RNAi-induced silencing of an effector confers transcriptional oscillation in another group of effectors in the root-knot nematode, Meloidogyne incognita

in Nematology
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The sophisticated parasitic tactic of sedentary endoparasitic nematodes seems to involve the simultaneous alteration of the expression of multitude of its effector genes in order to hijack the plant metabolic and developmental pathway. In concordance with this hypothesis, we have targeted some candidate effector genes of Meloidogyne incognita to understand the possible interaction among those effectors for successful infection of the host plant. In vitro RNAi strategy was used to knock down M. incognita-specific pioneer effector genes, such as msp-18, msp-20, msp-24, msp-33 and msp-16 (known to interact with plant transcription factor), to investigate their possible effect on the expression of key cell wall-degrading enzymes (CWDE) and vice versa. Supported by the phenotypic data, intriguingly our study revealed that induced suppression of these pioneer genes cause transcriptional alteration of CWDE genes in M. incognita. This remarkable finding may provide some useful links for future research on nematode effector interaction.

RNAi-induced silencing of an effector confers transcriptional oscillation in another group of effectors in the root-knot nematode, Meloidogyne incognita

in Nematology

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References

BakhetiaM.UrwinP.E.AtkinsonH.J. (2008). Characterisation by RNAi of pioneer genes expressed in the dorsal pharyngeal gland cell of Heterodera glycines and the effects of combinatorial RNAi. International Journal for Parasitology 381589-1597.

ByrdD.W.KirkpatrickT.BarkerK.R. (1983). An improved technique for clearing and staining plant tissues for detection of nematodes. Journal of Nematology 15142-143.

DavisE.L.HusseyR.S.MitchumM.G.BaumT.J. (2008). Parasitism proteins in nematode-plant interactions. Current Opinion in Plant Biology 11360-366.

DinhP.T.Y.ZhangL.BrownC.R.EllingA.A. (2014). Plant-mediated RNA interference of effector gene Mc16D10L confers resistance against Meloidogyne chitwoodi in diverse genetic backgrounds of potato and reduces pathogenicity of nematode offspring. Nematology 16669-682.

DuttaT.K.PowersS.J.KerryB.R.GaurH.S.CurtisR.H.C. (2011). Comparison of host recognition, invasion, development and reproduction of Meloidogyne graminicola and M. incognita on rice and tomato. Journal of Nematology 13509-520.

FireA.XuS.MontgomeryM.K.KostasS.A.DriverS.E.MelloC.C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391806-811.

GheysenG.MitchumM.G. (2011). How nematodes manipulate plant development pathways for infection. Current Opinion in Plant Biology 141-7.

HaegemanA.VanholmeB.GheysenG. (2009). Characterization of a putative endoxylanase in the migratory plant-parasitic nematode Radopholus similis. Molecular Plant Pathology 10389-401.

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

HamamouchN.LiC.HeweziT.BaumT.J.MitchumM.G.HusseyR.S.VodkinL.O.DavisE.L. (2012). The interaction of the novel 30C02 cyst nematode effector protein with a plant beta-1,3 endoglucanase may suppress host defence to promote parasitism. Journal of Experimental Botany 633683-3695.

HassanS.BehmC.A.MathesiusU. (2010). Effectors of plant parasitic nematodes that re-program root cell development. Functional Plant Biology 37933-942.

HeweziT.HoweP.MaierT.R.HusseyR.S.MitchumM.G.DavisE.L.BaumT.J. (2008). Cellulose binding protein from the parasitic nematode Heterodera schachtii interacts with Arabidopsis pectin methylesterase: cooperative cell wall modification during parasitism. Plant Cell 203080-3093.

HeweziT.HoweP.J.MaierT.R.HusseyR.S.MitchumM.G.DavisE.L.BaumT.J. (2010). Arabidopsis spermidine synthase is targeted by an effector protein of the cyst nematode Heterodera schachtii. Plant Physiology 152968-984.

HooperD.J. (1986). Extraction of free-living stages from soil. In: SoutheyJ.F. (Ed.). Laboratory methods for work with plant and soil nematodes. London, UKHer Majesty’s Stationery Office, Ministry of Agriculture, Fisheries & Food pp.  5-30.

HuangG.GaoB.MaierT.AllenR.DavisE.L.BaumT.J.HusseyR.S. (2003). A profile of putative parasitism genes expressed in the esophageal gland cells of the root-knot nematode Meloidogyne incognita. Molecular Plant-Microbe Interactions 16376-381.

HuangG.DongR.MaierT.AllenR.DavisE.L.BaumT.J.HusseyR.S. (2004). Use of solid-phase subtractive hybridization for the identification of parasitism gene candidates from the root-knot nematode Meloidogyne incognita. Molecular Plant Pathology 5217-222.

