The Arabidopsis thaliana papain-like cysteine protease RD21 interacts with a root-knot nematode effector protein

in Nematology
Restricted Access
Get Access to Full Text
Rent on DeepDyve

Have an Access Token?

Enter your access token to activate and access content online.

Please login and go to your personal user account to enter your access token.


Have Institutional Access?

Access content through your institution. Any other coaching guidance?


The root-knot nematode Meloidogyne chitwoodi secretes effector proteins into the cells of host plants to manipulate plant-derived processes in order to achieve successful parasitism. Mc1194 is a M. chitwoodi effector that is highly expressed in pre-parasitic second-stage juvenile nematodes. Yeast two-hybrid assays revealed Mc1194 specifically interacts with a papain-like cysteine protease (PLCP), RD21A in Arabidopsis thaliana. Mc1194 interacts with both the protease and granulin domains of RD21A. PLCPs are targeted by effectors secreted by bacterial, fungal and oomycete pathogens and the hypersusceptibility of rd21-1 mutants to M. chitwoodi indicates RD21A plays a role in plant-parasitic nematode infection.


International Journal of Fundamental and Applied Nematological Research



BarcalaM.FenollC.EscobarC. (2012). Laser microdissection of cells and isolation of high-quality RNA after cryosectioning. Methods in Molecular Biology 883, 87-95.

BartlemD.G.JonesM.G.K.HammesU.Z. (2013). Vascularization and nutrient delivery at root-knot nematode feeding sites in host roots. Journal of Experimental Botany 65, 1789-1798.

BatemanA.BennettH.P.J. (2009). The granulin gene family: from cancer to dementia. BioEssays 31, 1245-1254.

BebberD.P.HolmeS.T.GurrS.J. (2014). The global spread of crop pests and pathogens. Global Ecology Biogeography 23, 1398-1407.

BeersE.P.JonesA.M.DickermanA.W. (2004). The S8 serine, C1A cysteine and A1 aspartic protease families in Arabidopsis. Phytochemistry 65, 43-58.

BendtsenJ.D.NielsenH.von HeijneG.BrunakS. (2004). Improved prediction of signal peptides: SignalP 3.0. Journal of Molecular Biology 340, 783-795.

BernouxM.TimmersT.JauneauA.BriereC.De WitP.J.MarcoY.DeslandesL. (2008). RD19, an Arabidopsis cysteine protease required for RRS1-R-mediated resistance, is relocalized to the nucleus by the Ralstonia solanacearum PopP2 effector. Plant Cell 20, 2252-2264.

BhandariV.PalfreeR.G.BatemanA. (1992). Isolation and sequence of the granulin precursor cDNA from human bone marrow reveals tandem cysteine-rich granulin domains. Proceedings of the National Academy of Sciences of the United States of America 89, 1715-1719.

BlokV.C.JonesJ.T.PhillipsM.S.TrudgillD.L. (2008). Parasitism genes and host range disparities in biotrophic nematodes: the conundrum of polyphagy versus specialisation. BioEssays 30, 249-259.

BozkurtT.O.SchornackS.WinJ.ShindoT.IiyasM.OlivaR.CanoL.M.JonesA.M.HuitemaE.van der HoornR.A. (2011). Phytophthora infestans effector AVRblb2 prevents secretion of a plant immune protease at the haustorial interface. Proceedings of the National Academy of Sciences of the United States of America 108, 20832-20837.

BrignetiG.VoinnetO.LiW.X.JiL.H.DingS.W.BaulcombeD.C. (1998). Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana. EMBO Journal 17, 6739-6746.

CarterC.PanS.ZouharJ.AvilaE.L.GirkeT.RaikhelN.V. (2004). The vegetative vacuole proteome of Arabidopsis thaliana reveals predicted and unexpected proteins. Plant Cell 16, 3285-3303.

DavisE.L.HusseyR.S.MitchumM.G.BaumT.J. (2008). Parasitism proteins in nematode-plant interactions. Current Opinion in Plant Biology 11, 360-366.

de Almeida-EnglerJ.EnglerG.GheysenG. (2011). Unravelling the plant cell cycle in nematode feeding sites. In: JonesJ.T.GheysenG.FenollC. (Eds). Genomics and molecular genetics of plant-nematode interactions. Dordrecht, The Netherlands, Springer, pp.  349-368.

DinhP.T.Y.BrownC.R.EllingA.A. (2014). RNA interference of effector gene Mc16D10L confers resistance against Meloidogyne chitwoodi in Arabidopsis and potato. Phytopathology 104, 1098-1106.

