Limits...
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 representative morphology in H. vitripennis -Z15 cells after transfection with actin dsRNA. Cells were transfected with 2μg of actin dsRNA (A and C), or GFP dsRNA (B and D) and harvested 72hrs post transfection. Actin filaments in the cell membrane and cytoplasmic area were largely disturbed (arrows in A and C). (A) H. vitripennis cells showing partial disruption of the actin organization at the cell plasma membrane. Some filaments began to break and the cells failed to branch out. (B) H. vitripennis cells showing no changes in actin filament distribution and polymerization. Healthy isolated cells were connected through a densely branched actin filament network. (C) H. vitripennis cells showing severe disruption of actin filaments. The short fragments of actin filaments were scattered throughout the cytoplasm. Some actin fragments tended to aggregate into clusters below the plasma membrane and obvious twisted actin cables could be observed. (D) Actin filaments were found primarily in the cell cytoplasm as a continuous and organized net in the control cells. All observations were at 72hrs post transfection.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2902141&req=5

Figure 4: Actin representative morphology in H. vitripennis -Z15 cells after transfection with actin dsRNA. Cells were transfected with 2μg of actin dsRNA (A and C), or GFP dsRNA (B and D) and harvested 72hrs post transfection. Actin filaments in the cell membrane and cytoplasmic area were largely disturbed (arrows in A and C). (A) H. vitripennis cells showing partial disruption of the actin organization at the cell plasma membrane. Some filaments began to break and the cells failed to branch out. (B) H. vitripennis cells showing no changes in actin filament distribution and polymerization. Healthy isolated cells were connected through a densely branched actin filament network. (C) H. vitripennis cells showing severe disruption of actin filaments. The short fragments of actin filaments were scattered throughout the cytoplasm. Some actin fragments tended to aggregate into clusters below the plasma membrane and obvious twisted actin cables could be observed. (D) Actin filaments were found primarily in the cell cytoplasm as a continuous and organized net in the control cells. All observations were at 72hrs post transfection.

Mentions: Because actin is an abundant and essential component of eukaryotic cells, and actin filaments can be specifically labeled with fluorescent phallotoxin derivatives, such as phalloidin, we determined whether actin dsRNA treated cells showed phenotypic alterations that were consistent with the downregulation of actin mRNA accumulation. Microscopic observation of H. vitripennis -Z15 cells transfected with actin dsRNAs showed clear alterations in cell morphology during the experiment time frame (72 hrs) in comparison to control transfected cells (Figure 4). In actin dsRNA transfected cells, actin filaments were not apparent at the cell membranes at 72hr time point, especially at the cell periphery. The actin filaments were distorted and appeared broken in the cytoplasm, and cells were not connected to each other. Actin filaments often failed to develop and appeared to be shorter (Figure 4, panels A and C). In control cells transfected with GFP dsRNA (Figure 4, panels B and D), dense actin filaments were observed on the plasma membrane at the cell periphery, and tight junction strands formed well-developed networks.


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 representative morphology in H. vitripennis -Z15 cells after transfection with actin dsRNA. Cells were transfected with 2μg of actin dsRNA (A and C), or GFP dsRNA (B and D) and harvested 72hrs post transfection. Actin filaments in the cell membrane and cytoplasmic area were largely disturbed (arrows in A and C). (A) H. vitripennis cells showing partial disruption of the actin organization at the cell plasma membrane. Some filaments began to break and the cells failed to branch out. (B) H. vitripennis cells showing no changes in actin filament distribution and polymerization. Healthy isolated cells were connected through a densely branched actin filament network. (C) H. vitripennis cells showing severe disruption of actin filaments. The short fragments of actin filaments were scattered throughout the cytoplasm. Some actin fragments tended to aggregate into clusters below the plasma membrane and obvious twisted actin cables could be observed. (D) Actin filaments were found primarily in the cell cytoplasm as a continuous and organized net in the control cells. All observations were at 72hrs post transfection.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Actin representative morphology in H. vitripennis -Z15 cells after transfection with actin dsRNA. Cells were transfected with 2μg of actin dsRNA (A and C), or GFP dsRNA (B and D) and harvested 72hrs post transfection. Actin filaments in the cell membrane and cytoplasmic area were largely disturbed (arrows in A and C). (A) H. vitripennis cells showing partial disruption of the actin organization at the cell plasma membrane. Some filaments began to break and the cells failed to branch out. (B) H. vitripennis cells showing no changes in actin filament distribution and polymerization. Healthy isolated cells were connected through a densely branched actin filament network. (C) H. vitripennis cells showing severe disruption of actin filaments. The short fragments of actin filaments were scattered throughout the cytoplasm. Some actin fragments tended to aggregate into clusters below the plasma membrane and obvious twisted actin cables could be observed. (D) Actin filaments were found primarily in the cell cytoplasm as a continuous and organized net in the control cells. All observations were at 72hrs post transfection.
Mentions: Because actin is an abundant and essential component of eukaryotic cells, and actin filaments can be specifically labeled with fluorescent phallotoxin derivatives, such as phalloidin, we determined whether actin dsRNA treated cells showed phenotypic alterations that were consistent with the downregulation of actin mRNA accumulation. Microscopic observation of H. vitripennis -Z15 cells transfected with actin dsRNAs showed clear alterations in cell morphology during the experiment time frame (72 hrs) in comparison to control transfected cells (Figure 4). In actin dsRNA transfected cells, actin filaments were not apparent at the cell membranes at 72hr time point, especially at the cell periphery. The actin filaments were distorted and appeared broken in the cytoplasm, and cells were not connected to each other. Actin filaments often failed to develop and appeared to be shorter (Figure 4, panels A and C). In control cells transfected with GFP dsRNA (Figure 4, panels B and D), dense actin filaments were observed on the plasma membrane at the cell periphery, and tight junction strands formed well-developed networks.

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