Chengshu Wang

Shanghai Institute of Plant Physiology and Ecology

The requirement of macroautophagic/autophagic machinery for filamentous fungal development and pathogenicity has been recognized, but the underlying effects and mechanisms remain elusive. The insect pathogenic fungus Metarhizium robertsii infects hosts by cuticular penetration through the formation of the infection structure appressoria. Here, we s...

In contrast to the well-characterized gut microbiomes, the composition and function of the insect body-surface microbiotas are still elusive and highly underexplored. Here we report the dynamic features of the Drosophila melanogaster surface microbiomes. It was found that the microbiomes assembled on fly surfaces could defend insects against fungal...

Emerging evidence has shown that the plant and animal surface microbiomes can defend hosts against fungal parasite infections. The strategies employed by fungal pathogens to combat the antagonistic inhibition of insect surface bacteria are still elusive. In this study, we found that the potent antibiotic helvolic acid (HA) produced by the insect pa...

Insects can assemble defensive microbiomes on their body surfaces to defend against fungal parasitic infections. The strategies employed by fungal pathogens to combat host cuticular microbiotas remains unclear. Here, we report the identification and functional characterization of the defensin-like antimicrobial gene BbAMP1 encoded by the entomopath...

In addition to innate immunity in a physiological context, insects have evolved behavioral defenses against parasite attacks. Here, we report that Drosophila can sense the CFEM (common in fungal extracellular membrane) protein Mcdc9, which acts as a negative virulence factor of the entomopathogenic fungus Metarhizium robertsii. The individual delet...

The ascomycete insect pathogenic fungi such as Metarhizium species have been demonstrated with the abilities to form the rhizosphere or endophytic relationships with different plants for nutrient exchanges. In this study, after the evident infeasibility of bacterial disease development in the boxed sterile soils, we established a hydroponic system...

Purpose of Review Entomopathogenic fungi (EPF) play critical roles in regulating insect populations in nature, and some species of these fungi can cause diseases in immune-compromised humans. It is of biological and clinical importance to grasp the knowledge of EPF virulence control by sensing host and environment. Recent Findings This review summ...

Fungal chemical ecology is largely mediated by the metabolite(s) produced by individual biosynthetic gene clusters (BGCs) with antibiotic activities. We report a supercluster containing three BGCs that are jointly controlled by an embedded master regulator in the insect pathogen Metarhizium robertsii .

The genome of entomopathogenic fungus Tolypocladium inflatum Gams encodes 43 putative biosynthetic gene clusters for specialized metabolites, although genotype-phenotype linkages have been reported only for the cyclosporins and fumonisins. T. inflatum was cultured in defined minimal media, supplemented with or without one of nine different amino ac...

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 .

The exopolysaccharide galactosaminogalactan (GAG) has been well characterized in Aspergilli, especially the human pathogen Aspergillus fumigatus . It has been found that a five-gene cluster is responsible for GAG biosynthesis in Aspergilli to mediate fungal adherence, biofilm formation, immunosuppression or induction of host immune defences. Herein...

Fungal entomopathogens are largely facultative parasites and play an important role in controlling the density of insect populations in nature. A few species of these fungi have been used for biocontrol of insect pests. The pattern of the entomopathogen competition for insect individuals is still elusive. Here, we report the empirical competition f...

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. MrM...

The Woronin body (WB) is a peroxisome-derived dense-core vesicle, a self-assembling hexagonal crystal of a single protein Hex1. This organelle is specific to the ascomycete fungi belonging to the Pezizomycotina subphylum by functioning in sealing septal pores in response to mycelium damage and the control of cell heterogeneity. We retrieved all ava...

Fungal chemotaxonomy is an approach to classify fungi based on the fungal production profile of metabolites, especially the secondary metabolites. We found an atypical example that could question the reliability of fungal chemical classifications in this study, i.e., the more closely related entomopathogenic species Beauveria bassiana and Beauveria...

Amid the genomic data explosion, phylogenomic analysis has resolved the tree of life of different organisms, including fungi. Genome-wide clustering has also been conducted based on gene content data that can lighten the issue of the unequal evolutionary rate of genes. In this study, using different fungal species as models, we performed phylogenom...

