Micro RNA ‐induced gene silencing strategy for the delivery of siRNAs targeting Meloidogyne incognita in a model plant Nicotiana benthamiana

The chemical modification of DNA by methylation is a heritable trait and can be subsequently reversed without altering the original DNA sequence. Methylation can reduce or silence gene expression and is a component of a host’s defence response to foreign nucleic acids. In our study, we employed a plant transformation strategy using Nicotiana benthamiana Domin to study the heritable stability of the introduced transgenes. Through the introduction of the cauliflower mosaic virus (CaMV) 35S promoter and the green fluorescent protein (GFP) reporter gene, we demonstrated that this introduced promoter often triggers a homology-dependent gene-silencing (HDGS) response. These spontaneous transgene-silencing phenomena are due to methylation of the CaMV 35S promoter CAAT box during transgenic plant growth. This process is catalysed by SU(VAR)3–9 homologue 9 (SUVH9), histone deacetylase 1 (HDA1) and domains rearranged methylase 2 (DRM2). In particular, we showed from our data that SUVH9 is the key regulator of methylation activity in epigenetically silenced GFP transgenic lines; therefore, our findings demonstrate that an introduced viral promoter and transgene can be subject to a homology-dependent gene-silencing mechanism that can downregulate its expression and negatively influence the heritable stability of the transgene.

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High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus

BMC Biotechnology ◽

10.1186/1472-6750-9-96 ◽

2009 ◽

Vol 9 (1) ◽

pp. 96 ◽

Cited By ~ 38

Author(s):

Raffaele Lombardi ◽

Patrizia Circelli ◽

Maria Villani ◽

Giampaolo Buriani ◽

Luca Nardi ◽

...

Keyword(s):

Gene Silencing ◽

Transient Expression ◽

Nicotiana Benthamiana ◽

Silencing Suppressor ◽

High Level ◽

Hiv 1

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The virological model plant, Nicotiana benthamiana expresses a single functional RDR6 homeolog

Virology ◽

10.1016/j.virol.2019.08.017 ◽

2019 ◽

Vol 537 ◽

pp. 143-148 ◽

Cited By ~ 1

Author(s):

Márta Ludman ◽

Károly Fátyol

Keyword(s):

Nicotiana Benthamiana ◽

Model Plant

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419. Rational design of bifunctional siRNAs that silence genes and recruit the innate immune system to treat ectopic pregnancies

Reproduction Fertility and Development ◽

10.1071/srb08abs419 ◽

2008 ◽

Vol 20 (9) ◽

pp. 99

Author(s):

S. Tong ◽

M. P. Gantier ◽

M. Belhke ◽

B. R. G. Williams

Keyword(s):

Immune System ◽

Gene Silencing ◽

Rational Design ◽

Mononuclear Cells ◽

Human Peripheral Blood ◽

Micro Rna ◽

Peripheral Blood Mononuclear ◽

Sirna Sequence ◽

Sense Strand ◽

Ectopic Pregnancies

RNA interference (RNAi) is a new therapeutic approach, silencing genes to disrupt diseases. However, short interfering siRNAs (molecule used in RNAi) can have off-target effects, activating the immune system through RNA sensing toll-like receptors (TLR) 3, 7 and 8. We have previously proposed that in some diseases (cancers, ectopic pregnancies) it may be useful to enhance the immune response. A novel class of immunostimulatory siRNAs could be developed, silencing genes important to disease and recruiting the immune system to further aid disease clearance. We set out to develop a rational design strategy that enhances immunostimulatory properties to any siRNA sequence but maintains effective gene silencing. We screened a set of siRNAs targeting lamin. All were of the same sequence, except for different immunostimulatory motifs on the 3′ end of the sense strand. We also investigated a different design where we added a small micro-RNA like poly-uridine bulge (potentially immunostimulatory) on the sense strand. We used human peripheral blood mononuclear cells (PBMCs) to test for immunostimulation, and HEK 293-T-cells to test for lamin gene knockdown.Of all strategies tested, the poly-uridine bulge was best. It silenced the lamin gene as effectively as control, but caused a 2–3 fold increase of IFN-α and TNF-α. We verified this approach by adding the poly-uridine bulge onto an siRNA of low immunostimulatory potential targeting GFP. The bulge markedly enhanced immunostimulation in a dose response manner, and did not compromise gene knockdown. The addition of a poly-uridine bulge to siRNAs can increase immunostimulation without affecting gene silencing efficacy. Immunostimulatory siRNAs might be particularly efficacious to treat ectopic pregnancies where there are abundant immune cells, and functional TLR 7/8 in the trophoblast (unpublished observations). We now plan to test this immunostimulatory siRNA approach in an in vivo ectopic pregnancy model using JEG-3 cells xenographted in NOD-SCID mice.

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Chitin Biosynthesis Inhibition of Meloidogyne incognita by RNAi-Mediated Gene Silencing Increases Resistance to Transgenic Tobacco Plants

International Journal of Molecular Sciences ◽

10.3390/ijms21186626 ◽

2020 ◽

Vol 21 (18) ◽

pp. 6626

Author(s):

Vimalraj Mani ◽

Chinreddy Subramanyam Reddy ◽

Seon-Kyeong Lee ◽

Soyoung Park ◽

Hyoung-Rai Ko ◽

...

Keyword(s):

Gene Silencing ◽

Transgenic Tobacco ◽

Meloidogyne Incognita ◽

Chitin Synthase ◽

Egg Mass ◽

Transgenic Tobacco Plants ◽

Tobacco Plants ◽

Wide Range ◽

Transgenic Lines ◽

Plant Parasitic

Meloidogyne incognita is a devastating plant parasitic nematode that causes root knot disease in a wide range of plants. In the present study, we investigated host-induced RNA interference (RNAi) gene silencing of chitin biosynthesis pathway genes (chitin synthase, glucose-6-phosphate isomerase, and trehalase) in transgenic tobacco plants. To develop an RNAi vector, ubiquitin (UBQ1) promoter was directly cloned, and to generate an RNAi construct, expression of three genes was suppressed using the GATEWAY system. Further, transgenic Nicotiana benthamiana lines expressing dsRNA for chitin synthase (CS), glucose-6-phosphate isomerase (GPI), and trehalase 1 (TH1) were generated. Quantitative PCR analysis confirmed endogenous mRNA expression of root knot nematode (RKN) and revealed that all three genes were more highly expressed in the female stage than in eggs and in the parasitic stage. In vivo, transformed roots were challenged with M. incognita. The number of eggs and root knots were significantly decreased by 60–90% in RNAi transgenic lines. As evident, root galls obtained from transgenic RNAi lines exhibited 0.01- to 0.70-fold downregulation of transcript levels of targeted genes compared with galls isolated from control plants. Furthermore, phenotypic characteristics such as female size and width were also marginally altered, while effect of egg mass per egg number in RNAi transgenic lines was reduced. These results indicate the relevance and significance of targeting chitin biosynthesis genes during the nematode lifespan. Overall, our results suggest that further developments in RNAi efficiency in commercially valued crops can be applied to employ RNAi against other plant parasitic nematodes.

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