Abstract
We constructed a SSH (suppression subtractive hybridization) library based on two populations (Rs-C and Rs-P) of Radopholus similis from different host plants and exhibiting differences in pathogenicity on Musa paradisiaca and Anthurium andraeanum plants. In order to screen the clones with significant expression differences from the SSH library, a total of 2,400 clones was randomly selected and reverse northern blotting was performed on them. Out of the 2,400 clones, 89 clones showed significant expression differences. Out of sequencing these 89 clones, distinct sequences from 87 clones were obtained. Aligning the 87 distinct sequences against the non-redundant nucleotide database (nr) in NCBI, we found that five sequences were highly conserved with Rs-eng-1b. Two of five sequences with lengths of 467 base pairs (bp) (GW395922) and 742 bp (GW395923) were further employed to perform 5′ RACE-PCR and 3′ RACE-PCR, respectively. Subsequently, the complete length of Rs-eng-1b (EU414839) was obtained (1,427 bp). Our qPCR result showed that expression of Rs-eng-1b in the population Rs-C with high pathogenicity on host plants was approximately 2.7 times as much as the expression of Rs-eng-1b in the population Rs-P with low pathogenicity on host plants. Furthermore, the gene Rs-eng-1b from the Rs-C population also showed expression differences amongst four different development stages. The order of Rs-eng-1b relative expression abundance from high to low was females, juveniles, males, and eggs. We further used RNAi to test whether Rs-eng-1b of Rs-C population was responsible for pathogenicity which was the first RNAi work about Rs-eng-1b. The RNAi results showed that Rs-eng-1b expression had a positive correlation to pathogenicity of the population. The longer the RNAi treatment, the less pathogenic the nematode population was. Non-endogenous gfp dsRNA had no significant influence on the expression of Rs-eng-1b and pathogenicity of R. similis Rs-C population. In conclusion, all our evidence indicated Rs-eng-1b might be a crucial pathogenicity-related gene in R. similis.
Similar content being viewed by others
References
Bakhetia, M., Urwin, P. E., & Atkinson, H. J. (2007). qPCR analysis and RNAi define pharyngeal gland cell-expressed genes of Heterodera glycines required for initial interactions with the host. Molecular Plant-Microbe Interactions, 20, 306–312.
Barstead, R. (2001). Genome-wide RNAi. Current Opinion in Chemical Biology, 5, 63–66.
Brindley, P. J., Kalinna, B. H., Dalton, J. P., Day, S. R., Wong, J. Y. M., Smythe, M. L., & McManus, D. P. (1997). Proteolytic degradation of host hemoglobin by schistosomes. Molecular and Biochemical Parasitology, 89, 1–9.
Chen, Q., Rehman, S., Smant, G., & Jones, J. T. (2005). Functional analysis of pathogenicity proteins of the potato cyst nematode Globodera rostochiensis using RNAi. Molecular Plant-Microbe Interactions, 18, 621–625.
Cheng, J. C., Moore, T. B., & Sakamoto, K. M. (2003). RNA interference and human disease. Molecular Genetics and Metabolism, 80, 121–128.
Cheng, X. Y., Dai, S. M., Xiao, L., & Xie, B. Y. (2010). Influence of cellulase gene knockdown by dsRNA interference on the development and reproduction of the pine wood nematode, Bursaphelenchus xylophilus. Nematology, 12, 225–233.
Cotton, J., & Van Riel, H. (1993). Quarantine: Problems and Solution. In K. Evans, D. L. Trudgill, & J. M. Webster (Eds.), Plant Parasitic Nematodes in Temperate Agriculture (pp. 593–607). UK: CAB International.
Diatchenko, L., Lukyanov, S., Lau, Y. F., Chenchik, A., Moqadam, F., Huang, B., Lukyanov, S., Lukyanov, K., Gurskaya, N., Sverdlov, E. D., & Siebert, P. D. (1999). Suppression subtractive hybridization: a versatile method for identifying differentially expressed genes. Methods in Enzymology, 303, 349–380.
Fallas, G. A., & Sarah, J. L. (1994). Effect of storage temperature on the in vitro reproduction of Rahodpholus similis. Nematropica, 24, 175–177.
