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Host-induced silencing of Mi-msp-1 confers resistance to root-knot nematode Meloidogyne incognita in eggplant

  • Sonam Chaudhary
  • Tushar K. Dutta
  • Nidhi Tyagi
  • Tagginahalli N. Shivakumara
  • Pradeep K. Papolu
  • Kapil A. Chobhe
  • Uma RaoEmail author
Original Paper

Abstract

RNA interference (RNAi)-based host-induced gene silencing (HIGS) is emerging as a novel, efficient and target-specific tool to combat phytonematode infection in crop plants. Mi-msp-1, an effector gene expressed in the subventral pharyngeal gland cells of Meloidogyne incognita plays an important role in the parasitic process. Mi-msp-1 effector is conserved in few of the species of root-knot nematodes (RKNs) and does not share considerable homology with the other phytonematodes, thereby making it a suitable target for HIGS with minimal off-target effects. Six putative eggplant transformants harbouring a single copy RNAi transgene of Mi-msp-1 was generated. Stable expression of the transgene was detected in T1, T2 and T3 transgenic lines for which a detrimental effect on RKN penetration, development and reproduction was documented upon challenge infection with nematode juveniles. The post-parasitic nematode stages extracted from the transgenic plants showed long-term RNAi effect in terms of targeted downregulation of Mi-msp-1. These findings suggest that HIGS of Mi-msp-1 enhances nematode resistance in eggplant and protect the plant against RKN parasitism at very early stage.

Keywords

dsRNA siRNA Parasitism Southern hybridization RT-qPCR 

Notes

Acknowledgements

Ph.D. Student SC acknowledges her co-guide Dr. Vishakha Raina, School of Biotechnology, KIIT, Bhubaneswar, India. Current investigation was funded by Department of Biotechnology, Government of India (Grant No. BT/PR5908/AGR/36/727/2012).

Author contributions

SC performed all the experiments. TKD wrote the MS and analysed of the data. NT, TNS, PKP and KAC helped in performing experiments. UR conceived the experiment and edited the MS. All the authors read and approved the final MS.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11248_2019_126_MOESM1_ESM.pdf (946 kb)
Supplementary material 1 (PDF 945 kb)
11248_2019_126_MOESM2_ESM.pdf (117 kb)
Supplementary material 2 (PDF 117 kb)

