Glyoxal oxidase-mediated detoxification of reactive carbonyl species contributes to virulence, stress tolerance, and development in a pathogenic fungus.

Reactive carbonyl and oxygen species (RCS/ROS), often generated as metabolic byproducts, particularly under conditions of pathology, can cause direct damage to proteins, lipids, and nucleic acids. Glyoxal oxidases (Gloxs) oxidize aldehydes to carboxylic acids, generating hydrogen peroxide (H2O2). Although best characterized for their roles in lignin degradation, Glox in plant fungal pathogens are known to contribute to virulence, however, the mechanism underlying such effects are unclear. Here, we show that Glox in the insect pathogenic fungus, Metarhizium acridum, is highly expressed in mycelia and during formation of infection structures (appressoria), with the enzyme localizing to the cell membrane. MaGlox targeted gene disruption mutants showed RCS and ROS accumulation, resulting in cell toxicity, induction of apoptosis and increased autophagy, inhibiting normal fungal growth and development. The ability of the MaGlox mutant to scavenge RCS was significantly reduced, and the mutant exhibited increased susceptibility to aldehydes, oxidative and cell wall perturbing agents but not toward osmotic stress, with altered cell wall contents. The ΔMaGlox mutant was impaired in its ability to penetrate the host cuticle and evade host immune defense resulting in attenuated pathogenicity. Overexpression of MaGlox promoted fungal growth and conidial germination, increased tolerance to H2O2, but had little to other phenotypic effects. Transcriptomic analyses revealed downregulation of genes related to cell wall synthesis, conidiation, stress tolerance, and host cuticle penetration in the ΔMaGlox mutant. These findings demonstrate that MaGlox-mediated scavenging of RCS is required for virulence, and contributes to normal fungal growth and development, stress resistance.

MaEng1, an endo-1,3-glucanase, contributes to the conidiation pattern shift through changing the cell wall structure in Metarhizium acridum.

Microcycle conidiation has displayed the greater potential than normal conidiation in large-scale production of mycopesticides. Fungi require partial hydrolysis of the cell wall to achieve the necessary plasticity during their morphological changes. Therefore, various cell wall-associated hydrolases are crucial for fungal morphogenesis. Eng1, as an endo-ß-1,3-glucanase, is involved in the cell separation of fungi, but its role in morphological changes of entomopathogenic fungi is not yet clear. Here, the endo-ß-1,3-glucanase gene MaEng1 was characterized in the model entomopathogenic fungi M. acridum. MaEng1 possesses a typical carbohydrate hydrolase domain and belongs to the GH81 family. The functions of MaEng1 in fungal growth, stress tolerance, pathogenicity, and conidiation capacity were analyzed using targeted gene disruption. The results displayed that the absence of MaEng1 does not affect the fungal growth, stress tolerances, and pathogenicity in M. acridum. However, the knockout of MaEng1 led to the normal conidiation of M. acridum on the SYA medium, which can induce the microcycle conidiation. Moreover, the content of ß-1,3-glucan in the cell wall of the MaEng1-disruption strain were significantly reduced and the exposures of ß-1,3-glucan on the surface of the mature conidia and mycelia in ΔMaEng1 were declined, indicating that MaEng1 contributes to the conversion of conidiation mode in M. acridum by affecting the cell wall structure.

Tetracarboxylic acid transporter regulates growth, conidiation, and carbon utilization in Metarhizium acridum.

