Conidial Hydrophobicity Research Articles

Light enhances the production of conidia and influences their hydrophobicity in Tolypocladium inflatum

Insect fungal pathogens such as Beauveria bassiana, Metarhizium robertsii, and Tolypocladium inflatum have been used as insect biocontrol agents. Their infection mechanism involves non-specific adhesion to the host cuticle, which is controlled by hydrophobins, small proteins that form an amphipathic monolayer with rodlet morphology on diverse fungal structures. Light is an abiotic factor that may influence a wide range of cellular processes, including conidiogenesis, stress tolerance, and metabolite biosynthesis. Although its effects have been studied in many fungi, little is known about the effects of light on the hydrophobic properties of conidia. The aim of this work was to investigate the influence of visible light on the conidial hydrophobicity of three entomopathogenic fungal species. For this, conidia of B. bassiana, M. robertsii, and T. inflatum were grown either under light or in the dark, drop profiles of water and diiodomethane on conidial surfaces were analyzed, and conidial hydrophobicity was estimated from contact angle measurements. Moreover, conidial production was determined, and their genome was screened with sequences for hydrophobins. Conidia of B. bassiana and M. robertsii are more hydrophobic than conidia of T. inflatum. The light modified the surface tension of T. inflatum; therefore, conidia of T. inflatum became hydrophilic. However, light did not affect the conidial hydrophobicity of B. bassiana and M. robertsii. In addition, light modified the conidial production of B. bassiana and T. inflatum cultures, but it had no effect on the conidial production of M. robertsii. The T. inflatum genome contains two predicted proteins whose sequence is akin to that of proven class II hydrophobins from other ascomycetes. Presumably, these proteins are responsible for the conidial hydrophobicity properties in this fungus. Our study helps elucidate how light affects the conidial hydrophobicity of entomopathogenic fungi.

Protein disulfide isomerase 1 is required for RodA assembling-based conidial hydrophobicity of Aspergillus fumigatus.

The hydrophobic layer of Aspergillus conidia, composed of RodA, plays a crucial role in conidia transfer and immune evasion. It self-assembles into hydrophobic rodlets through intramolecular disulfide bonds. However, the secretory process of RodA and its regulatory elements remain unknown. Since protein disulfide isomerase (PDI) is essential for the secretion of many disulfide-bonded proteins, we investigated whether PDI is also involved in RodA secretion and assembly. By gene knockout and phenotypic analysis, we found that Pdi1, one of the four PDI-related proteins of Aspergillus fumigatus, determines the hydrophobicity and integrity of the rodlet layer of the conidia. Preservation of the thioredoxin-active domain of Pdi1 was sufficient to maintain conidial hydrophobicity, suggesting that Pdi1 mediates RodA assembly through its disulfide isomerase activity. In the absence of Pdi1, the disulfide mismatch of RodA in conidia may prevent its delivery from the inner to the outer layer of the cell wall for rodlet assembly. This was demonstrated using a strain expressing a key cysteine-mutated RodA. The dormant conidia of the Pdi1-deficient strain (Δpdi) elicited an immune response, suggesting that the defective conidia surface in the absence of Pdi1 exposes internal immunogenic sources. In conclusion, Pdi1 ensures the correct folding of RodA in the inner layer of conidia, facilitating its secretion into the outer layer of the cell wall and allowing self-assembly of the hydrophobic layer. This study has identified a regulatory element for conidia rodlet assembly.IMPORTANCEAspergillus fumigatus is the major cause of invasive aspergillosis, which is mainly transmitted by the inhalation of conidia. The spread of conidia is largely dependent on their hydrophobicity, which is primarily attributed to the self-assembly of the hydrophobic protein RodA on the cell wall. However, the mechanisms underlying RodA secretion and transport to the outermost layer of the cell wall are still unclear. Our study identified a critical role for Pdi1, a fungal protein disulfide isomerase found in regulating RodA secretion and assembly. Inhibition of Pdi1 prevents the formation of correct S-S bonds in the inner RodA, creating a barrier to RodA delivery and resulting in a defective hydrophobic layer. Our findings provided insight into the formation of the conidial hydrophobic layer and suggested potential drug targets to inhibit A. fumigatus infections by limiting conidial dispersal and altering their immune inertia.

