RESUMO
Reductive assimilation pathway involves ferric reductase and ferrous iron transporter, which is integral for fungal iron acquisition. A family of ferric reductase-like proteins has been functionally characterized in the filamentous entomopathogenic fungus Beauveria bassiana. In this investigation, two ferrous iron transporter-like proteins (Ftr) were functionally annotated in B. bassiana. BbFtr1 and BbFtr2 displayed high similarity in structure and were associated with the plasma and nuclear membrane. Their losses had no negatively influence on fungal growth on various nutrients and development under the iron-replete condition. Single mutants of BbFTR1 and BbFTR2 displayed the iron-availability dependent developmental defects, and double mutant exhibited the significantly impaired developmental potential under the iron-limited conditions. In insect bioassay, the double mutant also showed the weaker virulence than either of two single disruption mutants. These results suggested that two ferrous iron transporter-like proteins function independently in fungal physiologies under the iron-deficient condition. Intriguingly, a bZIP transcription factor BbHapX was required for expression of BbFTR1 and BbFTR2 under iron-depleted conditions. This study enhances our understanding of the iron uptake system in the filamentous entomopathogenic fungi.
Assuntos
Beauveria , Proteínas Fúngicas , Ferro , Beauveria/genética , Beauveria/patogenicidade , Beauveria/crescimento & desenvolvimento , Ferro/metabolismo , Virulência/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Animais , Regulação Fúngica da Expressão Gênica , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Reprodução Assexuada/genéticaRESUMO
Beauveria bassiana, the causative agent of arthropod, proliferates in the host hemolymph (liquid environment) and shits to saprotrophic growth on the host cadaver (aerial surface). In this study, we used transcriptomic analysis to compare the gene expression modes between these two growth phases. Of 10,366 total predicted genes in B. bassiana, 10,026 and 9985 genes were expressed in aerial (AM) and submerged (SM) mycelia, respectively, with 9853 genes overlapped. Comparative analysis between two transcriptomes indicated that there were 1041 up-regulated genes in AM library when compared with SM library, and 1995 genes were down-regulated, in particular, there were 7085 genes without significant change in expression between two transcriptomes. Furthermore, of 25 amidase genes (AMD), BbAMD5 has high expression level in both transcriptomes, and its protein product was associated with cell wall in aerial and submerged mycelia. Disruption of BbAMD5 significantly reduced mycelial hydrophobicity, hydrophobin translocation, and conidiation on aerial plate. Functional analysis also indicated that BbAmd5 was involved in B. bassiana blastospore formation in broth, but dispensable for fungal virulence. This study revealed the high similarity in global expression mode between mycelia grown under two cultivation conditions.
Assuntos
Beauveria , Proteínas Fúngicas , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Micélio , Transcriptoma , Beauveria/genética , Beauveria/crescimento & desenvolvimento , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Micélio/crescimento & desenvolvimento , Micélio/genética , Animais , Virulência/genética , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimentoRESUMO
Beauveria bassiana is one of the most extensively studied entomopathogenic fungi (EPF) and is widely used as a biocontrol agent against various insect pests. Proteins containing the MARVEL domain are conserved in eukaryotes, typically with four transmembrane structures. In this study, we identified the five MARVEL domain proteins in B. bassiana. Five MARVEL domain proteins were localized to cytomembrane and vacuoles in B. bassiana, but had different roles in maintaining the lipid-droplet homeostasis. These proteins were required for fungal virulence, but differentially contributed to fungal utilization of nutrients, stress tolerance, and development under aerial and submerged conditions. Notably, BbMARVEL2 was essential for conidial surface morphology. Additionally, these five MARVEL domain proteins contributed to fungal interaction with the host immune defense. This study provides new mechanistic insights into the life cycle of B. bassiana as a biocontrol agent.
