RESUMO
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-Ontor2 'T41' 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 µg·mL-1 of Dox, T41 had 68% of the wild type's (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 0.1-1 µg·mL-1 induced tor2 upregulation in T41 by up to 91% compared to 0 µg·mL-1 of Dox, resulting in significant increases in radial growth by 8-10% and conidiation by 8-27%. At 20 µg·mL-1 of Dox, which is 132% higher than T41's tor2 expression level at 0 µg·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 µg·mL-1 of Dox. T41 at 20 µg·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.
RESUMO
The quality of Beauveria bassiana conidia directly affects the virulence against insects. In this study, continuous subculturing of B. bassiana on both rice grains and potato dextrose agar (PDA) resulted in 55 and 49 % conidial yield reduction after 12 passages and 68 and 60 % virulence reduction after 20 and 12 passages at four d post-inoculation, respectively. The passage through Tenebrio molitor and Spodoptera exigua restored the virulence of rice and PDA subcultures, respectively. To explore the molecular mechanisms underlying the conidial quality and the decline of virulence after multiple subculturing, we investigated the conidial proteomic changes. Successive subculturing markedly increased the protein levels in oxidative stress response, autophagy, amino acid homeostasis, and apoptosis, but decreased the protein levels in DNA repair, ribosome biogenesis, energy metabolism, and virulence. The nitro blue tetrazolium assay verified that the late subculture's colony and conidia had a higher oxidative stress level than the early subculture. A 2A-type protein phosphatase and a Pleckstrin homology domain protein Slm1, effector proteins of the target of rapamycin (TOR) complex 1 and 2, respectively, were dramatically increased in the late subculture. These results suggest that TOR signalling might be associated with ageing in B. bassiana late subculture, in turn affecting its physiological characteristics and virulence.
