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. 2010 Nov;76(21):7202-9.
doi: 10.1128/AEM.01552-10. Epub 2010 Sep 10.

Interaction of Bacillus thuringiensis vegetative insecticidal protein with ribosomal S2 protein triggers larvicidal activity in Spodoptera frugiperda

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Interaction of Bacillus thuringiensis vegetative insecticidal protein with ribosomal S2 protein triggers larvicidal activity in Spodoptera frugiperda

Gatikrushna Singh et al. Appl Environ Microbiol. 2010 Nov.

Abstract

Vegetative insecticidal protein (Vip3A) is synthesized as an extracellular insecticidal toxin by certain strains of Bacillus thuringiensis. Vip3A is active against several lepidopteran pests of crops. Polyphagous pest, Spodoptera frugiperda, and its cell line Sf21 are sensitive for lyses to Vip3A. Screening of cDNA library prepared from Sf21 cells through yeast two-hybrid system with Vip3A as bait identified ribosomal protein S2 as a toxicity-mediating interacting partner protein. The Vip3A-ribosomal-S2 protein interaction was validated by in vitro pulldown assays and by RNA interference-induced knockdown experiments. Knockdown of expression of S2 protein in Sf21 cells resulted in reduced toxicity of the Vip3A protein. These observations were further extended to adult fifth-instar larvae of Spodoptera litura. Knockdown of S2 expression by injecting corresponding double-stranded RNA resulted in reduced mortality of larvae to Vip3A toxin. Intracellular visualization of S2 protein and Vip3A through confocal microscopy revealed their interaction and localization in cytoplasm and surface of Sf21 cells.

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Figures

FIG. 1.
FIG. 1.
Graph depicting cell mortality (%) at various time intervals in different concentrations of Vip3A. The mortalities of Sf21 cells exposed to different concentrations of Vip3A (100 ng, 250 ng, 500 ng, 750 ng, or 1 μg/ml) were determined for different time periods, as indicated.
FIG. 2.
FIG. 2.
(A) Microscopic view of Sf21 cells at 50, 55, and 60 h of growth in TNM-FH medium containing buffer. (i) Control Sf21 cells; (ii) Sf21 cells exposed to 500 ng of Vip3A; (iii) S2-silenced Sf21 exposed to 500 ng of Vip3A. (B) Histograms showing the percentages of dead versus live cells (see description of panel A).
FIG. 3.
FIG. 3.
(A) Mortalities of Sf21 cells exposed to Vip3A at various time period. Symbols: ⧫, mortality of Sf21 cells exposed to 500 ng of Vip3A/ml; ⋄, mortality of S2-silenced Sf21 cells exposed to 500 ng of Vip3A/ml. (B) Mortality of wild-type and S2-silenced Sf21 exposed to 500 ng of the insecticidal protein Cry1C/ml. Mortality was monitored by trypan blue staining at 5-h intervals for 40 h.
FIG. 4.
FIG. 4.
(A) Relative abundances of S2 transcripts in Sf21 cells and S2-silenced Sf21 cells exposed to 500 ng of Vip3A/ml. (i) RT-PCR analysis of S2 transcript at various time points; (ii) RT-PCR analysis of β-actin used as a control for corresponding samples; (iii) histogram depiction of the relative abundances of S2 transcript under conditions described above. (B) Northern blot analysis for S2-specific siRNA. Lane M, siRNA (21- and 23-mer) marker; lane 1, S2-silenced Sf21 cell line; lane 2, Sf21 cells.
FIG. 5.
FIG. 5.
In vitro binding study between the S2 ribosomal protein of Sf21 and Vip3A. Vip3A was expressed as His-Vip3A (lane 1), and ribosomal protein S2 was expressed as a GST fusion protein (lane 2). Lane 3 shows the purified GST. Lane 4, eluate from the Ni-NTA column with input His-Vip3A plus GST-S2; lane 5, flowthrough of input His-Vip3A plus GST-S2; lane 6, Vip3A protein eluate from the Ni-NTA column with input GST protein; lane 7, flowthrough of input His-Vip3A plus GST alone; lane M, marker.
FIG. 6.
FIG. 6.
Confocal microscopy images for the localization of Vip3A toxin with S2 protein in Sf21 insect cells. Images a, e, i, and m show views of the localization of Vip3A toxin from different fields labeled with FITC-conjugated anti-rabbit IgG. Images b, f, j, and n show views of the localization of S2 protein from different fields labeled with Cy3-conjugated anti-mouse IgG. Images c and g show the bright-field image. Images k and o reveal the DAPI staining of the nuclei of Sf21 cells. Images d, h, l, and p show the colocalization of Vip3A toxin and S2 by merging the three different images. The green arrows points to the colocalization of Vip3A toxin and S2 in the plasma membrane of the cell, and the red arrow points to the internalization of Vip3A toxin into the cell and colocalization at the cytoplasm.
FIG. 7.
FIG. 7.
(A) (i) Histogram showing the levels of S2 transcripts in different S. litura larva midguts after the injection of S2 dsRNA. (ii) RT-PCR analysis of the S2 transcript in the midguts of S. litura larvae. Lane 1, DEPC-water injected; lane 2, saline injected; lane 3, nonspecific dsRNA injected (2 μg); lane 4, S2 specific, dsRNA injected (2 μg). (iii) RT-PCR of β-actin used as a control of the respective samples. (B) Histogram showing the levels of S2 expression in different S. litura larvae midgut. (ii) Immunoblot analysis of S2 protein expression using anti-S2 antibody. Lane 1, DEPC-water injected; lane 2, saline injected; lane 3, nonspecific dsRNA injected (2 μg); lane 4, S2-specific dsRNA injected (2 μg). (C) Northern blot analysis for S2-specific siRNA in the midguts of S. litura larvae. Lane M, siRNA (21- and 23-mer) marker; lane 1, DEPC-water injected; lane 2, saline injected; lane 3, S2-specific dsRNA injected (2 μg).

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