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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 and sar1 dsRNA as effectors of RNAi in H. vitripennis Z-15 cells. A and B. Two μg of actin, sar 1, Lian-Aa1, GFP or arginine kinase dsRNAs were transfected into H. vitripennis cells. Cells were harvested 72hr post transfection and the sar1 or actin mRNAs were quantified by real time RT-PCR by comparative CT method (ΔΔCT method). Two sets of endogenous controls were used: sar1 mRNA (pale blue columns) and ribosomal RNA (lilac columns) to quantify actin mRNA (panel A); actin mRNA (blue columns) and ribosomal RNA (red columns) to quantify sar1 mRNA (panel B). RNAi effects are shown by the sar1 and actin mRNA reductions in sar1 and actin dsRNA transfected cells respectively, compared to cells treated with transfection reagent only (calibrators). The percentage knockdown for actin mRNA was 95% relative to the transfection reagent control, and 87% compared to the GFP control. For sar1 percentage knockdown was 75% relative to the transfection reagent and 87% compared to the GFP control (left columns in panels A and B respectively), when we used sar1 and actin mRNA as endogenous controls. Similar results were obtained using ribosomal RNA as endogenous controls. Numbers above the columns represent the fold differences of mRNA concentration compared to the control. Experiments were repeated three times. Of these, the cell transfection was performed twice in three replicates and once in two replicates. Real time RT-PCR samples were always loaded in duplicates. Error bars above the columns indicate the standard deviation among the 3 experiments.
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Figure 1: Actin and sar1 dsRNA as effectors of RNAi in H. vitripennis Z-15 cells. A and B. Two μg of actin, sar 1, Lian-Aa1, GFP or arginine kinase dsRNAs were transfected into H. vitripennis cells. Cells were harvested 72hr post transfection and the sar1 or actin mRNAs were quantified by real time RT-PCR by comparative CT method (ΔΔCT method). Two sets of endogenous controls were used: sar1 mRNA (pale blue columns) and ribosomal RNA (lilac columns) to quantify actin mRNA (panel A); actin mRNA (blue columns) and ribosomal RNA (red columns) to quantify sar1 mRNA (panel B). RNAi effects are shown by the sar1 and actin mRNA reductions in sar1 and actin dsRNA transfected cells respectively, compared to cells treated with transfection reagent only (calibrators). The percentage knockdown for actin mRNA was 95% relative to the transfection reagent control, and 87% compared to the GFP control. For sar1 percentage knockdown was 75% relative to the transfection reagent and 87% compared to the GFP control (left columns in panels A and B respectively), when we used sar1 and actin mRNA as endogenous controls. Similar results were obtained using ribosomal RNA as endogenous controls. Numbers above the columns represent the fold differences of mRNA concentration compared to the control. Experiments were repeated three times. Of these, the cell transfection was performed twice in three replicates and once in two replicates. Real time RT-PCR samples were always loaded in duplicates. Error bars above the columns indicate the standard deviation among the 3 experiments.

