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. Feeding StrategyExample Genera Infective StageResistant Stage Important hostsResistant StageNotes Ectoparasite Belonolaimus Xiphenema Trichodorus J2-adult J2-adult J2-adult Citrus, woody plants Vector viruses Semi-Endoparasites Rotylenchulus Tylenchulus 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 J3 J4 Coconut, rice J3 J4 J4 vectored by insects Seed Gall NematodesAnguina J2 Cereal, riceJ2 Foliar NematodesAphelenchoides J2-adultAdult riceAdult Plant parasitic nematodes

5. Adaptation for parasitism: Stylet

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

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

9. Tomato root system galled by root-knot nematode

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

11. Cyst filled with hundreds of embryonated eggs

12. Hatch

13. J-1 occurs in the egg

14. Preparasitic J2 Hatches from the egg

16. Feeding Sites Formation

17. Gland Cells

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

20. Feeding Sites Syncytium 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

21. Giant-Cells

23. Syncytium

24. Identifying nematode effectors

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

26. 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- in situ hybridization SCN Cellulases 2-Gland-specific expression

27. 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.

28. 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

29. Experimental Approaches for Functional Characterization of Nematode Effectors

30. 1-Developmental expression profile High expression level during parasitic stages

31. 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

32. 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

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

34. 4 Intracellular localization of the effectors Plasma membrane Cytoplasmic Nuclear

35. 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.

36. 5-Plant Expression of Parasitism Proteins WT10A07ox 10A06oxWT 32E03ox

37. 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.

38. 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

39. 8-Determination of Nematode Susceptibility Enhanced nematode susceptibility in the transgenic plants expressing nematode effectors

40. 10A06 interacts specifically with Spermidine Synthase 2 Bright Field YFP Overlay BiFC assay Hs-RFCP Lamin C Vector SD/-Leu/-TrpSD/-Leu/-Trp/-Ade/-His Bait Vector Lamin C 10A06 Prey Spermidine Synthase (SPDS2) Prey Spermidine Synthase (SPDS1) Bait Vector Lamin C Hs-RFCP SD/-Leu/-TrpSD/-Leu/-Trp/-Ade/-His 9-Search for Interacting Proteins

41. Pro-PK Pro-IAA16 4 dpi14 dpi7 dpi 10-Characterization of the interacting proteins Promoter lines, Overexpression, Mutant Lines, …

42. Functions of Nematode Effectors

43. 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 !

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

45. Chorismate Mutase (CM) Chorismate Prephenate Tyrosine Phenylalanine Shikimate Pathway Tryptophan CM Functions of Nematode Effectors

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

47. CLV1 CLV3 P P P P P Signal 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? Functions of Nematode Effectors

48. SCN SYV46 functions as CLAVATA3 wild-type clv3-1 mutantSYV46 in clv3-1 Does the cyst nematode use ‘ligand mimicry’ to alter plant cell development? Functions of Nematode Effectors

49. Cyst nematode effector 19C07 interacts with the Arabidopsis LAX3 auxin influx transporter 4-Auxin signaling Lee et al., 2011. Plant Physiology Functions of Nematode Effectors

50. 5-Suppression of host defenses Functions of Nematode Effectors Polyamine biosynthesis An effector 10AO7 specifically interacts and induces SPDS2 activity and alters spermidine level.

51. 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 Functions of Nematode Effectors

52. 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 http://onlinelibrary.wiley.com/doi/10.1111/mpp.12160/full#mpp12160-fig-0005 7- Control of Transcriptional Machinery

53. Nematode Resistance Genes Hs1 pro-1 Sugar beetSugar beet cyst nematode: Heterodera schachtii Mi-1TomatoRoot-knot nematodes: Meloidogyne incognita, M. javanica, M. arenaria; Potato aphid: White fly Hero ATomatoPotato cyst nematode: Globodera rostochiensis Globodera pallida pathotypes Rhg1 and Rhg4 SoybeanSoybean cyst nematode: Heterodera glycines type 0

54. 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)

55. 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

56. Broad Resistance of Mi-1 Gene resistance to the root- knot nematode Meloidogyne incognita Resistance to the potato aphid Macrosiphum euphorbiae