Assuntos
Beauveria , Animais , Virulência , Proteínas Fúngicas/metabolismo , Insetos/microbiologia , Proteínas com Domínio MARVEL/metabolismo , Esporos FúngicosRESUMO
Cla4, an orthologous p21-activated kinase crucial for non-entomopathogenic fungal lifestyles, has two paralogs (Cla4A/B) functionally unknown in hypocrealean entomopathogens. Here, we report a regulatory role of Cla4A in gene expression networks of Beauveria bassiana required for asexual and entomopathogenic lifecycles while Cla4B is functionally redundant. The deletion of cla4A resulted in severe growth defects, reduced stress tolerance, delayed conidiation, altered conidiation mode, impaired conidial quality, and abolished pathogenicity through cuticular penetration, contrasting with no phenotype affected by cla4B deletion. In ∆cla4A, 5288 dysregulated genes were associated with phenotypic defects, which were restored by targeted gene complementation. Among those, 3699 genes were downregulated, including more than 1300 abolished at the transcriptomic level. Hundreds of those downregulated genes were involved in the regulation of transcription, translation, and post-translational modifications and the organization and function of the nuclear chromosome, chromatin, and protein-DNA complex. DNA-binding elements in promoter regions of 130 dysregulated genes were predicted to be targeted by Cla4A domains. Samples of purified Cla4A extract were proven to bind promoter DNAs of 12 predicted genes involved in multiple stress-responsive pathways. Therefore, Cla4A acts as a novel regulator of genomic expression and stability and mediates gene expression networks required for insect-pathogenic fungal adaptations to the host and environment.
Assuntos
Beauveria , Proteínas Fúngicas , Regulação Fúngica da Expressão Gênica , Redes Reguladoras de Genes , Beauveria/genética , Beauveria/patogenicidade , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Animais , Insetos/microbiologia , Esporos Fúngicos/genética , Regiões Promotoras GenéticasRESUMO
Peroxisomes play important roles in fungal physiological processes. The RING-finger complex consists of peroxins Pex2, Pex10, and Pex12 and is essential for recycling of receptors responsible for peroxisomal targeting of matrix proteins. In this study, these three peroxins were functionally characterized in the entomopathogenic fungus Beauveria bassiana (Bb). These three peroxins are associated with peroxisomes, in which BbPex2 interacted with BbPex10 and BbPex12. Ablation of these peroxins did not completely block the peroxisome biogenesis, but abolish peroxisomal targeting of matrix proteins via both PTS1 and PTS2 pathways. Three disruptants displayed different phenotypic defects in growth on nutrients and under stress conditions, but have similar defects in acetyl-CoA biosynthesis, development, and virulence. Strikingly, BbPex10 played a less important role in fungal growth on tested nutrients than other two peroxins; whereas, BbPex2 performed a less important contribution to fungal growth under stresses. This investigation reinforces the peroxisomal roles in the lifecycle of entomopathogenic fungi and highlights the unequal functions of different peroxins in peroxisomal biology.
Assuntos
Beauveria , Proteínas de Membrana , Animais , Peroxinas , Proteínas de Membrana/metabolismo , Beauveria/genética , Beauveria/metabolismo , Insetos , Estágios do Ciclo de Vida , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismoRESUMO
The anti-ultraviolet (UV) role of a Rad4-Rad23-Rad33 complex in budding yeast relies on nucleotide excision repair (NER), which is mechanistically distinct from photorepair of DNA lesions generated under solar UV irradiation but remains poorly known in filamentous fungi. Here, two nucleus-specific Rad4 paralogs (Rad4A and Rad4B) and nucleocytoplasmic shuttling Rad23 ortholog are functionally characterized by multiple analyses of their null mutants in Metarhizium robertsii, an entomopathogenic fungus lacking Rad33. Rad4A was proven to interact with Rad23 and contribute significantly more to conidial UVB resistance (90%) than Rad23 (65%). Despite no other biological function, Rad4A exhibited a very high activity in photoreactivation of UVB-impaired/inactivated conidia by 5-h light exposure due to its interaction with Rad10, an anti-UV protein clarified previously to have acquired a similar photoreactivation activity through its interaction with a photolyase in M. robertsii. The NER activity of Rad4A or Rad23 was revealed by lower reactivation rates of moderately impaired conidia after 24-h dark incubation but hardly observable at the end of 12-h dark incubation, suggesting an infeasibility of its NER activity in the field where nighttime is too short. Aside from a remarkable contribution to conidial UVB resistance, Rad23 had pleiotropic effect in radial growth, aerial conidiation, antioxidant