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RNAi effects on actin mRNAs in Homalodisca vitripennis cells.

Rosa C, Kamita SG, Dequine H, Wuriyanghan H, Lindbo JA, Falk BW - J RNAi Gene Silencing (2010)

Bottom Line: RNAi effectors were delivered via lipid based transfection and real-time RT-PCR, RNA hybridization, and microscopic analyses were employed to verify RNAi effects.When actin dsRNAs were used, a 10-fold decrease in the target H. vitripennis actin mRNA level was seen in cells.Altered phenotypic effects also were evident in transfected cells, as were small interfering RNAs, hallmarks of RNAi.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology UC Davis, One Shields Ave, 95616 Davis CA, USA.

ABSTRACT
The xylem feeding leafhopper Homalodisaca vitripennis (H. vitripennis) is an unusually robust and efficient vector of Xylella fastidiosa, a Gram-negative bacterium which causes several very important plant diseases. Here we investigated RNA interference (RNAi) to target actin, a key component of insect cells and whole bodies, in H. vitripennis cells. RNAi effectors were delivered via lipid based transfection and real-time RT-PCR, RNA hybridization, and microscopic analyses were employed to verify RNAi effects. When actin dsRNAs were used, a 10-fold decrease in the target H. vitripennis actin mRNA level was seen in cells. Altered phenotypic effects also were evident in transfected cells, as were small interfering RNAs, hallmarks of RNAi. The use of H. vitripennis cells and RNAi offers new opportunities to research hemipterans, the most important insect vectors of plant pathogens.

No MeSH data available.


Related in: MedlinePlus

Actin dsRNA transfection of H. vitripennis -Z15 cells results in small interfering RNA (siRNA) accumulation. Cells were transfected with transfection reagent (c), 2μg of actin dsRNA (a), sar1 dsRNA (s), GFP dsRNA (g) and harvested 72hrs post transfection. Large and small RNA fractions were extracted (Ambion, mirVana PARIS) and 1μg of the small RNA fractions was separated by electrophoresis in a 7M urea 15% (w/v) polyacrylamide gel, and the gel was stained with ethidium bromide (Panel B). RNA was transferred onto a positively charged nylon membrane (NitroBind, Cast, Pure Nitrocellulose, GE) and UV cross-linked. 32PUTP labeled negative sense actin RNA transcripts were generated in vitro using T7 RNA polymerase (T7 MAXIscript, Ambion), fractionated and used as probe. Hybridization was performed using standard procedures (mirVana PARIS, Ambion). siRNAs were detected only in actin dsRNA treated cells (a) and not in the controls (c, s and g) (Panel A). Positions of marker siRNAs are labelled in Panel B and that of actin siRNAs (of ~22nt) in Panel A.
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Figure 3: Actin dsRNA transfection of H. vitripennis -Z15 cells results in small interfering RNA (siRNA) accumulation. Cells were transfected with transfection reagent (c), 2μg of actin dsRNA (a), sar1 dsRNA (s), GFP dsRNA (g) and harvested 72hrs post transfection. Large and small RNA fractions were extracted (Ambion, mirVana PARIS) and 1μg of the small RNA fractions was separated by electrophoresis in a 7M urea 15% (w/v) polyacrylamide gel, and the gel was stained with ethidium bromide (Panel B). RNA was transferred onto a positively charged nylon membrane (NitroBind, Cast, Pure Nitrocellulose, GE) and UV cross-linked. 32PUTP labeled negative sense actin RNA transcripts were generated in vitro using T7 RNA polymerase (T7 MAXIscript, Ambion), fractionated and used as probe. Hybridization was performed using standard procedures (mirVana PARIS, Ambion). siRNAs were detected only in actin dsRNA treated cells (a) and not in the controls (c, s and g) (Panel A). Positions of marker siRNAs are labelled in Panel B and that of actin siRNAs (of ~22nt) in Panel A.

Mentions: RNA interference is not only manifested by a decrease in the target mRNA accumulation, but also by the appearance of small interfering RNAs (siRNAs) corresponding to mRNA target sequence (Tomoyasu et al, 2008). Therefore the presence of siRNAs specific for actin mRNA was investigated by northern hybridization analysis. Actin-specific siRNAs were seen only in RNAs extracted from actin dsRNA transfected cells (a) and not in the controls (c, s and g) (Figure 3). Comparison of the positions of the actin siRNAs with those of oligonucleotide markers shows that the actin siRNAs were approximately 21nt. Taken together these data clearly showed that RNAi was induced in the H. vitripennis -Z15 cells.


