All Title Author
Keywords Abstract

PLOS ONE  2013 

Knocking-Down?Meloidogyne incognita?Proteases by Plant-Delivered dsRNA Has Negative Pleiotropic Effect on Nematode Vigor

DOI: 10.1371/journal.pone.0085364

Full-Text   Cite this paper   Add to My Lib

Abstract:

The root-knot nematode Meloidogyne incognita causes serious damage and yield losses in numerous important crops worldwide. Analysis of the M. incognita genome revealed a vast number of proteases belonging to five different catalytic classes. Several reports indicate that M. incognita proteases could play important roles in nematode parasitism, besides their function in ordinary digestion of giant cell contents for feeding. The precise roles of these proteins during parasitism however are still unknown, making them interesting targets for gene silencing to address protein function. In this study we have knocked-down an aspartic (Mi-asp-1), a serine (Mi-ser-1) and a cysteine protease (Mi-cpl-1) by RNAi interference to get an insight into the function of these enzymes during a host/nematode interaction. Tobacco lines expressing dsRNA for Mi-ser-1 (dsSER), Mi-cpl-1 (dsCPL) and for the three genes together (dsFusion) were generated. Histological analysis of galls did not show clear differences in giant cell morphology. Interestingly, nematodes that infected plants expressing dsRNA for proteases produced a reduced number of eggs. In addition, nematode progeny matured in dsSER plants had reduced success in egg hatching, while progeny resulting from dsCPL and dsFusion plants were less successful to infect wild-type host plants. Quantitative PCR analysis confirmed a reduction in transcripts for Mi-cpl-1 and Mi-ser-1 proteases. Our results indicate that these proteases are possibly involved in different processes throughout nematode development, like nutrition, reproduction and embryogenesis. A better understanding of nematode proteases and their possible role during a plant-nematode interaction might help to develop new tools for phytonematode control.

