TOR Signaling Tightly Regulated Vegetative Growth Conidiation Oxidative Stress Tolerance and Entomopathogenicity in the Fungus Beauveria bassiana

Abstract

Beauveria bassiana degenerates after repeated subcultures demonstrating declined conidiation and insect virulence. The target of rapamycin (TOR) kinase conserved among eukaryotes is the master regulator of cellular physiology and is likely involved in culture degeneration. Indeed the levels of TOR-associated proteins increase over successive subcultures. Here CRISPR/Cas9 locus engineering introduced the inducible Tet-On promoter upstream of the TOR kinase 2 gene tor2 in B. bassiana. The mutant PTet-On爐or2 慣41� was verified for the Tet-On integration via PCR analyses and provided a model for evaluating the fungal phenotypes according to the tor2 expression levels induced by doxycycline (Dox) concentrations. At 0 ?g?mL?1 of Dox T41 had 68% of the wild type抯 (WT) tor2 expression level hampered radial growth and relatively lower levels of oxidative stress tolerance conidiation and virulence against Spodoptera exigua compared to those under the presence of Dox. A low dose of Dox at 0.1�?g?mL?1 induced tor2 upregulation in T41 by up to 91% compared to 0 ?g?mL?1 of Dox resulting in significant increases in radial growth by 8�% and conidiation by 8�%. At 20 ?g?mL?1 of Dox which is 132% higher than T41抯 tor2 expression level at 0 ?g?mL?1 of Dox T41 showed an increased oxidative stress tolerance and a decrease in growth inhibition under iron replete by 62% but its conidiation significantly dropped by 47% compared to 0 ?g?mL?1 of Dox. T41 at 20 ?g?mL?1 of Dox had a strikingly increased virulence (1.2 day lower LT50) against S. exigua. The results reflect the crucial roles of TOR kinase in the vegetative growth conidiation pathogenicity and oxidative stress tolerance in B. bassiana. Since TOR upregulation is correlated with culture degeneration in multiple subcultures our data suggest that TOR signaling at relatively low levels plays an important role in growth and development but at moderate to high levels could contribute to some degenerated phenotypes e.g. those found in successive subcultures. ? 2023 by the authors.