Inductive Production of the Iron-Chelating 2-Pyridones Benefits the Producing Fungus To Compete for Diverse Niches.

Diverse 2-pyridone alkaloids have been identified with an array of biological and pharmaceutical activities, including the development of drugs. However, the biosynthetic regulation and chemical ecology of 2-pyridones remain largely elusive. Here, we report the inductive activation of the silent polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) (tenS) gene cluster for the biosynthesis of the tenellin-type 2-pyridones in the insect-pathogenic fungus Beauveria bassiana when cocultured with its natural competitor fungus Metarhizium robertsii. A pathway-specific transcription factor, tenR, was identified, and the overexpression of tenR well expanded the biosynthetic mechanism of 15-hydroxytenellin (15-HT) and its derivatives. In particular, a tandemly linked glycosyltransferase-methyltransferase gene pair located outside the tenS gene cluster was verified to mediate the rare and site-specific methylglucosylation of 15-HT at its N-OH residue. It was evident that both tenellin and 15-HT can chelate iron, which could benefit B. bassiana to outcompete M. robertsii in cocultures and to adapt to iron-replete and -depleted conditions. Relative to the wild-type strain, the deletion of tenS had no obvious negative effect on fungal virulence, but the overexpression of tenR could substantially increase fungal pathogenicity toward insect hosts. The results of this study well advance the understanding of the biosynthetic machinery and chemical ecology of 2-pyridones. IMPORTANCE Different 2-pyridones have been identified, with multiple biological activities but unclear chemical ecology. We found that the silent tenS gene cluster was activated in the insect pathogen Beauveria bassiana when the fungus was cocultured with its natural competitor Metarhizium robertsii. It was established that the gene cluster is regulated by a pathway-specific regulator, tenR, and the overexpression of this transcription factor expanded the biosynthetic machinery of the tenellin 2-pyridones. It was also found that the paired genes located outside the tenS cluster contribute to the site-specific methylglucosylation of the main compound 15-hydroxytenellin. Both tenellin and 15-hydroxytenellin can chelate and sequester iron to benefit the producing fungus to compete for different niches. This study well advances the biosynthetic mechanism and chemical ecology of 2-pyridones.

Bioactive Metabolites and Potential Mycotoxins Produced by Cordyceps Fungi: A Review of Safety.

Ascomycete Cordyceps fungi such as C.militaris, C. cicadae, and C.guangdongensis have been mass produced on artificial media either as food supplements or health additives while the byproducts of culture substrates are largely used as animal feed. The safety concerns associated with the daily consumption of Cordyceps fungi or related products are still being debated. On the one hand, the known compounds from these fungi such as adenosine analogs cordycepin and pentostatin have demonstrated different beneficial or pharmaceutical activities but also dose-dependent cytotoxicities, neurological toxicities and or toxicological effects in humans and animals. On the other hand, the possibility of mycotoxin production by Cordyceps fungi has not been completely ruled out. In contrast to a few metabolites identified, an array of biosynthetic gene clusters (BGCs) are encoded in each genome of these fungi with the potential to produce a plethora of as yet unknown secondary metabolites. Conservation analysis of BGCs suggests that mycotoxin analogs of PR-toxin and trichothecenes might be produced by Cordyceps fungi. Future elucidation of the compounds produced by these functionally unknown BGCs, and in-depth assessments of metabolite bioactivity and chemical safety, will not only facilitate the safe use of Cordyceps fungi as human food or alternative medicine, but will also benefit the use of mass production byproducts as animal feed. To corroborate the long record of use as a traditional medicine, future efforts will also benefit the exploration of Cordyceps fungi for pharmaceutical purposes.

A M35 family metalloprotease is required for fungal virulence against insects by inactivating host prophenoloxidases and beyond.

