Nematode-trapping Fungus Arthrobotrys Oligospora Research Articles

The Arf-GAPs, AoAge1 and AoAge2, regulate diverse cellular processes, conidiation, trap formation, and secondary metabolism in Arthrobotrys oligospora

Guanine nucleotide-binding proteins of the ADP ribosylation factor (Arf) family and their activating proteins (Arf-GAPs) are essential for diverse biological processes. Here, two homologous Arf-GAPs, Age1 (AoAge1) and Age2 (AoAge2), were identified in the widespread nematode-trapping fungus Arthrobotrys oligospora. Our results demonstrated that AoAge1, especially AoAge2, played crucial roles in mycelial growth, sporulation, trap production, stress response, mitochondrial activity, DNA damage, endocytosis, reactive oxygen species production, and autophagy. Notably, transcriptome data revealed that approximately 62.7% of the genes were directly or indirectly regulated by AoAge2, and dysregulated genes in Aoage2 deletion were enriched in metabolism, ribosome biogenesis, secondary metabolite biosynthesis, and autophagy. Furthermore, Aoage2 inactivation caused a substantial reduction in several compounds compared to the wild-type strain. Based on these results, a regulatory network for AoAge1 and AoAge2 was proposed and verified using a yeast two-hybrid assay. Based on our findings, AoAge1 and AoAge2 are essential for vegetative growth and mycelial development. Specifically, AoAge2 is required for sporulation and trapping morphogenesis. Our results demonstrated the critical functions of AoAge1 and AoAge2 in mycelial growth, diverse cellular processes, and pathogenicity, offering deep insights into the functions and regulatory mechanisms of Arf-GAPs in nematode-trapping fungi.

AoMedA has a complex regulatory relationship with AoBrlA, AoAbaA, and AoWetA in conidiation, trap formation, and secondary metabolism in the nematode-trapping fungus Arthrobotrys oligospora.

The asexual sporulation of filamentous fungi is an important mechanism for their reproduction, survival, and pathogenicity. In Aspergillus and several filamentous fungi, BrlA, AbaA, and WetA are the key elements of a central regulatory pathway controlling conidiation, and MedA is a developmental modifier that regulates temporal expression of central regulatory genes; however, their roles are largely unknown in nematode-trapping (NT) fungi. Arthrobotrys oligospora is a representative NT fungus, which can capture nematodes by producing adhesive networks (traps). Here, we characterized the function of AoMedA and three central developmental regulators (AoBrlA, AoAbaA, and AoWetA) in A. oligospora by gene disruption, phenotypic comparison, and multi-omics analyses, as these regulators are required for conidiation and play divergent roles in mycelial development, trap formation, lipid droplet accumulation, vacuole assembly, and secondary metabolism. A combined analysis of phenotypic traits and transcriptome showed that AoMedA and AoWetA are involved in the regulation of peroxisome, endocytosis, and autophagy. Moreover, yeast one-hybrid analysis showed that AoBrlA can regulate AoMedA, AoAbaA, and AoWetA, whereas AoMedA and AoAbaA can regulate AoWetA. Our results highlight the important roles of AoMedA, AoBrlA, AoAbaA, and AoWetA in conidiation, mycelia development, trap formation, and pathogenicity of A. oligospora and provide a basis for elucidating the relationship between conidiation and trap formation of NT fungi. IMPORTANCE Conidiation is the most common reproductive mode for many filamentous fungi and plays an essential role in the pathogenicity of fungal pathogens. Nematode-trapping (NT) fungi are a special group of filamentous fungi owing to their innate abilities to capture and digest nematodes by producing traps (trapping devices). Sporulation plays an important role in the growth and reproduction of NT fungi, and conidia are the basic components of biocontrol reagents for controlling diseases caused by plant-parasitic nematodes. Arthrobotrys oligospora is a well-known NT fungus and is a routinely used model fungus for probing the interaction between fungi and nematodes. In this study, the functions of four key regulators (AoMedA, AoBrlA, AoAbaA, and AoWetA) involved in conidiation were characterized in A. oligospora. A complex interaction between AoMedA and three central regulators was noted; these regulators are required for conidiation and trap formation and play a pleiotropic role in multiple intracellular activities. Our study first revealed the role of AoMedA and three central regulators in conidiation, trap formation, and pathogenicity of A. oligospora, which contributed to elucidating the regulatory mechanism of conidiation in NT fungi and helped in developing effective reagents for biocontrol of nematodes.