response, and cell wall integrity but no photoreactivation activity. However, Rad4B proved redundant in function. The high photoreactivation activity of Rad4A unveils its essentiality for M. robertsii's fitness to solar UV irradiation and is distinct from the yeast homolog's anti-UV role depending on NER. IMPORTANCE Resilience of solar ultraviolet (UV)-impaired cells is crucial for the application of fungal insecticides based on formulated conidia. Anti-UV roles of Rad4, Rad23, and Rad33 rely upon nucleotide excision repair (NER) of DNA lesions in budding yeast. Among two Rad4 paralogs and Rad23 ortholog characterized in Metarhizium robertsii lacking Rad33, Rad4A contributes to conidial UVB resistance more than Rad23, which interacts with Rad4A rather than functionally redundant Rad4B. Rad4A acquires a high activity in photoreactivation of conidia severely impaired or inactivated by UVB irradiation through its interaction with Rad10, another anti-UV protein previously proven to interact with a photorepair-required photolyase. The NER activity of either Rad4A or Rad23 is seemingly extant but unfeasible under field conditions. Rad23 has pleiotropic effect in the asexual cycle in vitro but no photoreactivation activity. Therefore, the strong anti-UV role of Rad4A depends on photoreactivation, unveiling a scenario distinct from the yeast homolog's NER-reliant anti-UV role.
Assuntos
Desoxirribodipirimidina Fotoliase , Metarhizium , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Desoxirribodipirimidina Fotoliase/genética , Desoxirribodipirimidina Fotoliase/metabolismo , Reparo do DNA , Proteínas de Saccharomyces cerevisiae/genética , Metarhizium/genética , Metarhizium/metabolismo , Raios Ultravioleta , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismoRESUMO
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.
Assuntos
Proteínas Fúngicas , Metarhizium , Animais , Esporos Fúngicos/genética , Virulência , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Insetos , Interações Hidrofóbicas e HidrofílicasRESUMO
Common in fungal extracellular membrane (CFEM) domain is unique in fungal proteins and some of which contribute to iron acquisition in yeast. However, their roles in iron acquisition remain largely unknown in filamentous fungi. In this study, 12 CFEM-containing proteins were bioinformatically identified in the filamentous entomopathogenic fungus Beauveria bassiana, and the roles of 11 genes were genetically characterized. Transmembrane helices were critical for their association with intracellular membranes, and their number varied among proteins. Eleven CFEM genes significantly contribute to vegetative growth under iron starvation and virulence. Notably, the virulence of most disruptants could be significantly weakened by a decrease in iron availability, in which the virulence of ΔBbcfem7 and 8 strains was partially recovered by exogenous hemin. ΔBbcfem7 and 8 mutants displayed defective competitiveness against the sister entomopathogenic fungus Beauveria brongniartii. All 11 disruptants displayed impaired growth in the antagonistic assay with the saprotrophic fungus Aspergillus niger, which could be repressed by exogenous ferric ions. These findings not only reveal the systematic contributions of CFEM proteins to acquire two forms of iron (i.e. heme and ferric ion) in the entire lifecycle of entomopathogenic fungi but also help to better understand the mechanisms of fungus-host and inter-fungus interactions.
Assuntos
Beauveria , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ferro/metabolismo , Esporos Fúngicos/metabolismo , Virulência/genéticaRESUMO
Sterol carrier protein 2 (SCP2) represents a family of proteins binding a variety of lipids and plays essential roles in cellular physiology. However, its physiological roles are largely unknown in filamentous fungi. In this study, we functionally characterized an orthologous Scp2 gene in the filamentous insect pathogenic fungus Beauveria bassiana (BbScp2). BbScp2 was verified to be a peroxisomal protein and displayed different affinities to various lipids, with strong affinity to palmitic acid (PA) and ergosterol (ES). No significant binding activity was detected between protein and oleic acid (OA) or linoleic acid (LA). Ablation of BbScp2 did not cause significant effects on fungal growth on various carbon sources, but resulted in a modest reduction in conidial (49%) and blastospore yield (45%). In addition, exogenous lipids could recover the defectives in conidiation of ΔBbScp2 mutant strain. BbScp2 was required for the cytomembrane functionality in germlings, and its loss resulted in a more significant decrease in virulence indicated by cuticle infection assay than intrahemocoel injection assay. Our findings indicate that Scp2 links the lipid trafficking to the asexual differentiation and virulence of B. bassiana.
