Expressing a fusion protein with protease and chitinase activities increases the virulence of the insect pathogen Beauveria bassiana

Entomopathogenic fungi, such as Beauveria bassiana and Metarhizium anisopliae are being developed as alternatives to chemical insecticides. They infect insects by direct penetration of the cuticle using a combination of physical pressure and extracellular hydrolytic enzymes such as proteases and chitinases. Previously we found that overexpression of a subtilisin-like protease (Pr1A) or a chitinase (Bbchit1) resulted in increased virulence of M. anisopliae and B. bassiana, respectively. In this study, we found that a mixture of the B. bassiana Pr1A homolog (CDEP1) and Bbchit1 degraded insect cuticle in vitro more efficiently than either CDEP1 or Bbchit1 alone. Based on this we produced three plasmid constructs; (1) Bbchit1, (2) CDEP1, and (3) a fusion gene of Bbchit1 linked to CDEP1 each under the control of the constitutive gpd promoter from Aspergillus nidulans. B. bassiana transformants secreting the fusion protein (CDEP1:Bbchit1) penetrated the cuticle significantly faster than the wild type or transformants overexpressing either Bbchit1 or CDEP1. Compared to the wild type, the transformant overexpressing CDEP1 showed a 12.5% reduction in LT50, without a reduction in LC50. The LT50 of the transformant expressing CDEP1:Bbchit1 was reduced by 24.9%. Strikingly, expression of CDEP1:Bbchit1 resulted in a 60.5% reduction in LC50, more than twice the reduction obtained by overexpression of Bbchit1 (28.5%). This work represents a significant step towards the development of hypervirulent insect pathogens for effective pest control.