Fire, A., Xu, S. Q., Montgomery, M. K., Kostas, S. A., Driver, S. E., & Mello, C. C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 391, 806–811.
Goddijn, O. J. M., Lindsey, K., Lee, F. M., Klap, J. C., & Sijmons, P. C. (1993). Differential gene expression in nematode-induced feeding structures of transgenic plants harbouring promoter-gusA fusion constructs. The Plant Journal, 4, 863–873.
Goellner, M., Wang, X., & Davis, E. L. (2001). Endo-beta-1, 4-glucanase expression in compatible plant-nematode interactions. The Plant Cell, 13, 2241–2255.
Gowen, S. R., Quénéhervé, P., & Fogain, R. (2005). Nematode parasites of bananas and plantains. In M. Luc, R. A. Sikora, & J. Bridge (Eds.), Plant Parasitic Nematodes in subtropical and tropical agriculture (2nd ed., pp. 611–644). UK: CABI Publishing.
Grenier, E., Blok, V. C., Jones, J. T., Fouville, D., & Mugniery, D. (2002). Identification of gene expression differences between Globodera pallida and G.‘mexicana’ by suppression subtractive hybridization. Molecular Plant Pathology, 3, 217–226.
Guo, S., & Kemphues, K. J. (1995). par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell, 81, 611–620.
Haegeman, A., Jacob, J., Vanholme, B., Kyndt, T., & Gheysen, G. (2008). A family of GHF5 endo-1, 4-beta-glucanases in the migratory plant-parasitic nematode Radopholus similis. Plant Pathology, 57, 581–590.
Haegeman, A., Vanholme, B., & Gheysen, G. (2009). Characterization of a putative endoxylanase in the migratory plant-parasitic nematode Radopholus similis. Molecular Plant Pathology, 10, 389–401.
Huang, G. Z., Dong, R. H., Maier, T., Allen, R., Davis, E. L., Baum, T. J., & Hussey, R. 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, 5, 217–222.
Jacob, J., Vanholme, B., Haegeman, A., & Gheysen, G. (2007). Four transthyretin-like genes of the migratory plant-parasitic nematode Radopholus similis: Members of an extensive nematode-specific family. Gene, 402, 9–19.
Jacob, J., Mitreva, M., Vanholme, B., & Gheysen, G. (2008). Exploring the transcriptome of the burrowing nematode Radopholus similis. Molecular Genetics and Genomics, 280, 1–17.
Jones, J. T., Furlanetto, C., & Kikuchi, T. (2005). Horizontal gene transfer from bacteria and fungi as a driving force in the evolution of plant parasitism in nematodes. Nematology, 7, 641–646.
Kamath, R. S., Martinez-Campos, M., Zipperlen, P., Fraser, A. G., & Ahringer, J. (2000). Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans. Genome Biology, 2, research0002.1–0002.10. Received September 12, 2000, from http://genomebiology.com/2000/2/1/research/0002.
Kawchuk, L. M., Martin, R. R., & McPherson, J. (1991). Sense and antisense RNA-mediated resistance to potato leafroll virus in Russet Burbank potato plants. Molecular Plant- Microbe Interactions, 4, 247–253.
Kuang, J., & Ashorn, C. L. (1993). At Least Two Kinases Phosphorylate the MPM-2 Epitope during Xenopus Oocyte Maturation. The Journal of Cell Biology, 123, 859–868.
Li, Y., Xie, H., Xue, C. L., Li, D. L., & Zhang, C. (2010). RNAi effect of Cathepsin B gene on reproduction of Radopholus similis. Scientia Agricultura Sinica, 43, 1–9.
Luc, M. (1987). A reappraisal of Tylenchina (Nemata). 7. The family Pratylenchidae Thorne, 1949. Revue Nématol, 10, 203–218.
Maine, E. M. (2001). RNAi as a tool for understanding germline development in Caenorhabditis elegans: Uses and cautions. Developmental Biology, 239, 177–189.
O'Bannon, J. H. (1977). Worldwide dissemination of Radopholus similis and its importance in crop production. Journal of Nematology, 9, 16–25.