Assuntos
Beauveria , Animais , Beauveria/genética , Proteínas de Transporte , Proteínas Fúngicas/genética , Insetos , Lipídeos , Esporos Fúngicos/genética , Virulência/genéticaRESUMO
Acyl-CoA synthetase (ACS) functions as a hub linking lipid metabolism with in cellular physiologies by producing active intermediate of catalyzes acyl-CoA. However, the biological roles of ACS are largely unknown in filamentous fungi. In this study, an ortholog of yeast Faa1, named BbFaa1, was functionally characterized in the filamentous entomopathogenic fungus Beauveria bassiana. BbFaa1 was associated with vesicular membrane, and its loss resulted in the impaired cytomembrane integrity. Notably, in ΔBbfaa1 mutant strain, the translocation of hydrophobins across cell membrane was significantly hampered, which resulted in the reduced hydrophobicity of aerial mycelia and conidia. In addition, loss of BbFaa1 significantly weakened fungal virulence. Our findings indicate that the metabolism of acyl-CoA synthetase Faa1 contributes to the cytomembrane functionality which cascades hydrophobin translocation and differentiation, thus affecting virulence of B. bassiana.
Assuntos
Beauveria , Animais , Beauveria/genética , Coenzima A/metabolismo , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Insetos/microbiologia , Saccharomyces cerevisiae , Esporos Fúngicos , VirulênciaRESUMO
Acetyl-coenzyme A (CoA) synthetase (Acs) links cellular metabolism and physiology by catalyzing acetate and CoA into acetyl-CoA. However, the biological roles of Acs are not well studied in entomopathogenic fungi. In this study, two Acs proteins (BbAcs1 and BbAcs2) was functionally characterized in the filamentous insect pathogenic fungus Beauveria bassiana. BbAcs1 and BbAcs2 localize in cytoplasm and peroxisome, respectively. BbAcs1 contributes to vegetative growth on fatty acids as carbon source, and BbAcs2 did not. Both genes did not contribute to fungal response to stresses. The BbAcs1 loss conferred a slight influence on conidiation, and did not result in the defects in blastospore formation. On the contrary, BbAcs2 significantly contributes to lipid metabolism in germlings, blastospore formation, and virulence. The results indicated that Acs2 played a more predominant role than Acs1 in B. bassiana, which links the acetyl-CoA metabolism with the lifestyle of entomopathogenic fungi.
Assuntos
Beauveria , Saccharomyces cerevisiae , Acetato-CoA Ligase/genética , Acetilcoenzima A , Beauveria/genética , Carbono , Coenzima A Ligases/genética , Ácidos GraxosRESUMO
AIMS: Peroxins Pex5 and Pex7 belong to the peroxisomal import machinery and recognize proteins containing peroxisomal targeting signal (PTS) type 1 and type 2, respectively. This study seeks to characterize these two peroxins in the entomopathogenic fungus Beauveria bassiana. METHODS AND RESULTS: The orthologs of Pex5 and Pex7 in B. bassiana (BbPex5 and BbPex7) were functionally analyzed via protein localization and gene disruption. BbPex5 and BbPex7 were associated with peroxisome and specifically required for PTS1 and PTS2 pathways, respectively, which were demonstrated to be involved in development, tolerance to oxidative stress and virulence. ΔBbPex5 mutant displayed additionally defectives that were undetected in ΔBbPex7 in vegetative growth and resistance to osmotic and cell wall-perturbing stresses. Notably, Woronin body major protein Hex1 with PTS1 linked this organelle to the development and virulence of B. bassiana, which indicates that Woronin body is associated with the roles of PTS1 pathway. CONCLUSION: Both PTS1 and PTS2 pathways are involved in broad physiological process, and the PTS1 pathway acts as a main peroxisomal import pathway. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the functional divergence of different peroxins and improves our understanding of organellar physiology involved in biocontrol potential of the entomopathogenic fungi.