Carbon sources and their utilization are vital for fungal growth and development. C4-dicarboxylic acids are important carbon and energy sources that function as intermediate products of the tricarboxylic acid cycle. Transport and regulation of C4-dicarboxylic acid uptake are mainly dependent on tetracarboxylic acid transporters (Dcts) in many microbes, although the roles of Dct genes in fungi have only been partially characterized. Here, we report on the functions of two Dct genes (Dct1 and Dct2) in the entomopathogenic fungus Metarhizium acridum. Our data showed that loss of the MaDct1 gene affected utilization of tetracarboxylic acids and other carbon sources. ΔMaDct1 mutants showed larger colony sizes with extensive mycelial growth but were delayed in conidiation with decreased conidia yield as compared to the wild-type parental strain. On the nutrient-deficient medium, SYA, the wild-type strain produced microcycle conidia, whereas the ΔMaDct1 mutant produced (normal) aerial conidia. In addition, ΔMaDct1 had decreased tolerance to cell wall perturbing agents, but increased tolerances to UV-B radiation and osmotic stress. Insect bioassays indicated that loss of MaDct1 did not affect pathogenicity. In contrast, no distinct phenotypic change was observed for the MaDct2 mutant in terms of growth and biocontrol characteristics. Transcriptomic profiling between wild type and ΔMaDct1 showed that differentially expressed genes were enriched in carbohydrate and amino acid metabolism, transport and catabolism, and signal transduction. These results demonstrate that MaDct1 regulates the conidiation pattern shift and mycelial growth by affecting utilization of carbon sources. These findings are helpful for better understanding the effect of intermediates of carbon metabolism on fungal growth and conidiation. KEY POINTS: • MaDct1 influences fungal growth and conidiation by affecting carbon source utilization. • MaDct1 regulates conidiation pattern shift under nutrient deficiency condition. • MaDct1 is involved in stress tolerance and has no effect on virulence. • MaDct2 has no effect on growth and biocontrol characteristic.

Arginine metabolism governs microcycle conidiation by changing nitric oxide content in Metarhizium acridum.

Microcycle conidiation commonly exists in filamentous fungi and has great potential for mass production of mycoinsecticides. L-Arginine metabolism is essential for conidiation and conditional growth and virulence, but its role in microcycle conidiation has not been explored. Here, a unique putative arginase (MaAGA) was characterized in the entomopathogenic fungus Metarhizium acridum. Conidial germination and thermotolerance were facilitated by the disruption of MaAGA. Despite little impact on fungal growth and virulence, the disruption resulted in normal conidiation after a 60-h incubation on microcycle conidiation medium (SYA) under normal culture conditions. In the MaAGA-disruption mutant (ΔMaAGA), intracellular arginine accumulation was sharply increased. Replenishment of the direct metabolites of arginase, namely ornithine and/or urea, was unable to restore the disruption mutant's microcycle conidiation on SYA. Interestingly, nitric oxide synthase (NOS) activity and nitric oxide (NO) levels of the ΔMaAGA strain were markedly decreased in the 60-h-old SYA cultures. Finally, adding Nω-nitro-L-arginine, an inhibitor of NOS, into the SYA converted the microcycle conidiation of the wild-type strain to normal conidiation. In contrast, adding sodium nitroprusside, an NO donor, into the SYA recovered the mutant's microcycle conidiation. The results indicate that arginine metabolism controls microcycle conidiation by changing the content of NO. KEY POINTS: • The MaAGA-disruption led to normal conidiation on microcycle conidiation medium SYA. • Nitric oxide (NO) level of the ΔMaAGA strain was markedly decreased. • Adding an NO donor into the SYA recovered the microcycle conidiation of ΔMaAGA.

MaNsdD regulates conidiation negatively by inhibiting the AbaA expression required for normal conidiation in Metarhizium acridum.

Conidiation necessary for filamentous fungal survival and dispersal proceeds in two fashions, namely, normal conidiation through conidiophores differentiated from hyphae and microcycle conidiation through conidial budding. Normal conidiation has been well studied, whereas mechanisms underlying microcycle conidiation are still largely unknown. Here, we report that a gene (MaNsdD) homologous to NsdD in Aspergillus nidulans serves as a suppressor of normal conidiation but a positive regulator of hyphal development in Metarhizium acridum. Disruption of MaNsdD (ΔMaNsdD) resulted in microcycle conidiation and significantly descended in conidial resistance to heat while improved to UV irradiation. Transcriptomic analysis revealed that many genes involved in conidiation, cell division and cell wall formation were differentially expressed in ΔMaNsdD, and likely associated with the conidiation process. We found that a gene (MaAbaA) homologous to the core asexual development regulator AbaA in A. nidulans was negatively controlled by MaNsdD. Disruption of MaAbaA led to the abolition of the conidiation process of M. acridum. These findings unravel a novel regulatory mechanism of microcycle conidiation and add knowledge to the asexual conidiation pathway of filamentous fungi.

Members of chitin synthase family in Metarhizium acridum differentially affect fungal growth, stress tolerances, cell wall integrity and virulence.