A diverse family of metalloproteases (MPs) is distributed in eukaryotes. However, the functions of MPs are still understudied. We report that seven MPs belonging to the M35 family are encoded in the genome of the insect pathogenic fungus Metarhizium robertsii. By gene deletions and insect bioassays, we found that one of the M35-family MPs, i.e. MrM35-4, is required for fungal virulence against insect hosts. MrM35-4 is a secretable enzyme and shows a proteolytic activity implicated in facilitating fungal penetration of insect cuticles. After gene rescue and overexpression, insect bioassays indicated that MrM35-4 contributes to inhibiting insect cuticular and hemocyte melanization activities. Enzymatic cleavage assays revealed that the recombinant prophenoloxidases PPO1 and PPO2 of Drosophila melanogaster could be clipped by MrM35-4 in a manner differing from a serine protease that can activate PPO activities. In addition, it was found that MrM35-4 is involved in suppressing antifungal gene expression in insects. Consistent with the evident apoptogenic effect of MrM35-4 on host cells, we found that the PPO mutant flies differentially succumbed to the infections of the wild-type and mutant strains of M. robertsii. Thus, MrM35-4 plays a multifaceted role beyond targeting PPOs during fungus-insect interactions, which represents a previously unsuspected strategy employed by Metarhizium to outmaneuver insect immune defenses.

A small secreted protein triggers a TLR2/4-dependent inflammatory response during invasive Candida albicans infection.

Candida albicans can switch from commensal to pathogenic mode, causing mucosal or disseminated candidiasis. The host relies on pattern-recognition receptors including Toll-like receptors (TLRs) and C-type lectin receptors (CLRs) to sense invading fungal pathogens and launch immune defense mechanisms. However, the complex interplay between fungus and host innate immunity remains incompletely understood. Here we report that C. albicans upregulates expression of a small secreted cysteine-rich protein Sel1 upon encountering limited nitrogen and abundant serum. Sel1 activates NF-κB and MAPK signaling pathways, leading to expression of proinflammatory cytokines and chemokines. Comprehensive genetic and biochemical analyses reveal both TLR2 and TLR4 are required for the recognition of Sel1. Further, SEL1-deficient C. albicans display an impaired immune response in vivo, causing increased morbidity and mortality in a bloodstream infection model. We identify a critical component in the Candida-host interaction that opens a new avenue to tackle Candida infection and inflammation.

Diverse effect of phosphatidylcholine biosynthetic genes on phospholipid homeostasis, cell autophagy and fungal developments in Metarhizium robertsii.

Phosphatidylcholine (PC) plays an important role in maintaining membrane integrity and functionality. In this study, two key genes (Mrpct and Mrpem) putatively involved in the cytidine diphosphate (CDP)-choline and phosphatidylethanolamine N-methyltransferase (PEMT) pathways for PC biosynthesis were characterized in the insect pathogenic fungus Metarhizium robertsii. The results indicated that disruption of Mrpct did not lead to any reduction of total PC content but impaired fungal virulence and increased cellular accumulation of triacylglycerol. Deletion of Mrpem reduced PC content and impaired fungal conidiation and infection structure differentiation but did not result in virulence defects. Lipidomic analysis revealed that deletion of Mrpct and Mrpem resulted in dissimilar effects on increase and decrease of PC moieties and other phospholipid species accumulations. Interestingly, we found that these two genes played opposite roles in activation of cell autophagy when the fungi were grown in a nutrient-rich medium. The connection between PC metabolism and autophagy was confirmed because PC content was drastically reduced in Mratg8Δ and that the addition of PC could rescue null mutant sporulation defect. The results of this study facilitate the understanding of PC metabolism on fungal physiology.

Fungal Cordycepin Biosynthesis Is Coupled with the Production of the Safeguard Molecule Pentostatin.

Cordycepin (COR) and pentostatin (PTN) are adenosine analogs with related bioactivity profiles as both mimic adenosine and can inhibit some of the processes that are adenosine dependent. Both COR and PTN are also natural products and were originally isolated from the fungus Cordyceps militaris and the bacterium Streptomyces antibioticus, respectively. Here, we report that not only is PTN produced by C. militaris but that biosynthesis of COR is coupled with PTN production by a single gene cluster. We also demonstrate that this coupling is an important point of metabolic regulation where PTN safeguards COR from deamination by inhibiting adenosine deaminase (ADA) activity. ADA is not inhibited until COR reaches self-toxic levels, at which point ADA derepression occurs allowing for detoxification of COR to 3'-deoxyinosine. Finally, we show that using our biosynthetic insights, we can engineer C. militaris to produce higher levels of COR and PTN.