Assuntos
Beauveria , Animais , Beauveria/genética , Beauveria/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Insetos , Receptor 2 de Sinal de Orientação para Peroxissomos , Receptores Citoplasmáticos e Nucleares/genética , VirulênciaRESUMO
Small heat shock proteins (sHsps) containing conserved α-crystallin domain play important roles in many cellular processes, but little is known about the functions of sHsps in filamentous entomopathogens. Here, three sHsps of Hsp20, Hsp30a, and Hsp30b were characterized in Beauveria bassiana, a filamentous fungal insect pathogen that serves as the main source of wide-spectrum fungal insecticides. The results demonstrated that these three genes are interrelated at the transcriptional level under normal and heat-shocked conditions. Meanwhile, all the deletion mutants showed significant but differential changes in cell wall integrity, antioxidant activity, hyphal tolerance to carbendazim fungicide, conidial tolerance to 45 °C wet heat and virulence. However, only Δhsp30b showed growth defects on rich and minimal media at 25 °C and Δhsp30a displayed the reduction in conidiophores and conidia. Moreover, the single deletion of hsp30a and hsp30b caused the decreases in hyphal growth at 34 °C and conidial tolerance to UV-B irradiation. Our findings provide a global insight into vital roles of hsp20, hsp30a, and hsp30b in asexual development, environmental adaptation, and fungal virulence of B. bassiana.
Assuntos
Beauveria , alfa-Cristalinas , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Esporos Fúngicos , Estresse Fisiológico/genética , Virulência/genéticaRESUMO
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.
Assuntos
Beauveria , Animais , Beauveria/genética , Beauveria/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Histonas/genética , Histonas/metabolismo , Insetos/metabolismo , MetilaçãoRESUMO
The photolyases PHR1 and PHR2 enable photorepair of fungal DNA lesions in the forms of UV-induced cyclobutane pyrimidine dimer (CPD) and (6-4)-pyrimidine-pyrimidone (6-4PP) photoproducts, but their regulation remains mechanistically elusive. Here, we report that the white collar proteins WC1 and WC2 mutually interacting to form a light-responsive transcription factor regulate photolyase expression required for fungal UV resistance in the insect-pathogenic fungus Metharhizum robertsii. Conidial UVB resistance decreased by 54% in Δwc1 and 67% in Δwc2. Five-hour exposure of UVB-inactivated conidia to visible light resulted in photoreactivation rates of 30% and 9% for the Δwc1 and Δwc2 mutants, contrasting to 79%-82% for wild-type and complemented strains. Importantly, abolished transcription of phr1 in Δwc-2 and of phr2 in Δwc1 resulted in incapable photorepair of CDP and 6-4PP DNA lesions in UVB-impaired Δwc2 and Δwc1 cells respectively. Yeast two-hybrid assays revealed interactions of either WC protein with both PHR1 and PHR2. Therefore, the essential roles for WC1 and WC2 in both photorepair of UVB-induced DNA lesions and photoreactivation of UVB-inactivated conidia rely upon their interactions with, and hence transcriptional activation of, PHR1 and PHR2. These findings uncover a novel WC-cored pathway that mediates filamentous fungal response and adaptation to solar UV irradiation.
Assuntos
Desoxirribodipirimidina Fotoliase , Proteínas Fúngicas , Metarhizium , Raios Ultravioleta , Dano ao DNA , Reparo do DNA , DNA Fúngico , Desoxirribodipirimidina Fotoliase/genética , Desoxirribodipirimidina Fotoliase/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Metarhizium/enzimologia , Metarhizium/genética , Metarhizium/efeitos da radiação , Dímeros de PirimidinaRESUMO
The upstream developmental activation (UDA) pathway comprises three fluG-cored cascades (fluG-flbA, fluG-flbE/B/D and fluG-flbC) that activate the key gene brlA of central developmental pathway (CDP) to initiate conidiation in aspergilli. However, the core role of fluG remains poorly understood in other fungi. Here, we report distinctive role of fluG in the insect-pathogenic lifecycle of Beauveria bassiana. Disruption of fluG resulted in limited conidiation defect, which was mitigated with incubation time and associated with time-course up-regulation/down-regulation of all flb and CDP genes and another fluG-like gene (BBA_06309). In ΔfluG, increased sensitivities to various stresses correlated with repression of corresponding stress-responsive genes. Its virulence through normal cuticle infection was attenuated greatly due to blocked secretion of cuticle-degrading enzymes and delayed formation of hyphal bodies (blastospores) to accelerate proliferation in vivo and host death. In submerged ΔfluG cultures mimicking insect haemolymph, largely increased blastospore production concurred with drastic up-regulation of the CDP genes brlA and abaA, which was associated with earlier up-regulation of most flb genes in the cultures. Our results unveil an essentiality of fluG for fungal adaptation to insect-pathogenic lifecycle and suggest the other fluG-like gene to act as an alternative player in the UDA pathway of B. bassiana.
