Gene Deletion and Elucidation of the dtx Biosynthesis Pathway.

To verify the functions of dtxS1 and its clustered genes, serial genes were deleted by homologous replacements. Because some dtxs have the same molecular masses (2, 4), we performed a combination of liquid chromatography (LC)-MS (Fig. S2 A–G), NMR (Table S2), and HPLC analysis to profile the dtxs produced by the wild-type (WT) and mutant strains. The data indicated that during growth in CD medium the WT strain produces eight dtxs, including dtx E-diol (ED), E, C, D, A, B₂, desmethyl-B, and B (Fig. 3A). Consistent with previous studies (13, 22), dtx E, A, and B were produced in the largest amounts (Fig. 3A and Table S3). As predicted, the dtxS1-null mutants were unable to produce dtxs either in the CD medium (Fig. 3B) or in silkworm larvae (Fig. S2H). Deletions of dtxS1 upstream genes (Fig. 2A) did not affect dtx production by the null mutants compared with the WT strain (Fig. S3), indicating that these genes are not involved in production and modification of dtxs. However, deletions of genes downstream of dtxS1 led to partial or complete loss of dtx production (Fig. 3 C–E).

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Fig. 3.

HPLC profiling of dtx production by the WT and gene deletion mutants of M. robertsii. When grown in CD broth, the WT strain could produce at least eight dtx derivatives (A). After the deletion of the NRPS gene dtxS1, the resulting mutant failed to synthesize the dtxs (B). The null mutant of dtxS2 could only produce dtx B, B₂, and desmethyl-B (C). The addition of dtx B to the cultures of ΔdtxS1 enabled the mutant to produce dtx E and A (B′) but not the dtx B₂ (A′). Dtx B did not allow ΔdtxS1ΔdtxS2 to produce dtx E and A (C′). The clustered dtxS3 gene functions in the conversion of α-ketoisocaproic acid to hydroxyisocaproic acid (HIC), the first substrate for dtx assembly. A mutant deleted in the dtxS3 gene did not produce dtxs (D). Conversely, the addition of HIC to ΔdtxS3 culture restored its ability to produce dtxs (D′). The dtxS4 gene was verified to involve in the decarboxylation of aspartic acid into β-Ala (E and E′), i.e., the null mutant produced dtxs only when β-Ala was added in medium.

The null mutants of MAA_10044 gene (designated as dtxS2), putatively encoding a cytochrome P450 (CYP) subfamily 65AF enzyme, could only produce dtx B, B₂, and desmethyl-B (Fig. 3C and Table S2). DtxS2 expressed using either a bacterium or yeast expression system lacked activity. To verify DtxS2 function, we generated a double mutant by deleting both dtxS1 and dtxS2 genes. When ΔdtxS1 (dtxS2 functional) and ΔdtxS1ΔdtxS2 were grown in medium supplemented with dtx B, ΔdtxS1 could convert dtx B into dtx E, C, D, and A, whereas ΔdtxS1ΔdtxS2 could not (Fig. 3 B′ and C′). Dtx B₂ was not produced by either ΔdtxS1 or ΔdtxS1ΔdtxS2 in dtx B feeding assays (Fig. 3A′). However, both mutants produced desmethyl-B. Thus, DtxS1 synthesizes dtx B and B₂, whereas DtxS2 is required to convert dtx B into other dtx derivatives (Fig. 2B).

The locus MAA_10045 (designated as dtxS3) putatively encodes an aldo-keto reductase and MAA_10046 (named as dtxS4) is predicted to be an aspartate decarboxylase. The null mutants of these two genes failed to produce any dtxs (Fig. 3 D and E). We hypothesized that DtxS3 converts α-ketoisocaproic acid from deaminated leucine into α-hydroxyisocaproic acid (HIC), the first substrate for dtx assembly (Fig. 2B). This was confirmed by adding HIC into the ΔdtxS3 culture to restore dtx production (Fig. 3D′). Decarboxylation of aspartic acid into β-Ala by DtxS4 was confirmed by adding β-Ala to the mutant culture because this rescued dtx biosynthesis by ΔdtxS4 (Fig. 3E′). Our previous study has shown that dtx E is not stable (24), and the degradation of dtx E to E-diol is a nonenzymatic process (Fig. 2B).

