Only a small subset of the SPRY domain gene family in Globodera pallida is likely to encode effectors, two of which suppress host defences induced by the potato resistance gene Gpa2

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Analysis of the genome sequence of the potato cyst nematode, Globodera pallida, has shown that a substantial gene family (approximately 300 sequences) of proteins containing a SPRY domain is present in this species. This is a huge expansion of the gene family as compared to other organisms, including other plant-parasitic nematodes. Some SPRY domain proteins from G. pallida and G. rostochiensis have signal peptides for secretion and are deployed as effectors. One of these SPRYSEC proteins has been shown to suppress host defence responses. We describe further analysis of this gene family in G. pallida. We show that only a minority (10%) of the SPRY domain proteins in this species have a predicted signal peptide for secretion and that the presence of a signal peptide is strongly correlated with the corresponding gene being expressed at the early stages of parasitism. The data suggest that while the gene family is greatly expanded, only a minority of SPRY domain proteins in G. pallida are SPRYSEC candidate effectors. We show that several new SPRYSECs from G. pallida are expressed in the dorsal gland cell and demonstrate that some, but not all, of the SPRYSECs can suppress the hypersensitive response induced by co-expression of the resistance gene Gpa2 and its cognate avirulence factor RBP-1 in Nicotiana benthamiana.

Only a small subset of the SPRY domain gene family in Globodera pallida is likely to encode effectors, two of which suppress host defences induced by the potato resistance gene Gpa2

in Nematology

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AbadP.GouzyJ.AuryJ.-M.Castagnone-SerenoP.DanchinE.G.J.DeleuryE.Perfus-BarbeochL.AnthouardV.ArtiguenaveF.BlokV.C. (2008). Genome sequence of the metazoan plant-parasitic nematode Meloidogyne incognita. Nature Biotechnology 26909-915.

ArmstrongM.R.WhissonS.C.PritchardL.BosJ.I.B.VenterE.AvrovaA.O.RehmanyA.P.BohmeU.BrooksK.CheravachI. (2005). An ancestral oomycete locus contains late blight avirulence gene Avr3a, encoding a protein that is recognized in the host cytoplasm. Proceedings of the National Academy of Sciences of the United States of America 1027766-7771.

BakkerE.DeesR.BakkerJ.GoverseA. (2006). Mechanisms involved in plant resistance to nematodes. In: TuzunS.BentE. (Eds). Multigenic and induced systemic resistance in plants. Heidelberg, GermanySpringer Academic Publishers pp.  314-334.

BendtsenD.J.NielsenH.von HeijneG.BrunakS. (2004). Improved prediction of signal peptides: SignalP 3.0. Journal of Molecular Biology 340783-795.

CaswellE.P.ThomasonI.J.MckinneyH.E. (1985). Extraction of cysts and eggs of Heterodera schachtii from soil with an assessment of extraction efficiency. Journal of Nematology 17337-340.

ChronisD.ChenS.LuS.HeweziT.CarpenterS.C.D.LoriaR.BaumT.J.WangX. (2013). A ubiquitin carboxyl extension protein secreted from a plant-parasitic nematode Globodera rostochiensis is cleaved in planta to promote plant parasitism. Plant Journal 74185-196.

CottonJ.A.LilleyC.J.JonesL.M.KikuchiT.ReidA.J.ThorpeP.TsaiI.J.BeasleyH.BlokV.C.CockP.J.A. (2014). The genome and life-stage specific transcriptomes of Globodera pallida elucidate key aspects of plant parasitism by a cyst nematode. Genome Biology 15R43.

EllenbyC. (1952). Resistance to the potato root eelworm, Heterodera rostochiensis Wollenweber. Nature 1701016.

Eves-van den AkkerS.LilleyC.J.JonesJ.T.UrwinP.E. (2014). Identification and characterisation of a hyper-variable apoplastic effector gene family of the potato cyst nematodes. PLoS Pathogens 10e1004391.

FinnR.D.BatemanA.ClementsJ.CoggillP.EberhardtR.Y.EddyS.R.HegerA.HetheringtonK.HolmL.MistryJ. (2014). The Pfam protein families database. Nucleic Acids ResearchDatabase Issue 42D222-D230.