Pathogenicity analysis and comparative genomics reveal the different infection strategies between the generalist Metarhizium anisopliae and the specialist Metarhizium acridum.

The fungal genera Metarhizium contain many important multiple species that are used as biocontrol agents and as model organisms for exploring insect-fungal interactions. Metarhizium spp. exhibit different traits of pathogenicity, suggesting that the pathogenesis can be quite distinctive. However, the underlying differences in their pathogenesis remain poorly understood. Pathogenicity analysis showed that Metarhizium anisopliae (strain CQMa421) displayed higher virulence against oriental migratory locusts, Locusta migratoria manilensis (Meyen), than the acridid-specific specie Metarhizium acridum (strain CQMa102). Relative to M. acridum, M. anisopliae possessed a higher conidial hydrophobicity, increased ability to penetrate the host, accelerated growth under hypoxia and enhanced ability for the utilization of different carbon sources. Different distributions of carbohydrate epitopes at cell wall surface of M. anisopliae might also contribute to successful evasion of host immune defenses. Comparative genomics showed that M. anisopliae has 98 more virulence-related secreted proteins (133) than M. acridum (35), which can be functionally classified as hydrolases, virulence effectors, cell wall degradation and stress tolerance-related proteins, and helpful to the cuticle penetration and host internal environment adaption. In addition, differences in genomic clusters specifically related to secondary metabolites, including the clusters of Indole-NRPS hybrid, T1PKS-NRPS like hybrid, Betalactone, Fungal-Ripp and NRPS-Terpene hybrid, may lead to differences in core virulence-related secondary metabolite genes in M. acridum (18) and M. anisopliae (36). The comparative study provided new insights into the different infection strategies between M. anisopliae and M. acridum, and further facilitate the identification of virulence-related genes for the improvement of mycoinsecticides. © 2023 Society of Chemical Industry.

Only one of three hydrophobins (Hyd1–3) contributes to conidial hydrophobicity and insect pathogenicity of Metarhizium robertsii

Class I/II hydrophobins constitute a family of small amphiphilic proteins that mediate cell hydrophobicity and adhesion to host or substrata and have pleiotropic effects in filamentous fungi. Here we report that only class I Hyd1 is essential for conidial hydrophobicity and insect pathogenicity among three hydrophobins (Hyd1–3) characterized in Metarhizium robertsii, an insect-pathogenic fungus. Aerial conidiation levels of three Δhyd1 mutants were much more reduced in 5-day-old cultures than in 7-day-old cultures, which were wettable (hydrophilic), but restored to a wild-type level in 15-day-old cultures. The Δhyd1 mutants were compromised in conidial quality, including significant decreases in hydrophobicity (58%), adhesion to insect cuticle (36%), insect pathogenicity via normal cuticle infection (37%), UVB resistance (20%), and heat tolerance (10%). In contrast, none of all examined phenotypes were affected in the null mutants of hyd2 and hyd3. Intriguingly, micromorphology and integrity of hydrophobin rodlet bundles on conidial coat were not affected in all mutant and wild-type strains, but the rodlet bundles were disordered in the absence of hyd1, suggesting a link of the disorder to the decreased hydrophobicity. Therefore, Hyd1 mediates the fungal hydrophobicity and plays an important role in conidial quality control and insect-pathogenic lifecycle. Class I Hyd2 and class II Hyd3 seem functionally redundant in M. robertsii.

