Search

The mitogen-activated protein kinase (MAPK) kinase Ste7 has a conserved Ser/Thr loop (S/T-X 4(6) -S/T) that can activate the MAPK Fus3 or Kss1 for the regulation of pheromone response and filamentous growth in model yeast. Here, we show that not only the loop but also four C-terminal Ser/Thr residues are essential for Ste7 to function in the Fus3 cascade of Beauveria bassiana, a filamentous fungal insect pathogen. Mutagenesis of either looped S216/T220 or C-terminal S362 resulted in the same severe defects in conidial germination, hyphal growth, aerial conidiation, and submerged blastospore production as the ste7 deletion, followed by a complete loss of virulence and similarly increased cell sensitivities to osmotic salts, oxidants, heat shock and UV-B irradiation. Mutagenesis of three other Ser/Thr residues (S391, S440, and T485) also caused severe defects in most of the mentioned phenotypes. These defects correlated well with dramatically reduced transcript levels of some phenotype-related genes. These genes encode a transcription factor (CreA) essential for carbon/nitrogen assimilation, developmental activators (BrlA, AbaA, and WetA) and upstream transcription factor (FluG) required for conidiation, P-type N + /K + ATPases (Ena1-5) required for intracellular N + /K + homeostasis, and antioxidant enzymes involved in multiple stress responses. Our study unveils that the loop and four C-terminal Ser/Thr residues are all vital for the regulatory role of Ste7 in the growth, conidiation, virulence, and/or stress tolerance of B. bassiana and perhaps other filamentous fungi.

Ambrosia beetles harbor fungal symbionts that serve as food sources for larvae and adults. These beetles lay their eggs along tunnels in xylem sapwood, which is the substrate for fungal growth. Symbiotic fungi of the genus Raffaelea found in invasive and indigenous ambrosia beetles include the highly virulent plant pathogen Raffaelea lauricola affecting members of the Lauraceae family. R. lauricola is responsible for the deaths of > 500 million trees since 2005. Infection by as few as 100 spores can kill a healthy tree within months. Our data show that R. lauricola is cold-adapted with optimal growth between 16 and 26 °C, with little to no growth at temperatures ≥ 30 °C. The fungus is halophilic and shows a dramatic decrease in growth at pH ≥ 6.8. Fungicide resistance profiling revealed sensitivity of R. lauricola to prochloraz, dichlorofluanid, most conazoles, dithiocarbamates, and zineb (zinc fungicide), whereas the related species Raffaelea arxii showed more limited fungicide sensitivity. Entomopathogenic fungi potentially useful for beetle control were generally highly resistant to most fungicides tested. Coupling pH decreased the concentration for 95% inhibition of fungal growth (IC 95 ) of the most potent R. lauricola fungicides by 3-4-fold. Use of avocado bark plug insect bioassays revealed that commercially available Beauveria bassiana can be used as a biological control agent capable of effectively killing the beetle vectors. These data provide simple and practical recommendations to specifically target R. lauricola while having minimal effects on other symbiotic and entomopathogenic fungi, the latter of which can be used to manage the beetle vectors.

The mitogen-activated protein kinase (MAPK) Mpk3/MpkC resembles the MAPK Hog1 but does not necessarily function in some filamentous fungi. Here, we compared functions of Mpk3 and Hog1 in Beauveria bassiana, a filamentous fungal insect pathogen, by multi-phenotypic analyses of their single/double deletion mutants. Growth defects of Δmpk3 were moderate on all 14 minimal media with different carbon or nitrogen sources and less severe than those of Δhog1 on most media tested. The double deletion mutant suffered significantly more severe growth defects than those observed in Δmpk3 and Δhog1, suggesting overlapping and collaborative roles of Mpk3 and Hog1 in uptake of six carbon and four nitrogen sources during normal growth. Despite little impact on conidiation capacity, mpk3 deletion slowed down conidial germination as much as hog1 or double deletion. Conidial resistance to UV-B irradiation decreased less in Δmpk3 than in Δhog1 or in the double mutant. The fungal virulence was similarly attenuated for all deletion mutants against Galleria mellonella larvae through normal cuticle infection. Intriguingly, the Δmpk3 mutant displayed null response to high osmolarity and fludioxonil fungicide, to which both Δhog1 and double mutants were hypersensitive and highly resistant, respectively, but it was more sensitive to a 3-h heat shock at 40 °C than Δhog1 during normal incubation. Western blot hybridization demonstrated that Mpk3 could collaborate with Hog1 in response to heat shock rather than to the chemical stresses. Altogether, Mpk3 collaborates with Hog1 only in response to heat shock and functions in sustaining the pest control potential of B. bassiana.