The indolizidine alkaloid swainsonine (SW) is a deadly mycotoxin to livestock that can be produced by different plant-associated fungi including the endophytic entomopathogenic fungi Metarhizium species. The SW biosynthetic gene cluster has been identified but the genetic mechanism of SW biosynthesis remains obscure. To unveil the SW biosynthetic p...

Fungal G-protein coupled receptors (GPCRs) play essential roles in sensing environmental cues including host signals. The study of GPCR in mediating fungus-insect interactions is still limited. Here we report the evolution of GPCR genes encoded in the entomopathogenic Metarhizium species and found the expansion of Pth11-like GPCRs in the generalist...

Fungal chemodiversity is well known in part due to the production of diverse analogous compounds by a single biosynthetic gene cluster (BGCs). Usually, similar metabolites are produced by closely related fungal species. Here we report a rare case of the production of the cyclodepsipeptide beauveriolides (BVDs) in three insect pathogenic fungi. We f...

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...

Metarhizium is a group of insect-pathogenic fungi that can produce insecticidal metabolites, such as destruxins. Interestingly, the acridid-specific fungus Metarhizium acridum (MAC) can kill locusts faster than the generalist fungus Metarhizium robertsii (MAA) even without destruxin. However, the underlying mechanisms of different pathogenesis betw...

Entomopathogenic fungi are one of the key regulators of insect populations in nature. Some species such as Beauveria bassiana with a wide host range have been developed as promising alternatives to chemical insecticides for the biocontrol of insect pests. However, the long-term persistence of the released strains, the effect on non-target hosts and...

Many prokaryotic and eukaryotic proteins contain domains of unknown function (DUFs). A DUF3129 family of proteins is widely encoded in the genomes of fungal pathogens. A few studies in plant and insect pathogens indicated that the DUF3129 genes are required for fungal penetration of host cuticles with an unclear mechanism. We found that a varied nu...

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 an...

The cycloundecapeptide cyclosporin A (CsA) was first isolated from the insect-pathogenic fungus Tolypocladium inflatum for its antifungal activity and later developed as an immunosuppressant drug. However, the full biosynthetic mechanism of CsA remains unknown and has puzzled researchers for decades. In this study, the biosynthetic gene cluster is...

The ecological importance of the duplication and diversification of gene clusters that synthesize secondary metabolites in fungi remains poorly understood. Here, we demonstrated that the duplication and subsequent diversification of a gene cluster produced two polyketide synthase gene clusters in the cosmopolitan fungal genus Metarhizium. Diversifi...

Assays of appressoria of WT (the wild-type strain of M. robertsii) and three independent isolates (ΔPks2 #1, ΔPks2 #2, and ΔPks2 #3) of M. robertsii’s Pks2 KO mutant ΔPks2. (A) Collapse rates of appressoria in the PEG8000 solution [80% (w/v)]. The assays were repeated three times with three replicates per repeat. Data are expressed as the mean ± SE...

1H NMR spectrum of Compound I (shown in Fig 8) in DMSO-d6. (PDF)

Pks2 cannot complement the Pks1 KO mutant (ΔPks1) in M. robertsii. (A) qRT-PCR confirmation of overexpression of Pks2 in ΔPks1. ΔPks1-Pks2OE: ΔPks1 with Pks2 overexpressed. 1, 2, 3: three independent isolates of ΔPks1-Pks2OE. The expression level in ΔPks1 is set to 1. Values with different letters are significantly different (P < 0.05, Tukey’s test...

