A Polydnavirus Protein Tyrosine Phosphatase Negatively Regulates the Host Phenoloxidase Pathway

Abstract

Polydnavirus (PDV) is a parasitic factor of endoparasitic wasps and contributes greatly to overcoming the immune response of parasitized hosts. Protein tyrosine phosphatases (PTPs) regulate a wide variety of biological processes at the post-transcriptional level in mammals, but knowledge of PDV PTP action during a parasitoid−host interaction is limited. In this study, we characterized a PTP gene, CvBV_12-6, derived from Cotesia vestalis bracovirus (CvBV), and explored its possible regulatory role in the immune response of the host Plutella xylostella. Our results from qPCR show that CvBV_12-6 was highly expressed in hemocytes at an early stage of parasitization. To explore CvBV_12-6 function, we specifically expressed CvBV_12-6 in Drosophila melanogaster hemocytes. The results show that Hml-Gal4 > CvBV_12-6 suppressed the phenoloxidase activity of hemolymph in D. melanogaster, but exerted no effect on the total count or the viability of the hemocytes. In addition, the Hml-Gal4 > CvBV_12-6 flies exhibited decreased antibacterial abilities against Staphylococcus aureus. Similarly, we found that CvBV_12-6 significantly suppressed the melanization of the host P. xylostella 24 h post parasitization and reduced the viability, but not the number, of hemocytes. In conclusion, CvBV_12-6 negatively regulated both cellular and humoral immunity in P. xylostella, and the related molecular mechanism may be universal to insects.

Keywords: phenoloxidase; polydnavirus; protein tyrosine phosphatase.

Figure 1

The conserved motifs and expression patterns of CvBV_12-6. (A) Image showing the conserved domain in the CvBV_12-6 protein and the amino acid sequence of each motif. X represents any amino acid. Conserved domain sequence: PTP NR6 and PTP N11 were from Homo sapiens; PTP_meg2 and PTP_99A were from D. melanogaster; PTP H1 and PTP H2 were from MdBV. (B) The transcriptional dynamics of CvBV_12-6 in P. xylostella 6, 12, 24, 48, 72, 96, and 120 h post parasitization (pp). (C) The expression pattern of CvBV_12-6 in different tissues of parasitized P. xylostella. Data are presented as the mean ± SD based on three independent experiments. Differences among samples were tested via Tukey’s test, and different letters indicate significant differences at a p value < 0.05.

Figure 2

Eukaryotic expression of CvBV_12-6 and tyrosine phosphatase activity detection. (A) The expression of CvBV_12-6 in the Sf9 cell line was detected by western blotting using an HA tag monoclonal antibody, and actin was used as the internal control. (B) The tyrosine phosphatase activity of CvBV_12-6 in Sf9 cells was measured by the amount of phosphate released after the incubation of the total protein and synthesized phosphopeptides. The cell lysate was extracted 48 h post infection. The GFP was used as the negative control, and sodium vanadate was used as the protein tyrosine phosphatase inhibitor (I). Data are presented as the mean ± SD. Differences among samples were evaluated for significance via Tukey’s test (***, significant difference at a p value < 0.001).

Figure 3

The immunosuppression of CvBV_12-6 in D. melanogaster. (A,B) The effect of CvBV_12-6 on the number (A) and the viability of hemocytes (B) in D. melanogaster. (C) The effect of CvBV_12-6 on the PO activity in D. melanogaster. The hemolymph was collected from third instar D. melanogaster larvae, and the PO activity was determined as a change in absorbance at 490 nm per mg protein per min. (D) The antibacterial activity of male D. melanogaster to S. aureus. Thirty adult Hml-GAL4 strain males and 200 adult UAS-CvBV_12-6 strain virgins were mated for 1 day and then transferred to a new food bottle every 2 h. Hml-GAL4 flies crossed with W1118 flies were the controls. Data are presented as the mean ± SD from three independent experiments. Each treatment group included 20 larvae (n = 15). Differences among samples were evaluated for significance via Tukey’s test (NS, no significance; *, significant difference at a p value < 0.05; **, significant difference at a p value < 0.01).

Figure 4

The effect of CvBV_12-6 on hemocytes of the P. xylostella host. (A) The biosynthesis of dsRNA of CvBV_12-6 was detected by agarose gel electrophoresis. (B) The RNA interference efficiency was measured by qPCR. (C,D) A standard curve was used to calculate the titer of the recombinant baculovirus as determined by qPCR. (E,F) The effect of CvBV_12-6 on the hemocyte count and the hemocyte viability in P. xylostella. The hemolymph of P. xylostella was collected 24 h after parasitization, RNAi or injection of NPV. Then, 19.5 µL of trypan blue working solution was mixed with 0.5 μL hemolymph, and then added to a cell counting plate. Count Star 1.0 software was used to automatically analyze the cell number and viability. dsCvBV_12-6-p: dsCvBV_12-6 was injected into third instar larvae, and parasitization was performed immediately; dsGFP-p was used as the negative control. NPV-CvBV_12-6: injection of NPV-CvBV_12-6 into third instar larva; NPV-GFP was used as the negative control. Each treatment group included more than 30 larvae (n >15). Data are presented as the mean ± SD. Differences among samples were evaluated for significance via Tukey’s test (***, significant difference for a p value < 0.001).

Figure 5

CvBV_12-6 inhibited the melanization response of the P. xylostella host. (A) One microgram of dsCvBV_12-6 or dsGFP was injected into third instar P. xylostella larvae, and parasitization was performed immediately. The hemolymph was collected, and the PO activity was measured 24 h pp. (B) NPV-CvBV_12-6 or NPV-GFP was injected into third instar P. xylostella larvae, and the PO activity was measured 24 h post injection. Data are presented as the mean ± SD. Differences among samples were evaluated for significance via Tukey’s test (**, significant difference at a p value < 0.01).