References

[1]  Chitwood DJ (2003) Research on plant-parasitic nematode biology conducted by the United States Department of Agriculture-Agricultural Research Service. Pest Manag Sci 59: 748-753. doi:10.1002/ps.684. PubMed: 12846325.
[2]  Trudgill DL, Blok VC (2001) Apomictic, polyphagous root-knot nematodes: exceptionally successful and damaging biotrophic root pathogens. Annu Rev Phytopathol 39: 53-77. doi:10.1146/annurev.phyto.39.1.53. PubMed: 11701859.
[3]  Davis EL, Hussey RS, Mitchum MG, Baum TJ (2008) Parasitism proteins in nematode-plant interactions. Curr Opin Plant Biol 11: 360-366. doi:10.1016/j.pbi.2008.04.003. PubMed: 18499507.
[4]  Huang G, Gao B, Maier T, Allen R, Davis EL 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: 376-381. doi:10.1094/MPMI.2003.16.5.376. PubMed: 12744507.
[5]  Souza DS, de Souza JDA Jr., Grossi-de-Sá M, Rocha TL, Fragoso RR et al. (2011) Ectopic expression of a Meloidogyne incognita dorsal gland protein in tobacco accelerates the formation of the nematode feeding site. Plant Sci 180: 276-282. doi:10.1016/j.plantsci.2010.09.003. PubMed: 21421371.
[6]  Gheysen G, Mitchum MG (2011) How nematodes manipulate plant development pathways for infection. Curr Opin Plant Biol, 14: 415–21. PubMed: 21458361.
[7]  Tort J, Brindley PJ, Knox D, Wolfe KH, Dalton JP (1999) Proteinases and associated genes of parasitic helminths. Adv Parasitol 43: 161-266. PubMed: 10214692.
[8]  Castagnone-Sereno P, Deleury E, Danchin EG, Perfus-Barbeoch L, Abad P (2011) Data-mining of the Meloidogyne incognita degradome and comparative analysis of proteases in nematodes. Genomics 97: 29-36. doi:10.1016/j.ygeno.2010.10.002. PubMed: 20951198.
[9]  Abad P, Gouzy J, Aury JM, Castagnone-Sereno P, Danchin EG et al. (2008) Genome sequence of the metazoan plant-parasitic nematode Meloidogyne incognita. Nat Biotechnol 26: 909-915. doi:10.1038/nbt.1482. PubMed: 18660804.
[10]  Neveu C, Abad P, Castagnone-Sereno P (2003) Molecular cloning and characterization of an intestinal cathepsin L protease from the plant-parasitic nematode Meloidogyne incognita. Physiological and Molecular Plant Pathology 63: 159-165. doi:10.1016/j.pmpp.2003.10.005.
[11]  Shingles J, Lilley CJ, Atkinson HJ, Urwin PE (2007) Meloidogyne incognita: molecular and biochemical characterisation of a cathepsin L cysteine proteinase and the effect on parasitism following RNAi. Exp Parasitol 115: 114-120. doi:10.1016/j.exppara.2006.07.008. PubMed: 16996059.
[12]  Fragoso RR, Batista JA, Neto OB, Grossi de Sá MF (2005) Isolation and characterization of a cDNA encoding a serine proteinase from the root-knot nematode Meloidogyne incognita. Exp Parasitol 110: 123-133. doi:10.1016/j.exppara.2005.02.010. PubMed: 15888294.
[13]  Fragoso RR, Louren?o IT, Batista JA, Oliveira-Neto OB, Silva MC et al. (2009) Meloidogyne incognita: molecular cloning and characterization of a cDNA encoding a cathepsin D-like aspartic proteinase. Exp Parasitol 121: 115-123. doi:10.1016/j.exppara.2008.09.017. PubMed: 18952081.
[14]  Vieira P, Danchin EG, Neveu C, Crozat C, Jaubert S et al. (2011) The plant apoplasm is an important recipient compartment for nematode secreted proteins. J Exp Bot 62: 1241-1253. doi:10.1093/jxb/erq352. PubMed: 21115667.
[15]  Beynon RJ, Bond JS (1994) Proteolytic enzimes: a pratical approach: IRL Press, Oxford university Press Molecular.
[16]  Baulcombe D (2005) RNA silencing. Trends Biochem Sci 30: 290-293. doi:10.1016/j.tibs.2005.04.012. PubMed: 15950871.
[17]  Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE et al. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391: 806-811. doi:10.1038/35888. PubMed: 9486653.
[18]  Rosso MN, Jones JT, Abad P (2009) RNAi and functional genomics in plant parasitic nematodes. Annu Rev Phytopathol 47: 207-232. doi:10.1146/annurev.phyto.112408.132605. PubMed: 19400649.
[19]  Urwin PE, Lilley CJ, Atkinson HJ (2002) Ingestion of double-stranded RNA by preparasitic juvenile cyst nematodes leads to RNA interference. Mol Plant Microbe Interact 15: 747-752. doi:10.1094/MPMI.2002.15.8.747. PubMed: 12182331.
[20]  Rosso MN, Dubrana MP, Cimbolini N, Jaubert S, Abad P (2005) Application of RNA interference to root-knot nematode genes encoding esophageal gland proteins. Mol Plant Microbe Interact 18: 615-620. doi:10.1094/MPMI-18-0615. PubMed: 16042006.
