RNAi effects on actin mRNAs in Homalodisca vitripennis cells

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.

Keywords: Homalodisca vitripennis; Pierce's disease; RNA interference; glassy-winged sharpshooter.

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 C_T 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.

Figure 2.

A. Transfection of actin dsRNA in H. vitripennis -Z15 cells results in a reduction in the level of actin mRNA. 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 0.5μg of the large fraction RNAs were separated by electrophoresis in a denaturing 1.5% (w/v) agarose gel. RNA was transferred to a positively-charged nylon membrane (NitroBind, Cast, Pure Nitrocellulose, GE) and UV cross-linked. ³²P-UTP-labeled negative sense actin RNA transcripts were generated in vitro using T7 RNA Polymerase (T7 MAXIscript, Ambion), and used as probe. Hybridization was performed using standard procedures (PerfectHyb Plus, Hybridization Buffer, SIGMA/ALDRICH). Reduction of the actin RNA was visible in actin dsRNA treated cells (a), compared to the controls (c, s and g). The ribosomal RNAs after staining the membrane with Methylene Blue are shown in top panel, to indicate equivalent sample loading. B. Diagrammatic representation of H. vitripennis actin mRNA (Gen Bank accession AY588061) (orange arrow) compared to the input actin dsRNA (blue segment). Position of the northern hybridization probe is indicated as a continuous black line, and position of the primers used in real time PCR is indicated by two black arrows. The conserved and variable regions of the actin mRNA are also indicated at the 5' and 3' termini of the actin mRNA, respectively.

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.

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.