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Transcriptional silencing of RNAi constructs against nematode genes in Arabidopsis

Tina Kyndt (UGent) , Hongli Ji (UGent) , Bartel Vanholme (UGent) and Godelieve Gheysen (UGent)
(2013) NEMATOLOGY. 15(5). p.519-528
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Abstract
In this research, Arabidopsis thaliana plants were transformed with hairpin constructs targeting cyst nematode (Heterodera schachtii) genes, driven by the cauliflower mosaic virus (CaMV) 35S promoter: two housekeeping genes (the splicing factor Hs-U2AF and the vacuolar Hs-H(+)ATPase) and one candidate effector gene (the ubiquitin extension protein Hs-ubi). Expression of the dsRNA appeared to be extremely variable between and within homozygous T3 lines and even between tissues. Infection experiments showed up to 50% reduction in nematode infection for some transgenic lines. The results varied not only between lines containing the same construct but also between independent repetitions of the experiment. Further focusing on the Hs-U2AF-RNAi lines revealed large variations and a general decline of construct expression levels over the generations. Bisulphite sequencing of a 197 bp part of the CaMV 35S promoter revealed substantial methylation in this region and a negative correlation between the methylation level and expression of the hairpin construct. Taken together, our results show that host-generated RNAi can suffer from high levels of transcriptional silencing of the construct, leading to varying expression levels within and between transgenic lines.
Keywords
Hs-H+ATPase, Hs-U2AF, Heterodera schachtii, gene expression, hairpin constructs, Hs-ubi, methylation, T-DNA, PLANT-PARASITIC NEMATODES, DOUBLE-STRANDED-RNA, HETERODERA-GLYCINES, DNA METHYLATION, TRANSGENIC PLANTS, CAENORHABDITIS-ELEGANS, INTERFERENCE, HOST, EXPRESSION, PROTEINS

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Please use this url to cite or link to this publication:

Chicago
Kyndt, Tina, Hongli Ji, Bartel Vanholme, and Godelieve Gheysen. 2013. “Transcriptional Silencing of RNAi Constructs Against Nematode Genes in Arabidopsis.” Nematology 15 (5): 519–528.
APA
Kyndt, T., Ji, H., Vanholme, B., & Gheysen, G. (2013). Transcriptional silencing of RNAi constructs against nematode genes in Arabidopsis. NEMATOLOGY, 15(5), 519–528.
Vancouver
1.
Kyndt T, Ji H, Vanholme B, Gheysen G. Transcriptional silencing of RNAi constructs against nematode genes in Arabidopsis. NEMATOLOGY. 2013;15(5):519–28.
MLA
Kyndt, Tina, Hongli Ji, Bartel Vanholme, et al. “Transcriptional Silencing of RNAi Constructs Against Nematode Genes in Arabidopsis.” NEMATOLOGY 15.5 (2013): 519–528. Print.
@article{4124071,
  abstract     = {In this research, Arabidopsis thaliana plants were transformed with hairpin constructs targeting cyst nematode (Heterodera schachtii) genes, driven by the cauliflower mosaic virus (CaMV) 35S promoter: two housekeeping genes (the splicing factor Hs-U2AF and the vacuolar Hs-H(+)ATPase) and one candidate effector gene (the ubiquitin extension protein Hs-ubi). Expression of the dsRNA appeared to be extremely variable between and within homozygous T3 lines and even between tissues. Infection experiments showed up to 50\% reduction in nematode infection for some transgenic lines. The results varied not only between lines containing the same construct but also between independent repetitions of the experiment. Further focusing on the Hs-U2AF-RNAi lines revealed large variations and a general decline of construct expression levels over the generations. Bisulphite sequencing of a 197 bp part of the CaMV 35S promoter revealed substantial methylation in this region and a negative correlation between the methylation level and expression of the hairpin construct. Taken together, our results show that host-generated RNAi can suffer from high levels of transcriptional silencing of the construct, leading to varying expression levels within and between transgenic lines.},
  author       = {Kyndt, Tina and Ji, Hongli and Vanholme, Bartel and Gheysen, Godelieve},
  issn         = {1388-5545},
  journal      = {NEMATOLOGY},
  keyword      = {Hs-H+ATPase,Hs-U2AF,Heterodera schachtii,gene expression,hairpin constructs,Hs-ubi,methylation,T-DNA,PLANT-PARASITIC NEMATODES,DOUBLE-STRANDED-RNA,HETERODERA-GLYCINES,DNA METHYLATION,TRANSGENIC PLANTS,CAENORHABDITIS-ELEGANS,INTERFERENCE,HOST,EXPRESSION,PROTEINS},
  language     = {eng},
  number       = {5},
  pages        = {519--528},
  title        = {Transcriptional silencing of RNAi constructs against nematode genes in Arabidopsis},
  url          = {http://dx.doi.org/10.1163/15685411-00002698},
  volume       = {15},
  year         = {2013},
}

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