Sheng-Hua Ying

Genome Sequencing and Comparative Transcriptomics of the Model Entomopathogenic Fungi Metarhizium anisopliae and M. acridum

Metarhizium spp. are being used as environmentally friendly alternatives to chemical insecticides, as model systems for studying insect-fungus interactions, and as a resource of genes for biotechnology. We present a comparative analysis of the genome sequences of the broad-spectrum insect pathogen Metarhizium anisopliae and the acridid-specific M. acridum. Whole-genome analyses indicate that the genome structures of these two species are highly syntenic and suggest that the genus Metarhizium evolved from plant endophytes or pathogens. Both M. anisopliae and M. acridum have a strikingly larger proportion of genes encoding secreted proteins than other fungi, while ∼30% of these have no functionally characterized homologs, suggesting hitherto unsuspected interactions between fungal pathogens and insects. The analysis of transposase genes provided evidence of repeat-induced point mutations occurring in M. acridum but not in M. anisopliae. With the help of pathogen-host interaction gene database, ∼16% of Metarhizium genes were identified that are similar to experimentally verified genes involved in pathogenicity in other fungi, particularly plant pathogens. However, relative to M. acridum, M. anisopliae has evolved with many expanded gene families of proteases, chitinases, cytochrome P450s, polyketide synthases, and nonribosomal peptide synthetases for cuticle-degradation, detoxification, and toxin biosynthesis that may facilitate its ability to adapt to heterogenous environments. Transcriptional analysis of both fungi during early infection processes provided further insights into the genes and pathways involved in infectivity and specificity. Of particular note, M. acridum transcribed distinct G-protein coupled receptors on cuticles from locusts (the natural hosts) and cockroaches, whereas M. anisopliae transcribed the same receptor on both hosts. This study will facilitate the identification of virulence genes and the development of improved biocontrol strains with customized properties.

Genomic perspectives on the evolution of fungal entomopathogenicity in Beauveria bassiana

The ascomycete fungus Beauveria bassiana is a pathogen of hundreds of insect species and is commercially produced as an environmentally friendly mycoinsecticide. We sequenced the genome of B. bassiana and a phylogenomic analysis confirmed that ascomycete entomopathogenicity is polyphyletic, but also revealed convergent evolution to insect pathogenicity. We also found many species-specific virulence genes and gene family expansions and contractions that correlate with host ranges and pathogenic strategies. These include B. bassiana having many more bacterial-like toxins (suggesting an unsuspected potential for oral toxicity) and effector-type proteins. The genome also revealed that B. bassiana resembles the closely related Cordyceps militaris in being heterothallic, although its sexual stage is rarely observed. A high throughput RNA-seq transcriptomic analysis revealed that B. bassiana could sense and adapt to different environmental niches by activating well-defined gene sets. The information from this study will facilitate further development of B. bassiana as a cost-effective mycoinsecticide.

Additive contributions of two manganese-cored superoxide dismutases (MnSODs) to antioxidation, UV tolerance and virulence of Beauveria bassiana.

