Abstract
Background
Genetic engineering of entomopathogenic fungi offers an innovative approach to improve their virulence against a broad spectrum of arthropod hosts and increase their resilience to stressful environmental factors. In this research, transformation of Beauveria bassiana protoplasts with a constitutively expressed endogenous gene encoding a chitinase (BbChit1) was achieved with the aim to improve fungal virulence. The relative gene expression and the number of gene copies in the chitinase-overexpressing mutants (OEBbChit1) were assessed, followed by phenotypic characterization of blastospores in terms of virulence, insect cellular immune response, colony morphology, growth, and tolerance to multiple chemical cell stressors.
Results
Blastospores of OEBbChit1 mutants conferred faster mortality rates and lower lethal doses when compared to the parental wild-type (WT) after being topically inoculated to larvae of the model insect host Galleria mellonella. Moreover, infections by the OEBbChit1 mutant triggered an increased production of total hemocytes relative to the WT and mock control, indicating induction of the insect cellular immune response. Genomic analysis of the OEBbChit1 mutants revealed that the number of gene copies encoding the chitinase was 4 and 2 for mutants OEBbChit1-3 and OEBbChit1-5, respectively. Furthermore, the chitinase-encoding gene was significantly upregulated by ~ 3 to 5 fold relative to the WT, confirming the overexpression of this enzyme in the mutants.
Conclusions
Collectively, these findings provide compelling evidence of genetic improvement of B. bassiana by overexpression of chitinase-encoding gene (BbChit1) with 2–4 copies integrated into its genome leading to enhanced virulence without detrimental pleiotropic effects in fungal development.