Functional identification of the DHN melanin synthesis gene cluster and its role in UV-C tolerance in citrus postharvest pathogenic fungus Penicillium digitatum

The filamentous fungus Penicillium digitatum brings out great losses in citrus fruits by causing citrus green mold disease during the postharvest period. Previously, we obtained a T-DNA insertion mutant N2130 of P. digitatum, which produced albino conidia. To understand the role of green-grey conidial pigment in P. digitatum, we identified the insertion site and deeply explored the 1,8-dihydroxynaphtsalene (DHN)-melanin synthesis gene cluster in this phytopathogen. In this study, we deleted five genes in P. digitatum, PdPksP, PdAbr1, PdArp1, PdArp2, and PdAyg1, and the experiments were further performed on phenotype analyses, including pigmentation, UV-C tolerance, virulence, growth rate, conidiation, stress (osmotic-, oxidative-, cell wall disturbing-, and high temperature-) tolerance, fungicide resistance, and conidial hydrophobicity. The results showed that the five deletion mutants (ΔPdPksP, ΔPdAbr1, ΔPdArp1, ΔPdArp2 and ΔPdAyg1) produced albino, brownish, brown, reddish-brown, and Yellowish green conidia, respectively. In addition, the survival colony forming units (CFUs) of the deletion mutants, under the treatment of UV-C radiation (261.4 mJ/cm2), were 0.3- to 0.6-fold of those surviving in wild-type strain N1. Moreover, after 522.8 mJ/cm2-UV-C-irradiation on conidia, the deletion mutants showed a larger decrease in pathogenicity on Valencia Orange fruits compared with strain N1. However, there were no significant differences among other phenotypes tested in this study. Collectively, our research reported the DHN-melanin synthesis pathway in P. digitatum for the first time, and revealed that DHN-melanin is important for P. digitatum to tolerate UV-C irradiation.

N-terminal zinc fingers of MaNCP1 contribute to growth, stress tolerance, and virulence in Metarhizium acridum

C2H2 zinc finger proteins (ZFPs) are a class of important transcriptional regulators in eukaryotes involved in multiple biological regulation processes. Here, MaNCP1, a C2H2 ZFP, was functionally characterized in the model entomopathogenic fungus Metarhizium acridum. Deletion of MaNCP1 delayed conidial germination and hyphal growth, decreased the conidial yield and reduced the tolerances to UV-B irradiation and heat-shock. The N-terminal zinc fingers (ZFs) of MaNCP1 made the main contributions to these traits. In addition, disruption of MaNCP1 altered the conidial surface structure and decreased the conidial hydrophobicity. Bioassays showed that the virulence of the MaNCP1-disruption strain (ΔMaNCP1) was reduced in topical inoculation compared to the WT or the mutant complemented strain (CP), and the N-terminal C2H2 ZFs made a major contribution to virulence. Furthermore, the ΔMaNCP1 and C2H2 ZFs deletion mutants (MaNCP1∆N and MaNCP1∆N+C) impaired cuticular penetration. RNA-seq showed that several cuticle-degrading genes were down-regulated in the ΔMaNCP1 background, suggesting that MaNCP1 plays vital roles in regulating insect cuticle penetration. In summary, MaNCP1 affected the growth, stress tolerances and virulence of M. acridum, and the N-terminal C2H2 ZFs played indispensable roles in these important biocontrol traits. These results provide further insights into the functions of C2H2 ZFPs in entomopathogenic fungi.

Host-Pathogen Interactions between Metarhizium spp. and Locusts.

The progress in research on the interactions between Metarhizium spp. and locusts has improved our understanding of the interactions between fungal infection and host immunity. A general network of immune responses has been constructed, and the pathways regulating fungal pathogenicity have also been explored in depth. However, there have been no systematic surveys of interaction between Metarhizium spp. and locusts. The pathogenesis of Metarhizium comprises conidial attachment, germination, appressorial formation, and colonization in the body cavity of the host locusts. Meanwhile, the locust resists fungal infection through humoral and cellular immunity. Here, we summarize the crucial pathways that regulate the pathogenesis of Metarhizium and host immune defense. Conidial hydrophobicity is mainly affected by the contents of hydrophobins and chitin. Appressorial formation is regulated by the pathways of MAPKs, cAMP/PKA, and Ca2+/calmodulin. Lipid droplets degradation and secreted enzymes contributed to fungal penetration. The humoral response of locust is coordinated by the Toll pathway and the ecdysone. The regulatory mechanism of hemocyte differentiation and migration is elusive. In addition, behavioral fever and density-dependent population immunity have an impact on the resistance of hosts against fungal infection. This review depicts a prospect to help us understand host–pathogen interactions and provides a foundation for the engineering of entomopathogenic fungi and the discovery of insecticidal targets to control insect pests.