Assuntos
Beauveria , Animais , Beauveria/genética , Proteínas Fúngicas , Insetos , Esporos Fúngicos/genética , VirulênciaRESUMO
FK506-sensitive proline rotamases (FPRs), also known as FK506-binding proteins (FKBPs), can mediate immunosuppressive drug resistance in budding yeast but their physiological roles in filamentous fungi remain opaque. Here, we report that three FPRs (cytosolic/nuclear 12.15-kD Fpr1, membrane-associated 14.78-kD Fpr2 and nuclear 50.43-kD Fpr3) are all equally essential for cellular Ca2+ homeostasis and contribute significantly to calcineurin activity at different levels in the insect-pathogenic fungus Beauveria bassiana although the deletion of fpr1 alone conferred resistance to FK506. Radial growth, conidiation, conidial viability and virulence were less compromised in the absence of fpr1 or fpr2 than in the absence of fpr3, which abolished almost all growth on scant media and reduced growth moderately on rich media. The Δfpr3 mutant was more sensitive to Na+ , K+ , Mn2+ , Ca2+ , Cu2+ , metal chelate, heat shock and UVB irradiation than was Δfpr2 while both mutants were equally sensitive to Zn2+ , Mg2+ , Fe2+ , H2 O2 and cell wall-perturbing agents. In contrast, the Δfpr1 mutant was less sensitive to fewer stress cues. Most of 32 examined genes involved in DNA damage repair, Na+ /K+ detoxification or osmotolerance and Ca2+ homeostasis were downregulated sharply in Δfpr2 and Δfpr3 but rarely so affected in Δfpr1, coinciding well with their phenotypic changes. These findings uncover important, but differential, roles of three FPRs in the fungal adaptation to insect host and environment and provide novel insight into their essential roles in calcium signalling pathway.
Assuntos
Beauveria/metabolismo , Beauveria/patogenicidade , Mariposas/microbiologia , Peptidilprolil Isomerase/metabolismo , Animais , Beauveria/genética , Beauveria/crescimento & desenvolvimento , Calcineurina/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Resposta ao Choque Térmico , Homeostase , Pressão Osmótica , Peptidilprolil Isomerase/genética , Esporos Fúngicos/crescimento & desenvolvimento , Estresse Fisiológico , VirulênciaRESUMO
Adherence of conidia to insect integument is crucial for initiation of fungal infection through cuticular penetration and was previously reported to rely upon the Metarhizium-type adhesin Mad1 rather than Mad2, another adhesin crucial for conidial adherence of Metarhizium anisopliae to plant root surface. Mad1 and Mad2 have since been considered to function in fungal insect pathogenesis and plant root colonization respectively. Here, three adhesins were characterized in Beauveria bassiana, including Adh1/Mad1, Adh2/Mad2, and Adh3 known as filamentous hemagglutinin/adhesin and virulence factor in animal-pathogenic bacteria. Among those, only Adh2 was found to play a substantial role in sustaining the fungal virulence and some phenotypes associated with biological control potential. Disruption of adh2 resulted in decreased conidial adherence to insect wing cuticle, attenuated virulence via normal cuticle infection or cuticle-bypassing infection (injection), reduced blastospore production in an insect hemolymph-mimicking broth, largely reduced conidiation capacity, impaired conidial quality indicative of lowered viability, hydrophobicity, and UV resistance, but no growth defects on rich and scant media under normal or stressful culture conditions. The main phenotypic changes correlated well with repressed expression of developmental activator genes required for aerial conidiation and submerged blastospore production and of key hydrophobin genes essential for hydrophobin synthesis and assembly into rodlet bundles of conidial coat crucial for conidial adherence. In contrast, either adh1 or adh3 disruption caused insignificant changes in all phenotypes examined. These findings offer novel insight into a significance of Adh2, but a dispensability of Adh1 or Adh3, for insect-pathogenic lifecycle of B. bassiana. KEY POINTS: ⢠Three adhesins (Adh1-3) of Beauvera bassiana are functionally characterized. ⢠Adh2 plays a role in sustaining virulence and lifecycle-related cellular events. ⢠Either Adh1 or Adh3 is dispensable for insect-pathogenic lifecycle of B. bassiana.
