Transcriptional silencing of RNAi constructs against nematode genes in Arabidopsis

Abstract

In this research, Arabidopsis thaliana plants were transformed with hairpin constructs targeting cyst nematode (Heterodera schachtii) genes, driven by the cauUflower mosaic virus (CaMV) 35S promoter: two housekeeping genes (the splicing factor Hs-U2AF and the vacuolar Hs-W^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.

The Pratylenchus penetrans Transcriptome as a Source for the Development of Alternative Control Strategies: Mining for Putative Genes Involved in Parasitism and Evaluation of in planta RNAi

“…However, in planta delivery, such as that carried out here, is less effective than targeting the same genes by dsRNA soaking [ 37 , 38 ] and perhaps provides a more pragmatic assessment of this technology for control. The observed differences are likely a consequence of silencing efficiency using dsRNA delivered by the plant versus soaking nematode experiments [ 37 , 38 ], as other studies have indicated different levels of variability in silencing efficacy among transgenic events, expression levels of dsRNA, and lines bearing the same type of construct [ 52 ]. Furthermore, as root lesion nematodes are migratory nematodes, able to move in and out of the roots, the continuous exposure of nematodes to dsRNA delivered by the plant can be arrested or reduced for the periods of time that the nematodes will exit the roots, as a consequential effect of the dsRNA exposure, or ordinary movements of the nematodes, may allow for some degree of recovery.…”

The novel cyst nematode effector protein 30D08 targets host nuclear functions to alter gene expression in feeding sites

“…Pre-immune (g); anti-30D08 antibody (h). New Phytologist is useful for confirming dsRNA expression in select lines for analysis; however, it does not always correlate with RNA silencing efficiency of the target gene (Patel et al, 2008;Kyndt et al, 2013). Transgenic 30D08 RNAi lines exhibited similar morphological features to that of wild-type, plants confirming no offtarget effects in the growth and development of transgenic plants.…”

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

“…Position effects frequently are reported in potato and can override the regulatory control of the pro moter associated with the transgene, such that the tran script level, as well as the temporal and spatial expression patterns of the transgene are altered (Meiyalaghan et al, 2006;Barrell & Conner, 2009;Jacobs et al, 2009;Bar rell et a l, 2013). It has been shown that in A. thaliana methylation of the 35S promoter can cause epigenetic si lencing of transgenic RNAi constructs, leading to strongly varying levels of small RNAs between lines (Kyndt et al, 2013). Thus, it is possible that in some cases one or more 16D10i-2 RNAi transgene copies were partially or fully deactivated in similar ways, thereby resulting in double or multiple insertion lines that produce small RNAs at a level equivalent to what would be expected in single insertion lines.…”