Function discovery of a non-ribosomal peptide synthetase-like encoding gene in the nematode-trapping fungus Arthrobotrys oligospora.

In this study, the function of a non-ribosomal peptide synthetase-like (NRPS-like) encoding gene AOL_s00188g306 (g306) was investigated to reveal the association between NRPS and nematocidal activity in the nematode-trapping fungus Arthrobotrys oligospora. Sequence analysis indicated that the encoded product of g306 is an adenylation domain of non-ribosomal peptide synthetases and extended short-chain dehydrogenase/reductase domain-containing proteins, and displays a wide substrate spectrum. The Δg306 mutants were more sensitive to chemical stressors than the wild type. Disruption of g306 impeded the nematocidal efficiency of A. oligospora. Metabolomics analysis showed that secondary metabolite biosynthesis and lipid metabolism were altered in the mutants. The phenotypic changes in the mutants can be attributed to the down-regulation of various metabolites, including fatty acyls, prenol lipids, steroidsand steroid derivative, and amino acid derivatives, identified in the present study. This study investigated the association between the non-ribosomal polypeptide-encoding gene g306 and nematicidal activity in A. oligospora, providing a reference for resolving the predation mechanism of nematode-trapping fungus.

Nematode-induced trap formation regulated by the histone H3K4 methyltransferase AoSET1 in the nematode-trapping fungus Arthrobotrys oligospora.

The methylation of lysine 4 of histone H3 (H3K4), catalyzed by the histone methyltransferase KMT2/SET1, has been functionally identified in many pathogenic fungi but remains unexplored in nematode-trapping fungi (NTFs). Here, we report a regulatory mechanism of an H3K4-specific SET1 orthologue, AoSET1, in the typical nematode-trapping fungus Arthrobotrys oligospora. When the fungus is induced by the nematode, the expression of AoSET1 is up-regulated. Disruption of AoSet1 led to the abolishment of H3K4me. Consequently, the yield of traps and conidia of ΔAoSet1 was significantly lower than that of the WT strain, and the growth rate and pathogenicity were also compromised. Moreover, H3K4 trimethylation was enriched mainly in the promoter of two bZip transcription factor genes (AobZip129 and AobZip350) and ultimately up-regulated the expression level of these two transcription factor genes. In the ΔAoSet1 and AoH3K4A strains, the H3K4me modification level was significantly decreased at the promoter of transcription factor genes AobZip129 and AobZip350. These results suggest that AoSET1-mediated H3KEme serves as an epigenetic marker of the promoter region of the targeted transcription factor genes. Furthermore, we found that AobZip129 negatively regulates the formation of adhesive networks and the pathogenicity of downstream AoPABP1 and AoCPR1. Our findings confirm that the epigenetic regulatory mechanism plays a pivotal role in regulating trap formation and pathogenesis in NTFs, and provide novel insights into the mechanisms of interaction between NTFs and nematodes.

Recent Advances in Life History Transition with Nematode-Trapping Fungus Arthrobotrys oligospora and Its Application in Sustainable Agriculture.

Parasitic nematodes cause great annual loss in the agricultural industry globally. Arthrobotrys oligospora is the most prevalent and common nematode-trapping fungus (NTF) in the environment and the candidate for the control of plant- and animal-parasitic nematodes. A. oligospora is also the first recognized and intensively studied NTF species. This review highlights the recent research advances of A. oligospora as a model to study the biological signals of the switch from saprophytism to predation and their sophisticated mechanisms for interacting with their invertebrate hosts, which is of vital importance for improving the engineering of this species as an effective biocontrol fungus. The application of A. oligospora in industry and agriculture, especially as biological control agents for sustainable purposes, was summarized, and we discussed the increasing role of A. oligospora in studying its sexual morph and genetic transformation in complementing biological control research.

AoSte12 Is Required for Mycelial Development, Conidiation, Trap Morphogenesis, and Secondary Metabolism by Regulating Hyphal Fusion in Nematode-Trapping Fungus Arthrobotrys oligospora.

