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

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.

Metabolomic Analysis Demonstrates the Impacts of Polyketide Synthases PKS14 and PKS15 on the Production of Beauvericins, Bassianolide, Enniatin A, and Ferricrocin in Entomopathogen Beauveria bassiana.

Beauveria bassiana is a globally distributed entomopathogenic fungus that produces various secondary metabolites to support its pathogenesis in insects. Two polyketide synthase genes, pks14 and pks15, are highly conserved in entomopathogenic fungi and are important for insect virulence. However, understanding of their mechanisms in insect pathogenicity is still limited. Here, we overexpressed these two genes in B. bassiana and compared the metabolite profiles of pks14 and pks15 overexpression strains to those of their respective knockout strains in culture and in vivo using tandem liquid chromatography-mass spectrometry (LC-MS/MS) with Global Natural Products Social Molecular Networking (GNPS). The pks14 and pks15 clusters exhibited crosstalk with biosynthetic clusters encoding insect-virulent metabolites, including beauvericins, bassianolide, enniatin A, and the intracellular siderophore ferricrocin under certain conditions. These secondary metabolites were upregulated in the pks14-overexpressing strain in culture and the pks15-overexpressing strain in vivo. These data suggest that pks14 and pks15, their proteins or their cluster components might be directly or indirectly associated with key pathways in insect pathogenesis of B. bassiana, particularly those related to secondary metabolism. Information about interactions between the polyketide clusters and other biosynthetic clusters improves scientific understanding about crosstalk among biosynthetic pathways and mechanisms of pathogenesis.

Microbial polyketides and their roles in insect virulence: from genomics to biological functions.

Covering: May 1966 up to January 2022Entomopathogenic microorganisms have potential for biological control of insect pests. Their main secondary metabolites include polyketides, nonribosomal peptides, and polyketide-nonribosomal peptide (PK-NRP) hybrids. Among these secondary metabolites, polyketides have mainly been studied for structural identification, pathway engineering, and for their contributions to medicine. However, little is known about the function of polyketides in insect virulence. This review focuses on the role of bacterial and fungal polyketides, as well as PK-NRP hybrids in insect infection and killing. We also discuss gene distribution and evolutional relationships among different microbial species. Further, the role of microbial polyketides and the hybrids in modulating insect-microbial symbiosis is also explored. Understanding the mechanisms of polyketides in insect pathogenesis, how compounds moderate the host-fungus interaction, and the distribution of PKS genes across different fungi and bacteria will facilitate the discovery and development of novel polyketide-derived bio-insecticides.

The polyketide synthase PKS15 has a crucial role in cell wall formation in Beauveria bassiana.

Entomopathogenic fungi utilize specific secondary metabolites to defend against insect immunity, thereby enabling colonization of their specific hosts. We are particularly interested in the polyketide synthesis gene pks15, which is involved in metabolite production, and its role in fungal virulence. Targeted disruption of pks15 followed by genetic complementation with a functional copy of the gene would allow for functional characterization of this secondary metabolite biosynthesis gene. Using a Beauveria bassiana ∆pks15 mutant previously disrupted by a bialophos-resistance (bar) cassette, we report here an in-cis complementation at bar cassette using CRISPR/Cas9 gene editing. A bar-specific short guide RNA was used to target and cause a double-strand break in bar, and a donor DNA carrying a wild-type copy of pks15 was co-transformed with the guide RNA. Isolate G6 of ∆pks15 complemented with pks15 was obtained and verified by PCR, Southern analyses and DNA sequencing. Compared to ∆pks15 which showed a marked reduction in sporulation and insect virulence, the complementation in G6 restored with insect virulence, sporulation and conidial germination to wild-type levels. Atomic force and scanning electron microscopy revealed that G6 and wild-type conidial wall surfaces possessed the characteristic rodlet bundles and rough surface while ∆pks15 walls lacked the bundles and were relatively smoother. Conidia of ∆pks15 were larger and more elongated than that of G6 and the wild type, indicating changes in their cell wall organization. Our data indicate that PKS15 and its metabolite are likely not only important for fungal virulence and asexual reproduction, but also cell wall formation.