Chitin is an important component of the fungal cell wall with a family of chitin synthases mediating its synthesis. Here, we report on the genetic characterization of the full suite of seven chitin synthases (MaChsI-VII) identified in the insect pathogenic fungus, Metarhizium acridum. Aberrant distribution of chitin was most evident in targeted gene knockouts of MaChsV and MaChsVII. Mutants of MaChsI, MaChsIII, MaChsIV showed delayed conidial germination, whereas ΔMaChsII and ΔMaChsV mutants germinated more rapidly when compared to the wild-type parent. All MaChs genes impacted conidial yield, but differentially affected stress tolerances. Inactivation of MaChsIII, MaChsV, MaChsVII resulted in cell wall fragility, and ΔMaChsV and ΔMaChsVII mutants showed high sensitivity to Congo red and calcofluor white, suggesting that the three genes are required for cell wall integrity. In addition, ΔMaChsIII and ΔMaChsVII mutants showed the highest sensitivities to heat and UV-B stress. Three of seven chitin synthase genes, MaChsIII, MaChsV, MaChsVII, were found to contribute to fungal virulence. Compared with the wild-type strain, ΔMaChsIII and ΔMaChsV mutants were reduced in virulence by topical inoculation, while the ΔMaChsVII mutant showed more severe virulence defects. Inactivation of MaChsIII, MaChsV, or MaChsVII impaired appressorium formation, affected growth of in insecta produced hyphal bodies, and altered the surface properties of conidia and hyphal bodies, resulting in defects in the ability of the mutant strains to evade insect immune responses. These data provide important links between the physiology of the cell wall and the ability of the fungus to parasitize insects and reveal differential functional consequences of the chitin synthase family in M. acridum growth, stress tolerances, cell wall integrity and virulence.

Dipeptidase PEPDA Is Required for the Conidiation Pattern Shift in Metarhizium acridum.

Filamentous fungi conduct two types of conidiation, typical conidiation from mycelia and microcycle conidiation (MC). Fungal conidiation can shift between the two patterns, which involves a large number of genes in the regulation of this process. In this study, we investigated the role of a dipeptidase gene pepdA in conidiation pattern shift in Metarhizium acridum, which is upregulated in MC pattern compared to typical conidiation. Results showed that disruption of the pepdA resulted in a shift of conidiation pattern from MC to typical conidiation. Metabolomic analyses of amino acids showed that the levels of 19 amino acids significantly changed in ΔpepdA mutant. The defect of MC in ΔpepdA can be rescued when nonpolar amino acids, α-alanine, ß-alanine, or proline, were added into sucrose yeast extract agar (SYA) medium. Digital gene expression profiling analysis revealed that PEPDA mediated transcription of sets of genes which were involved in hyphal growth and development, sporulation, cell division, and amino acid metabolism. Our results demonstrated that PEPDA played important roles in the regulation of MC by manipulating the levels of amino acids in M. acridum. IMPORTANCE Conidia, as the asexual propagules in many fungi, are the start and end of the fungal life cycle. In entomopathogenic fungi, conidia are the infective form essential for their pathogenicity. Filamentous fungi conduct two types of conidiation, typical conidiation from mycelia and microcycle conidiation. The mechanisms of the shift between the two conidiation patterns remain to be elucidated. In this study, we demonstrated that the dipeptidase PEPDA, a key enzyme from the insect-pathogenic fungus Metarhizium acridum for the hydrolysis of dipeptides, is associated with a shift of conidiation pattern. The conidiation pattern of the ΔpepdA mutant was restored when supplemented with the nonpolar amino acids rather than polar amino acids. Therefore, this report highlights that the dipeptidase PEPDA regulates MC by manipulating the levels of amino acids in M. acridum.

O-mannosyltransferase MaPmt2 contributes to stress tolerance, cell wall integrity and virulence in Metarhizium acridum.

As a conserved post-translational modification, O-mannosyltransferase families play important roles in many cellular processes. Three subfamilies (MaPmt1, MaPmt2 and MaPmt4) are grouped in Metarhizium acridum according to sequence homology. The functions of MaPmt1 and MaPmt4 have been characterized in M. acridum previously. In this study, the functions of another member belonging to the Pmt2 subfamily, MaPmt2, were identified through RNAi strategy. The three MaPmt2 knockdown mutants showed dramatically decreased expression of MaPmt2. Phenotypic analyses showed that the mutants exhibited decreased tolerances to wet-heat, UV-B irradiation and cell wall perturbing chemicals. Further studies revealed that the mutants presented thinner cell walls observed by transmission electron microscope combined with changed cell wall components. Besides, knockdown of MaPmt2 decelerated conidial germination and decreased conidial yield. Compared with the wild-type strain, the MaPmt2 knockdown mutants caused impaired virulence only by topical inoculation. Results illustrated that the decreased virulence by inoculation could result from the delayed conidial germination on locust wings, reduced appressorium formation, as well as reduced turgor pressure in MaPmt2 knockdown mutants.