DoyleE.A.LambertK.N. (2002). Cloning and characterization of an esophageal-gland-specific pectate lyase from the root-knot nematode Meloidogyne javanica. Molecular Plant-Microbe Interactions 15, 549-556.

EllingA.A. (2013). Major emerging problems with minor Meloidogyne species. Phytopathology 103, 1092-1102.

EllingA.A.JonesJ.T. (2014). Functional characterization of nematode effectors in plants. Methods in Molecular Biology 1127, 113-124.

GheysenG.MitchumM.G. (2011). How nematodes manipulate plant development pathways for infection. Current Opinion in Plant Biology 14, 415-421.

GuC.ShababM.StrasserR.WoltersP.J.ShindoT.NiemerM.KaschaniF.MachL.van der HoornR.A. (2012). Post-translational regulation and trafficking of the granulin-containing protease RD21 of Arabidopsis thaliana. PLoS ONE 7, e32422.

HaegemanA.MantelinS.JonesJ.T.GheysenG. (2012). Functional roles of effectors of plant-parasitic nematodes. Gene 492, 19-31.

HayashiY.YamadaK.ShimadaT.MatsushimaR.NishizawaM.Hara-NishimuraI. (2001). A proteinase-storing body that prepares for cell death or stresses in the epidermal cells of Arabidopsis. Plant Cell Physiology 42, 894-899.

HeweziT.BaumT.J. (2013). Manipulation of plant cells by cyst and root-knot nematode effectors. Molecular Plant-Microbe Interactions 26, 9-16.

HofmannJ.GrundlerF. (2007). Identification of reference genes for qRT-PCR studies of gene expression in giant cells and syncytia induced in Arabidopsis thaliana by Meloidogyne incognita and Heterodera schachtii. Nematology 9, 317-323.

HusseyR.S. (1989). Disease-inducing secretions of plant-parasitic nematodes. Annual Review of Phytopathology 27, 123-141.

HusseyR.S.BarkerK.R. (1973). A comparison of methods of collecting inocula of Meloidogyne spp., including a new technique. Plant Disease Reports 57, 1025-1028.

JaouannetM.Perfus-BarbeochL.DeleuryE.MaglianoM.EnglerG.VieiraP.DanchinE.G.da RochaM.CoquillardP.AbadP. (2012). A root-knot nematode-secreted protein is injected into giant cells and targeted to the nuclei. New Phytologist 194, 924-931.

JonesM.G.K.GotoD.B. (2011). Root-knot nematodes and giant cells. In: JonesJ.GheysenG.FenollC. (Eds). Genomics and molecular genetics of plant-nematode interactions. Dordrecht, The Netherlands, Springer, pp.  83-102.

KaschaniF.ShababM.BozkurtT.ShindoT.SchornackS.GuC.IiyasM.WinJ.KamounS.van der HoornR.A. (2010). An effector-targeted protease contributes to defense against Phytophthora infestans and is under diversifying selection in natural hosts. Plant Physiology 154, 1794-1804.

KrügerJ.ThomasC.M.GolsteinC.DixonM.S.SmokerM.TangS.MulderL.JonesJ.D. (2002). A tomato cysteine protease required for Cf-2-dependent disease resistance and suppression of autonecrosis. Science 296, 744-747. Almeida-EnglerJ. (2013). Nematode feeding sites: unique organs in plant roots. Planta 238, 807-818.

LamplN.AlkanN.DavydovO.FluhrR. (2013). Set-point control of RD21 protease activity by AtSerpin1 controls cell death in Arabidopsis. Plant Journal 74, 498-510.

LivakK.J.SchmittgenT.D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2ΔΔCT method. Methods 25, 402-408.

Lozano-TorresJ.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 109, 10119-10124.

Marchler-BauerA.ZhengC.ChitsazF.DerbyshireM.K.GeerL.Y.GeerR.C.GonzalesN.R.GwadzM.HurwitzD.I.LanczyckiC.J. (2013). CDD: conserved domains and protein three-dimensional structure. Nucleic Acids Research 41, D348-D352.

MartinK.KopperudK.ChakrabartyR.BanerjeeR.BrooksR.GoodinM.M. (2009). Transient expression in Nicotiana benthamiana fluorescent marker lines provides enhanced definition of protein localization, movement and interactions in planta. Plant Journal 59, 150-162.