Nematode-trapping (NT) fungi are a unique group of carnivorous microorganisms that can capture and digest nematodes by producing ingenious trapping devices (traps). Arthrobotrys oligospora, a representative NT fungus, can develop adhesive three-dimensional networks for nematode predation. Hyphal fusion is indispensable for the trap formation of A. oligospora. Here, we characterized an orthologous Ste12 protein (AoSte12) in A. oligospora via gene disruption, DNA affinity purification sequencing (DAP-Seq), and multi-omics approaches. The disruption of the Aoste12 gene caused an increase in hyphal fusion and resulted in defects in mycelial growth, conidiation, trap morphology, and stress resistance, as well as reducing the number of nuclei and lipid droplet accumulation. Moreover, transcriptome and DAP-Seq analysis revealed that AoSte12 was involved in cellular processes associated with growth, cell fusion, the tricarboxylic acid cycle, vesicles, actin filaments, and lipid metabolism. In addition, combining metabolome with transcriptome and DAP-Seq analysis indicated that AoSte12 was involved in the mitogen-activated protein kinase signaling pathway, lipid metabolism, and secondary metabolites. A yeast two-hybrid assay revealed that AoSte12 can interact with diverse proteins, such as the MAK-2 orthologue protein Fus3, the vacuolar sorting protein Pep3, and UDP-glycosyltransferase. Our results suggest that AoSte12 plays an indispensable role in hyphal fusion and thus regulates sporulation and trap morphogenesis. These results provide deep insights into the connection between hyphal fusion and trap formation in NT fungi. IMPORTANCE Nematode-trapping (NT) fungi are an important natural enemy of nematodes and can capture their prey by producing traps. Hyphal anastomosis and fusion are important for mycelial growth and the colony morphological development of filamentous fungi and are also crucial for the trap morphogenesis of NT fungi. Arthrobotrys oligospora can form complex three-dimensional networks (traps) when sensing the presence of nematodes. This study revealed that AoSte12 is indispensable for hyphal fusion and that it regulates mycelial growth, conidiation, trap morphogenesis, stress resistance, the number of nuclei, and lipid droplet accumulation in A. oligospora. In addition, DNA affinity purification sequencing, transcriptome, and metabolome analyses further revealed that AoSte12 is involved in the mitogen-activated protein kinase pathway, lipid metabolism, and secondary metabolism. Overall, these findings expand the important role of AoSte12 in NT fungus A. oligospora and provide a broad foundation for elucidating the regulatory mechanism of trap development and the lifestyle transitions of pathogenic fungi.

Roles of the Fungal-Specific Lysine Biosynthetic Pathway in the Nematode-Trapping Fungus Arthrobotrys oligospora Identified through Metabolomics Analyses.

In higher fungi, lysine is biosynthesized via the α-aminoadipate (AAA) pathway, which differs from plants, bacteria, and lower fungi. The differences offer a unique opportunity to develop a molecular regulatory strategy for the biological control of plant parasitic nematodes, based on nematode-trapping fungi. In this study, in the nematode-trapping fungus model Arthrobotrys oligospora, we characterized the core gene in the AAA pathway, encoding α-aminoadipate reductase (Aoaar), via sequence analyses and through comparing the growth, and biochemical and global metabolic profiles of the wild-type and Aoaar knockout strains. Aoaar not only has α-aminoadipic acid reductase activity, which serves fungal L-lysine biosynthesis, but it also is a core gene of the non-ribosomal peptides biosynthetic gene cluster. Compared with WT, the growth rate, conidial production, number of predation rings formed, and nematode feeding rate of the ΔAoaar strain were decreased by 40-60%, 36%, 32%, and 52%, respectively. Amino acid metabolism, the biosynthesis of peptides and analogues, phenylpropanoid and polyketide biosynthesis, and lipid metabolism and carbon metabolism were metabolically reprogrammed in the ΔAoaar strains. The disruption of Aoaar perturbed the biosynthesis of intermediates in the lysine metabolism pathway, then reprogrammed amino acid and amino acid-related secondary metabolism, and finally, it impeded the growth and nematocidal ability of A. oligospora. This study provides an important reference for uncovering the role of amino acid-related primary and secondary metabolism in nematode capture by nematode-trapping fungi, and confirms the feasibility of Aoarr as a molecular target to regulate nematode-trapping fungi to biocontrol nematodes.