Transcription Factor Mavib-1 Negatively Regulates Conidiation by Affecting Utilization of Carbon and Nitrogen Source in Metarhizium acridum.

Conidium is the main infection unit and reproductive unit of pathogenic fungi. Exploring the mechanism of conidiation and its regulation contributes to understanding the pathogenicity of pathogenic fungi. Vib-1, a transcription factor, was reported to participate in the conidiation process. However, the regulation mechanism of Vib-1 in conidiation is still unclear. In this study, we analyzed the function of Vib-1 and its regulation mechanism in conidiation through knocking out and overexpression of Vib-1 in entomopathogenic fungus Metarhizium acridum. Results showed that the colonial growth of Mavib-1 disruption mutant (ΔMavib-1) was significantly decreased, and conidiation was earlier compared to wild type (WT), while overexpression of Mavib-1 led to a delayed conidiation especially when carbon or nitrogen sources were insufficient. Overexpression of Mavib-1 resulted in a conidiation pattern shift from microcycle conidiation to normal conidiation on nutrient-limited medium. These results indicated that Mavib-1 acted as a positive regulator in vegetative growth and a negative regulator in conidiation by affecting utilization of carbon and nitrogen sources in M. acridum. Transcription profile analysis demonstrated that many genes related to carbon and nitrogen source metabolisms were differentially expressed in ΔMavib-1 and OE strains compared to WT. Moreover, Mavib-1 affects the conidial germination, tolerance to UV-B and heat stresses, cell wall integrity, conidial surface morphology and conidial hydrophobicity in M. acridum. These findings unravel the regulatory mechanism of Mavib-1 in fungal growth and conidiation, and enrich the knowledge to conidiation pattern shift of filamentous fungi.

Isoeugenol affects expression pattern of conidial hydrophobin gene RodA and transcriptional regulators MedA and SomA responsible for adherence and biofilm formation in Aspergillus fumigatus.

Aspergillus fumigatus is one of the major pathogenic fungal species, causing life-threatening infections. Due to a limited spectrum of available antifungals, exploration of new drug targets as well as potential antifungal molecules has become pertinent. Rodlet layer plays an important role in adherence of fungal conidia to hydrophobic cell surfaces in host, which also leads to A. fumigatus biofilm formation, contributing factor to fungal pathogenicity. From decades, natural sources have been known for the development of new active molecules. The present study investigates effect of isoeugenol on genes responsible for hydrophobins (RodA), adhesion as well as biofilm formation (MedA and SomA) of A. fumigatus. Minimum inhibitory concentrations (MIC and IC50) of isoeugenol against A. fumigatus were determined using broth microdilution assay. The IC50 results showed reduced hydrophobicity and biofilm formation as well as eradication after treatment with the compound and electron micrograph data corroborated these findings. The qRT-PCR showed a significant downregulation of genes RodA, MedA, SomA and pksP involved in hydrophobicity and biofilm formation. SwissADME studies potentiated drug-like propensity for isoeugenol which formed four hydrogen bonds with low binding energy (− 4.54 kcal/mol) at the catalytic site of RodA protein studied via AutoDock4. Hence, the findings conclude that isoeugenol inhibits conidial hydrophobicity and biofilm formation of A. fumigatus and further investigations are warranted in this direction.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00203-022-02817-w.

Conserved and Noncanonical Activities of Two Histone H3K36 Methyltransferases Required for Insect-Pathogenic Lifestyle of Beauveria bassiana.