MitchumM.G.HusseyR.S.BaumT.J.WangX.EllingA.A.WubbenM.DavisE.L. (2013). Nematode effector proteins: an emerging paradigm of parasitism. New Phytologist 199, 879-894.

MukhtarM.S.CarvunisA.R.DrezeM.EppleP.SteinbrennerJ.MooreJ.TasanM.GalliM.HaoT.NishimuraM.T. (2011). Independently evolved virulence effectors converge onto hubs in a plant immune system network. Science 333, 596-601.

OndzighiC.A.ChristopherD.A.ChoE.J.ChangS.C.StaehelinL.A. (2008). Arabidopsis protein disulfide isomerase-5 inhibits cysteine proteases during trafficking to vacuoles before programmed cell death of the endothelium in developing seeds. Plant Cell 20, 2205-2220.

RooneyH.C.E.van’t KloosterJ.W.van der WitP.J. (2005). Cladosporium Avr2 inhibits tomato Rcr3 protease required for Cf-2-dependent disease resistance. Science 308, 1783-1786.

RozeE.HanseB.MitrevaM.VanholmeB.BakkerJ.SmantG. (2008). Mining the secretome of the root-knot nematode Meloidogyne chitwoodi for candidate parasitism genes. Molecular Plant Pathology 9, 1-10.

ShababM.ShindoT.GuC.KaschaniF.PansuriyaT.ChinthaR.HarzenA.ColbyT.KamounS.van der HoornR.A. (2008). Fungal effector protein AVR2 targets diversifying defense-related cys proteases of tomato. Plant Cell 20, 1169-1183.

ShindoT.van der HoornR.A.L. (2008). Papain-like cysteine proteases: key players at molecular battlefields employed by both plants and their invaders. Molecular Plant Pathology 9, 119-125.

ShindoT.Misas-VillamilJ.C.HörgerA.C.SongJ.van der HoornR.A. (2012). A role in immunity for Arabidopsis cysteine protease RD21, the ortholog of the tomato immune protease C14. PLoS ONE 7, e29317.

SongJ.WinJ.TianM.SchornackS.KaschaniF.IiyasM.van der HoornR.A.KamounS. (2009). Apoplastic effectors secreted by two unrelated eukaryotic plant pathogens target the tomato defense protease Rcr3. Proceedings of the National Academy of Sciences of the United States of America 106, 1654-1659.

TianM.WinJ.SongJ.van der HoornR.A.van der KnaapE.KamounS. (2007). A Phytophthora infestans cystatin-like protein targets a novel tomato papain-like apoplastic protease. Plant Physiology 143, 364-377.

van der LindeK.HemetsbergerC.KastnerC.KaschaniF.van der HoornR.A.KumlehnJ.DoehlemannG. (2012). A maize cystatin suppresses host immunity by inhibiting apoplastic cysteine proteases. Plant Cell 24, 1285-1300.

van EsseH.P.van’t KloosterJ.W.BoltonM.D.YadetaK.A.van WitP.J.ThommaB.P. (2008). The Cladosporium fulvum virulence protein Avr2 inhibits host proteases required for basal defense. Plant Cell 20, 1948-1963. Almeida-EnglerJ.Castagnone-SerenoP. (2011). The plant apoplasm is an important recipient compartment for nematode secreted proteins. Journal of Experimental Botany 62, 1241-1253.

WangJ.LeeC.ReplogleA.JoshiS.KorkinD.HusseyR.BaumT.J.DavisE.L.WangX.MitchumM.G. (2010). Dual roles for the variable domain in protein trafficking and host-specific recognition of Heterodera glycines CLE effector proteins. New Phytologist 187, 1003-1017.

WangX.MeyersD.YanY.BaumT.J.SmantG.HusseyR.DavisE.L. (1999). In planta localization of a beta-1,4-endoglucanase secreted by Heterodera glycines. Molecular Plant-Microbe Interactions 12, 64-67.

WeigelD.GlazebrookJ. (2006). Transformation of agrobacterium using the freeze-thaw method. In: Cold Spring Harbor Protocols 2006, DOI:10.1101/pdb.prot4666.

YamadaK.MatsushimaR.NishimuraM.Hara-NishimuraI. (2001). A slow maturation of a cysteine protease with a granulin domain in the vacuoles of senescing Arabidopsis leaves. Plant Physiology 127, 1626-1634.

