Functional Characterization Of Eight Small G Proteins In Ras Superfamily And Their Contributions To Biological Control Potential Of Beauveria Bassiana

Beauveria bassiana is a classic entomopathogenic fungus that has been widely applied for biological control of insect pests in agriculture and forestry. The fungal potential against insect pests is dependent on the yield, quality, adverse tolerance and virulence of aerial conidia as a main form of the fungal inoculum. The fungal beneficial traits are linked with many cellular events, such as conidial germination, vegetative growth, conidiation, stress responses and host infection. In eukaryotes, small G proteins in the Ras superfamilty share two molecular switches for GTP or GDP binding and are known to regulate cellular events including cell growth, differentiation and apoptosis, gene expression, and construction of cellular framework. However, most of small G proteins have not been explored in filamentous fungal entomopathogens. This study sought to elucidate the functions of eight small G proteins (Miro, novel Ras3 and six Rho GTPases) in B. bassiana by constructing and analyzing single-gene deletion/complementary mutants and point-mutated strains. The main results are summarized below.Miro GTPase controls mitochondrial distribution and mobilitythat affect stress tolerance and virulence in B. bassiana. Miro homologues are small, mitochondria-targeting Rho GTPases in the Ras superfamily across organisms but are generally unexplored in filamentous fungi. A Miro orthologue (bMiro) identified in B. bassiana was proven to anchor on mitochondrial outer membrane in a manner depending completely upon a C-terminal transmembrane domain. Deletion of bmiro resulted in aggregated distribution, reduced mass and suppressed mobility of mitochondria in hyphal cells but little change in mitochondrial morphology, accompanied with a remarkable decrease in intracellular ATP content. The deletion mutant became 42%,37%,19% and 10% more tolerant to Ca2+, Mn2+, Zn2+ and Mg22+ than wild-type, respectively, during cultivation in a minimal medium under normal conditions. The deletion mutant also showed mild defects in conidial germination, vegetative growth, thermotolerance, UV-B resistance and virulence despite null response to oxidative and osmotic stresses. All these phenotypic changes were restored by targeted gene complementation. Taken together, bMiro plays a critical role in regulating mitochondrial distribution and movement required for the release of energy from ATP for the transport of metal ions and hence contributes significantly to the biological control potential of B. bassiana against insect pests.A novel Ras GTPase (Ras3) regulates conidiation, multiple stress tolerance and virulence by acting upstream of Hogl signaling pathway in B.bassiana. Two Ras ATPases (Ras1 and Ras2) are well known to regulate antagonistically or cooperatively various cellular events in many fungi. In this study, a novel Ras homologue (Ras3) was found in B. bassiana. Ras3 was revealed to possess five domains and two GTP/GDP switches typical for the Ras family and was proven to localize on plasma membrane despite the position change of a membrane-targeting cysteine in the C-terminal motif CAAX. Deletion of ras3 altered temporal transcription pattern of rasl instead of ras2. Compared with wild-type, Aras3 grew significantly faster in a rich medium but slower in some minimal media, and produced far fewer conidia with impaired quality, which was represented by slower germination, attenuated virulence, reduced thermotolerance and decreased UV-B resistance. Moreover, Aras3 was much more sensitive to the oxidative stress of menadione than of H2O2 and to the stress of high osmolarity than of cell wall perturbation during growth. The high sensitivity of Aras3 to menadione was concurrent with reductions in both gene transcripts and total activity of superoxide dismutases. Intriguingly, the high osmosensitivity was concurrent with not only reduced transcripts of a critical transcription factor (Msn2) and most signaling proteins in high-osmolarity-glycerol (HOG) pathway of ArasJbut nearly undetectable phosphorylation signal of Hogl hallmarking the pathway. All the changes were restored by targeted ras3 complementation. Taken together, Ras3 is involved in the Hog1 pathway required for osmoregulation and hence can positively regulate conidiation, germination, multiple stress tolerance and virulence linked to the biological control potential of the filamentous insect pathogen.Functional characterization of six small Rho GTPases in B. bassiana. The Rho family comprises six small G proteins that belong to the Ras superfamily. Of those, four coding genes (rho2, rho3, rho4 and cdc42) were individually deleted from B. bassiana and complemented in the corresponding deletion mutants while the the deletion of either rholor racA failed in many attempts perhaps due to its lethality and hence was alternated by dominant active (DA) and dominant negative (DN) point mutation, yielding the mutant strains DArhol, DNrhol, DAracA and DNracA. Among the mutants, only DNrhol, Arho4 and Acdc42 exhibited consistently severity-varying growth defects on a rich medium or several minimal media with altered carbon/nitrogen sorces. Aerial conidiation defect was very severe in DNrhol and mild in Acdc42. Conidial germination was largely delayed in UNrho1, followed by formation of thicker hypae with increased septa and shorter cells. In Arho4, hyphae were broken into septum-free, tubular cells with each forming one or two terminal buds which were enlarged into oval-shaped, blastospore-like cells, suggesting an inhibition of its polar growth. Morphological change was inconspicuous in Acdc42 hyphae except for abnormal elongation of tip cell. Growth, condiation and germinatin defects were absent in the rest mutants. In stress assays with metal ions, the mutants Arho2, Arho4, DArhol, DNrhol and DNracA were more sensitive to only Cu2+ than wild-type whereas all deletion and point-mutated mutants showed greater tolerance or null response to Mn2+, Ca2+, Fe2+, Mg24" and Zn2+. Only was Arho4 significantly more sensitive to oxidants H2O2 and menadione but all the mutans were more tolerant or not responsive to cell wall perturbation and high osmolarity. Moreover, all the mutants except Arho2 and Arho3 were more sensitive to 40℃ heat shock during normal growth, and their conidia except those produced by Arho4 were significantly less tolerant to a UV-B irradiation at 0.2 J/cm2. In standardized bioassays, virulence was attenuated greatly in Arho4 and mildly in Arho2 but was not affected in the rest mutants. All phenotypica changes were restored by targed gene complementation. All together, the Rho proteins contribute differentially to the biological control potential of B. bassiana with the main contribion of Rho4 to virulence, antioxidation and thermotolerance, of Rho1 and Cdc42 to conidiation, germination, thermotolerance and UV-B resistance, of Rho2 and Rho3 to germination and UV-B resistance, and of RacA to thermotolerance and UV-B resistance.