Omics data reveal the unusual asexual-fruiting nature and secondary metabolic potentials of the medicinal fungus Cordyceps cicadae.

BACKGROUND: Ascomycete Cordyceps species have been using as valued traditional Chinese medicines. Particularly, the fruiting bodies of Cordyceps cicadae (syn. Isaria cicadae) have long been utilized for the treatment of chronic kidney disease. However, the genetics and bioactive chemicals in this fungus have been largely unexplored. RESULTS: In this study, we performed comprehensive omics analyses of C. cicadae, and found that, in contrast to other Cordyceps fungi, C. cicadae produces asexual fruiting bodies with the production of conidial spores instead of the meiotic ascospores. Genome sequencing and comparative genomic analysis indicate that the protein families encoded by C. cicadae are typical of entomopathogenic fungi, including the expansion of proteases and chitinases for targeting insect hosts. Interestingly, we found that the MAT1-2 mating-type locus of the sequenced strain contains an abnormally truncated MAT1-1-1 gene. Gene deletions revealed that asexual fruiting of C. cicadae is independent of the MAT locus control. RNA-seq transcriptome data also indicate that, compared to growth in a liquid culture, the putative genes involved in mating and meiosis processes were not up-regulated during fungal fruiting, further supporting asexual reproduction in this fungus. The genome of C. cicadae encodes an array of conservative and divergent gene clusters for secondary metabolisms. Based on our analysis, the production of known carcinogenic metabolites by this fungus could be potentially precluded. However, the confirmed production of oosporein raises health concerns about the frequent consumption of fungal fruiting bodies. CONCLUSIONS: The results of this study expand our knowledge of fungal genetics that asexual fruiting can occur independent of the MAT locus control. The obtained genomic and metabolomic data will benefit future investigations of this fungus for medicinal uses.

Transumbilical single-incision laparoscopic vaginoplasty hybrid transperineal approach using a sigmoid colon segment: initial twenty-five cases.

OBJECTIVE: To validate the technical feasibility and anatomical and functional outcomes of transumbilical single-incision laparoscopic (SIL) sigmoid vaginoplasty hybrid transperineal approach in women with Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome. METHODS: Twenty-five patients who underwent transumbilical SIL sigmoid vaginoplasty hybrid transperineal approach were retrospectively evaluated. Operation time and postoperative complications were analyzed. The length and breadth of the neovagina, lubrication and the Female Sexual Function Index were evaluated to assess the anatomical and functional outcomes of the surgery. RESULTS: All the cases were successfully performed without any intraoperative morbidity. The mean operative time and postoperative hospital stay were 162 ± 38 min and 11 ± 3 days, respectively. The postoperative complications included one case of a stress ulcer, one case of a neovaginal ulcer and two cases of umbilical incision infection. Sixteen women had regular intercourse and were satisfied with both the surgical outcome and subsequent sexual activity. CONCLUSION: Transumbilical SIL sigmoid vaginoplasty hybrid transperineal approach offers a feasible scarless method with cosmetic advantage for women with MRKH syndrome to establish new functional vagina.

Functional convergence and divergence of mating-type genes fulfilling in Cordyceps militaris.

Fungal sexual lives are considerably diversified in terms of the types of mating systems and mating-control gene functions. Sexual fruiting bodies of the ascomycete fungus Cordyceps militaris have been widely consumed as edible and medicinal mushrooms, whereas the regulation of fruiting-body development and sex in this fungus remain elusive. Herein, we performed the comprehensive functional analyses of mating-type (MAT) genes in C. militaris. Interspecies functional convergence was evident that MAT1-1 and MAT1-2-1 null mutants were sterile and lost the ability to produce stromata in outcrosses with the opposite mating-type partner. In contrast to other fungal species, functional divergence of MAT1-1-1 and MAT1-1-2 was also observed that ΔMAT1-1-1 produced barren stromata in outcrosses, whereas ΔMAT1-1-2 generated fruiting bodies morphologically similar to that of the parental strain but with sterile perithecia. The homothallic-like transformants MAT1-2::MAT1-1-1 (haploidic MAT1-2 isolate transformed with the MAT1-1-1 gene) produced sterile stromata, whereas the MAT1-1::MAT1-2-1 (haploidic MAT1-1 isolate transformed with the MAT1-2-1 gene) mutant was determined to be completely fruitless. The findings relating to the fully fertile gene-complementation mutants suggest that the genomic location is not essential for the MAT genes to fulfill their functions in C. militaris. Comparison of the production of bioactive constituents cordycepin and adenosine provides experimental support that the fungal sexual cycle is an energy consuming process. The results of the present study enrich our knowledge of both convergent and divergent controls of fungal sex.