Functional and characteristic analysis of an appressorium-specific promoter PMagas1 in Metarhizium acridum.

Entomopathogenic fungi have been used as important biological control agents throughout the world. To improve the biocontrol efficacy of entomopathogenic fungi, many genes have been used to improve fungal virulence or tolerance to adverse conditions via modulating their expression with strong promoters. The Magas1 gene is specifically expressed during appressorium formation and contributes to the virulence in Metarhizium acridum. In this study, we analyzed the functional region of the promoter of Magas1 gene (PMagas1) in M. acridum using 5'-deletion technique with enhanced green fluoresces protein (EGFP) as a reporter. Results showed the full length of the PMagas1 was at least 897 bp. Two regions (-897 to -611 bp and -392 to -328 bp) were essential for the activity of PMagas1. An engineered M. acridum strain was constructed with PMagas1 driving the expression of a subtilisin-like proteinase gene Pr1A (PMagas1-PR1A). Bioassay showed that the virulence was significantly increased in PMagas1-PR1A strain compared to wild type strain. Pmagas1 promoter is suitable for the overexpression of some genes during the infection of entomopathogenic fungi, which avoids the waste of nutritional resources and the influence on other fungal characteristics during the saprophytic process of constitutive promoter.

The phosphatase gene MaCdc14 negatively regulates UV-B tolerance by mediating the transcription of melanin synthesis-related genes and contributes to conidiation in Metarhizium acridum.

Reversible phosphorylation of proteins regulated by protein kinases and phosphatases mediate multiple biological events in eukaryotes. In this study, a dual-specificity cell division cycle 14 phosphatase, MaCdc14, was functionally characterized in Metarhizium acridum. Deletion of MaCdc14 decreased branch numbers, affected septum formation and resulted in multiple nuclei in each hyphal compartment, indicating nuclear division and cytokinesis defects. The spore production capacity was severely impaired with decreased conidial yield and delayed conidiation in MaCdc14-deletion mutant (ΔMaCdc14). The transcription levels of conidiation-related genes were significantly changed after MaCdc14 inactivation. The morphology of conidia was uneven in size and the germination rate of conidia was increased in ΔMaCdc14. In addition, ΔMaCdc14 displayed significantly enhanced conidial tolerance to ultraviolet (UV) irradiation but had no significant effect on the thermotolerance, the sensitivities to cell wall damage reagents, osmotic and oxidative stresses, and virulence compared to the wild-type strain and complementary transformant. Furthermore, the pigmentation of ΔMaCdc14 was increased by the upregulated expression of melanin synthesis-related genes, which may result in the enhanced UV-B tolerance of ΔMaCdc14. In summary, MaCdc14 negatively regulated UV-B tolerance by mediating the transcription of melanin synthesis-related genes, contributed to conidiation by regulating the expression levels of conidiation-related genes and also played important roles in cytokinesis and morphogenesis in Metarhizium acridum.

MaPacC, a pH-responsive transcription factor, negatively regulates thermotolerance and contributes to conidiation and virulence in Metarhizium acridum.

PacC is a pH-responsive transcription factor gene highly expressed at alkaline pH and plays distinct roles in environmental fitness, conidiation and virulence of different fungi. Here, we show biological functions of orthologous MaPacC in the locust-specific fungal pathogen Metarhizium acridum. Disruption of MapacC slowed down the fungal growth only under alkaline conditions. Intriguingly, the fungal thermotolerance was enhanced by the MapacC deletion, accompanied by transcriptional upregulation of some heat shock-responsive genes. The disruptant suffered a reduction in conidial yield and a change in conidial surface structure, but showed little change in cell wall integrity. The virulence of the disruptant against a locust species was markedly attenuated due to delayed appressorium formation, repressed expression of some insect cuticle hydrolases and slowed growth in locust hemolymph. The phenoloxidase activity and nodules of the locusts infected by the disruptant were also boosted. All of these phenotypic changes were restored by targeted gene complementation. Our results indicate that MaPacC acts a negative regulator of thermotolerance and contributes to the virulence of M. acridum by an involvement in hyphal penetration through insect cuticle and evasion from insect immunity.

