

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

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 root-knot nematode

Meloidogyne incognita

Resistance to the potato aphid Macrosiphum

euphorbiae

Nematodes

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