ZhangB.TremousaygueD.DenancéN.van EsseH.P.HorgerA.C.DabosP.GoffnerD.ThommaB.P.van der HoornR.A.TuominenH. (2014). PIRIN2 stabilizes cysteine protease XCP2 and increases susceptibility to the vascular pathogen Ralstonia solanacearum in Arabidopsis. Plant Journal 79, 1009-1019.


  • qRT-PCR analysis of Mc1194 in Meloidogyne chitwoodi life stages. The developmental expression pattern of Mc1194 was determined in pre-parasitic second-stage juveniles (pre-J2), parasitic J2 (par-J2), mixed sample of third- and fourth-stage juveniles (J3-J4) and adult female nematodes. Data are averages ± standard error of two biologically independent experiments, each consisting of three technical replicates. Meloidogyne chitwoodi internal transcribed spacer 2 rRNA (ITS) was used an internal control to normalise gene expression. Gene expression values were calculated using the 2ΔΔCT method. ∗∗ = P<0.001.

    View in gallery
  • Mc1194 interacts with RD21A in yeast and plant cells. A: Yeast two-hybrid screening revealed Mc1194 interacts with RD21A. To confirm this putative interaction, yeast cells containing the RD21A prey plasmid were co-transformed with the Mc1194 bait vector, empty bait vector or bait vector containing the human Lamin C gene. RD21A plasmids co-transformed with Mc1194 were able to grow on selective media. Conversely, yeast cells co-transformed with RD21A in addition to empty or Lamin C vector failed to grow; B: To determine if RD21A and Mc1194 interact in planta, bimolecular fluorescence complementation was performed. Constructs expressing nEYFP-Mc1194 and cEYFP-RD21A were transiently expressed in tobacco epidermal cells. YFP fluorescence was detected 2 days post infiltration. Scale bar = 20 μm. This figure is published in colour in the online edition of this journal, which can be accessed via

    View in gallery
  • Effects of RD21A gene knockout on Meloidogyne chitwoodi parasitism. The RD21A knockout mutant allele, rd21-1 and wild-type (Col-0) plants were infected with approximately 200 sterilised M. chitwoodi second-stage juveniles. At 7 days post inoculation (dpi) the number of galls were counted. At 28 dpi eggs masses were visualised by phloxine B staining and counted. Values represent mean ± SE of two independent biological replicates. ∗∗ = P<0.001.

    View in gallery
  • Effects of Meloidogyne chitwoodi parasitism on RD21A and AtSerpin1 gene expression. A: qRT-PCR analysis of RD21A expression in gall tissue dissected from Arabidopsis thaliana roots at 7, 14 and 21 dpi with M. chitwoodi. Relative normalised gene expression was calculated in infected tissue relative to uninfected root tissue. Values represent the mean ± standard error from two independent biological replicates with three technical replicates each. ∗∗ = P<0.01; B: qRT-PCR analysis of the protease inhibitor, AtSerpin1 expression in gall tissue dissected from A. thaliana roots at 7, 14 and 21 dpi with M. chitwoodi. Values represent the mean ± SE from two independent biological replicates with three technical replicates each. ∗∗ = P<0.01.

    View in gallery
  • Investigating RD21A domains involved in the RD21A-Mc1194 interaction. A: To gain an insight into which RD21A protein domains are involved in the interaction with Mc1194, the translated insert from yeast two-hybrid prey plasmids pGADT7 were aligned with the full-length RD21A protein sequence. The catalytic domain of RD21A is shown in red, the proline-rich region is shown in blue and the granulin domain is shown in green. The alignment indicated that Mc1194 is interacting with the C-terminal region of the RD21A catalytic domain, the proline-rich region and the granulin domain; B: To determine if Mc1194 interacts with the RD21A domains in planta, bimolecular fluorescence complementation was performed. Constructs expressing nEYFP-Mc1194 were transiently expressed in tobacco cells with cEYFP-RD21A catalytic domain and proline-rich region or cEYFP-RD21A granulin domain. Scale bars = 20 μm; C: The ability of Mc1194 to interact with the granulin domain from other papain-like cysteine proteases was determined. The granulin domain from RD21B, RD21C and XBCP3 were cloned into prey plasmids and co-transformed with Mc1194 in the bait plasmid. All co-transformations grew on the selective media indicating an interaction between the granulin domain and Mc1194. This figure is published in colour in the online edition of this journal, which can be accessed via

    View in gallery


Content Metrics

Content Metrics

All Time Past Year Past 30 Days
Abstract Views 26 26 11
Full Text Views 4 4 4
PDF Downloads 0 0 0
EPUB Downloads 0 0 0