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Nematodes

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

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  1. Nematodes

  2. 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”

  3. 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

  4. Plant parasitic nematodes

  5. Adaptation for parasitism: Stylet

  6. Cyst Nematodes (Heterodera & Globodera) Root-Knot Nematodes (Meloidogyne) Sedentary endoparasites Family Heteroderidae

  7. Rows of stunted, chlorotic soybean plants damaged by soybean cyst nematode

  8. Tomato root system galled by root-knot nematode

  9. Life cycle Six stages (egg, 4 juvenile stages, and adult)

  10. Cyst filled with hundreds of embryonated eggs

  11. Hatch

  12. J-1 occurs in the egg

  13. Preparasitic J2 Hatches from the egg

  14. Feeding Sites Formation

  15. Gland Cells

  16. - Extensive endoreduplication - Increased cytoplasmic density - Cell wall degradation - Breakdown of large vacuoles - Increased numbers of organelles - High metabolic activity

  17. 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

  18. Giant-Cells

  19. Giant-Cells

  20. Syncytium

  21. Identifying nematode effectors

  22. Parasitism Genes: Nematode Effectors The genetic determinants that enable a nematode to infect plants • Parasitome ParasitismGenes Parasitism Proteins

  23. 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

  24. 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.

  25. 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

  26. Experimental Approaches for Functional Characterization of Nematode Effectors

  27. 1-Developmental expression profile High expression level during parasitic stages

  28. 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

  29. 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

  30. Secretion of cellulase (green fluorescence) associated with cell wall degradation along the migratory path of the J2 Wang, et al. 1999; 12:64-67

  31. 4 Intracellularlocalization of the effectors Plasma membrane Cytoplasmic Nuclear

  32. 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

  33. 5-Plant Expression of Parasitism Proteins WT 10A06ox WT 10A07ox 32E03ox WT

  34. 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.

  35. 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.

  36. 8-Determination of Nematode Susceptibility Enhanced nematode susceptibility in the transgenic plants expressing nematode effectors

  37. 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

  38. 10-Characterization of the interacting proteins Promoter lines, Overexpression, Mutant Lines, … 4 dpi 7 dpi 14 dpi Pro-PK Pro-IAA16

  39. Functions of Nematode Effectors

  40. 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

  41. Functions of Nematode Effectors Nematodes need to change plant metabolism in the infected cells! 2-Metabolic Pathway Enzymes ChorismateMutase

  42. ShikimatePathway Chorismate Tryptophan CM Tyrosine Phenylalanine Prephenate Functions of Nematode Effectors Chorismate Mutase (CM)

  43. Functions of Nematode Effectors Nematodes need to alter plant cell development? 3-Small bioactive peptides CLAVATA3-like peptide Unknown peptide < 3KDa

  44. 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?

  45. 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?

  46. 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

  47. 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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