Intracellular trehalose accumulation is relevant to fungal life and pathogenicity. Trehalose-6-phosphate synthase (TPS) is known to control the first step of trehalose synthesis, but functions of multiple TPS genes in some filamentous fungi are variable. Here, we examined the functions of two TPS genes (tpsA and tpsB) in Beauveria bassiana, a fungal insect pathogen widely applied in arthropod pest control. Intracellular TPS activity and trehalose content decreased by 71-75 and 72-80% in ΔtpsA, and 21-30 and 15-45% in ΔtpsB, respectively, and to undetectable levels in ΔtpsAΔtpsB, under normal and stressful conditions. The three mutants lost 33, 50, and 98% of conidiation capacity in standard cultures. Conidial quality indicated by viability, density, intracellular trehalose content, cell wall integrity, and hydrophobicity was more impaired in ΔtpsA than in ΔtpsB and mostly in ΔtpsAΔtpsB, which was also most sensitive to nutritional, chemical, and environmental stresses and least virulent to Galleria mellonella larvae. Almost all of phenotypic defects in ΔtpsAΔtpsB approached to the sums of those observed in ΔtpsA and ΔtpsB and were restored by targeted gene complementation. Altogether, TpsA and TpsB play complementary roles in sustaining trehalose synthesis, conidiation capacity, conidial quality, multiple stress tolerance, and virulence, highlighting a significance of both for the fungal adaptation to environment and host.

Protein O-mannosyltransferases (Pmts) belong to a highly conserved protein family responsible for the initiation of O-glycosylation of many proteins. Pmts contain one dolichyl-phosphate-mannose-protein mannosyltransferases (PMT) domain and three MIR motifs (mannosyltransferase, inositol triphosphate, and ryanodine receptor) that are essential for activity in yeast. We report that in the insect fungal pathogen, Beauveria bassiana, deletion of the C-terminal Pmt1 MIR-containing region (Pmt1∆ 311-902 ) does not alter O-mannosyltransferase activity, but does increase total cell wall protein O-mannosylation levels and results in phenotypic changes in fungal development and cell wall stability. B. bassiana mutants harboring the Pmt1 ∆ 311-902 mutation displayed a significant increase in conidiation with up-regulation of conidiation-associated genes and an increase in biomass accumulation as compared to the wild-type parent. However, decreased vegetative growth and blastospore production was noted, and Pmt1 ∆ 311-902 mutants were altered in cell wall composition and cell surface features. Insect bioassays revealed little effect on virulence for the Pmt1 ∆ 311-902 strain via cuticle infection or intrahemocoel injection assays, although differences in hyphal body differentiation in the host hemolymph and up-regulation of virulence-associated genes were noted. These data suggest novel roles for Pmt1 in negatively regulating conidiation and demonstrate that the C-terminal Pmt1 MIR-containing region is dispensable for enzymatic activity and organismal virulence.