The reducing clade IIb polyketide synthase PKS14 acts as a virulence determinant of the entomopathogenic fungus Beauveria bassiana.

The reducing clade IIb polyketide synthase gene, pks14, is preserved throughout the evolution of entomopathogenic fungi. We examined the functions of pks14 in Beauveria bassiana using targeted gene disruption, and pks14 disruption was verified by Southern blot and PCR analyses. The radial growth, cell dry weight and conidial germination of Δpks14 were comparable to that of the wild type. Our sequence and gene expression analyses of the pks14 biosynthetic cluster demonstrated: (i) cotranscription and constitutive expression of nearly all the genes of the aforementioned cluster including the C2H2 zinc finger transcription regulator gene, but not pks14 and the cytochrome P450 gene; (ii) expression of the pks14 gene in the insect-containing culture condition only; and (iii) a KAR9-like gene in direct proximity with pks14 is the only gene showing co-regulation. The Δpks14-infected Spodoptera exigua larvae survived significantly longer than those infected by the wild type, indicating a marked reduction in the virulence of Δpks14 against the insect. LT50 of Δpks14 was increased by 1.55 days. Hyphal body formation was decreased in the hemolymph of insects infected by Δpks14 as compared with those inoculated by the wild type. Our results suggest that PKS14-catalyzed polyketide enhances virulence and pathogenicity of B. bassiana on insects.

Targeted disruption of the polyketide synthase gene pks15 affects virulence against insects and phagocytic survival in the fungus Beauveria bassiana.

The reducing clade III polyketide synthase genes, including pks15, are highly conserved among entomopathogenic fungi. To examine the function of pks15, we used targeted disruption to investigate the impact of Beauveria bassiana pks15 on insect pathogenesis. Southern analysis verified that the Δpks15 mutant was disrupted by a single integration of the transformation cassette at the pks15 locus. The Δpks15 mutant had a slight reduction in radial growth, and it produced fewer spores. Our insect bioassays indicated the Δpks15 mutant to be significantly reduced in virulence against beet armyworms compared to wild type (WT), which could be partially accounted for by its markedly decreased ability to survive phagocytosis. Total haemocyte count decreased sharply by 50-fold from days 1-3 post-inoculation in insects infected with WT, compared to a 5-fold decrease in the Δpks15 mutant. The mutant also produced fewer hemolymph hyphal bodies than WT by 3-fold. In co-culture studies with amoebae that have phagocytic ability similar to that of insect haemocytes, at 48 h the mortality rate of amoebae engulfing Δpks15 decreased by 72 %, and Δpks15 CFU decreased by 83 % compared to co-culture with WT. Thus, the Δpks15 mutant had a reduced ability to cope with phagocytosis and highly reduced virulence in an insect host. These data elucidate a mechanism of insect pathogenesis associated with polyketide biosynthesis.

Expression of mosquito-larvicidal toxin genes under the control of a native promoter in Enterobacter amnigenus An11.

Enterobacter amnigenus An11, that can colonize the gut of mosquito larva, is an alternative toxin-producing host to be used as a mosquito control since it is able to float in the feeding zone of mosquito larvae. To produce mosquito-larvicidal toxins in this bacterium, a native promoter has been identified from its genomic DNA. The promoter exhibited consensus sequences for -35 and -10 regions of bacterial promoters and constitutively drove the expression of gfp. This promoter was inserted into recombinant plasmids upstream of promoter-free cyt2Aa2 from Bacillus thuringiensis and mtx2 from Bacillus sphaericus. Results demonstrated that Cyt2Aa2 and Mtx2 are constitutively produced without induction. The recombinant E. amnigenus showed toxicity against mosquito larvae, demonstrating a potential to be applied in a mosquito control program.