References

  1. Atkinson HJ, Lilley CJ, Urwin PE (2012) Strategies for transgenic nematode control in developed and developing world crops. Curr Opin Biotechnol 23:251–256CrossRefGoogle Scholar
  2. Charlton WL, Harel HYM, Bakhetia M, Hibbard JK, Atkinson HJ, McPherson MJ (2010) Additive effects of plant expressed double-stranded RNAs on root-knot nematode development. Int J Parasitol 40:855–864CrossRefGoogle Scholar
  3. Chaudhary S, Dutta TK, Shivakumara TN, Rao U (2019) RNAi of an esophageal gland specific Mi-msp-1 gene alters the early stage infection behaviour of root-knot nematode, Meloidogyne Incognita. J Gen Plant Pathol.  https://doi.org/10.1007/s10327-019-00837-x Google Scholar
  4. Danchin EGJ, Arguel MJ, Campan-Fournier A, Perfus-Barbeoch L, Magliano M, Rosso M-N et al (2013) Identification of novel target genes for safer and more specific control of root-knot nematodes from a pan-genome mining. Plos Pathogen 9:e1003745CrossRefGoogle Scholar
  5. de Souza Júnior JDA, Coelho RR, Lourenço IT, da Rocha Fragoso R, Viana AAB, de Macedo LLP et al (2013) Knocking-down Meloidogyne incognita proteases by plant-delivered dsRNA has negative pleiotropic effect on nematode vigor. PLoS One 8:e85364CrossRefGoogle Scholar
  6. Ding X, Shields J, Allen R, Hussey R (2000) Molecular cloning and characterisation of a venom allergen AG5-like cDNA from Meloidogyne incognita. Int J Parasitol 30:77–81CrossRefGoogle Scholar
  7. Dinh PT, Zhang L, Brown CR, Elling AA (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 16:669–682CrossRefGoogle Scholar
  8. Duarte A, Maleita C, Egas C, Abrantes I, Curtis R (2017) Significant effects of RNAi silencing of the venom allergen-like protein (Mhi-vap-1) of the root-knot nematode Meloidogyne hispanica in the early events of infection. Plant Pathol 66:1329–1337CrossRefGoogle Scholar
  9. Dutta TK, Banakar P, Rao U (2015a) The status of RNAi-based transgenic research in plant nematology. Front Microbiol 5:760CrossRefGoogle Scholar
  10. Dutta TK, Papolu PK, Banakar P, Choudhary D, Sirohi A, Rao U (2015b) Tomato transgenic plants expressing hairpin construct of a nematode protease gene conferred enhanced resistance to root-knot nematodes. Front Microbiol 6:260Google Scholar
  11. Dutta TK, Khan MR, Phani V (2019) Plant-parasitic nematode management via biofumigation using brassica and non-brassica plants: current status and future prospects. Curr Plant Biol.  https://doi.org/10.1016/j.cpb.2019.02.001 Google Scholar
  12. Elling AA (2013) Major emerging problems with minor Meloidogyne species. Phytopathol 103:1092–1102CrossRefGoogle Scholar
  13. Fuller VL, Lilley CJ, Urwin PE (2008) Nematode resistance. New Phytol 180:27–44CrossRefGoogle Scholar
  14. Gao B, Allen R, Maier T, Davis EL, Baum TJ, Hussey RS (2001) Molecular characterisation and expression of two venom allergen-like protein genes in Heterodera glycines. Int J Parasitol 31:1617–1625CrossRefGoogle Scholar
  15. Gleason CA, Liu QL, Williamson VM (2008) Silencing a candidate nematode effector gene corresponding to the tomato resistance gene Mi-1 leads to acquisition of virulence. Mol Plant-Microbe Interact 21:576–585CrossRefGoogle Scholar
  16. Grishok A, Tabara H, Mello CC (2000) Genetic requirements for inheritance of RNAi in C. elegans. Science 287:2494–2497CrossRefGoogle Scholar
  17. Hewezi T, Baum TJ (2013) Manipulation of plant cells by cyst and root-knot nematode effectors. Mol Plant-Microbe Interact 26:9–16CrossRefGoogle Scholar
  18. Huang G, Gao B, Maier T, Allen R, Davis EL, Baum TJ et al (2003) A profile of putative parasitism genes expressed in the esophageal gland cells of the root-knot nematode Meloidogyne incognita. Mol Plant-Microbe Interact 16:376–381CrossRefGoogle Scholar
  19. Huang G, Dong R, Maier T, Allen R, Davis EL, Baum TJ et al (2004) Use of solid-phase subtractive hybridization for the identification of parasitism gene candidates from the root-knot nematode Meloidogyne incognita. Mol Plant Pathol 5:217–222CrossRefGoogle Scholar
  20. Huang G, Allen R, Davis EL, Baum TJ, Hussey RS (2006) Engineering broad root-knot resistance in transgenic plants by RNAi silencing of a conserved and essential root-knot nematode parasitism gene. Proc Natl Acad Sci USA 103:14302–14306CrossRefGoogle Scholar
  21. Kerschen A, Napoli CA, Jorgensen RA, Müller AE (2004) Effectiveness of RNA interference in transgenic plants. FEBS Lett 566:223–228CrossRefGoogle Scholar
  22. Li XQ, Wei JZ, Tan A, Aroian RV (2007) Resistance to root-knot nematode in tomato roots expressing a nematicidal Bacillus thuringiensis crystal protein. Plant Biotechnol J 5:455–464CrossRefGoogle Scholar
  23. Lilley CJ, Davies LJ, Urwin PE (2012) RNA interference in plant parasitic nematodes: a summary of the current status. Parasitology 139:630–640CrossRefGoogle Scholar
  24. Lozano-Torres JL, Wilbers RHP, Warmerdam S, Finkers-Tomczak A, Diaz-Granados A, van Schaik CC et al (2014) Apoplastic venom allergen-like proteins of cyst nematodes modulate the activation of basal plant innate immunity by cell surface receptors. Plos Pathogen 10:e1004569CrossRefGoogle Scholar