Mdj1 is a member of the Hsp40 family containing a DnaJ or J domain. Here, we have examined the functions of an Mdj1 orthologue (56.68 kDa) in Beauveria bassiana, a filamentous fungal insect pathogen widely applied in biological control of insect pests. Deletion of mdj1 in B. bassiana resulted in significant growth defects on a variety of complex and minimal media. The Δmdj1 mutant exhibited not only a drastic reduction (92 %) in aerial conidiation during optimal cultivation but also a remarkable decrease (77 %) in submerged blastospore production. Compared to wild-type, the deletion mutant was significantly more sensitive to the stresses of cell wall perturbation, high osmolarity, oxidation, carbendazim fungicide, several metal ions, and acidic/alkaline pH during conidial germination and/or colony growth. In the mutant, conidial thermotolerance and UV-B resistance decreased by 61 and 25 %, respectively, and virulence to Galleria mellonella larvae was largely attenuated. Transcript levels of many phenotype-related genes were drastically suppressed in the absence of mdj1, accompanied with impaired cell walls and reduced intracellular anti-stress molecules, including superoxide dismutases, catalases, glycerol, trehalose, and mannitol. These data indicate that Mdj1 plays a vital role in normal fungal development and contributes significantly to the biological control potential of B. bassiana against insect pests.

The yeast form (blastospore) of the dimorphic insect-pathogenic fungus Beauveria bassiana can be rapidly produced using liquid fermentation methods but is generally unable to survive rapid dehydration processes or storage under non-refrigerated conditions. In this study, we evaluated the influence of two convective drying methods, various modified atmosphere packaging systems, and storage temperatures on the desiccation tolerance, storage stability, and virulence of blastospores of B. bassiana ESALQ 1432. All blastospore formulations were dried to <5 % water content equivalent to aw < 0.3. The viability of B. bassiana blastospores after air drying and spray drying was greater than 80 %. Vacuum-packaged blastospores remained viable longer when stored at 4 °C compared with 28 °C with virtually no loss in viability over 9 months regardless the drying method. When both oxygen and moisture scavengers were added to sealed packages of dried blastospore formulations stored at 28 °C, viability was significantly prolonged for both air- and spray-dried blastospores. The addition of ascorbic acid during spray drying did not improve desiccation tolerance but enhanced cell stability (∼twofold higher half-life) when stored at 28 °C. After storage for 4 months at 28 °C, air-dried blastospores produced a lower LC80 and resulted in higher mortality to whitefly nymphs (Bemisia tabaci) when compared with spray-dried blastospores. These studies identified key storage conditions (low aw and oxygen availability) that improved blastospore storage stability at 28 °C and will facilitate the commercial development of blastospores-based bioinsecticides.

The filamentous fungus Beauveria bassiana is an economically important pathogen of numerous arthropod pests and is able to grow in submerged culture as filaments (mycelia) or as budding yeast-like blastospores. In this study, we evaluated the effect of dissolved oxygen and high glucose concentrations on blastospore production by submerged cultures of two isolates of B. bassiana, ESALQ1432 and GHA. Results showed that maintaining adequate dissolved oxygen levels coupled with high glucose concentrations enhanced blastospore yields by both isolates. High glucose concentrations increased the osmotic pressure of the media and coincided with higher dissolved oxygen levels and increased production of significantly smaller blastospores compared with blastospores produced in media with lower concentrations of glucose. The desiccation tolerance of blastospores dried to less than 2.6 % moisture was not affected by the glucose concentration of the medium but was isolate dependent. Blastospores of isolate ESALQ1432 produced in media containing 140 g glucose L(-1) showed greater virulence toward whitefly nymphs (Bemisia tabaci) as compared with blastospores produced in media containing 40 g glucose L(-1). These results suggest a synergistic effect between glucose concentration and oxygen availability on changing morphology and enhancing the yield and efficacy of blastospores of B. bassiana, thereby facilitating the development of a cost-effective production method for this blastospore-based bioinsecticide.