Relative germination rates of three independent isolates of the M. robertsii Pks2 KO mutant under three abiotic stresses. All assays were repeated three times with three replicates per repeat. Within each row (strains), values appended by different letters are significantly different (P < 0.05, Tukey’s test in One-way ANOVA). (PDF)

Primers used in this study. (PDF)

The .nwk files for the CONSEL assays. (ZIP)

Conidial pigmentation of WT and three independent isolates of the Pks2 KO (knock out) mutant in M. robertsii. Scale bars represent 10 mm. Note: no difference in conidial pigments in seen between the three independent isolates of the mutant and the WT. (PDF)

13C NMR spectrum of Compound I (shown in Fig 8) in DMSO-d6. (PDF)

NMR spectroscopic data for Compound I (1-acetyl-2,4,6,8-tetrahydroxy-9,10-anthraquinone) in DMSO-d6. (PDF)

Plasmids and fungal strains used in this study. (PDF)

Phylogenetic analysis of domains in the PKSs analyzed in Fig 1. (A) AT domain; (B) PP-binding domain, (C) PS-DH domain, (D) TE domain. The Genbank accession number for each full-length amino acid sequence of each PKS is shown after the species name. The clade containing Metarhizium PKS1s is highlighted in pink, PKS2s highlighted in red. Numbers at...

The clades (a to e) assigned for comparison of topologies of alternative (constrained) trees with the obtained tree. Shown is the obtained tree (Fig 1A). The clade a in pink is the PKS1 clade, and the clade c in red is the PKS2 clade. The results of the topology comparison are shown in S2 Table. (PDF)

Estimation of gene duplication and loss events of the Pks genes in the fungal species shown in Fig 1A by reconciling the raw Pks gene’s ML tree (Fig 1A) with the species tree (S4 Fig) using NOTUNG with a duplication-loss (DL) model (1.5 for a duplication, 0.0 for a conditional duplication and 1.0 for a loss.). Red circles indicate duplication event...

Phylogenetic analysis of KS domains in the PKSs analyzed in Fig 1A and additional 18 PKSs in M. robertsii. The Genbank accession number for each full-length amino acid sequence of each PKS is shown after the species name. The clade containing Metarhizium’s PKS1s is highlighted in pink, PKS2s highlighted in red and additional M. robertsii PKSs in bl...

Ka/Ks values of each Pks1 and Pks2 gene in Metarhizium species. (A) Pks1s. (B) Pks2s. (PDF)

RT-PCR confirmation of expression of M. robertsii’s Pks2 (Left panel) and Arp1 (Right panel) in M. album. M: DNA ladder (Genray, Shanghai); 1: The wild-type M. album; 2, 3, 4: three independent M. album transformants expressing M. robertsii’s Pks2 and Arp1. (PDF)

Confirmation of the insertions of M. robertsii’s Pks1 and Pks2 into the genome of A. nidulans strain LO8030. PCR reactions were conducted with primers MAA_Pks1_RT_F/R for Pks1 and MAA_Pks2_RT_F/R for Pks2 (see S9 Table for information about the primers). T1 to T5 represents five independent transformants expressing Pks1 or Pks2; CK: the positive co...

Manual annotation of the Pks1 gene in M. acridum and M. album. (A) A schematic diagram showing the reannotation of the Pks1 genes in M. acridum and M. album. Wrong annotation (the boxed area) of the genomic region (MAA_05385 in M. acridum; MAM_08215 in M. album) corresponding to EthD and PKS1 by NCBI is shown at the bottom; note: a gene containing...

Species tree of the 31 fungal species analyzed in Fig 1. The species tree was constructed using the concatenated alignment (S2 Dataset) of the 20 best scoring single-copy genes (S8 Table). Numbers at nodes represent the bootstrap values of Maximum Likelihood (left), Neighbor-Joining (middle), and the Bayesian posterior probabilities (right). A hyph...

Estimation of gene duplication and loss events of the Pks genes in the fungal species shown in Fig 1A by reconciling the rearranged Pks gene ML tree (Fig 2A) with the species tree (S4 Fig) using NOTUNG with a duplication-loss (DL) model (1.5 for a duplication, 0.0 for a conditional duplication and 1.0 for a loss.). Red circles indicate duplication...

Phylogenetic analyses of Arp1 (A), Arp2 (B) and Abr2 (C) in Metarhizium and other fungi that have gene clusters similar to Pks1-gc or Pks2-gc. Numbers at nodes represent bootstrap values of Maximum Likelihood (left), Neighbor-Joining (middle) and Bayesian posterior probabilities (right). Hyphen (-) indicates no support value in the corresponding me...