Transgenic plants expressing the AaIT/GNA fusion protein show increased resistance and toxicity to both chewing and sucking pests.

The adoption of pest-resistant transgenic plants to reduce yield losses and decrease pesticide use has been successful. To achieve the goal of controlling both chewing and sucking pests in a given transgenic plant, we generated transgenic tobacco, Arabidopsis, and rice plants expressing the fusion protein, AaIT/GNA, in which an insecticidal scorpion venom neurotoxin (Androctonus australis toxin, AaIT) is fused to snowdrop lectin (Galanthus nivalis agglutinin, GNA). Compared with transgenic tobacco and Arabidopsis plants expressing AaIT or GNA, transgenic plants expressing AaIT/GNA exhibited increased resistance and toxicity to one chewing pest, the cotton bollworm, Helicoverpa armigera. Transgenic tobacco and rice plants expressing AaIT/GNA showed increased resistance and toxicity to two sucking pests, the whitefly, Bemisia tabaci, and the rice brown planthopper, Nilaparvata lugens, respectively. Moreover, in the field, transgenic rice plants expressing AaIT/GNA exhibited a significant improvement in grain yield when infested with N. lugens. This study shows that expressing the AaIT/GNA fusion protein in transgenic plants can be a useful approach for controlling pests, particularly sucking pests which are not susceptible to the toxin in Bt crops.

Comparative genomics and transcriptomics analyses reveal divergent lifestyle features of nematode endoparasitic fungus Hirsutella minnesotensis.

Hirsutella minnesotensis [Ophiocordycipitaceae (Hypocreales, Ascomycota)] is a dominant endoparasitic fungus by using conidia that adhere to and penetrate the secondary stage juveniles of soybean cyst nematode. Its genome was de novo sequenced and compared with five entomopathogenic fungi in the Hypocreales and three nematode-trapping fungi in the Orbiliales (Ascomycota). The genome of H. minnesotensis is 51.4 Mb and encodes 12,702 genes enriched with transposable elements up to 32%. Phylogenomic analysis revealed that H. minnesotensis was diverged from entomopathogenic fungi in Hypocreales. Genome of H. minnesotensis is similar to those of entomopathogenic fungi to have fewer genes encoding lectins for adhesion and glycoside hydrolases for cellulose degradation, but is different from those of nematode-trapping fungi to possess more genes for protein degradation, signal transduction, and secondary metabolism. Those results indicate that H. minnesotensis has evolved different mechanism for nematode endoparasitism compared with nematode-trapping fungi. Transcriptomics analyses for the time-scale parasitism revealed the upregulations of lectins, secreted proteases and the genes for biosynthesis of secondary metabolites that could be putatively involved in host surface adhesion, cuticle degradation, and host manipulation. Genome and transcriptome analyses provided comprehensive understanding of the evolution and lifestyle of nematode endoparasitism.

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.

Hindgut innate immunity and regulation of fecal microbiota through melanization in insects.

