Expressing Parasitoid Venom Protein VRF1 in an Entomopathogen Beauveria bassiana Enhances Virulence toward Cotton Bollworm Helicoverpa armigera.

Despite entomopathogenic fungi being used in various insect pest control, it is recognized that they could replace more chemical insecticides if they were more efficient. We have found that cotton bollworm Helicoverpa armigera responded to the infection of entomopathogenic fungus Beauveria bassiana by activating the Toll pathway. Koinobiont wasps also regulate host immunity and development to ensure the survival of their progeny. Previously, venom protein VRF1 was identified in Microplitis mediator. It enters H. armigera hemocytes, suppresses the expression of antimicrobial peptides (AMPs) by inhibiting the Toll pathway, and prevents parasite offspring from being encapsulated. With this in mind, we thought that it might be feasible to increase the virulence of B. bassiana by embedding VRF1 into its genome. Compared with that of wild-type (WT) B. bassiana, the median lethal dose (LD50) of the transformant expressing VRF1 (named BbVRF1) decreased approximately 2.36-fold, and the median time to lethality (LT50) was shortened to 84% when infecting H. armigera (a natural host of M. mediator). The AMP expression level of hemocytes in H. armigera infected with BbVRF1 strain was significantly downregulated compared to that in the control group infected with the WT. In addition, the LD50 of BbVRF1 against the fall armyworm Spodoptera frugiperda (an unnatural host of M. mediator) was decreased 3.45-fold and the LT50 was shortened to 73%, showing a greater virulence. Our research indicated that BbVRF1, an engineered strain of B. bassiana, has greater efficacy against pest insects both within and outside its host range (M. mediator), expanding the utilization of parasitoid wasp virulence effectors. IMPORTANCE Mycoinsecticides are essential for the development of integrated pest management as substitutes to chemical insecticides, but their usage is limited by their inferior virulence. Thus, genetically engineered bioinsecticides, including recombinant entomopathogenic fungi, have been regarded as a breakthrough to rapidly control pests. Deep knowledge of parasitoid wasps allows us to take advantage of this natural enemy of pest insects beyond raising them for field release. Our transformant BbVRF1 (Beauveria bassiana integrated with a venom protein VRF1 from Microplitis mediator) showed a higher virulence in H. armigera and S. frugiperda, demonstrating its potential for managing natural or unnatural hosts of M. mediator. This result provides a new strategy regarding which venom protein of parasitoid wasps can become part of the arsenal with which to equip entomopathogenic fungi. Utilizing parasitoid wasps with this approach could easily overcome the difficulties of artificial culture and enhance the virulence of other biocontrol agents.

Rho 1 participates in parasitoid wasp eggs maturation and host cellular immunity inhibition.

Endoparasitoid wasps introduce venom into their host insects during the egg-laying stage. Venom proteins play various roles in the host physiology, development, immunity, and behavior manipulation and regulation. In this study, we identified a venom protein, MmRho1, a small guanine nucleotide-binding protein derived from ovary in the endoparasitoid wasp Microplitis mediator and found that knockdown of its expression by RNA interference caused down-regulation of vitellogenin and juvenile hormone, egg production, and cocoons formation in the female wasps. We demonstrated that MmRho1 entered the cotton bollworm's (host) hemocytes and suppressed cellular immune responses after parasitism using immunofluorescence staining. Furthermore, wasp MmRho1 interacted with the cotton bollworm's actin cytoskeleton rearrangement regulator diaphanous by yeast 2-hybrid and glutathione s-transferase pull-down. In conclusion, this study indicates that MmRho1 plays dual roles in wasp development and the suppression of the host insect cellular immune responses.