Expression of a Toll Signaling Regulator Serpin in a Mycoinsecticide for Increased Virulence

Abstract

bSerpins are ubiquitously distributed serine protease inhibitors that covalently bind to target proteases to exert their activities. Serpins regulate a wide range of activities, particularly those in which protease-mediated cascades are active. The Drosophila melanogaster serpin Spn43Ac negatively controls the Toll pathway that is activated in response to fungal infection. The entomopathogenic fungus Beauveria bassiana offers an environmentally friendly alternative to chemical pesticides for insect control. However, the use of mycoinsecticides remains limited in part due to issues of efficacy (low virulence) and the recalcitrance of the targets (due to strong immune responses). Since Spn43Ac acts to inhibit Toll-mediated activation of defense responses, we explored the feasibility of a new strategy to engineer entomopathogenic fungi with increased virulence by expression of Spn43Ac in the fungus. Compared to the 50% lethal dose (LD 50 ) for the wild-type parent, the LD 50 of B. bassiana expressing Spn43Ac (strain Bb::S43Ac-1) was reduced ϳ3-fold, and the median lethal time against the greater wax moth (Galleria mellonella) was decreased by ϳ24%, with the more rapid proliferation of hyphal bodies being seen in the host hemolymph. In vitro and in vivo assays showed inhibition of phenoloxidase (PO) activation in the presence of Spn43Ac, with Spn43Ac-mediated suppression of activation by chymotrypsin, trypsin, laminarin, and lipopolysaccharide occurring in the following order: chymotrypsin and trypsin > laminarin > lipopolysaccharide. Expression of Spn43Ac had no effect on the activity of the endogenous B. bassianaderived cuticle-degrading protease (CDEP-1). These results expand our understanding of Spn43Ac function and confirm that suppression of insect immune system defenses represents a feasible approach to engineering entomopathogenic fungi for greater efficacy.

The Herbivore-Induced Plant Volatiles Methyl Salicylate and Menthol Positively affect Growth and Pathogenicity of Entomopathogenic Fungi

“…However, these EPFs encountered low pathogenicity, conidia viability and instability due to the effect of UVB irradiance, and as consequence, there are only few EPF products being used in the field3031. In this regards, to overcome these challenges, many methods of molecular biology3233, chemical biology343536, and biophysics3738 have been investigated to increase the use of EPFs under adverse field conditions and some studies look promising39. Therefore, our findings in this study are important for understanding the multi-trophic interactions in ecosystems as well as for developing new and easier means for increasing conidial production and improving the pathogenicity of EPFs.…”

Improving mycoinsecticides for insect biological control

“…These proteins act to suppress or dampen immune responses in the absence of challenge, but can be quickly degraded when stimulated, i.e., in the presence of an infectious agent, to allow for rapid activation of innate immune responses. Expression of an insect serpin (Spn43Ac) in B. bassiana resulted in immune suppression, including inhibition of host prophenoloxidase activation, allowing for more rapid proliferation of the fungus in the host hemolymph, and subsequently greater fungal virulence towards the test organism, G. mellonella (Yang et al 2014). These data indicate that fungal strains can be engineered to target specific aspects of the host immune system resulting in more effective strains.…”

Tenebrionid secretions and a fungal benzoquinone oxidoreductase form competing components of an arms race between a host and pathogen

“…These findings have led to several genetic engineering approaches to improve mycoinsecticide performance by targeting specific defenses, e.g., cuticular compounds and insect immune pathways (73)(74)(75)(76). This paper adds benzoquinones to the list of insect cuticular defenses and presents BbBqrA as a potential adaptive response of B. bassiana to counteract the insect's attempt to escape fungal infection.…”