Set2 and Ash1 are histone methyltransferases (KMTs) in the KMT3 family normally used to catalyze methylation of histone H3K36 (H3K36me) but remain unexplored in fungal insect pathogens. Here, we report broader/greater roles of Set2 and Ash1 in mono-/di-/trimethylation (me1/me2/me3) of H3K4 than of H3K36 in Beauveria bassiana and function similarly to Set1/KMT2, which has been reported to catalyze H3K4me3 as an epigenetic mark of cre1 (carbon catabolite repressor) to upregulate the classes I and II hydrophobin genes hyd1 and hyd2 required for conidial hydrophobicity and adherence to insect cuticle. H3K4me3 was more attenuated than H3K36me3 in the absence of set2 (72% versus 67%) or ash1 (92% versus 12%), leading to sharply repressed or nearly abolished expression of cre1, hyd1 and hyd2, as well as reduced hydrophobicity. Consequently, the delta-set2 and delta-ash1 mutants were differentially compromised in radial growth on various media or under different stresses, aerial conidiation under normal culture conditions, virulence, and cellular events crucial for normal cuticle infection and hemocoel colonization, accompanied by transcriptional repression of subsets of genes involved in or required for asexual development and multiple stress responses. These findings unravel novel roles of Set2 and Ash1 in the co-catalysis of usually Set1-reliant H3K4me3 required for fungal insect-pathogenic lifestyle.

Genome-Wide Insight into Profound Effect of Carbon Catabolite Repressor (Cre1) on the Insect-Pathogenic Lifecycle of Beauveriabassiana

Carbon catabolite repression (CCR) is critical for the preferential utilization of glucose derived from environmental carbon sources and regulated by carbon catabolite repressor A (Cre1/CreA) in filamentous fungi. However, a role of Cre1-mediated CCR in insect-pathogenic fungal utilization of host nutrients during normal cuticle infection (NCI) and hemocoel colonization remains explored insufficiently. Here, we report an indispensability of Cre1 for Beauveria bassiana’s utilization of nutrients in insect integument and hemocoel. Deletion of cre1 resulted in severe defects in radial growth on various media, hypersensitivity to oxidative stress, abolished pathogenicity via NCI or intrahemocoel injection (cuticle-bypassing infection) but no change in conidial hydrophobicity and adherence to insect cuticle. Markedly reduced biomass accumulation in the Δcre1 cultures was directly causative of severe defect in aerial conidiation and reduced secretion of various cuticle-degrading enzymes. The majority (1117) of 1881 dysregulated genes identified from the Δcre1 versus wild-type cultures were significantly downregulated, leading to substantial repression of many enriched function terms and pathways, particularly those involved in carbon and nitrogen metabolisms, cuticle degradation, antioxidant response, cellular transport and homeostasis, and direct/indirect gene mediation. These findings offer a novel insight into profound effect of Cre1 on the insect-pathogenic lifestyle of B. bassiana.

SET1/KMT2-governed histone H3K4 methylation coordinates the lifecycle in vivo and in vitro of the fungal insect pathogen Beauveria bassiana.

Biological control potential of insect-pathogenic fungi against pests is an overall output of various cellular processes regulated by signalling and epigenetic networks. In Beauveria bassiana, mono/di/trimethylation of histone H3 Lys 4 (H3K4me1/me2/m3) was abolished by inactivation of the histone lysine methyltransferase SET1/KMT2, leading to marked virulence loss, reductions in conidial hydrophobicity and adherence to insect cuticle, impeded proliferation in vivo, severe defects in growth and conidiation, and increased sensitivities to cell wall perturbation, H2 O2 and heat shock. Such compromised phenotypes correlated well with transcriptional abolishment or repression of carbon catabolite-repressing transcription factor Cre1, classes I and II hydrophobins Hyd1 and Hyd2 required for cell hydrophobicity, key developmental regulators, and stress-responsive enzymes/proteins. Particularly, expression of cre1, which upregulates hyd4 upon activation by KMT2-mediated H3K4me3 in Metarhizium robertsii, was nearly abolished in the Δset1 mutant, leading to abolished expression of hyd1 and hyd2 as homologues of hyd4. These data suggest that the SET1-Cre1-Hyd1/2 pathway function in B. bassiana like the KMT2-Cre1-Hyd4 pathway elucidated to mediate pathogenicity in M. robertsii. Our findings unveil not only a regulatory role for the SET1-cored pathway in fungal virulence but also its novel role in mediating asexual cycle in vitro and stress responses in B. bassiana.