MaPmt1, a protein O-mannosyltransferase, contributes to virulence through governing the appressorium turgor pressure in Metarhizium acridum.

O-glycosylation is a very important post-translational modification of protein and involved in many cell processes in fungi. There exist three protein O-manosyltransferanse genes (MaPmt1, MaPmt2, MaPmt4) in Metarhizium acridum based on sequence homology. Here, MaPmt1, a gene for Pmt1 O-manosyltransferanse in M. acridum, was characterized and functionally analyzed through targeted gene disruption and complementation methods. Deletion of MaPmt1 had no effect on conidial germination, but slightly increased the conidial yield and significantly impaired fungal tolerances to UV-B radiation and wet-heat. Deletion of MaPmt1 made the fungus become more sensitive to cell wall disturbing agents and exhibit a thinner cell wall with changed components. Insect bioassays showed that disruption of MaPmt1 attenuated the fungal virulence significantly by topical inoculation but not by injection, indicating that MaPmt1 is required for penetration during the infection of M. acridum. Interestingly, deletion of MaPmt1 did not affect appressorium formation but significantly decreased appressorium turgor pressure. Moreover, the decreased virulence of MaPmt1 disruptant is mainly due to the reduced appressorium turgor pressure, which may be resulted from the declined glycerol concentration, combined with the weakened cell wall that could not hold the normal appressorium turgor pressure to penetrate the host cuticle.

The transmembrane protein MaSho1 negatively regulates conidial yield by shifting the conidiation pattern in Metarhizium acridum.

Sho1 is an important membrane sensor upstream of the HOG-MAPK signaling pathway, which plays critical roles in osmotic pressure response, growth, and virulence in fungi. Here, a Sho1 homolog (MaSho1), containing four transmembrane domains and one Src homology (SH3) domain, was characterized in Metarhizium acridum, a fungal pathogen of locusts. Targeted gene disruption of MaSho1 impaired cell wall integrity, virulence, and tolerances to UV-B and oxidative stresses, while none of them was affected when the SH3 domain was deleted. Intriguingly, disruption of MaSho1 significantly increased conidial yield, which was not affected in the SH3 domain mutant. Furthermore, it was found that deletion of MaSho1 led to microcycle conidiation of M. acridum on the normal conidiation medium. Deletion of MaSho1 significantly shortened the hyphal cells but had no effect on conidial germination. Digital gene expression profiling during conidiation indicated that differential expression of genes was associated with mycelial development, cell division, and differentiation between the wild type and the MaSho1 mutant. These data suggested that disruption of MaSho1 shifted the conidiation pattern by altering the transcription of genes to inhibit mycelial growth, thereby promoting the conidiation of M. acridum.

MaPmt4, a protein O-mannosyltransferase, contributes to cell wall integrity, stress tolerance and virulence in Metarhizium acridum.

In eukaryotic cells, protein O-glycosylation is an essential protein modification. Analysis of the Metarhizium acridum genome database revealed a total of three O-glycoside mannosyltransferase homologs (Pmt1, Pmt2 and Pmt4), closely related to Saccharomyces cerevisiae Pmt1, Pmt2, and Pmt4. In this study, the functions of MaPmt4, encoding a protein O-mannosyltransferase in M. acridum, were characterized using disruption and complementation strategies. Disruption of MaPmt4 delayed the conidial germination and reduced the fungal tolerances to heat shock and UV-B irradiation, but did not affect conidial yield. Inactivation of MaPmt4 displayed increased sensitivity to cell wall-perturbing agents, formed thinner cell walls, and changed composition of fungal cell wall, demonstrating that MaPmt4 was also important to maintain fungal cell wall integrity. Bioassays by topical inoculation and intrahemocoel injection showed that the MaPmt4 deletion mutant exhibited greatly reduced virulence. The subsequent examination revealed that the inactivation of MaPmt4 impaired appressorium formation, decreased fungal growth in locust hemolymph in vitro, and boosted insect immune responses, the latter in part potentially owing to the changes in conidial surface structures, and thus attenuated the virulence of MaPmt4 deletion mutant. Furthermore, the results of comparative proteomics showed that MaPmt4 played important roles in fungal cell wall integrity, stress tolerances, and virulence via broad genetic pathways.