Construction of knockout (KO) or knockdown (KD) mutants of Pks1 and Pks2 genes in Metarhizium. (A) A schematic diagram of gene disruption based on homologous recombination showing a map of a disruption plasmid and its relative position in the fungal genome. Note: only around the region (~1.2kb) corresponding to the N-terminus of a Pks gene was dele...

Conidial pigmentation of two of three independent isolates of a Pks1 gene’s KD (Knock down) or KO (knock out) mutant. One of the three isolates for each mutant is shown in Fig 6. Scale bars represent 1 mm in M. album and 10 mm in M. anisopliae, M. brunneum, M. guizhouense, M. majus, M. acridum and M. album. (PDF)

LC-MS analysis of Compound I (shown in Fig 8) from the A. nidulans transformant expressing Pks1. This figure is supplemental to Fig 8C. Molecular weight of Compound I was detected by LC-MS analysis at m/z 315 [M+H]+, and 651 [M+Na]+. (PDF)

Information about proteins and their domain structures used in the analyses conducted in Fig 1. (PDF)

Statistics of comparison of topologies of the constrained trees (S3 Fig) with the obtained tree (Fig 1). (PDF)

GT50 (Time taken for 50% of conidia to germinate) values of three independent isolates of a Pks1 mutant in seven Metarhizium species and their respective wild-type strains (WT) under optimal conditions (grown at 26°C in 1/2 SDY). Within each row (species), values appended by different letters are significantly different (P < 0.05, Tukey’s test in O...

Relative germination rates of two of three independent isolates (#2 and #3) of a Pks1 mutant in seven Metarhizium species under three abiotic stresses. All assays were repeated three times with three replicates per repeat. (PDF)

Genbank accession numbers of the 20 genes in the 31 fungal species shown in Fig 1A for constructing a species phylogenetic tree (shown in S4 Fig). (PDF)

The concatenated alignment of 20 genes for constructing species tree. (FASTA)

NOTUNG assays using DL or DTL model with different parameter combinations. (PDF)

Alignment of PKS domains. (PDF)

Logos and sequences of overrepresented motifs in the promoters of the Pks1 and Pks2 genes in Metarhizium. (PDF)

Nitrogen starvation can induce cellular triacylglycerol (TAG) accumulation in different organisms with an unclear mechanism. In this study, we performed nutrient starvation and lipid droplet (LD) proteomics analyses of the filamentous fungus Metarhizium robertsii. Our results indicated that nitrogen starvation activated cell autophagic activity but...

Hypocrealean fungi (Ascomycota) are known for their diversity of lifestyles. Their vital influences on agricultural and natural ecosystems have resulted in a number of sequenced genomes, which provide essential data for genomic analysis. Totally, 45 hypocrealean fungal genomes constructed a phylogeny. The phylogeny showed that plant pathogens in Ne...

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 Met...

Skin immunity protects animals from airborne pathogen infection. Unlike mammals, arthropods, including insects, undergo periodic ecdysis to grow and develop. Newly molted insects emerge with unsclerotized thin cuticles but successfully escape pathogenic infections during the post-molt period. Here we show that prophenoloxidases (PPOs) in molting fl...

The topic of ‘fungal stress’ is central to a myriad of important disciplines, including medical mycology, chronobiology, plant and insect pathology, industrial microbiology, material sciences, and astrobiology. The International Symposium on Fungal Stress (ISFUS) brought together researchers, who study fungal stress in a variety of fields. The seco...

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, respective...

The lysin motif (LysM) containing proteins can bind chitin and are ubiquitous in various organisms including fungi. In plant pathogenic fungi, a few LysM proteins have been characterized as effectors to suppress chitin-induced immunity in plant hosts and therefore contribute to fungal virulence. The effector mechanism is still questioned in fungus-...

LysM domain-containing proteins identified from 13 species of entomopathogenic fungi. (XLSX)

LysM domain-containing proteins identified from the selected plant and mammalian pathogenic fungi. (XLSX)