Essential Role of COP9 Signalosome Subunit 5 (Csn5) in Insect Pathogenicity and Asexual Development of Beauveria bassiana.

Csn5 is a subunit ofthe COP9/signalosome complex in model fungi. Here, we report heavier accumulation of orthologous Csn5 in the nucleus than in the cytoplasm and its indispensability to insect pathogenicity and virulence-related cellular events of Beauveria bassiana. Deletion of csn5 led to a 68% increase in intracellular ubiquitin accumulation and the dysregulation of 18 genes encoding ubiquitin-activating (E1), -conjugating (E2), and -ligating (E3) enzymes and ubiquitin-specific proteases, suggesting the role of Csn5 in balanced ubiquitination/deubiquitination. Consequently, the deletion mutant displayed abolished insect pathogenicity, marked reductions in conidial hydrophobicity and adherence to the insect cuticle, the abolished secretion of cuticle penetration-required enzymes, blocked haemocoel colonisation, and reduced conidiation capacity despite unaffected biomass accumulation. These phenotypes correlated well with sharply repressed or abolished expressions of key hydrophobin genes required for hydrophobin biosynthesis/assembly and of developmental activator genes essential for aerial conidiation and submerged blastospore production. In the mutant, increased sensitivities to heat shock and oxidative stress also correlated with reduced expression levels of several heat-responsive genes and decreased activities of antioxidant enzymes. Altogether, Csn5-reliant ubiquitination/deubiquitination balance coordinates the expression of those crucial genes and the quality control of functionally important enzymes, which are collectively essential for fungal pathogenicity, virulence-related cellular events, and asexual development.

Selection of Beauveria bassiana (Hypocreales: Cordycipitaceae) strains to control Xyleborus affinis (Curculionidae: Scolytinae) females.

BackgroundXyleborus affinis Eichhoff (Coleoptera: Curculionidae) is an ambrosia beetle reported to affect avocado trees (Persea americana Mill.). The use of the entomopathogenic fungus (EPF) Beauveria bassiana (Bals.-Criv.) Vuill. for ambrosia beetle control represents an alternative to insecticides.MethodsThis study was designed in two stages to select B. bassiana strains with potential to control X. affinis females. In the first stage, 19 B. bassiana Mexican strains from EPF collection, isolated from Coleoptera (CHE-CNRCB, http://www.gob.mx/senasica/documentos/coleccion-de-hongos-entomopatogenos), were tested. Analyses included radial growth rate, conidial yield, spore germination, and germ tube length. Results were analysed by Principal Component Analysis (PCA) to identify clusters within favourable growth phenotypes. For the second stage, 10 selected strains were re-analysed for virulence-related metabolic characteristic, including cell wall-bound cuticle-degrading enzymes–Pr1-like proteases and β-N-acetyl glucosaminidases (NAGase) chitinases, conidial hydrophobicity and monopolar germination parameters. A second PCA analysis was run for those virulence parameters analysed, and upon results strains CHE-CNRCB 44, 171, 431 and 485 were selected and tested against X. affinis females. Females were treated with a 1 × 108 conidia mL−1 suspension (recommended rate), using a Potter Tower.ResultsAll strains showed insecticidal activity, inducing up to 58% mortality; about 30% dead beetles developed aerial mycelia (CHE-CNRCB 485) and the fastest mortality rate was t0 = 1.95 (CHE-CNRCB 44).ConclusionSince all selected strains showed virulence against X. affinis females, results indicated the possibility of selecting B. bassiana strains based on multiple metabolic attributes, as a preliminary test to perform bioassays against order-related target insects.