The biocontrol potential of entomopathogenic fungi against arthropod pests depends on not only their virulence to target pests but tolerance to outdoor high temperature and solar UV irradiation. Two Beauveria bassiana superoxide dismutases (SODs), BbSod2 and BbSod3, were characterized as cytosolic and mitochondrial manganese-cored isoenzymes (MnSODs) dominating the total SOD activity of the fungal entomopathogen under normal growth conditions. To probe their effects on the biocontrol potential of B. bassiana, ΔBbSod2, ΔBbSod3, and three hairpin RNA-interfered (RNAi) mutants with the transcripts of both BbSod2 and BbSod3 being suppressed by 91–97% were constructed and assayed for various phenotypic parameters in conjunction with ΔBbSod2/BbSod2, ΔBbSod3/BbSod3 and wild-type (control strains). In normal cultures, the knockout and RNAi mutants showed significant phenotypic alterations, including delayed sporulation, reduced conidial yields, and impaired conidial quality, but little change in colony morphology. Their mycelia or conidia became much more sensitive to menadione or H2O2-induced oxidative stress but had little change in sensitivity to the hyperosmolarity of NaCl and the high temperature of 45°C. Accompanied with the decreased antioxidative capability, conidial tolerances to UV-A and UV-B irradiations were reduced by 16.8% and 45.4% for ΔBbSod2, 18.7% and 44.7% for ΔBbSod3, and ∼33.7% and ∼63.8% for the RNAi mutants, respectively. Their median lethal times (LT50s) against Myzus persicae apterae, which were topically inoculated under a standardized spray, were delayed by 18.8%, 14.5% and 37.1%, respectively. Remarkably, the effects of cytosolic BbSod2 and mitochondrial BbSod3 on the phenotypic parameters important for the fungal bioncontrol potential were additive, well in accordance with the decreased SOD activities and the increased superoxide levels in the knockout and RNAi mutants. Our findings highlight for the first time that the two MnSODs co-contribute to the biocontrol potential of B. bassiana by mediating cellular antioxidative response.

Novel blastospore-based transformation system for integration of phosphinothricin resistance and green fluorescence protein genes into Beauveria bassiana

A novel system was developed for efficient transformation of the fungal biocontrol agent Beauveriabassiana. Competent blastospores were prepared and stored in LiAc- and glycerol-inclusive suspension at −76 °C for sequential use in transformation. The system was successfully applied to integrating phosphinothricin resistance gene bar and enhanced green fluorescence protein gene egfp into B. bassiana via blastospore absorption of a plasmid vectoring bar and egfp. A frequency of 24 transformants per microgram of DNA was achieved. The blastospore-based transformation system has proven to be very convenient and would be highly potential for use in genetic manipulation of B. bassiana and other filamentous species.

Differentiated functions of Ras1 and Ras2 proteins in regulating the germination, growth, conidiation, multi-stress tolerance and virulence of Beauveria bassiana

Ras1 and Ras2 are two distinct Ras GTPases in Beauveria bassiana, an entomopathogenic fungus whose biocontrol potential against insect pests depends largely on virulence and multi-stress tolerance. The functions of both proteins were characterized for the first time by constructing dominant-active (GTP-bound) Ras1(G19V) and dominant-negative (GDP-bound) Ras1(D126A) and integrating them and normal Ras1 into wild type and ΔRas2 for a series of phenotypic and transcriptional analyses. The resultant mutants showed gradient changes of multiple phenotypes but little difference in conidial thermotolerance. Expression of Ras1(D126A) caused vigorous hyphal growth, severely defective conidiation, and increased tolerances to oxidation, cell wall disturbance, fungicide and UV-A/UV-B irradiations, but affected slightly germination, osmosensitivity and virulence. These phenotypes were antagonistically altered by mRas1(G19V) expressed in either wild type or ΔRas2, which was severely defective in conidial germination and hyphal growth and displayed intermediate changes in other mentioned phenotypes between paired mutants expressing Ras1(G19V) or Ras1(D126A) in wild type and ΔRas2. Their growth, UV tolerance or virulence was significantly correlated with cellular response to oxidation or cell wall disturbance. Transcriptional changes of 35 downstream effector genes involved in conidiation and multi-stress responses also related to most of the phenotypic changes among the mutants. Our findings highlight that Ras1 and Ras2 regulate differentially or antagonistically the germination, growth, conidiation, multi-stress tolerance and virulence of B. bassiana, thereby exerting profound effects on the fungal biocontrol potential.