Assuntos
Beauveria , Metarhizium , Animais , Beauveria/genética , Proteínas Fúngicas/genética , Insetos , Metarhizium/genética , Esporos FúngicosRESUMO
The Mbp1 protein functions as a DNA-binding protein in the MluI cell cycle box-binding complex and plays significant roles in yeast development. In this study, an ortholog of yeast Mbp1, BbMbp1, was characterized in a filamentous insect mycopathogen, Beauveria bassiana. BbMbp1 plays an important role in morphological changes under aerial and liquid environments. On the aerial surface, BbMbp1 was indispensable for the biogenesis of conidiophores and conidiation. Under submerged conditions, the ∆BbMbp1 mutant displayed abnormal spore-producing structures, with a dramatic decrease in blastospore yield (~95%). The virulence of the ∆BbMbp1 mutant was notably weakened, which might be due to the defect in in vivo blastospore formation in the insect. Moreover, disruption of BbMbp1 resulted in a substantial reduction in hyphal growth on cadavers. Comparative transcriptomics revealed that BbMbp1 mediated different transcriptomes during the formation processes of conidia and blastospores. Yeast one-hybrid assays demonstrated that BbMbp1 was required for transcriptional control of a cell wall protein gene, BbCwp, and an integral membrane protein gene, BbImp that played significant roles in conidiation and blastospore formation respectively. Our results demonstrate that BbMbp1 contributes to the morphological transitions in the pathogenic and saprophytic growth of B. bassiana via different genetic pathways.
Assuntos
Beauveria/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Animais , Beauveria/patogenicidade , Ciclo Celular/fisiologia , Divisão Celular , Parede Celular/metabolismo , Biologia Computacional , Proteínas Fúngicas/genética , Insetos/microbiologia , Esporos Fúngicos/metabolismo , Transcriptoma , Virulência/genéticaRESUMO
Ubi4 is a polyubiquitin precursor well characterized in yeasts but unexplored in insect mycopathogens. Here, we report that orthologous Ubi4 plays a core role in ubiquitin- and asexual lifestyle-required cellular events in Beauveria bassiana. Deletion of ubi4 led to abolished ubiquitin accumulation, blocked autophagic process, severe defects in conidiation and conidial quality, reduced cell tolerance to oxidative, osmotic, cell wall perturbing and heat-shock stresses, decreased transcript levels of development-activating and antioxidant genes, but light effect on radial growth under normal conditions. The deletion mutant lost insect pathogenicity via normal cuticle infection and was severely compromised in virulence via cuticle-bypassing infection due to a block of dimorphic transition critical for acceleration of host mummification. Proteomic and ubiquitylomic analyses revealed 1081 proteins differentially expressed and 639 lysine residues significantly hyper- or hypo-ubiquitylated in the deletion mutant, including dozens of ubiquitin-activating, conjugating and ligating enzymes, core histones, and many more involved in proteasomes, autophagy-lysosome process and protein degradation. Singular deletions of seven ubiquitin-conjugating enzyme genes exerted differential Ubi4-like effects on conidiation level and conidial traits. These findings uncover an essential role of Ubi4 in ubiquitin transfer cascade and its pleiotropic effects on the in vitro and in vivo asexual cycle of B. bassiana.