Molecular biology of the entomopathogenic fungus Beauveria bassiana : insect-cuticle degrading enzymes and development of a new selection marker for fungal transformation

Abstract

The entomopathogenic fungus, Beauveria bassiana, is attracting increased attention as potential biological control agent against insect pests. The research of this thesis focused on a B. bassiana strain, ARSEF 252 that is highly pathogenic for the Colorado potato beetle, Leptinotarsa decemlineata. Because of both the strong evidence supporting the role of cuticle-degrading proteases (PR1 and PR2) as well as phospholipase B (PLB) in fungal pathogens and their correlations to virulence observed in either M. anisopliae or C. albicans, these genes were cloned from B. bassiana 252. Two PLB-encoding genes (plb1 and plb2, 57% identity) and PR2-encoding genes (try1 and try2, 22.4% identity) are detected in B. bassiana 252. The predicted TRY1peptide is homologous to the trypsin-like proteases from fungi, whereas the putative TRY2 peptide is homologous to that from insects. The structure similarity of TRY2 protease to insect enzymes might allow the fungal cells to evade host “non-self” recognition and thus might represents one important virulence determinant. The antisera against PR1 and PLB2 were raised, respectively. B. bassiana was grown in poor medium containing insoluble beetle cuticle as the sole nitrogen and carbon source. Equivalent studies on M. anisophlie have shown that this is an effective way of inducing the production of cuticle-degrading enzymes. As a control, B. bassiana was also grown in rich medium. Expression of PR1 was not detected in rich medium by either RT-PCR or western blot analysis, whereas in cuticle medium, the expression of the pr1 and the secretion of the enzyme were highly increased. The formation of PLB2 was not influenced by carbon or nitrogen sources. These results indicate that the infectious process that involves enzyme production and penetration of host cuticle occurs only when a nutritional relationship with the host is necessary. Because B. bassiana252 is naturally resistant to many selection antibiotics, a new selectable marker, sorR, was developed for transformation assay. It is based on the sensitivity of B. bassiana252 to fungicide soraphen A. Soraphen A is a macrocyclic polyketide, which is produced by the myxobacterium Sorangium cellulosum. Soraphen A specifically inhibits the eukaryotic Acetyl-CoA Carboxylase (ACC) but not prokaryotic ACC. In fungi, three ACC functional domains are located on a multifunctional polypeptide derived from the expression of a single gene. In contrast, these three domains of E. coli ACC reside on four distinct polypeptides, which are the products of the separate genes. These four peptides function together in an enzymatic complex. The distinct structure of the ACC enzymes of eukaryotes and prokaryotes is believed to be the reason for their different sensitivity to soraphen A. The sorR marker is constructed based on the expression of complete E. coli ACC polypeptides in fungus, which confer soraphen A resistance. P. pastoris was chosen for marker development since the transformation procedure for B. bassiana252 was not yet available. The presence of the four E. coli acc genes under the control of the fungal expression signals confers soraphen A resistance to P. pastoris. As the sorR marker consists of four E. coli acc expression cassettes, it might be an advantage for the application of this system to split the marker cassette and transform its elements in subsequent transformation experiments. Therefore, a stepwise transformation procedure for sorR marker was developed in order to confirm this hypothesis. The transformant containing three E. coli acc expression cassettes was as sensitive to soraphen A as the recipient strain. Furthermore, this strain obtained soraphen A resistance only after the transformation of the fourth E. coli acc expression cassette in the last step. This new selection marker system has been submitted to apply a patent.