A carbon responsive G‐protein coupled receptor modulates broad developmental and genetic networks in the entomopathogenic fungus, Beauveria bassiana

In fungi, G-protein coupled receptors (GPCRs) link ligand/nutrient sensing to growth, mating, developmental/life-stage activation and pathogenesis. A GPCR was characterized from the entomopathogenic fungus, Beauveria bassiana (BbGPCR3), which links nutrient sensing to stress response and development. ΔBbGPCR3 mutants grew slower on various carbohydrates and displayed increased sensitivity to osmotic, oxidative and cell wall stresses. Gene expression profiling revealed a set of heat-shock and antioxidant factors that failed to be induced under oxidative stress and aberrant regulation of compatible solute-forming enzymes and cell wall biosynthesis/remodelling proteins in ΔBbGPCR3 after osmotic stress. Glucose-specific developmental defects included reduced (> 90%) conidiation and reduced dimorphic transition to the production of yeast-like blastospores, effects suppressed in media containing trehalose or glycerol, but not by addition of cyclic AMP. Insect bioassays revealed reduced virulence in topical assays but no effect in intrahaemoceol injection assays, indicating that BbGPCR3 was important in sensing signals during the initial interaction with the host but dispensable for post-penetration events. Comparative gene expression profiling of ΔBbGPCR3 mutants grown in glucose media compared with wild-type/glucose and ΔBbGPCR3/trehalose grown cells revealed sets of genes misregulated and recovered, respectively. These data link BbGPCR3 to broad developmental and genetic networks that include the major MAP kinase pathways.

Distinct contributions of one Fe- and two Cu/Zn-cofactored superoxide dismutases to antioxidation, UV tolerance and virulence of Beauveria bassiana.

Beauveria bassiana, a filamentous entomopathogen, has five distinct superoxide dismutases (SODs), including cytosolic and mitochondrial MnSODs (Sod2/3) which have proved contributing primarily to intracelluar SOD activity and additively to antioxidation and virulence. Here we characterized cytosolic Cu/ZnSOD (Sod1), mitochondrial FeSOD (Sod4) and cell wall-anchored Cu/ZnSOD (Sod5). The latter two are unexplored despite existence in many filamentous fungi, and their subcellular localization was well confirmed with specifically stained cells expressing Sod4::eGFP or Sod5::eGFP fusion. Total SOD activity decreased by ∼15% in Δsod1 but increased by 11-20% in three sod4 knockdown mutants (Δsod4 was lethal) when co-cultivated with menadone and H2O2. Surprisingly, total catalase activity decreased much more in the sod4 mutants (69-75%) than in Δsod1 (27-33%) under normal and oxidative conditions. However, Δsod5 showed little change in either SOD or catalase activity. Transcript levels of SOD partners and five catalases also changed more dramatically in the sod4 mutants than in Δsod1 and Δsod5. As a consequence of global effect, intracellular ROS levels induced by both oxidants were higher in Δsod1 than in the sod4 mutants and Δsod5. All the mutants were differentially more sensitive to the two oxidants and UV-A/UV-B irradiations and less virulent to Galleria mellonella larvae but not responsive to high osmolarity, cell wall stress and high temperature. Taken together with previously characterized Sod2 and Sod3, our results provide full insight into the SOD family, unveiling the interactions of each SOD with other partners and catalases in the antioxidant reaction associated with the fungal biocontrol potential.

Three Mitogen-Activated Protein Kinases Required for Cell Wall Integrity Contribute Greatly to Biocontrol Potential of a Fungal Entomopathogen