Effects on hyphal morphology and development by the putative copper radical oxidase glx1 in Trichoderma virens suggest a novel role as a cell wall associated enzyme

Trichoderma spp. have been characterized for their capacity to act as biological control agents against several pathogens through the activity of secondary metabolites and cell wall degrading enzymes. However, only T. reesei has been widely studied for the ability to assimilate lignocellulose substrates. Protein analysis by SDS-PAGE of culture filtrate of T. virens revealed the presence of an unknown ∼77 kDa band protein (GLX1) that showed sequence homology to glyoxal-like oxidase genes involved in lignin degradation. The analysis and biochemical characterization of the 1,119 amino acid coded protein showed the presence of five carbohydrate-binding modules (CBMs) with affinity for colloidal chitin, and a functional glyoxal oxidase catalytic domain that is involved in the production of hydrogen peroxide when methylglyoxal was used as a substrate. The silencing of the glx1 gene resulted in mutants with more than 90% expression reduction and the absence of glyoxal oxidase catalytic activity. These mutants showed delayed hyphal growth, reduced colony and conidial hydrophobicity, but showed no changes in their biocontrol ability. Most significantly, mutants exhibited a loss of growth directionality resulting in a curled phenotype that was eliminated in the presence of exogenous H2O2. Here we present evidence that in T. virens, glx1 is not involved in the breakdown of lignin but instead is responsible for normal hyphal growth and morphology and likely does this through free radical production within the fungal cell wall. This is the first time that a glyoxal oxidase protein has been isolated and characterized in ascomycete fungi.

Surface properties, adhesion and biofilm formation on different surfaces by Scedosporium spp. and Lomentospora prolificans

In the present work, some surface properties of the fungi Scedosporium apiospermum, S. aurantiacum, S. minutisporum, and Lomentospora prolificans and their capability to adhere to and form a biofilm on diverse surfaces were evaluated. All four species had high conidial surface hydrophobicity and elevated electronegative zeta potentials. Abundant quantities of melanin were detected at the conidial surface, whereas sialic acid was absent. The numbers of non-germinated and germinated conidia adhered to poly-L-lysine-covered slides was higher than on glass after 4 h of fungi–surface contact. Additionally, after 72 h of interaction a typical biofilm structure had formed. Mature biofilms were also observed after 72 h on a nasogastric catheter (made from polyvinyl chloride), a late bladder catheter (siliconized latex), and a nasoenteric catheter (polyurethane). Interestingly, biofilm biomass increased considerably when the catheters had previously been incubated with serum. These results confirm that Scedosporium/Lomentospora spp. are capable of forming biofilms on diverse abiotic surfaces.

Rtt109-dependent histone H3 K56 acetylation and gene activity are essential for the biological control potential of Beauveria bassiana.

Rtt109 is a histone acetyltransferase that catalyzes histone H3K56 acetylation required for genomic stability, DNA damage repair and virulence-related gene activity in yeast-like human pathogens but remains functionally unknown in fungal insect pathogens. This study seeks to elucidate the catalytic activity of a Rtt109 orthologue and its possible role in sustaining the biological control potential of Beauveria bassiana, a fungal entomopathogen. Deletion of rtt109 in B. bassiana abolished histone H3K56 acetylation and triggered histone H2A-S129 phosphorylation. Consequently, the deletion mutant showed increased sensitivity to the stresses of DNA damage, oxidation, cell wall perturbation, high osmolarity and heat shock during colony growth, severe conidiation defects under normal culture conditions, reduced conidial hydrophobicity, decreased conidial UV-B resistance, and attenuated virulence through normal cuticle infection. These phenotypic changes correlated well with reduced transcript levels of many genes that encode the families of H2A-S129 dephosphorylation-related protein phosphatases, DNA damage-repairing factors, antioxidant enzymes, heat-shock proteins, key developmental activators, hydrophobins and cuticle-degrading Pr1 proteases respectively. Rtt109 can acetylate H3K56 and dephosphorylate H2A-S129 in direct and indirect ways respectively, and hence has an essential role in sustaining the genomic stability and global gene activity required for conidiation capacity, environmental fitness and pest control potential in B. bassiana. © 2018 Society of Chemical Industry.

Role of Hydrophobins in Aspergillus fumigatus.