GabriëlsS.H.E.J.VossenJ.H.EkengrenS.K.van OoijenG.Abd-El-HaliemA.M.van den BergG.C.M.RaineyD.Y.MartinG.B.TakkenF.L.W.de WitP.J.G.M. (2007). An NB-LRR protein required for HR signalling mediated by both extra- and intracellular resistance proteins. Plant Journal 5014-28.

GaoB.L.AllenR.MaierT.DavisE.L.BaumT.J.HusseyR.S. (2003). The parasitome of the phytonematode Heterodera glycines. Molecular Plant-Microbe Interactions 16720-726.

GilroyE.M.TaylorR.M.HeinI.BoevinkP.SadanandomA.BirchP.R. (2011). CMPG1-dependent cell death follows perception of diverse pathogen elicitors at the host plasma membrane and is suppressed by Phytophthora infestans RXLR effector AVR3a. New Phytologist 190653-666.

GrundlerF.M.W.BetkaM.WyssU. (1991). Influence of changes in the nurse cell system (syncytium) on sex determination and development of Heterodera schachtii: total amounts of proteins and amino acids. Phytopathology 8170-74.

GuindonS.DufayardJ.F.LefortV.AnisimovaM.HordijkW.GascuelO. (2010). New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Systematic Biology 59307-321.

HaegemanA.MantelinS.JonesJ.T.GheysenG. (2012). Secretions of plant parasitic nematodes. Gene 49219-31.

HocklandS.NiereB.GrenierE.BlokV.PhillipsM.Den NijsL.AnthoineG.PickupJ.VianeN. (2012). An evaluation of the implications of virulence in non-European populations of Globodera pallida and G. rostochiensis for potato cultivation in Europe. Nematology 141-13.

JacobJ.MitrevaM.VanholmeB.GheysenG. (2008). Exploring the transcriptome of the burrowing nematode Radopholus similis. Molecular Genetics and Genomics 2801-17.

JanssenR.BakkerJ.GommersF.J. (1991). Mendelian proof for a gene-for-gene relationship between virulence of Globodera rostochiensis and the H1 resistance gene in Solanum tuberosum ssp. andigena CPC 1673. Revue de Nématologie 14213-219.

JaouannetM.Perfus-BarbeochL.DeleuryE.MaglianoM.EnglerG.VieiraP.DanchinE.G.J.Da RochaM.CoquillardP.AbadP. (2012). A root-knot nematode-secreted protein is injected into giant cells and targeted to the nuclei. New Phytologist 194924-931.

JonesJ.D.G.DanglJ.L. (2006). The plant immune system. Nature 444323-329.

JonesJ.T.CurtisR.H.WightmanP.J.BurrowsP.R. (1996). Isolation and characterization of a putative collagen gene from the potato cyst nematode Globodera pallida. Parasitology 113581-588.

JonesJ.T.FurlanettoC.BakkerE.BanksB.BlokV.C.ChenQ.PriorA. (2003). Characterisation of a chorismate mutase from the potato cyst nematode Globodera pallida. Molecular Plant Pathology 443-50.

JonesJ.T.KumarA.PylypenkoL.A.ThirugnanasambandamA.CastelliL.ChapmanS.CockP.J.GrenierE.LilleyC.J.PhillipsM.S. (2009). Identification and functional characterisation of effectors in Expressed Sequence Tags from various life cycle stages of the potato cyst nematode Globodera pallida. Molecular Plant Pathology 10815-828.

JonesJ.T.GheysenG.FenollC. (Eds) (2011). Genomics and molecular genetics of plant-nematode interactions. Heidelberg, GermanySpringer Academic Publishers.

KaloshianI.DesmondO.J.AtamianH.S. (2011). Disease resistance-genes and defense responses during incompatible interactions. In: JonesJ.T.GheysenG.FenollC. (Eds). Genomics and molecular genetics of plant-nematode interactions. Heidelberg, GermanySpringer Academic Publishers pp.  309-324.

KamounS.HamadaW.HuitemaE. (2003). Agrosuppression: a bioassay for the hypersensitive response suited to high-throughput screening. Molecular Plant-Microbe Interactions 167-13.

KarimiM.InzeD.DepickerA. (2002). Gateway vectors for Agrobacterium-mediated plant transformation. Trends in Plant Science 7193-195.

KatohK.StandleyD.M. (2013). MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular Biology and Evolution 30772-780.