Mentions: H. vitripennis -Z15 cells were transfected with dsRNAs specific for H. vitripennis arginine kinase (AY588062) (Wang et al, 2009), actin, sar1 or Lian-Aa1 (a non-LTR retrotransposon) as well as for the green fluorescent protein (GFP), or with transfection reagent alone. At 72hr time point, realtime RT-PCR analyses showed that actin mRNA was decreased by 95% in cells transfected with actin dsRNA, in comparison to mock-transfected control cells (Figure 1). However, the other dsRNAs also induced some reduction (~50-60%) in actin mRNA relative to the mock control. When these same cells were analyzed for the sar1 mRNA, the sar1 dsRNA transfected cells had 75% reduction relative to the transfection reagent control, but in contrast to results for actin mRNA cells transfected with the other dsRNAs showed approximately twice the amount of sar1 mRNA relative to the control cells. Thus off-target effects were seen for both actin and sar1 analyses. If control transfection reagent treated cells are not considered and only dsRNA treated cells are compared, then reductions of 87% for the actin mRNA and 87% for the sar1 mRNA were seen when compared to the control GFP dsRNA transfected 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 and sar1 dsRNA as effectors of RNAi in H. vitripennis Z-15 cells. A and B. Two μg of actin, sar 1, Lian-Aa1, GFP or arginine kinase dsRNAs were transfected into H. vitripennis cells. Cells were harvested 72hr post transfection and the sar1 or actin mRNAs were quantified by real time RT-PCR by comparative CT method (ΔΔCT method). Two sets of endogenous controls were used: sar1 mRNA (pale blue columns) and ribosomal RNA (lilac columns) to quantify actin mRNA (panel A); actin mRNA (blue columns) and ribosomal RNA (red columns) to quantify sar1 mRNA (panel B). RNAi effects are shown by the sar1 and actin mRNA reductions in sar1 and actin dsRNA transfected cells respectively, compared to cells treated with transfection reagent only (calibrators). The percentage knockdown for actin mRNA was 95% relative to the transfection reagent control, and 87% compared to the GFP control. For sar1 percentage knockdown was 75% relative to the transfection reagent and 87% compared to the GFP control (left columns in panels A and B respectively), when we used sar1 and actin mRNA as endogenous controls. Similar results were obtained using ribosomal RNA as endogenous controls. Numbers above the columns represent the fold differences of mRNA concentration compared to the control. Experiments were repeated three times. Of these, the cell transfection was performed twice in three replicates and once in two replicates. Real time RT-PCR samples were always loaded in duplicates. Error bars above the columns indicate the standard deviation among the 3 experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Actin and sar1 dsRNA as effectors of RNAi in H. vitripennis Z-15 cells. A and B. Two μg of actin, sar 1, Lian-Aa1, GFP or arginine kinase dsRNAs were transfected into H. vitripennis cells. Cells were harvested 72hr post transfection and the sar1 or actin mRNAs were quantified by real time RT-PCR by comparative CT method (ΔΔCT method). Two sets of endogenous controls were used: sar1 mRNA (pale blue columns) and ribosomal RNA (lilac columns) to quantify actin mRNA (panel A); actin mRNA (blue columns) and ribosomal RNA (red columns) to quantify sar1 mRNA (panel B). RNAi effects are shown by the sar1 and actin mRNA reductions in sar1 and actin dsRNA transfected cells respectively, compared to cells treated with transfection reagent only (calibrators). The percentage knockdown for actin mRNA was 95% relative to the transfection reagent control, and 87% compared to the GFP control. For sar1 percentage knockdown was 75% relative to the transfection reagent and 87% compared to the GFP control (left columns in panels A and B respectively), when we used sar1 and actin mRNA as endogenous controls. Similar results were obtained using ribosomal RNA as endogenous controls. Numbers above the columns represent the fold differences of mRNA concentration compared to the control. Experiments were repeated three times. Of these, the cell transfection was performed twice in three replicates and once in two replicates. Real time RT-PCR samples were always loaded in duplicates. Error bars above the columns indicate the standard deviation among the 3 experiments.
Mentions: H. vitripennis -Z15 cells were transfected with dsRNAs specific for H. vitripennis arginine kinase (AY588062) (Wang et al, 2009), actin, sar1 or Lian-Aa1 (a non-LTR retrotransposon) as well as for the green fluorescent protein (GFP), or with transfection reagent alone. At 72hr time point, realtime RT-PCR analyses showed that actin mRNA was decreased by 95% in cells transfected with actin dsRNA, in comparison to mock-transfected control cells (Figure 1). However, the other dsRNAs also induced some reduction (~50-60%) in actin mRNA relative to the mock control. When these same cells were analyzed for the sar1 mRNA, the sar1 dsRNA transfected cells had 75% reduction relative to the transfection reagent control, but in contrast to results for actin mRNA cells transfected with the other dsRNAs showed approximately twice the amount of sar1 mRNA relative to the control cells. Thus off-target effects were seen for both actin and sar1 analyses. If control transfection reagent treated cells are not considered and only dsRNA treated cells are compared, then reductions of 87% for the actin mRNA and 87% for the sar1 mRNA were seen when compared to the control GFP dsRNA transfected 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