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Nematodes Parijat S Juvale Department of Plant Pathology and Microbiology Room No. 429, Bessey Hall [email protected] 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.1001050 http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001050 Adaptation for parasitism: Stylet 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 Sedentary endoparasites Family Heteroderidae Cyst Nematodes (Heterodera & Globodera) Root-Knot Nematodes (Meloidogyne) SCN and RKN distribution in USA Soybean root showing SCN cysts Tomato root system galled by root-knot nematode Life cycle Six stages (egg, 4 juvenile stages, and adult) 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 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 Detection and collection of the gland cells Signal peptide prediction N-terminal sequence that targets proteins to ER and the secretory pathway MNWMHYCLIACFSIYYFNTVESSTINSVTVQVNKIEN NEKGRQFNLKFTNQVYERVCHVDFRVDLPDTAKLDK YSKMVPIPDTCGQYALPKSLDLLPGETFDAQLTLLGH DGKPNVTVLNTNNIPTSKQCKK- 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 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. 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 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 Plastids 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 5-Plant Expression of Parasitism Proteins A Plasmodium‐like virulence effector of the soybean cyst nematode suppresses plant innate immunity New Phytologist 21643, 6 JUN 2016 DOI: 10.1111/nph.14047 http://onlinelibrary.wiley.com/doi/10.1111/nph.14047/full#nph14047-fig-0003 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) 10AO6 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. 5-Suppression of host defenses A Plasmodium‐like virulence effector of the soybean cyst nematode suppresses plant innate immunity New Phytologist 21643, 6 JUN 2016 DOI: 10.1111/nph.14047 http://onlinelibrary.wiley.com/doi/10.1111/nph.14047/full#nph14047-fig-0004 6- 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