KikuchiT.CottonJ.A.DalzellJ.J.HasegawaK.KanzakiN.McVeighP.TakanashiT.TsaiI.J.AssefaS.A.CockP.J.A. (2011). Genomic insights into the origin of parasitism in the emerging plant pathogen Bursaphelenchus xylophilus. PLoS Pathogens 7e1002219.

LeeC.ChronisD.KenningC.PeretB.HeweziT.DavisE.L.BaumT.J.HusseyR.S.BennetM.MitchumM.G. (2011). The novel cyst nematode effector protein 19C07 interacts with the Arabidopsis auxin influx transporter LAX3 to control feeding site development. Plant Physiology 155866-880.

Lozano-TorresJ.L.WilbersR.H.P.GawronskiP.BoshovenJ.C.Finkers-TomczakA.CordewenerJ.H.G.AmericaA.H.P.OvermarsH.A.Van’t KloosterJ.W.BaranowskiL. (2012). Dual disease resistance mediated by the immune receptor Cf-2 in tomato requires a common virulence target of a fungus and a nematode. Proceedings of the National Academy of Sciences of the United States of America 10910119-10124.

MaierT.R.HeweziT.PengJ.BaumT.J. (2013). Isolation of whole esophageal gland cells from plant-parasitic nematodes for transcriptome analyses and effector identification. Molecular Plant-Microbe Interactions 2631-35.

MilneI.LindnerD.BayerM.HusmeierD.McGuireG.MarshallD.F.WrightF. (2009). TOPALi v2: a rich graphical interface for evolutionary analyses of multiple alignments on HPC clusters and multi-core desktops. Bioinformatics 25126-127.

MitchumM.G.HusseyR.S.BaumT.J.WangX.EllingA.A.WubbenM.DavisE.L. (2013). Nematode effector proteins: an emerging paradigm of parasitism. New Phytologist 199879-894.

OppermanC.H.BirdD.M.WilliamsonV.M.RokhsarD.S.BurkeM.CohnJ.CromerJ.DienerS.GajanJ.GrahamS. (2008). Sequence and genetic map of Meloidogyne hapla: a compact nematode genome for plant parasitism. Proceedings of the National Academy of Sciences of the United States of America 10514802-14807.

PerfettoL.GherardiniP.F.DaveyN.E.DiellaF.Helmer-CitterichM.CesareniG. (2013). Exploring the diversity of SPRY/B30.2-mediated interactions. Trends in Biochemical Sciences 3838-46.

PhillipsM.S.TrudgillD.L. (1998). Variation of virulence, in terms of quantitative reproduction of Globodera pallida populations, from Europe and South America, in relation to resistance from Solanum vernei and S. tuberosum ssp. andigena CPC 2802. Nematologica 44409-423.

PontingC.SchultzJ.BorkP. (1997). SPRY domains in ryanodine receptors (Ca2+-release channels). Trends in Biochemical Sciences 22193-194.

PostmaW.J.SlootwegE.J.RehmanS.Finkers-TomczakA.TytgatT.O.G.van GelderenK.Lozano-TorresJ.L.RoosienJ.PompR.van SchaikC. (2012). The effector SPRYSEC-19 of Globodera rostochiensis suppresses CC-NB-LRR mediated disease resistance in plants. Plant Physiology 160944-954.

RehmanS.PostmaW.TytgatT.PrinsP.QinL.OvermarsH.VossonJ.SpiridonL.N.PetrescuA.J.GoverseA. (2009). A secreted SPRY domain-containing protein from the plant parasitic nematode Globodera rostochiensis interacts with a CC-NB-LRR protein from a susceptible tomato. Molecular Plant-Microbe Interactions 22330-340.

RobertsonL.RobertsonW.M.SobczakM.BakkerJ.TetaudE.ArinagayayamM.R.FergusonM.A.J.FairlambA.H.JonesJ.T. (2000). Cloning, expression and functional characterisation of a thioredoxin peroxidase from the potato cyst nematode Globodera rostochiensis. Molecular and Biochemical Parasitology 11141-49.

SaccoM.A.KoropackaK.GrenierE.JaubertM.J.BlanchardA.GoverseA.SmantG.MoffettP. (2009). The cyst nematode SPRYSEC protein RBP-1 elicits Gpa2- and RanGAP2-dependent plant cell death. PLoS Pathogens 5e1000564.