The Multifaceted Gene 275 Embedded in the PKS-PTS Gene Cluster Was Involved in the Regulation of Arthrobotrisin Biosynthesis, TCA Cycle, and Septa Formation in Nematode-Trapping Fungus Arthrobotrys oligospora.

The predominant nematode-trapping fungus Arthrobotrys oligospora harbors a unique polyketide synthase-prenyltransferase (PKS-PTS) gene cluster AOL_s00215g responsible for the biosynthesis of sesquiterpenyl epoxy-cyclohexenoids (SECs) that are involved in the regulation of fungal growth, adhesive trap formation, antibacterial activity, and soil colonization. However, the function of one rare gene (AOL_s00215g275 (275)) embedded in the cluster has remained cryptic. Here, we constructed two mutants with the disruption of 275 and the overexpression of 275, respectively, and compared their fungal growth, morphology, resistance to chemical stress, nematicidal activity, transcriptomic and metabolic profiles, and infrastructures, together with binding affinity analysis. Both mutants displayed distinct differences in their TCA cycles, SEC biosynthesis, and endocytosis, combined with abnormal mitochondria, vacuoles, septa formation, and decreased nematicidal activity. Our results suggest that gene 275 might function as a separator and as an integrated gene with multiple potential functions related to three distinct genes encoding the retinoic acid induced-1, cortactin, and vacuolar iron transporter 1 proteins in this nematode-trapping fungus. Our unexpected findings provide insight into the intriguing organization and functions of a rare non-biosynthetic gene in a biosynthetic gene cluster.

Aoatg11 and Aoatg33 are indispensable for mitophagy, and contribute to conidiation, the stress response, and pathogenicity in the nematode-trapping fungus Arthrobotrys oligospora

Mitophagy is one of the most important cellular processes to ensure mitochondrial quality control, which aims to transport damaged, dysfunctional, or excess mitochondria for degradation and reuse. Here, we determined the function of AoAtg11 and AoAtg33, two orthologous autophagy-related proteins involved in yeast mitophagy, in the typical nematode-trapping fungus Arthrobotrys oligospora. Deletion of Aoatg11 and Aoatg33 impairs mitophagy, mitochondrial morphology and activity, autophagy, cell apoptosis, reactive oxygen species levels, lipid droplet accumulation, and endocytosis. These combined effects resulted in slow vegetative growth; reduced conidiation, trap formation, cell nucleus, and extracellular protease activity; increased susceptibility to the stress response; and arthrobotrisin production in the ΔAoatg11 and ΔAoatg33 mutants, compared with the wild-type strain. In addition, the absence of Aoatg11 caused an endoplasmic reticulum stress response. Transcriptome analysis revealed that many differentially expressed genes in the ΔAoatg11 mutants were involved in various important cellular processes, such as lipid metabolism, the TCA cycle, mitophagy, nitrogen metabolism, endocytosis, and the MAPK signaling pathway. In conclusion, our study revealed that Aoatg11 and Aoatg33 mediate autophagy and mitophagy in A. oligospora, and provides a basis for elucidating the links between mitophagy and fungal vegetative growth, conidiation, and pathogenicity.

A Putative Adhesin-encoding Gene AOL_s00007g5 is involved in the Mycelial Growth and Development of Nematode-trapping Fungus Arthrobotrys oligospora

A Putative Adhesin-encoding Gene AOL_s00007g5 is involved in the Mycelial Growth and Development of Nematode-trapping Fungus Arthrobotrys oligospora

AMPK Is Involved in Regulating the Utilization of Carbon Sources, Conidiation, Pathogenicity, and Stress Response of the Nematode-Trapping Fungus Arthrobotrys oligospora.