HuangG.DongR.AllenR.DavisE.L.BaumT.J.HusseyR.S. (2005). Developmental expression and molecular analysis of two Meloidogyne incognita pectate lyase genes. International Journal for Parasitology 35685-692.

HuangG.AllenR.DavisE.L.BaumT.J.HusseyR.S. (2006a). Engineering broad root-knot resistance in transgenic plants by RNAi silencing of a conserved and essential root-knot nematode parasitism gene. Proceedings of the National Academy of Sciencesof the United States of America 10314302-14306.

HuangG.DongR.AllenR.DavisE.L.BaumT.J.HusseyR.S. (2006b). A root-knot nematode secretory peptide functions as a ligand for a plant transcription factor. Molecular Plant-Microbe Interactions 19463-470.

JanowskiB.A.YoungerS.T.HardyD.B.RamR.HuffmanK.E.CoreyD.R. (2007). Activating gene expression in mammalian cells with promoter-targeted duplex RNAs. Nature Chemical Biology 3166-173.

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.

JaubertS.LaffaireJ.-B.AbadP.RossoM.-N. (2002). A polygalacturonase of animal origin isolated from the root-knot nematode Meloidogyne incognita. FEBS Letters 522109-112.

LedgerT.N.JaubertS.BosselutN.AbadP.RossoM.-N. (2006). Characterization of a new β-1,4-endoglucanase gene from the root-knot nematode Meloidogyne incognita and evolutionary scheme for phytonematode family 5 glycosyl hydrolases. Gene 382121-128.

LeeC.ChronisD.KenningC.PeretB.HeweziT.DavisE.L.BaumT.J.HusseyR.S.BennettM.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.

LiL.C.OkinoS.T.ZhaoH.PookotD.PlaceR.F.UrakamiS.EnokidaH.DahiyaR. (2006). Small dsRNAs induce transcriptional activation in human cells. Proceedings of the National Academy of Sciences of the United States of America 10317337-17342.

LilleyC.J.DaviesL.J.UrwinP.E. (2012). RNA interference in plant parasitic nematodes: a summary of the current status. Parasitology 139630-640.

LivakK.J.SchmittgenT.D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25402-408.

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.

Mitreva-DautovaM.RozeE.OvermarsH.De GraaffL.SchotsA.HelderJ.GoverseA.BakkerJ.SmantG. (2006). A symbiont-independent endo-1,4-β-xylanase from the plant-parasitic nematode Meloidogyne incognita. Molecular Plant-Microbe Interactions 19521-529.

NaitoY.YamadaT.MatsumiyaT.Ui-TeiK.SaigoK.MorishitaS. (2005). dsCheck: highly sensitive off-target search software for double-stranded RNA-mediated RNA interference. Nucleic Acids Research 33W589-W591.

NiuJ.H.JianH.XuJ.ChenC.GuoQ. (2012). RNAi silencing of the Meloidogyne incognita Rpn7 gene reduces nematode parasitic success. European Journal of Plant Pathology 134131-144.

NiuJ.H.LiuP.LiuQ.ChenC.GuoQ.YinJ.YangG.JianH. (2016). Msp40 effector of root-knot nematode manipulates plant immunity to facilitate parasitism. Scientific Reports 619443.

RaoU.MauchlineT.H.DaviesK.G. (2012). The 16S rRNA gene of Pasteuria penetrans provides an early diagnostic of infection of root-knot nematodes (Meloidogyne spp.). Nematology 14799-804.

RossoM.-N.FaveryB.PiotteC.ArthaudL.De BoerJ.M.HusseyR.S.BakkerJ.BaumT.J.AbadP. (1999). Isolation of a cDNA encoding a β-1,4-endoglucanase in the root-knot nematode Meloidogyne incognita and expression analysis during plant parasitism. Molecular Plant-Microbe Interactions 12585-591.

SmantG.StokkermansJ.P.W.G.YanY.De BoerJ.M.BaumT.J.WangX.HusseyR.S.GommersF.J.HenrissatB.DavisE.L. (1998). Endogenous cellulases in animals: isolation of β-1,4-endoglucanase genes from two species of plant-parasitic cyst nematodes. Proceedings of the National Academy of Sciences of the United States of America 954906-4911.

UrwinP.E.LilleyC.J.AtkinsonH.J. (2002). Ingestion of double-stranded RNA by pre-parasitic juvenile cyst nematodes leads to RNA interference. Molecular Plant-Microbe Interactions 15747-752.

WangC.L.LowerS.WilliamsonV.M. (2009). Application of Pluronic gel to the study of root-knot nematode behaviour. Nematology 11453-464.

XueB.HamamouchN.LiC.HuangG.HusseyR.S. (2013). The 8D05 parasitism gene of Meloidogyne incognita is required for successful infection of host roots. Phytopathology 103175-181.