SaundersD.G.O.BreenS.WinJ.SchornackS.HeinI.BozkurtT.O.ChampouretN.VleeshouwersV.G.A.A.BirchP.R.J.GilroyE.M. (2012). Host protein BSL1 associates with Phytophthora infestans RXLR effector AVR2 and the Solanum demissum immune receptor R2 to mediate disease resistance. Plant Cell 243420-3434.

SchomakerC.H.BeenT.H. (2013). Plant growth and population dynamics. In: PerryR.N.MoensM. (Eds). Plant nematology2nd edition. Wallingford, UKCAB International pp.  301-330.

SiY.PengL.PinghuaL.BrutnellT.P. (2013). Model-based clustering for RNA-seq data. Bioinformatics 30197-205.

SlootwegE.RoosienJ.SpiridonL.N.PetrescuA.-J.TamelingW.JoostenM.PompR.van SchaikC.DeesR.BorstJ.W. (2010). Nucleocytoplasmic distribution is required for activation of resistance by the potato NB-LRR receptor Rx1 and is balanced by its functional domains. Plant Cell 224195-4215.

SobczakM.GolinowskiW. (2011). Cyst nematodes and syncytia. In: JonesJ.T.GheysenG.FenollC. (Eds). Genomics and molecular genetics of plant-nematode interactions. Heidelberg, GermanySpringer Academic Publishers pp.  61-82.

SteentoftC.VakhrushevS.Y.JoshiH.J.KongY.Vester-ChristensenM.B.SchjoldagerK.T.-B.G.LavrsenK.DabelsteenS.PedersenN.B.Marcos-SilvaL. (2013). Precision mapping of the human O-GalNAc glycoproteome through SimpleCell technology. EMBO Journal 321478-1488.

SullivanM.J.InserraR.N.FrancoJ.Moreno-LeheudéI.GrecoN. (2007). Potato cyst nematodes: plant host status and their regulatory impact. Nematropica 37193-201.

ThomasC.M.TangS.Hammond-KosackK.JonesJ.D.G. (2000). Comparison of the hypersensitive response induced by the tomato Cf-4 and Cf-9 genes in Nicotiana spp. Molecular Plant-Microbe Interactions 13465-469.

ThorpeP.MantelinS.CockP.J.A.BlokV.C.CokeM.C.CottonJ.A.Eves-van den AkkerS.GuzeevaE.LilleyC.J.ReidA.J. (2014). Characterisation of the full effector complement of the potato cyst nematode Globodera pallida. BMC Genomics 15923.

TurnerS.J.EvansK. (1998). The origins, global distribution and biology of potato cyst nematodes (Globodera rostochiensis (Woll.) and Globodera pallida Stone). In: MarksR.J.BrodieB.B. (Eds). Potato cyst nematodes. Wallingford, UKCAB International pp.  7-26.

van der FitsL.DeakinE.A.HogeJ.H.C.MemelinkJ. (2000). The ternary transformation system: constitutive virG on a compatible plasmid dramatically increases Agrobacterium-mediated plant transformation. Plant Molecular Biology 43495-502.

VieiraP.DanchinE.G.J.NeveuC.CrozatC.JaubertS.HusseyR.S.EnglerG.AbadP.de Almeida-EnglerJ.Castagnone-SerenoP. (2011). The plant apoplasm is an important recipient compartment for nematode secreted proteins. Journal of Experimental Botany 621241-1253.

WangX.MitchumM.G.GaoB.LiC.DiabH.BaumT.J.HusseyR.S.DavisE.L. (2005). A parasitism gene from a plant-parasitic nematode with function similar to CLAVATA3/ESR (CLE) of Arabidopsis thaliana. Molecular Plant Pathology 6187-191.

WaterhouseA.M.ProcterJ.B.MartinD.M.A.ClampM.BartonG.J. (2009). Jalview Version 2 a multiple sequence alignment editor and analysis workbench. Bioinformatics 251189-1191.

Figures

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    Phylogeny for SPRY domain proteins of Globodera pallida and other plant-parasitic nematodes. The distance tree was mid-point rooted. Distances used were based on maximum-likelihood estimated parameters (see text for details) for the SPRY domain only. Numbers at branching points indicate bootstrap percentages (when ⩾50%) derived from 100 replicates. Sequences of SPRY domain proteins from G. pallida (blue), Meloidogyne incognita (green) and Bursaphelenchus xylophilus (red) are represented. SPRYSEC candidate effectors from G. pallida are shown in light blue and G. pallida proteins lacking a predicted signal peptide shown in dark blue. A major clade is present at the top of the tree containing the SPRY domain proteins from some G. pallida sequences and other nematodes while a large G. pallida specific expansion is present below this clade.