High-yield production of spider short-chain insecticidal neurotoxin Tx4(6-1) in Pichia pastoris and bioactivity assays in vivo.

Short-chain insecticidal neurotoxin Tx4(6-1) from the spider Phoneutria nigriventer can be prepared by reversed-phase high-performance liquid-chromatography (HPLC) fractionation of PhTx4, but this is difficult and represents an obstacle preventing analyses of its insecticidal activity against agricultural insect pests. Herein, we performed secretory expression of recombinant Tx4(6-1) using Pichia pastoris strain X33 as the host, and screened transformants using enzyme-linked immunosorbent assay (ELISA). In flasks, ∼5 mg/l rTx4(6-1) was expressed as a secreted protein following induction with methanol, and this was increased to 45 mg/l rTx4(6-1) in a fed-batch reactor. Approximately 4 mg of high-purity rTx4(6-1) was purified from a 400 ml fed-batch culture supernatant by Ni+-nitriloacetic acid affinity chromatography, followed by carboxymethyl (CM) sepharose ion-exchange chromatography. Purified rTx4(6-1) was determined by mass spectrometry (MS) analysis, revealing a molecular weight (MW) of 7660.5 Da, larger than the expected size due to O-linked glycosylation. Insect bioactivity tests of rTx4(6-1)-treated fifth-instar silkworm larvae (Bombyx mori Linnaeus) showed neurotoxin symptoms such as contraction paralysis, abdominal contraction, and mouth movement syndrome, with a half lethal dose at 12 h post-injection of ∼4.5-8.5 µg/g body weight. Dietary toxicity was not observed in silkworm larvae.

Mid1 affects ion transport, cell wall integrity, and host penetration of the entomopathogenic fungus Metarhizium acridum.

Calcium signaling plays important roles in stress tolerance and virulence in fungi. Mid1, an accessory protein of Cch1 calcium channel, has been discussed in baker's yeast and some filamentous fungi. However, functions of the Mid1 gene in entomopathogenic fungi are not clear. In this study, the Mid1 gene was functionally characterized by deleting it in the entomopathogenic fungus Metarhizium acridum. The growth of the ΔMaMid1 mutant was similar as the wild type on normal growth medium, but inhibited by exogenous Ca2+, Fe2+, Mg2+, Mn2+, Li+, and calcium chelator ethylene glycol tetraacetic acid (EGTA). Cation transportation-related genes were upregulated and intracellular calcium concentration was decreased in ΔMaMid1. Deletion of the MaMid1 gene impaired the tolerance to cell wall-disrupting agents but had no impact on heat or ultraviolet irradiation tolerance compared with the wild type. Bioassays showed that ΔMaMid1 had decreased virulence, with defects in the ability to penetrate the host cuticle. Compared with the wild type, appressorium formation on locust wings and fungal growth in the insect hemocoel were significantly decreased in the ΔMaMid1 mutant in a bioassay through topical inoculation. The phenotypes of ΔMaMid1 were fully restored in a complementation strain. Taken together, our study demonstrates that the MaMid1 affects intracellular ion homeostasis and contributes to virulence by affecting the initial penetration process in M. acridum.

The homeobox gene MaH1 governs microcycle conidiation for increased conidial yield by mediating transcription of conidiation pattern shift-related genes in Metarhizium acridum.

Conidiation capacity and conidial quality are very important for the production and application of mycopesticides. Most filamentous ascomycetous fungi have two distinct patterns of conidiation. Conidiation through microcycle conidiation proceeds to more rapidly achieve a maximum of conidial yield than normal conidiation and hence is of greater merit for exploitation in mass production of fungal insect pathogens, such as Metarhizium acridum. In this study, the mechanism underlying the conidiation pattern shift in M. acridum was explored by characterization of the fungal homeobox gene MaH1. MaH1 was evidently localized to the nuclei of hyphae and transcriptionally expressed at a maximal level when conidiation began. Intriguingly, deletion of MaH1 in M. acridum resulted in a shift of normal conidiation to microcycle conidiation on one-quarter strength Sabouraud's dextrose agar medium, and hence accelerated conidiation and increased conidial yield. In the deletion mutant, moreover, conidia became larger in size and hyphae cells were shorter in length while conidial virulence and stress tolerance were not altered. As revealed by digital gene expression profiling, MaH1 controlled the shift of conidiation patterns by mediating transcription of a set of genes related to hyphal growth, cell differentiation, conidiation, and some important signaling pathways. These findings indicate that MaH1 and its downstream genes can be exploited to increase the conidial yield for more efficient production of mycopesticides.