Many insects eat the green leaves of plants but excrete black feces in an as yet unknown mechanism. Insects cannot avoid ingesting pathogens with food that will be specifically detected by the midgut immune system. However, just as in mammals, many pathogens can still escape the insect midgut immune system and arrive in the hindgut, where they are excreted out with the feces. Here we show that the melanization of hindgut content induced by prophenoloxidase, a key enzyme that induces the production of melanin around invaders and at wound sites, is the last line of immune defense to clear bacteria before feces excretion. We used the silkworm Bombyx mori as a model and found that prophenoloxidase produced by hindgut cells is secreted into the hindgut contents. Several experiments were done to clearly demonstrate that the blackening of the insect feces was due to activated phenoloxidase, which served to regulate the number of bacteria in the hindgut. Our analysis of the silkworm hindgut prophenoloxidase discloses the natural secret of why the phytophagous insect feces is black and provides insight into hindgut innate immunity, which is still rather unclear in mammals.

Insertion of an esterase gene into a specific locust pathogen (Metarhizium acridum) enables it to infect caterpillars.

An enduring theme in pathogenic microbiology is poor understanding of the mechanisms of host specificity. Metarhizium is a cosmopolitan genus of invertebrate pathogens that contains generalist species with broad host ranges such as M. robertsii (formerly known as M. anisopliae var. anisopliae) as well as specialists such as the acridid-specific grasshopper pathogen M. acridum. During growth on caterpillar (Manduca sexta) cuticle, M. robertsii up-regulates a gene (Mest1) that is absent in M. acridum and most other fungi. Disrupting M. robertsii Mest1 reduced virulence and overexpression increased virulence to caterpillars (Galleria mellonella and M. sexta), while virulence to grasshoppers (Melanoplus femurrubrum) was unaffected. When Mest1 was transferred to M. acridum under control of its native M. robertsii promoter, the transformants killed and colonized caterpillars in a similar fashion to M. robertsii. MEST1 localized exclusively to lipid droplets in M. robertsii conidia and infection structures was up-regulated during nutrient deprivation and had esterase activity against lipids with short chain fatty acids. The mobilization of stored lipids was delayed in the Mest1 disruptant mutant. Overall, our results suggest that expression of Mest1 allows rapid hydrolysis of stored lipids, and promotes germination and infection structure formation by M. robertsii during nutrient deprivation and invasion, while Mest1 expression in M. acridum broadens its host range by bypassing the regulatory signals found on natural hosts that trigger the mobilization of endogenous nutrient reserves. This study suggests that speciation in an insect pathogen could potentially be driven by host shifts resulting from changes in a single gene.

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 heterogeneous 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.

A phosphoketolase Mpk1 of bacterial origin is adaptively required for full virulence in the insect-pathogenic fungus Metarhizium anisopliae.

Pentose metabolism through the phosphoketolase pathway has been well characterized in bacteria. In this paper, we report the identification of a phosphoketolase homologue Mpk1 in the insect-pathogenic fungus Metarhizium anisopliae. Phylogenetic analysis showed that fungal phosphoketolases are of bacterial origin and diverged into two superfamilies. Frequent gene loss or lack of acquisition is evident in specific fungal lineages or species. The mpk1 gene is highly expressed when grown in trehalose-rich insect haemolymph but poorly induced by insect cuticle or carbohydrate-rich plant root exudate. In addition, mpk1 gene expression and enzyme activity could be upregulated by different sugars including xylose, trehalose, glucose or sucrose. mpk1 null mutants generated by homologous recombination grew similar to the wild type of M. anisopliae on medium amended with xylose as a sole carbon source. However, insect (tobacco hornworm, Manduca sexta) bioassays showed significantly reduced virulence in Deltampk1. The results of this study suggest that the horizontally transferred Mpk1 in M. anisopliae plays an important niche adaptation role for fungal propagation in insect haemocoel. Following the carbohydrate flux from plants to plant-feeding insects and insect pathogenic fungi, a tritrophic relationship is discussed in association with the requirement of fungal phosphoketolase pathway.

Differentially expressed genes in resistant and susceptible Bombyx mori strains infected with a densonucleosis virus.