The detection of both dtx B and B₂ in the ΔdtxS2 cultures confirmed that the A3 domain could interchangeably incorporate either Ile (highly biased) for dtx B or Val for B₂ biosynthesis. Phylogenetic analysis of DtxS1 A domains demonstrated that they clustered according to substrate structures, e.g., A3 (for Val/Ile) and A4 (Val) grouped together (Fig. S1B). The C domains of NRPSs catalyze peptide bond formation (25). The topology of DtxS1 C domains is different from the A domain tree (Fig. S1C).

Effects of dtxs on Fungal Virulence.

Injection of culture filtrates of the WT, ΔdtxS1, and ΔdtxS2 confirmed the insecticidal effects of dtxs (Fig. S4A). To verify the contribution of dtx to fungal virulence, insect bioassays were conducted by injecting locusts and silkworms with spore suspensions of the WT and mutants (Fig. S4 B and C). Median lethal times (LT₅₀) for locusts were estimated at 105.4 ± 1.4, 106.5 ± 1.7, and 112.4 ± 3.2 h for the WT, ΔdtxS1, and ΔdtxS2, respectively. Log-rank tests revealed that the difference between the WT and ΔdtxS1 was not significant (χ² = 0.26; P = 0.61), whereas the differences between the WT and ΔdtxS2 (χ² = 5.91; P = 0.015) and between ΔdtxS1 and ΔdtxS2 (χ² = 5.91, P = 0.043) were significant. A similar trend was observed in bioassays using silkworms as LT₅₀ values were 79.3 ± 0.5, 81.0 ± 0.6 and 88.0 ± 1.7 h for the WT, ΔdtxS1, and ΔdtxS2, respectively. However, differences were significant between the WT and ΔdtxS1 (χ² = 4.92; P = 0.027), as well as between the WT and ΔdtxS2 (χ² = 17.12; P = 0) and ΔdtxS1 and ΔdtxS2 (χ² = 15.13; P = 0). Thus, the effects of dtx on fungal virulence varied with insect species but, intriguingly, the differences were significantly greater between the WT and ΔdtxS2 than between the WT and ΔdtxS1, i.e., ΔdtxS2 (with the ability to produce dtx B and B₂) was less virulent than ΔdtxS1.

Microscopic observations indicated that the dtxs could counteract insect host immune defenses (Fig. 4). Following injection of silkworm larvae, Metarhizium spores were rapidly encapsulated by hemocytes. The germinated WT spores in silkworm hemocoel quickly escaped (16–20 h postinjection) from hemocyte encapsulation (Fig. 4A), but spores from both the ΔdtxS1 and ΔdtxS2 mutants were unable to escape (Fig. 4 B and B′). Thus, the WT cells propagated more quickly than the mutants (Fig. 4 C and D). Forty-eight hours postinjection, the average number of WT cells in insects was fourfold higher than in the treatment with ΔdtxS1 and eightfold higher than in the insects treated with ΔdtxS2. Furthermore, the hemocyte numbers were higher (P < 0.05) in ΔdtxS2-treated insects than those in insects injected with the WT and ΔdtxS1 spores, respectively (Fig. 4E). The data were generally consistent with the bioassay results, suggesting that ΔdtxS2 is less virulent than the WT and ΔdtxS1. In addition, unlike insects infected with the mutants, the hemolymph collected from the WT-injected insects lost melanization activity (Fig. 4F). Thus, the dtxs, especially A and E, may suppress both cellular and humoral immune responses to facilitate fungal colonization in insects.

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Fig. 4.

Time point observations of fungal development and insect immune responses to the WT and mutant strains. Within sixteen hours of injection, WT spores germinated and the germ tube (arrow) escaped from hemocyte encapsulation (A). However, the ΔdtxS1 spores were heavily melanized, and most of the germ tubes (arrow) were continuously attacked by hemocytes (B). ΔdtxS2 showed similar observations (B′). In contrast to mutants, the WT demonstrated massive growth and yeast-type budding producing hyphal-bodies (HB) up to 36 h postinjection (C). After 48 h, the production of HBs was counted using a hemocytometer (D). In comparison with the untreated insects (4.2 × 10⁷ hemocytes/μL), the number of hemocytes was reduced fourfold in the insects injected with the WT or ΔdtxS1 spores and threefold in ΔdtxS2 treated insects (E). At this time point (48 h), the insects were bled and the hemolymph was individually collected and left exposed to air for 1 h (F). The blood collected from 80% insects infected with the WT remained unmelanized and resembled freshly collected hemolymph from the untreated larvae (the first tube). However, the blood collected from 75% of ΔdtxS1 and 70% of ΔdtxS2 mutant-treated insects was melanized to a similar extent as controls (hemolymph collected from untreated insects and left for 1 h exposed to air). HE, hemocyte; HB, hyphal-body. (Scale bar, 5 μm.)