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    Comparison of expression profiles, inferred from RNAseq data, of Globodera pallida SPRY domain proteins with (A) and without (B) a predicted signal peptide. Y axis figures represent Reads Per Kilobase per Million (RPKM). All SPRY domain proteins predicted from the G. pallida genome are included in this analysis. Each line represents expression pattern of an individual sequence. Sequences with a predicted signal peptide (30 sequences) are upregulated at second-stage juvenile (J2) or early parasitic stages, whilst the vast majority of sequences without a predicted signal peptide (269 sequences) are expressed constitutively or at the adult male stage. This figure is published in colour in the online edition of this journal, which can be accessed via http://booksandjournals.brillonline.com/content/journals/15685411.

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    Localisation of expression of some genes encoding some SPRYSEC candidate effectors by in situ hybridisation to Globodera pallida preparasitic second-stage juveniles (J2). Sections of nematodes were incubated with antisense probes designed based on DNA coding sequence for the following gene loci: A: GPLIN_000892900; B: GpSPRY-17I9-1; C: GpSPRY-414-2. All are expressed in the dorsal pharyngeal gland cell (arrows). No staining was observed with sense control probes (not shown). Globodera pallida J2 are approximately 30 μm in diam.

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    SPRYSEC effector candidates GpSPRY-12N3 and GpSPRY-33H17 suppress the hypersensitive response induced by Gpa2/RBP-1. A: Cell death symptoms induced in Nicotiana benthamiana by co-expression of R2/Avr2 (3 days post infiltration (dpi)), R3a/Avr3aKI (2 dpi), Cf-4/Avr4 (4 dpi), Cf-9/Avr9 (4 dpi) Rx/PVX-CP (3 dpi), Gpa2/RBP-1 (7 dpi), an autoactive form of Mi1.2 (Mi1.2T557S; 3 dpi), or the Phytophthora infestans PAMP elicitor INF1 (3 dpi) in leaves expressing either the free enhanced green fluorescent protein (eGFP) as a control or Globodera pallida SPRYSEC candidate effectors GpSPRY-12N3 or GpSPRY-33H17 fused to eGFP at the N or C terminus or lacking a GFP fusion. Asterisks indicate combinations where the symptoms are significantly suppressed by the candidate effector compared to eGFP. B and C: Graphs show the percentage of infiltration sites developing a clear hypersensitive response (HR) over time, at 6-9 dpi depending on the experiment, mediated by Gpa2/RBP-1 in N. benthamiana leaves expressing either free eGFP as a control or G. pallida SPRYSEC candidate effectors GpSPRY-12N3 (B) or GpSPRY-33H17 (C) fused to eGFP at the C-terminus. Experiments were done at least twice with blocks of 12 plants infiltrated on two leaves each; error bars indicate ±SE. Asterisks above the error bars indicate a significant difference (t-test at P<0.05) from the free eGFP control evaluated at the same time point.

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    A tag is required for SPRYSECs to suppress the hypersensitive response (HR) induced by Gpa2/RBP-1. A: No suppression of the HR induced by Gpa2/RBP-1 in Nicotiana benthamiana leaves is observed at 4 days post infiltration (dpi) in the presence of free enhanced green fluorescent protein (eGFP) or untagged SPRYSEC GpSPRY-12N3 compared with spots expressing GpSPRY-12N3 tagged with either eGFP or HA tag. Infiltrated regions are approximately 1 cm in diam. B: Graph shows the percentage of infiltration sites developing a clear HR over time (4-6 dpi) mediated by Gpa2/RBP-1 in N. benthamiana leaves expressing either free eGFP as a control or Globodera pallida SPRYSEC candidate effector GpSPRY-12N3 tagged with either eGFP or HA tag. Experiments were done at least twice with blocks of 12 plants infiltrated on two leaves each; error bars indicate ±SE. Asterisks above the error bars indicate a significant difference (t-test at P<0.05) from the free eGFP control evaluated at the same time point.

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