[21]  Huang G, Dong R, Allen R, Davis EL, Baum TJ et al. (2006) A root-knot nematode secretory peptide functions as a ligand for a plant transcription factor. Mol Plant Microbe Interact 19: 463-470. doi:10.1094/MPMI-19-0463. PubMed: 16673933.
[22]  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 U S A 103: 14302-14306. doi:10.1073/pnas.0604698103. PubMed: 16985000.
[23]  Dubreuil G, Deleury E, Magliano M, Jaouannet M, Abad P et al. (2011) Peroxiredoxins from the plant parasitic root-knot nematode, Meloidogyne incognita, are required for successful development within the host. Int J Parasitol 41: 385-396. doi:10.1016/j.ijpara.2010.10.008. PubMed: 21145323.
[24]  Yadav BC, Veluthambi K, Subramaniam K (2006) Host-generated double stranded RNA induces RNAi in plant-parasitic nematodes and protects the host from infection. Mol Biochem Parasitol 148: 219-222. doi:10.1016/j.molbiopara.2006.03.013. PubMed: 16678282.
[25]  Hewezi T, Howe P, Maier TR, Hussey RS, Mitchum MG et al. (2008) Cellulose binding protein from the parasitic nematode Heterodera schachtii interacts with Arabidopsis pectin methylesterase: cooperative cell wall modification during parasitism. Plant Cell 20: 3080-3093. doi:10.1105/tpc.108.063065. PubMed: 19001564.
[26]  Patel N, Hamamouch N, Li C, Hewezi T, Hussey RS et al. (2010) A nematode effector protein similar to annexins in host plants. J Exp Bot 61: 235-248. doi:10.1093/jxb/erp293. PubMed: 19887499.
[27]  Klink VP, Kim KH, Martins V, Macdonald MH, Beard HS et al. (2009) A correlation between host-mediated expression of parasite genes as tandem inverted repeats and abrogation of development of female Heterodera glycines cyst formation during infection of Glycine max. Planta 230: 53-71. doi:10.1007/s00425-009-0926-2. PubMed: 19347355.
[28]  Sindhu AS, Maier TR, Mitchum MG, Hussey RS, Davis EL et al. (2009) Effective and specific in planta RNAi in cyst nematodes: expression interference of four parasitism genes reduces parasitic success. J Exp Bot 60: 315-324. PubMed: 19015219.
[29]  Charlton WL, Harel HY, Bakhetia M, Hibbard JK, Atkinson HJ et al. (2010) Additive effects of plant expressed double-stranded RNAs on root-knot nematode development. Int J Parasitol 40: 855-864. doi:10.1016/j.ijpara.2010.01.003. PubMed: 20100489.
[30]  Li J, Todd TC, Oakley TR, Lee J, Trick HN (2010) Host-derived suppression of nematode reproductive and fitness genes decreases fecundity of Heterodera glycines Ichinohe. Planta 232: 775-785. doi:10.1007/s00425-010-1209-7. PubMed: 20582434.
[31]  Li J, Todd TC, Trick HN (2010) Rapid in planta evaluation of root expressed transgenes in chimeric soybean plants. Plant Cell Rep 29: 113-123. doi:10.1007/s00299-009-0803-2. PubMed: 20012965.
[32]  Youssef RM, Kim KH, Haroon SA, Matthews BF (2013) Post-transcriptional gene silencing of the gene encoding aldolase from soybean cyst nematode by transformed soybean roots. Exp Parasitol 134: 266-274. doi:10.1016/j.exppara.2013.03.009. PubMed: 23541467.
[33]  Hussey RS, Barker AA (1973) Comparation methods of colleting inocula of Meloidogyne spp. including a new technique Plant Disease Reporter 57: 1025-1028.
[34]  Vrain TC (1977) A technique for the collection of larvae of Meloidogyne spp. and a comparison of eggs and larvae as inocula. J Nematol 9: 249-251. PubMed: 19305604.
[35]  Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215: 403-410. doi:10.1016/S0022-2836(05)80360-2. PubMed: 2231712.
[36]  Dubreuil G, Magliano M, Dubrana MP, Lozano J, Lecomte P et al. (2009) Tobacco rattle virus mediates gene silencing in a plant parasitic root-knot nematode. J Exp Bot 60: 4041-4050. doi:10.1093/jxb/erp237. PubMed: 19625337.
[37]  Zhao S, Fernald RD (2005) Comprehensive algorithm for quantitative real-time polymerase chain reaction. J Comput Biol 12: 1047-1064. doi:10.1089/cmb.2005.12.1047. PubMed: 16241897.
[38]  Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29: e45. doi:10.1093/nar/29.9.e45. PubMed: 11328886.
[39]  Karimi M, Inzé D, Depicker A (2002) GATEWAY? vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7: 193-195. doi:10.1016/S1360-1385(02)02251-3. PubMed: 11992820.