The eukaryotic calcineurin (CN) pathway comprising catalytic A (CnA) and regulatory B subunits (CnB) is crucial for many biological processes but functionally unexplored in entomopathogenic fungi. Here, we characterise three CN subunits (CnA1, CnA2 and CnB) and a downstream CN-responsive zinc finger transcription factor (Crz1) in Beauveria bassiana. CN-mediated phosphatase activity decreased by 16-38 % in all deletion mutants compared with wild type. Growth and conidiation were most defective in ΔcnB, which showed a large proportion of abnormally branched germlings but were less defective in ΔcnA1 and ΔcnA2. Conidiation defects also occurred in Δcrz1, uniquely accompanied with slower germination. Compared with wild type, the four deletion mutants became, to varying degrees, more sensitive to Ca(2+), Mn(2+), Zn(2+), Mg(2+), two oxidants, three cell wall stressors, carbendazim, heat shock and ultraviolet (UV)-B irradiation. They were also less virulent to Spodoptera litura larvae. Only ΔcnB and Δcrz1 were less tolerant to high osmolarity. The altered phenotypes of the deletion mutants were associated with lower intracellular mannitol and trehalose levels, reduced overall activity of superoxide dismutases and catalases, altered cell wall composition and down-regulation of numerous phenotype-influencing genes. Additionally, the transcription of six cascaded genes in two stress-responsive mitogen-activated protein kinase (MAPK) pathways and the phosphorylation of hallmarking Hog1 and Slt2 were largely down-regulated in all the deletion mutants under osmotic and cell wall stresses, respectively. All the changes were restored by gene complementation. Taken together, three calcineurin subunits and Crz1 play vital, but variable, roles in B. bassiana responses to environmental stresses during development and host signals during infection.

SNF1/AMPK protein kinase plays important roles in fungal development and activation of catabolite-repressed genes. In this study, we characterized the role of the Beauveria bassiana SNF1 ortholog. The vegetative growth of the ΔBbSNF1 mutant was reduced by 16 to 50 % on diverse carbon/nitrogen sources. Transcriptional analysis revealed a collection of proteases and chitinases that were not induced when the mutant was grown on complex carbon/nitrogen sources. BbSNF1 also contributes to extracellular acidification. The ΔBbSNF1 mutant had enhanced production of lactic, pyruvic, and citric acid, but oxalic acid production was partially repressed. Transcriptional analysis showed that a set of genes involved in acid biosynthesis and secretion was changed in the disruption mutant, indicating that BbSNF1 controls the production of different organic acids with different mechanisms. Deletion of BbSNF1 resulted in a significant reduction in conidiation (57-75 %) and blastospore yield (80-95 %) in the mutant. Additionally, BbSNF1 regulates the morphology of blastospore-forming structures and the in vitro blastospore size. Insect bioassays revealed that the ΔBbSNF1 strain exhibited an approximately doubled median lethal time in topical bioassays, but the decreased virulence in intrahemocoel assays (~20 % change) was not as great as in the topical bioassays. These data suggest that BbSNF1 is important in penetration through the host cuticle. Moreover, a series of genes regulated by BbSNF1 and associated with blastospore formation were primarily involved in metabolism, cell cycle, and transportation. In conclusion, the SNF1/AMPK kinase contributes to the biocontrol potential of B. bassiana by mediating cellular differentiation and utilization of carbon/nitrogen sources.

A cell wall protein, CWP10, resolved from the conidial formic acid extract of a Metarhizium anisopliae isolate, was characterized as a new 9.9-kDa protein with a 32-aa signal peptide with a central hydrophobic region (ca. 10 residues) at its N-terminus. This protein was proven neither to be hydrophobic nor glycosylated and encoded by a 363-bp, single-copy gene with three introns. CWP10 was existent in the conidial extracts of seven of 18 tested M. anisopliae isolates and much more abundant (immunogold-labeled) on conidial walls than in cytoplasm. Integrating the gene into a CWP10-absent strain of Beauveria bassiana led to excellent expression of CWP10 in aerial conidia, increasing net conidial hydrophobicity by 10.8% or adhesion to hydrophobic Teflon by 1.3-fold. However, the expressed protein had no effect on conidial tolerance to thermal and ultraviolet stresses. This is the first report on a non-hydrophobic cell-wall protein enhancing conidial hydrophobicity and adhesion of the fungal species.