Analysis of NRPS Genes and Gene Deletion.

The preliminary analysis showed that in contrast to M. robertsii strain ARSEF 23, the M. acridum strain CQMa 102 does not produce dtxs. Using the published genome data (15), we performed comparative sequence analysis to determine the nonorthologous NRPS genes in the M. robertsii genome. The predicted NRPS genes were collected from each fungal species, and the A domain(s) from each NRPS were aligned with Clustal X 2.0 and subjected to phylogenetic analysis with MAGA 5.0 (42) using a Jones–Taylor–Thornton substitution model, a pair-wised deletion for gaps/missing data, and 1,000 bootstrap replications test. To verify gene function and determine the biosynthetic pathway, serial gene deletions were conducted through homologous replacements (43). Briefly, the flanking regions of the target genes were amplified using different primer pairs (Table S6) and ligated into the binary vector of pDHt containing the herbicide resistance bar gene for Agrobacterium-mediated transformations.

Dtx Induction.

Different strains/species of Metarhizium (Table S4) and gene knockout mutants were cultured in CD broth for induction of dtx biosynthesis (13, 44). To verify the functions of DtxS2, DtxS3, and DtxS4 activities, the substrates of dtx B, α-HIC (Sigma), and β-Ala (Sigma) were added into the cultures of corresponding null mutants at a final concentration of 10 mM. After 7 d of incubation at 25 °C and 200 rpm, the culture filtrates were collected by filtration through Whatman No. 1 filter paper for dtx extraction as described previously (44). The crude extracts were dissolved in 1 mL of neat methanol and filtrated through a 0.22-μm membrane before HPLC and HPLC-MS analysis. The experiments were repeated twice with three replicates per treatment. To detect in insecta dtx production, the mycosed larvae silkworm were freeze-dried and ground to powder for toxin extraction.

HPLC, MS, and NMR Analysis.

The chromatographic profiles of crude extract samples were detected with a LC-20AD HPLC system (Shimadzu Scientific Instruments), equipped with a C18 reverse phase column (particle size: 5μm; length: 4.6 × 250 mm) and a SPD-20A UV/Vis detector. The mobile phase was a linear gradient of deionized water and methanol from 90:10 for 2 min, 55:45 for 2 min, 39:61 for 22 min, 5:95 for 5 min, and 90:10 for 8 min at a flow rate of 0.1 mL/min and was monitored at a wavelength of 215 nm (13). Quantification of dtxs was determined by integration with calibration curves obtained from dtx A standard (Sigma) and purified dtx B and Dtx E. Detection of in insecta dtxs production was performed by high-performance ion chromatography. The molecular masses of different components were determined by Q-TOF LC-MS analysis using the Agilent G6520A accurate-mass system with a poroshell 300 sb-C8 column as follows: drying gas temperature 325 °C, drying gas flow 10 L/mL, and nebulizer pressure 30 psi. To verify the structures of ΔdtxS2 products, H and C NMR spectra were recorded on a Bruker ARX 600 spectrometer (600/150 MHz) and a Bruker AM-400 (400/100 MHz) instrument with tetramethylsilane as a calibration standard.

Insect Bioassays.

Two insect species were included in virulence assays: fifth instar larvae of the silkworm (Bombyx mori) and adult locusts (Locusta migratoria). The silkworms were each injected with 10 μL of suspension of 5 × 10⁵ WT or mutant spores/mL through the second proleg, and the locust were injected with 10 μL of 1 × 10⁶ spores/mL through the intersegmental membrane between two abdominal terga. Each treatment had 3 replicates with 10 insects each, and the experiments were repeated twice. Mortality was recorded every 12 h. Extra silkworm larvae were injected for bleeding for microscopic observation of encapsulation and fungal development within the insect hemocoel (43). The median lethal time (LT₅₀) of different fungal strains was estimated and compared with the Kaplan–Meier method (log-rank pairwise comparison) using the SPSS 17 program (SPSS).

We thank Prof. Raymond J. St. Leger (University of Maryland) for critical reading of the manuscript. The study was supported by Ministry of Science and Technology of China Grants 2009CB118904 and 2009AA10Z112 and Chinese Academy of Sciences Grants KSCX2-EW-N-06, DBSH-2011-047, and KSCX2-EW-G-16.