Rangasamy, D., Greaves, I., & Tremethick, D. J. (2004). RNA interference demonstrates a novel role for H2A.Z in chromosome segregation. Nature Structural & Molecular Biology, 11, 650–655.
Rosso, M. N., Jones, J. T., & Abad, P. (2009). RNAi and functional genomics in plant parasitic nematodes. Annual Review of Phytopathology, 47, 207–232.
Shen, G. S., & Liu, L. X. (2004). The advances in the research of suppression subtractive hybridization. Chinese Journal of Veterinary Science, 24, 511–514.
Shingles, J., Lilley, C. J., Atkinson, H. J., & Urwin, P. E. (2007). Meloidogyne incognita: Molecular and biochemical characterisation of a cathepsin L cysteine proteinase and the effect on parasitism following RNAi. Experimental Parasitology, 115, 114–120.
Smant, G., Stokkermans, J. P. W. G., Yan, Y., de Boer, J. M., Baum, T. J., Wang, X., Hussey, R. S., Gommers, F. J., Henrissat, B., Davis, E. L., Helder, J., Schots, A., & Bakker, J. (1998). Endogenous cellulases in animals: Isolation of β-1, 4-endoglucanase genes from two species of plant-parasitic cyst nematodes. Proc Natl Acad Sci USA, 95, 4906–4911.
Smith, I. M., & Charles, L. M. F. (1998). Distribution Maps of Quarantine Pests for Europe. CABI and EPPO: CABI Publishing.
Tchernitsa, O. L., Zuber, J., Sers, C., Brinckmann, R., Britsch, S. K., Adams, V., & Schafer, R. (1999). Gene expression profiling of fibroblasts resistant toward oncogene-mediated transformation reveals preferential transcription of negative growth regulators. Oncogene, 18, 5448–5454.
Tijsterman, M., May, R. C., Simmer, F., Okihara, K. L., & Plasterk, R. H. A. (2004). Genes Required for Systemic RNA Interference in Caenorhabditis elegans. Current Biology, 14, 111–116.
Tsang, M. M. C., Hara, A. H., & Sipes, B. S. (2004). Efficacy of hot water drenches of A.andraeanum plants against the burrowing nematode Radopholus similis and plant thermotolerance. Annals of Applied Biology, 145, 309–316.
Urwin, P. E., Lilley, C. J., & Atkinson, H. J. (2002). Ingestion of double-stranded RNA by preparasitic juveniles cyst nematodes leads to RNA interference. Molecular Plant-Microbe Interactions, 15, 747–752.
Vercauteren, I., de Almeida Engler, J., De Groodt, R., & Gheysen, G. (2002). An Arabidopsis thaliana Pectin Acetylesterase Gene Is Upregulated in Nematode Feeding Sites Induced by Root-knot and Cyst Nematodes. Molecular Plant-Microbe Interactions, 15, 404–407.
Viglierchio, D. R., & Schmitt, R. V. (1983). On the methodology of nematode extraction from field samples: baermann funnel modifications. Journal of Nematology, 15, 438–444.
von Stein, O., Thies, W., & Hofmann, M. (1997). A high throughput screening for rarely transcribed differentially expressed genes. Nucleic Acids Research, 25, 2598–2602.
Williams, K. J. O., & Siddiqi, M. R. (1973). Radopholus similis. C.I.H. Descriptions of plant-parasitic nematodes. Set 2, No.27. Commonwealth Agricultural Bureaux, Farnham Royal, UK.
Acknowledgments
This work was funded by National Natural Science Foundation of China (31071665) and Special Fund for Agro-Scientific Research in the Public Interest (200903040). The support of Fuliang Xie (East Carolina University, USA) is gratefully acknowledged. Thanks to Ph.D QL Zhu for the contribution of vector PYL 322-d1-GFPn.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Zhang, C., Xie, H., Xu, CL. et al. Differential expression of Rs-eng-1b in two populations of Radopholus similis (Tylenchida: Pratylecnchidae) and its relationship to pathogenicity. Eur J Plant Pathol 133, 899–910 (2012). https://doi.org/10.1007/s10658-012-0015-4
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10658-012-0015-4