Bck1, Mkk1 and Slt2 are three mitogen-activated protein (MAP) kinases constituting cell wall integrity (CWI) pathway that may control multi-stress responses via crosstalk with high-osmolarity glycerol (HOG) pathway in budding yeast. In this study, Bck1, Mkk1 and Slt2 orthologues in Beauveria bassiana were confirmed as the three-module cascade essential for CWI because cell wall impairment occurred in the hyphae and conidia of Δbck1, Δmkk1 and Δslt2 examined in multiple experiments. Strikingly, all the deletion mutants became more sensitive to hyperosmotic NaCl and sorbitol with the Western blot of Hog1 phosphorylation being weakened in Δbck1 and absent in Δmkk1 and Δslt2. Apart from crossing responses to cell wall perturbation and high osmolarity, three deletion mutants exhibited faster growth and conidiation on nutrition-rich medium, much less virulence to Galleria mellonella larvae, and higher sensitivity to nutritional, fungicidal, thermal and UV-B irradiative stresses, accompanied with less accumulation of intracellular mannitol and trehalose. Moreover, Δmkk1 and Δslt2 were equally more sensitive to all the stresses of different types except wet-heat stress than wild type and more or less different from Δbck1 in sensitivity to most of the stresses despite their null responses to two oxidants. All the changes in three deletion mutants were restored by each targeted gene complementation. Taken together, the CWI-required Bck1, Mkk1 and Slt2 are all positive, but differential, regulators of multi-stress tolerance and virulence perhaps due to interplay with the HOG pathway essential for osmoregulation, thereby contributing greatly to the biocontrol potential of the fungal entomopathogen.

The transcriptional co‐activator multiprotein bridging factor 1 from the fungal insect pathogen, Beauveria bassiana, mediates regulation of hyphal morphogenesis, stress tolerance and virulence

Multiprotein bridging factors (MBFs) are evolutionarily highly conserved cofactors that link TATA-binding protein and the associated basal transcription machinery to transcription factors. The filamentous fungus, Beauveria bassiana, has a multipotential lifestyle capable of growing as a saprophyte, plant endophyte and insect pathogen. Deletion of the single B. bassiana MBF homologue (BbMBF1) affected fungal growth and hyphal morphogenesis, stress response and virulence. Compared with wild type, the ΔBbMBF1 strain displayed increased sensitivity to UV-irradiation and to oxidative, osmotic and heat stress, and decreased virulence in both topical and intrahaemocoel injection bioassays using the greater wax moth, Galleria mellonella larvae. Although only minor radial growth effects were seen for the ΔBbMBF1 strain, aberrant hyphal morphogenesis was observed, which could be rescued by growth in rich broth media. Transcriptional analysis during stress response showed altered gene expression in ΔBbMBF1 during growth under osmotic, oxidative and thermal stress conditions. Genome-wide expression analyses during growth under unstressed and thermal stress conditions revealed global gene expression changes and a set of putative targets for MBF1 mediated gene expression control. Our data indicate that BbMBF1 acts as a key regulatory cofactor controlling stress responses and virulence and that MBF1 dependent and independent pathways control proper hyphal morphogenesis.

P‐type calcium ATPase functions as a core regulator of Beauveria bassiana growth, conidiation and responses to multiple stressful stimuli through cross‐talk with signalling networks

P-type Ca(2+) -ATPase (Pmr1) is a core element in calcium-calcineurin pathway and evidence for its cross-talk with other signalling pathways in filamentous fungi is of scarcity. Here, we characterized the striking functions of a Golgi Ca(2+) -ATPase (Bbpmr1) in Beauveria bassiana (fungal entomopathogen) by multi-phenotypic and transcriptional analyses under normal and stressful conditions. Bbpmr1 inactivation caused severe defects in nutritional uptake, growth, conidiation and germination under normal conditions, drastic reductions in cell tolerances to oxidative, hyperosmotic, cell wall disturbing and fungicidal stresses and toxic metal ions during colony growth and/or conidial germination, and half loss of the fungal biocontrol potential represented by conidial virulence, thermotolerance and UV-B resistance. Accompanied with the multi-phenotypic defects, four important genes associated with asexual development were repressed by ≥ 75% in ΔBbpmr1 versus wild type, and all or most of stress-responsive genes encoding 14 cascaded proteins in MAPK pathways, two Ras GTPases, two protein kinases, Ssk1-type response regulator, TOR signalling protein, and many downstream enzymes and proteins were greatly downregulated in ΔBbpmr1 under the chemical stresses. Conclusively, Bbpmr1 regulates positively fundamental aspects on B. bassiana biology and environmental adaptation through wide cross-talk with cellular signalling networks including MAPK cascades and those upstream or independent of the cascades.