[40]  Gallois P, Marinho P (1995) Leaf disk transformation usin Agrobacterium tumefaciens - expression of heterologous genes in tobacco. In: H. Jones. Plant gene trasnfer and expression protocols. Totowa: Human Press. pp. 38-48.
[41]  Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473-497. doi:10.1111/j.1399-3054.1962.tb08052.x.
[42]  Atkinson HJ, Urwin PE, Clarke MC, McPherson MJ (1996) Image Analysis of the Growth of Globodera pallida and Meloidogyne incognita on Transgenic Tomato Roots Expressing Cystatins. J Nematol 28: 209-215. PubMed: 19277136.
[43]  Craig H, Isaac RE, Brooks DR (2007) Unravelling the moulting degradome: new opportunities for chemotherapy? Trends Parasitol 23: 248-253. doi:10.1016/j.pt.2007.04.003. PubMed: 17459772.
[44]  Britton C, Murray L (2002) A cathepsin L protease essential for Caenorhabditis elegans embryogenesis is functionally conserved in parasitic nematodes. Mol Biochem Parasitol 122: 21-33. doi:10.1016/S0166-6851(02)00066-X. PubMed: 12076767.
[45]  Hashmi S, Britton C, Liu J, Guiliano DB, Oksov Y et al. (2002) Cathepsin L is essential for embryogenesis and development of Caenorhabditis elegans. J Biol Chem 277: 3477-3486. doi:10.1074/jbc.M106117200. PubMed: 11707440.
[46]  Dowen RH, Engel JL, Shao F, Ecker JR, Dixon JE (2009) A family of bacterial cysteine protease type III effectors utilizes acylation-dependent and -independent strategies to localize to plasma membranes. J Biol Chem 284: 15867-15879. doi:10.1074/jbc.M900519200. PubMed: 19346252.
[47]  Steeves RM, Todd TC, Essig JS, Trick HN (2006) Transgenic soybeans expressing siRNAs specific to a major sperm protein gene suppress Heterodera glycines reproduction. Functional Plant Biology 33: 991-999. doi:10.1071/FP06130.
[48]  Fanelli E, Di Vito M, Jones JT, De Giorgi C (2005) Analysis of chitin synthase function in a plant parasitic nematode, Meloidogyne artiellia, using RNAi. Gene 349: 87-95. doi:10.1016/j.gene.2004.11.045. PubMed: 15777697.
[49]  Matsunaga Y, Kawano K, Iwasaki T, Kawano T (2012) RNA interference-mediated growth control of the southern root-knot nematode Meloidogyne incognita. Biosci Biotechnol Biochem 76: 378-380. doi:10.1271/bbb.110661. PubMed: 22313763.
[50]  Birmingham A, Anderson EM, Reynolds A, Ilsley-Tyree D, Leake D et al. (2006) 3' UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nat Methods 3: 199-204. doi:10.1038/nmeth854. PubMed: 16489337.
[51]  Bolter CJ, Jongsma MA (1995) Colorado Potato Beetles (Leptinotarsa-Decemlineata) Adapt to Proteinase-Inhibitors Induced in Potato Leaves by Methyl Jasmonate. Journal of Insect Physiology 41: 1071-1078. doi:10.1016/0022-1910(95)00073-4.
[52]  Gatehouse LN, Shannon AL, Burgess EP, Christeller JT (1997) Characterization of major midgut proteinase cDNAs from Helicoverpa armigera larvae and changes in gene expression in response to four proteinase inhibitors in the diet. Insect Biochem Mol Biol 27: 929-944. PubMed: 9501417.
[53]  Wu YR, Llewellyn D, Mathews A, Dennis ES (1997) Adaptation of Helicoverpa armigera (Lepidoptera: Noctuidae) to a proteinase inhibitor expressed in transgenic tobacco. Molecular Breeding 3: 371-380. doi:10.1023/A:1009681323131.
[54]  Bakhetia M, Urwin PE, Atkinson HJ (2008) Characterisation by RNAi of pioneer genes expressed in the dorsal pharyngeal gland cell of Heterodera glycines and the effects of combinatorial RNAi. Int J Parasitol, 38: 1589–97. PubMed: 18579145.
[55]  Janowski BA, Younger ST, Hardy DB, Ram R, Huffman KE et al. (2007) Activating gene expression in mammalian cells with promoter-targeted duplex RNAs. Nat Chem Biol 3: 166-173. doi:10.1038/nchembio860. PubMed: 17259978.
[56]  Li LC, Okino ST, Zhao H, Pookot D, Place RF et al. (2006) Small dsRNAs induce transcriptional activation in human cells. Proc Natl Acad Sci U S A 103: 17337-17342. doi:10.1073/pnas.0607015103. PubMed: 17085592.
[57]  Dalzell JJ, McMaster S, Fleming CC, Maule AG (2010) Short interfering RNA-mediated gene silencing in Globodera pallida and Meloidogyne incognita infective stage juveniles. Int J Parasitol 40: 91-100. doi:10.1016/j.ijpara.2009.07.003. PubMed: 19651131.

Full-Text

comments powered by Disqus