Bumblebee venom serine protease increases fungal insecticidal virulence by inducing insect melanization

Abstract

Insect-killing (entomopathogenic) fungi have high potential for controlling agriculturally harmful pests. However, their pathogenicity is slow, and this is one reason for their poor acceptance as a fungal insecticide. The expression of bumblebee, Bombus ignitus, venom serine protease (VSP) by Beauveria bassiana (ERL1170) induced melanization of yellow spotted longicorn beetles (Psacothea hilaris) as an over-reactive immune response, and caused substantially earlier mortality in beet armyworm (Spodopetra exigua) larvae when compared to the wild type. No fungal outgrowth or sporulation was observed on the melanized insects, thus suggesting a self-restriction of the dispersal of the genetically modified fungus in the environment. The research is the first use of a multi-functional bumblebee VSP to significantly increase the speed of fungal pathogenicity, while minimizing the dispersal of the fungal transformant in the environment.

Figure 1. Tailing of B. ignitus (Bi) VSP fragment with B. bassiana (Bb) chitinase signal for extracellular secretion and integration of the fusion form into a fungal transformation vector.

(a) A schematic diagram of the fusion of Bb chitinase signal and Bi VSP fragments. The 5′-end of Bi serine protease domain was tailed by 3′-end of Bb signal by four-round serial PCR. The fusion form of PCR product was confirmed by sequencing. Bb signal, shadowed; and Bi VSP, not-shadowed. (b) A map of fungal transformation vector, pAB-BbsVSP including the fusion form of Bb signal and Bi VSP fragment. The plasmid retains the BAR and AMP resistance genes of the parent plasmid pABeG (BAR is a selectable marker providing resistance to glufosinate).

Figure 2. Expression of bumble bee venom serine protease (VSP) in an insect-killing fungus, B. bassiana ERL1170 (transformant: BbsVSP-#181).

(a) Solid culture of wild type (Wt) and BbsVSP-#181 on fourth-strength Sabouraud dextrose agar (SDA/4) for 7 days. (b) RT-PCR analysis of VSP in BbsVSP-#181 (#181). M, Marker. (c) Western blot analysis of liquid-cultured BbsVSP-#181 supernatant using an antiserum to bumble bee VSP. (d) Fibrinolytic activity of BbsVSP-#181 supernatant. Serine proteases are known to have fibrinolytic activity. (e) Melanization activity of BbsVSP-#181 spores (conidia) against yellow spotted longicorn beetles 4 days post injection. Beetles were injected with conidia at 40 µl (1×10⁷ conidia ml⁻¹) per larva. Phosphate buffered saline (PBS) solution was used as a base for all the treatments.

Figure 3. Insecticidal activity of wild type (Wt) and transformant BbsVSP-#181 (#181) against beet armyworm larvae in laboratory conditions.

(a) Percentage (%) of dead beet armyworm larvae after the spray of Wt and BbsVSP-#181 spores at 1×10⁷ conidia ml⁻¹ (N = 27). Siloxane solution (0.03%) as a surfactant was used as a base for all the treatments. (b) Symptoms of beet armyworms in 4, 7 and 10 days after the treatment. BbsVSP-#181-treated beet armyworms turned black in 4 days and no stage development was observed. But beet armyworms in the wild type treatment developed to fourth instars with mycosis in 7 days and completely mycotized in 10 days.