RNAi effects on actin mRNAs in Homalodisca vitripennis cells.

Rosa C, Kamita SG, Dequine H, Wuriyanghan H, Lindbo JA, Falk BW - J RNAi Gene Silencing (2010)

Actin dsRNA transfection of H. vitripennis -Z15 cells results in small interfering RNA (siRNA) accumulation. Cells were transfected with transfection reagent (c), 2μg of actin dsRNA (a), sar1 dsRNA (s), GFP dsRNA (g) and harvested 72hrs post transfection. Large and small RNA fractions were extracted (Ambion, mirVana PARIS) and 1μg of the small RNA fractions was separated by electrophoresis in a 7M urea 15% (w/v) polyacrylamide gel, and the gel was stained with ethidium bromide (Panel B). RNA was transferred onto a positively charged nylon membrane (NitroBind, Cast, Pure Nitrocellulose, GE) and UV cross-linked. 32PUTP labeled negative sense actin RNA transcripts were generated in vitro using T7 RNA polymerase (T7 MAXIscript, Ambion), fractionated and used as probe. Hybridization was performed using standard procedures (mirVana PARIS, Ambion). siRNAs were detected only in actin dsRNA treated cells (a) and not in the controls (c, s and g) (Panel A). Positions of marker siRNAs are labelled in Panel B and that of actin siRNAs (of ~22nt) in Panel A.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2902141&req=5

Figure 3: Actin dsRNA transfection of H. vitripennis -Z15 cells results in small interfering RNA (siRNA) accumulation. Cells were transfected with transfection reagent (c), 2μg of actin dsRNA (a), sar1 dsRNA (s), GFP dsRNA (g) and harvested 72hrs post transfection. Large and small RNA fractions were extracted (Ambion, mirVana PARIS) and 1μg of the small RNA fractions was separated by electrophoresis in a 7M urea 15% (w/v) polyacrylamide gel, and the gel was stained with ethidium bromide (Panel B). RNA was transferred onto a positively charged nylon membrane (NitroBind, Cast, Pure Nitrocellulose, GE) and UV cross-linked. 32PUTP labeled negative sense actin RNA transcripts were generated in vitro using T7 RNA polymerase (T7 MAXIscript, Ambion), fractionated and used as probe. Hybridization was performed using standard procedures (mirVana PARIS, Ambion). siRNAs were detected only in actin dsRNA treated cells (a) and not in the controls (c, s and g) (Panel A). Positions of marker siRNAs are labelled in Panel B and that of actin siRNAs (of ~22nt) in Panel A.
Mentions: RNA interference is not only manifested by a decrease in the target mRNA accumulation, but also by the appearance of small interfering RNAs (siRNAs) corresponding to mRNA target sequence (Tomoyasu et al, 2008). Therefore the presence of siRNAs specific for actin mRNA was investigated by northern hybridization analysis. Actin-specific siRNAs were seen only in RNAs extracted from actin dsRNA transfected cells (a) and not in the controls (c, s and g) (Figure 3). Comparison of the positions of the actin siRNAs with those of oligonucleotide markers shows that the actin siRNAs were approximately 21nt. Taken together these data clearly showed that RNAi was induced in the H. vitripennis -Z15 cells.

Bottom Line: RNAi effectors were delivered via lipid based transfection and real-time RT-PCR, RNA hybridization, and microscopic analyses were employed to verify RNAi effects.When actin dsRNAs were used, a 10-fold decrease in the target H. vitripennis actin mRNA level was seen in cells.Altered phenotypic effects also were evident in transfected cells, as were small interfering RNAs, hallmarks of RNAi.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology UC Davis, One Shields Ave, 95616 Davis CA, USA.

ABSTRACT
The xylem feeding leafhopper Homalodisaca vitripennis (H. vitripennis) is an unusually robust and efficient vector of Xylella fastidiosa, a Gram-negative bacterium which causes several very important plant diseases. Here we investigated RNA interference (RNAi) to target actin, a key component of insect cells and whole bodies, in H. vitripennis cells. RNAi effectors were delivered via lipid based transfection and real-time RT-PCR, RNA hybridization, and microscopic analyses were employed to verify RNAi effects. When actin dsRNAs were used, a 10-fold decrease in the target H. vitripennis actin mRNA level was seen in cells. Altered phenotypic effects also were evident in transfected cells, as were small interfering RNAs, hallmarks of RNAi. The use of H. vitripennis cells and RNAi offers new opportunities to research hemipterans, the most important insect vectors of plant pathogens.

No MeSH data available.


Related in: MedlinePlus