The protein phosphatase gene MaPpt1 acts as a programmer of microcycle conidiation and a negative regulator of UV-B tolerance in Metarhizium acridum.

The Ser/Thr protein phosphatase Ppt1 (yeast)/PP5 (humans) has been implicated in signal transduction-mediated growth and differentiation, DNA damage/repair, cell cycle progression, and heat shock responses. Little, however, is known concerning the functions of Ppt1/PP5 in filamentous fungi. In this study, the Ppt1 gene MaPpt1 was characterized in the insect pathogenic fungus, Metarhizium acridum. The MaPpt1 protein features a three-tandem tetratricopeptide repeat (TPR) domain and a peptidyl-prolyl cis-trans isomerase-like (PP2Ac) domain. Subcellular localization using an MaPpt1::eGFP fusion protein revealed that MaPpt1 was localized in the cytoplasm of spores, but gathered at the septa in growing hyphae. Targeted gene inactivation of MaPpt1 in M. acridum resulted in unexpected reprogramming of normal aerial conidiation to microcycle conidiation. Although overall vegetative growth was unaffected, a significant increase in conidial yield was noted in ΔMaPpt1. Stress-responsive phenotypes and virulence were largely unaffected in ΔMaPpt1. Exceptionally, ΔMaPpt1 displayed increased UV tolerance compared to wild type. Digital gene expression data revealed that MaPpt1 mediates transcription of sets of genes involved in conidiation, polarized growth, cell cycle, cell proliferation, DNA replication and repair, and some important signaling pathways. These data indicate a unique role for Ppt1 in filamentous fungal development and differentiation.

High-level expression and purification of active scorpion long-chain neurotoxin BjαIT from Pichia pastoris.

As an insect-selective neurotoxin, scorpion long-chain BjαIT is a promising prospect for insecticidal application; however, the difficulty of obtaining natural BjαIT represents the major obstacle preventing analysis of its insecticidal activity against agricultural insect pests. Here, we screened recombinant Pichia pastoris transformants showing high levels of secretory recombinant (r)BjαIT. Secreted rBjαIT was expressed at levels as high as 340 mg/L following methanol induction in a fed-batch reactor, with ∼21 mg of pure rBjαIT obtained from 200-mL fed-batch culture supernatant by Ni2+-nitriloacetic acid affinity chromatography and CM Sepharose ion-exchange chromatography. Injection of purified rBjαIT induced neurotoxicity symptoms in locust (Locusta migratoria) larvae, and the half-lethal dose of rBjαIT for locusts at 24-h post-injection ranged from 11 to 14 µg/g body weight. These results demonstrated that large amounts of active rBjαIT were efficiently prepared from P. pastoris, suggesting this system as efficacious for determining rBjαIT insecticidal activity against other agricultural insect pests.

Recombinant production of the insecticidal scorpion toxin BjαIT in Escherichia coli.

Scorpion long-chain insect neurotoxins have important potential application value in agricultural pest control. The difficulty of obtaining natural toxins is the major obstacle preventing analyses of their insecticidal activity against more agricultural insect pests. Here we cloned the insect neurotoxin BjαIT gene into the pET32 expression vector and expressed the resulting thioredoxin (Trx)-BjαIT fusion protein in Escherichia coli. Soluble Trx-BjαIT was expressed at a high level when induced at 18 °C with 0.1 mM isopropyl ß-d-1-thiogalactopyranoside, and it was purified by Ni2+-nitriloacetic acid affinity chromatography. After cleaving the Trx tag with recombinant enterokinase, the digestion products were purified by CM Sepharose FF ion-exchange chromatography, and 1.5 mg of purified recombinant BjαIT (rBjαIT) was obtained from 100 ml of induced bacterial cells. Injecting rBjαIT induced obvious neurotoxic symptoms and led to death in locust (Locusta migratoria) larvae. Dietary toxicity was not observed in locusts. The results demonstrate that active rBjαIT could be obtained efficiently from an E. coli expression system, which is helpful for determining its insecticidal activity against agricultural insect pests.