We investigated variations in the gene expression of Bombyx mori following infection with a densonucleosis virus (BmDNV-Z). Two B. mori near-isogenic lines, Jingsong and Jingsong.nsd-Z.NIL, which are highly susceptible and completely resistant to BmDNV-Z, respectively, were used in this study. The infection profiles of BmDNV-Z in the midguts of the B. mori Jingsong and Jingsong.nsd-Z.NIL larvae revealed that the virus invaded the midguts of both of these strains. However, its proliferation was notably inhibited in the midgut of the resistant strain. By using the suppression subtractive hybridization method, three cDNA libraries were constructed to compare BmDNV-Z responsive gene expression between the two silkworm lines. In total, 151 differentially expressed genes were obtained. Real-time qPCR analysis confirmed that 11 genes were significantly up-regulated in the midgut of the Jingsong.nsd-Z.NIL strain following BmDNV-Z infection. Our results imply that these up-regulated genes might be involved in B. mori immune responses against BmDNV infection.

MOS1 osmosensor of Metarhizium anisopliae is required for adaptation to insect host hemolymph.

Entomopathogenic fungi such as Metarhizium anisopliae infect insects by direct penetration of the cuticle, after which the fungus adapts to the high osmotic pressure of the hemolymph and multiplies. Here we characterize the M. anisopliae Mos1 gene and demonstrate that it encodes the osmosensor required for this process. MOS1 contains transmembrane regions and a C-terminal Src homology 3 domain similar to those of yeast osmotic adaptor proteins, and homologs of MOS1 are widely distributed in the fungal kingdom. Reverse transcription-PCR demonstrated that Mos1 is up-regulated in insect hemolymph as well as artificial media with high osmotic pressure. Transformants containing an antisense vector directed to the Mos1 mRNA depleted transcript levels by 80%. This produced selective alterations in regulation of genes involved in hyphal body formation, cell membrane stiffness, and generation of intracellular turgor pressure, suggesting that these processes are mediated by MOS1. Consistent with a role in stress responses, transcript depletion of Mos1 increased sensitivity to osmotic and oxidative stresses and to compounds that interfere with cell wall biosynthesis. It also disrupted developmental processes, including formation of appressoria and hyphal bodies. Insect bioassays confirmed that Mos1 knockdown significantly reduces virulence. Overall, our data show that M. anisopliae MOS1 mediates cellular responses to high osmotic pressure and subsequent adaptations to colonize host hemolymph.

A scorpion neurotoxin increases the potency of a fungal insecticide.

The low virulence of the insecticidal fungus Metarhizium anisopliae has stymied its widespread use in controlling insect pests. We show that high-level expression of an insect-specific neurotoxin from the scorpion Androctonus australis in hemolymph by M. anisopliae increases fungal toxicity 22-fold against tobacco hornworm (Manduca sexta) caterpillars and ninefold against adult yellow fever mosquitoes (Aedes aegypti) without compromising host specificity. Prelethal effects include reduced mobility and feeding of the insects targeted.

A collagenous protective coat enables Metarhizium anisopliae to evade insect immune responses.

The ubiquitous fungal pathogen Metarhizium anisopliae kills a wide range of insects. Host hemocytes can recognize and ingest its conidia, but this capacity is lost on production of hyphal bodies. We show that the unusual ability of hyphal bodies to avoid detection depends on a gene (Mcl1) that is expressed within 20 min of the pathogen contacting hemolymph. A mutant disrupted in Mcl1 is rapidly attacked by hemocytes and shows a corresponding reduction of virulence to Manduca sexta. Mcl1 encodes a three domain protein comprising a hydrophilic, negatively charged N-terminal region with 14 cysteine residues, a central region comprising tandem repeats (GXY) characteristic of collagenous domains, and a C-terminal region that includes a glycosylphosphatidylinositol-dependent cell wall attachment site. Immunofluorescence assay showed that hyphal bodies are covered by the N-terminal domains of MCL1. The collagen domain became antibody accessible after treatment with DTT, suggesting that the N termini are linked by interchain disulfide bonds and are presented on the cell surface by extended collagenous fibers. Studies with staining reagents and hemocyte monolayers showed that MCL1 functions as an antiadhesive protective coat because it masks antigenic structural components of the cell wall such as beta-glucans, and because its hydrophilic negatively charged nature makes it unattractive to hemocytes. A survey of 54 fungal genomes revealed that seven other species have proteins with collagenous domains suggesting that MCL1 is a member of a patchily distributed gene family.