YamamuraY.ShimW.B. (2008). The coiled-coil protein-binding motif in Fusarium verticillioides Fsr1 is essential for maize stalk rot virulence. Microbiology 1541637-1645.

YangY.JittayasothornY.ChronisD.WangX.CousinsP.ZhongG.Y. (2013). Molecular characteristics and efficacy of 16D10 siRNAs in inhibiting root-knot nematode infection in transgenic grape hairy roots. PLoS ONE 8e69463.

ZhangL.DaviesL.J.EllingA.A. (2015). A Meloidogyne incognita effector is imported into the nucleus and exhibits transcriptional activation activity in planta. Molecular Plant Pathology 1648-60.

Figures

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    Phylogenetic tree showing the relationship among targeted dsRNA sequences of CWDEs and pioneer genes of Meloidogyne incognita using neighbour-joining method in MEGA6 platform. Different clusters can be seen based on similarity in the nucleotide sequences of those genes. Numbers in the figure indicate distance.

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    Target-specific silencing of CWDE and pioneer genes in Meloidogyne incognita second-stage juveniles (J2). Effect of in vitro silencing of Mi-xyl-1, Mi-xyl-3, Mi-pg-1, Mi-eng-1, Mi-pel, msp-16, msp-18, msp-20, msp-24 and msp-33 genes on the transcript abundance of corresponding genes in M. incognita J2 at 24 h. Expression was quantified using 2−ΔΔCT method and fold change values were log2-transformed. Gene expression levels were normalised using two internal reference genes, 18S rRNA and actin. Bars represent mean expression levels and standard error from two biological and three technical replicates. Asterisks indicate significant differential expression (P<0.05) in comparison with the control worms (treated with soaking buffer). gfp was used as the non-effector control.

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    Phenotypic effect of in vitro silencing of CWDE and pioneer genes on Meloidogyne incognita infectivity. Penetration ability of (A) Mi-xyl-1, Mi-xyl-3, Mi-pg-1, Mi-eng-1, Mi-pel and (B) msp-16, msp-18, msp-20, msp-24, msp-33 dsRNA-treated worms in tomato root in PF-127 medium at 24, 48 and 72 h. Each bar represents the mean ± standard error (n = 3), and bars with different letters denote a significant difference at P<0.05. C: Stained nematodes in infected tomato root at 24 h post infection. Roots were stained with acid fuchsin (Byrd et al., 1983). Nematodes treated with gfp dsRNA and soaking buffer were used as the control. 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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    Effect of in vitro silencing of pioneer genes on CWDE expression and vice versa in Meloidogyne incognita second-stage juveniles (J2). A: Effect of in vitro silencing of msp-16, msp-18, msp-20, msp-24 and msp-33 genes on the transcript abundance of Mi-xyl-1, Mi-xyl-3, Mi-pg-1, Mi-eng-1 and Mi-pel genes in M. incognita J2 at 24 h; B: Effect of in vitro silencing of Mi-xyl-1, Mi-xyl-3, Mi-pg-1, Mi-eng-1 and Mi-pel genes on the transcript abundance of msp-16, msp-18, msp-20, msp-24 and msp-33 genes in M. incognita J2 at 24 h. Expression was quantified using the 2−ΔΔCT method and fold change values were log2-transformed. Gene expression levels were normalised using two internal reference genes, 18S rRNA and actin. Bars represent mean expression levels and standard error from two biological and three technical replicates. Asterisks indicate significant differential expression (P<0.05) in comparison with the worms treated with soaking buffer. Nematodes treated with gfp, flp-1 and sbp-1 dsRNA were used as the non-effector control.

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    Amplification and in vitro dsRNA synthesis of CWDE and pioneer genes from the cDNA of Meloidogyne incognita second-stage juveniles. A: PCR products of CWDE genes (top panel) and their corresponding dsRNA (bottom panel). A1: Mi-xyl-1 (610 bp), A2: Mi-xyl-3 (610 bp), A3: Mi-eng-1 (585 bp), A4: Mi-pel (452), A5: Mi-pg-1 (547 bp); B: PCR products of pioneer genes (top panel) and their corresponding dsRNA (bottom panel). B1: msp-16 (150 bp), B2: msp-18 (598 bp), B3: msp-20 (456 bp), B4: msp-24 (324 bp), B5: msp-33 (116 bp); C: DsRNA of flp-1 (232 bp, C1), gfp (750 bp, C2) and sbp-1 (877 bp, C3). M: 100 bp DNA Ladder.

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    Multiple sequence alignment of CWDE and pioneer genes of Meloidogyne incognita using multAlin (www.multalin.toulouse.inra.fr) bioinformatic tool.

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