  25. Luo S, Liu S, Kong L, Peng H, Huang W, Jian H et al (2018) Two venom allergen-like proteins, HaVAP 1 and HaVAP 2, are involved in the parasitism of Heterodera avenae. Mol Plant Pathol.  https://doi.org/10.1111/mpp.12768 Google Scholar
  26. Majumdar R, Rajasekaran K, Cary JW (2017) RNA interference (RNAi) as a potential tool for control of mycotoxin contamination in crop plants: concepts and considerations. Front Plant Sci 8:200Google Scholar
  27. Mitchum MG, Hussey RS, Baum TJ, Wang X, Elling AA, Wubben M, Davis EL (2013) Nematode effector proteins: an emerging paradigm of parasitism. New Phytol 199:879–894CrossRefGoogle Scholar
  28. Moens M, Perry RN, Starr JL (2009) Meloidogyne species—a diverse group of novel and important plant parasites. In: Perry RN, Moens M, Starr JL (eds) Root-knot nematodes, UK. CAB International Publishers, Wallingford, pp 1–17Google Scholar
  29. Naito Y, Yamada T, Matsumiya T, Ui-Tei K, Saigo K, Morishita S (2005) dsCheck: highly sensitive off-target search software for double-stranded RNA-mediated RNA interference. Nucleic Acids Res 33:W589–W591CrossRefGoogle Scholar
  30. Niu J, Liu P, Liu Q, Chen C, Guo Q, Yin J et al (2016) Msp40 effector of root-knot nematode manipulates plant immunity to facilitate parasitism. Sci Rep 6:19443CrossRefGoogle Scholar
  31. Pak J, Maniar JM, Mello CC, Fire A (2012) Protection from feed-forward amplification in an amplified RNAi mechanism. Cell 151:885–899CrossRefGoogle Scholar
  32. Palomares-Rius JE, Escobar C, Cabrera J, Vovlas A, Castillo P (2017) Anatomical alterations in plant tissues induced by plant-parasitic nematodes. Front Plant Sci 8:1987CrossRefGoogle Scholar
  33. Papolu PK, Gantasala NP, Kamaraju D, Banakar P, Sreevathsa R, Rao U (2013) Utility of host delivered RNAi of two FMRF amide like peptides, flp-14 and flp-18, for the management of root knot nematode, Meloidogyne incognita. PLoS One 8:e80603CrossRefGoogle Scholar
  34. Papolu PK, Dutta TK, Tyagi N, Urwin PE, Lilley CJ, Rao U (2016) Expression of a cystatin transgene in eggplant provides resistance to root-knot nematode, Meloidogyne incognita. Front Plant Sci 7:1122CrossRefGoogle Scholar
  35. Roderick H, Urwin PE, Atkinson HJ (2018) Rational design of biosafe crop resistance to a range of nematodes using RNA interference. Plant Biotechnol J 16:520–529CrossRefGoogle Scholar
  36. Rosso M-N, Jones JT, Abad P (2009) RNAi and functional genomics in plant parasitic nematodes. Ann Rev Phytopathol 47:207–232CrossRefGoogle Scholar
  37. Shivakumara TN, Papolu PK, Dutta TK, Kamaraju D, Chaudhary S, Rao U (2016) RNAi-induced silencing of an effector confers transcriptional oscillation in another group of effectors in the root-knot nematode, Meloidogyne incognita. Nematology 18:857–870CrossRefGoogle Scholar
  38. Shivakumara TN, Chaudhary S, Kamaraju D, Dutta TK, Papolu PK, Banakar P et al (2017) Host-induced silencing of two pharyngeal gland genes conferred transcriptional alteration of cell wall-modifying enzymes of Meloidogyne incognita vis-à-vis perturbed nematode infectivity in eggplant. Front Plant Sci 8:473CrossRefGoogle Scholar
  39. Sindhu AS, Maier TR, Mitchum MG, Hussey RS, Davis EL, Baum T (2009) Effective and specific in planta RNAi in cyst nematodes: expression interference of four parasitism genes reduces parasitic success. J Exp Bot 60:315–324CrossRefGoogle Scholar
  40. Southey JF (1986) Laboratory methods for work with plant and soil nematodes. Ministry of Agriculture, Fisheries, and Food Reference Book, London, p 402Google Scholar
  41. Wang X, Li H, Hu Y, Fu P, Xu J (2007) Molecular cloning and analysis of a new venom allergen-like protein gene from the root-knot nematode Meloidogyne incognita. Exp Parasitol 117:133–140CrossRefGoogle Scholar
  42. Xie J, Li S, Mo C, Wang G, Xiao X, Xiao Y (2016) A novel Meloidogyne incognita effector Misp12 suppresses plant defense response at latter stages of nematode parasitism. Front Plant Sci 7:964Google Scholar
  43. Xue B, Hamamouch N, Li C, Huang G, Hussey RS, Baum TJ, Davis EL (2013) The 8D05 parasitism gene of Meloidogyne incognita is required for successful infection of host roots. Phytopathol 103:175–181CrossRefGoogle Scholar
  44. Yang Y, Jittayasothorn Y, Chronis D, Wang X, Cousins P, Zhong G-Y (2013) Molecular characteristics and efficacy of 16D10 siRNAs in inhibiting root-knot nematode infection in transgenic grape hairy roots. PLoS One 8:e69463CrossRefGoogle Scholar
  45. Zhang F, Peng D, Ye X, Yu Z, Hu Z, Ruan L et al (2012) In vitro uptake of 140 kDa Bacillus thuringiensis nematicidal crystal proteins by the second stage juvenile of Meloidogyne hapla. PLoS One 7:e38534CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • Sonam Chaudhary
    • 1
    • 2
  • Tushar K. Dutta
    • 1
  • Nidhi Tyagi
    • 1
  • Tagginahalli N. Shivakumara
    • 1
  • Pradeep K. Papolu
    • 1
  • Kapil A. Chobhe
    • 3
  • Uma Rao
    • 1
    Email author
  1. 1.Division of NematologyICAR-Indian Agricultural Research InstituteNew DelhiIndia
  2. 2.School of BiotechnologyKalinga Institute of Industrial TechnologyBhubaneswarIndia
  3. 3.Division of Soil Science and Agricultural ChemistryNew DelhiIndia

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