Resistance of Aspergillus fumigatus conidia to desiccation and their capacity to reach the alveoli are partly due to the presence of a hydrophobic layer composed of a protein from the hydrophobin family, called RodA, which covers the conidial surface. In A. fumigatus there are seven hydrophobins (RodA–RodG) belonging to class I and III. Most of them have never been studied. We constructed single and multiple hydrophobin-deletion mutants until the generation of a hydrophobin-free mutant. The phenotype, immunogenicity, and virulence of the mutants were studied. RODA is the most expressed hydrophobin in sporulating cultures, whereas RODB is upregulated in biofilm conditions and in vivo Only RodA, however, is responsible for rodlet formation, sporulation, conidial hydrophobicity, resistance to physical insult or anionic dyes, and immunological inertia of the conidia. None of the hydrophobin plays a role in biofilm formation or its hydrophobicity. RodA is the only needed hydrophobin in A. fumigatus, conditioning the structure, permeability, hydrophobicity, and immune-inertia of the cell wall surface in conidia. Moreover, the defect of rodlets on the conidial cell wall surface impacts on the drug sensitivity of the fungus.

The histone acetyltransferase Mst2 sustains the biological control potential of a fungal insect pathogen through transcriptional regulation.

Histone lysine acetylation orchestrates transcriptional activity essential for diverse cellular events across organisms, but it remains poorly understood how an acetylated lysine affects cellular functions in filamentous fungal pathogens. Here, we show the functions of a histone acetyltransferase that is phylogenetically close to Mst2 in fission yeast and specifically acetylates histone H3K14 in Beauveria bassiana, a fungal insect pathogen widely applied in arthropod pest management. Deletion of mst2 in B. bassiana resulted in moderate growth defects on rich and minimal media, delayed conidiation, and drastic reduction (75%) in conidiation capacity under normal culture conditions. The Δmst2 conidia suffered slower germination, decreased hydrophobicity, attenuated virulence, and reduced thermotolerance and UV-B resistance. The Δmst2 mutant also displayed increased sensitivities to DNA damaging, oxidative, cell wall perturbing, and osmotic stresses during conidial germination and colony growth at optimal 25°C. Intriguingly, the phenotypic changes were accompanied with transcriptional repression of related gene sets, which are required for asexual development and conidial hydrophobicity or cascaded for CWI and HOG pathways, and encode the families of superoxide dismutases (SOD), catalases, heat-shock proteins, and trehalose or mannitol-metabolizing enzymes. Consequently, total SOD and catalase activities, trehalose and mannitol contents, and hydrophobicity were remarkably lowered in the hyphal cells or conidia of Δmst2. All of these changes were well restored by targeted mst2 complementation. Our results indicate that Mst2 enables to mediate global gene transcription and/or post-translation through H3K14 acetylation and plays an essential role in sustaining the biological control potential of B. bassiana against arthropod pests.

Discovery of a new intravacuolar protein required for the autophagy, development and virulence of Beauveria bassiana.

High proportions of hypothetical proteins exist in genomic databases of fungi, including putative secretory proteins (PSPs) likely involved in fungal invasion and virulence. Here we characterize one of many PSPs revealed in the previous transcriptome of Beauveria bassiana (a fungal insect pathogen) infecting a global lepidopteran pest and name it vacuole-localized protein 4 (VLP4) because this small, domain-lacking protein (22.96 kDa) was specifically localized in the vacuoles of hyphal cells. Deletion of VLP4 resulted in repression of almost all genes acting in autophagy and central development pathways. Consequently, the deletion mutant formed no autophagosome in hyphal vacuoles and displayed severe defects in aerial conidiation. conidial hydrophobicity to the insect surface, and secretion of cuticle-degrading Pr1 proteases required for normal cuticle infection. Blastospore formation was inhibited in the submerged mutant culture mimic to insect haemolymph, and formation of hyphal bodies in vivo was delayed. The fungal virulence was attenuated in the absence of VLP4. These phenotypic defects were well restored by targeted gene complementation. Our findings unveil a vital role of VLP4 in B. bassiana and call attention to many more PSPs for new insights into the interactions of fungal insect pathogens with insects.