ABSTRACTAMP-activated protein kinase (AMPK), a heterotrimeric complex, can sense energy and nutritional status in eukaryotic cells, thereby participating in the regulation of multiple cellular processes. In this study, we characterized the function of the catalytic α-subunit (SNF1) and the two regulatory β- and γ-subunits (GAL83 and SNF4) of AMPK in a representative nematode-trapping fungus, Arthrobotrys oligospora, by gene knockout, phenotypic analysis, and RNA sequencing. The ability of the AMPK complex mutants (including ΔAosnf1, ΔAogal83, and ΔAosnf4) to utilize a nonfermentable carbon source (glycerol) was reduced, and the spore yields and trap formation were remarkably decreased. Moreover, AMPK plays an important role in regulating stress response and nematode predation efficiency. Transcriptomic profiling between the wild-type strain and ΔAosnf1 showed that differentially expressed genes were enriched for peroxisome, endocytosis, fatty acid degradation, and multilipid metabolism (sphingolipid, ether lipid, glycerolipid, and glycerophospholipid). Meanwhile, a reduced lipid droplet accumulation in ΔAosnf1, ΔAogal83, and ΔAosnf4 mutants was observed, and more vacuoles appeared in the mycelia of the ΔAosnf1 mutant. These results highlight the important regulatory role of AMPK in the utilization of carbon sources and lipid metabolism, as well as providing novel insights into the regulatory mechanisms of the mycelia development, conidiation, and trap formation of nematode-trapping (NT) fungi.IMPORTANCE NT fungi are widely distributed in various ecosystems and are important factors in the control of nematode populations in nature; their trophic mycelia can form unique infectious devices (traps) for capturing nematodes. Arthrobotrys oligospora is a representative NT fungi which can develop complex three-dimensional networks (adhesive networks) for nematode predation. Here, we demonstrated that AMPK plays an important role in the glycerol utilization, conidiation, trap formation, and nematode predation of A. oligospora, which was further confirmed by transcriptomic analysis of the wild-type and mutant strains. In particular, our analysis indicated that AMPK is required for lipid metabolism, which is primarily associated with energy regulation and is essential for trap formation. Therefore, this study extends the functional study of AMPK in NT fungi and helps to elucidate the molecular mechanism of the regulation of trap development, as well as laying the foundation for the development of efficient nematode biocontrol agents.

The Arf-GAP Proteins AoGcs1 and AoGts1 Regulate Mycelial Development, Endocytosis, and Pathogenicity in Arthrobotrys oligospora.

Small GTPases from the ADP-ribosylation factor (Arf) family and their activating proteins (Arf-GAPs) regulate mycelial development, endocytosis, and virulence in fungi. Here, we identified two orthologous Arf-GAP proteins, AoGcs1 and AoGts1, in a typical nematode-trapping fungus Arthrobotrys oligospora. The transcription of Aogcs1 and Aogts1 was highly expressed in the sporulation stage. The deletion of Aogcs1 and Aogts1 caused defects in DNA damage, endocytosis, scavenging of reactive oxygen species, lipid droplet storage, mitochondrial activity, autophagy, serine protease activity, and the response to endoplasmic reticulum stress. The combined effects resulted in slow growth, decreased sporulation capacity, increased susceptibility to chemical stressors and heat shock, and decreased pathogenicity of the mutants compared with the wild-type (WT) strain. Although deletion of Aogcs1 and Aogts1 produced similar phenotfypic traits, their roles varied in conidiation and proteolytic activity. The ΔAogts1 mutant showed a remarkable reduction in conidial yield compared with the WT strain but not in proteolytic activity; in contrast, the ΔAogcs1 mutant showed an increase in proteolytic activity but not in sporulation. In addition, the growth of ΔAogcs1 and ΔAogts1 mutants was promoted by rapamycin, and the ΔAogts1 mutant was sensitive to H-89. Collectively, the ΔAogts1 mutant showed a more remarkable difference compared with the WT strain than the ΔAogcs1 mutant. Our study further illustrates the importance of Arf-GAPs in the growth, development, and pathogenicity of nematode-trapping fungi.

Functional Analysis of Two Affinity cAMP Phosphodiesterases in the Nematode-Trapping Fungus Arthrobotrys oligospora.

Phosphodiesterases are essential regulators of cyclic nucleotide signaling with diverse physiological functions. Two phosphodiesterases, PdeH and PdeL, have been identified from yeast and filamentous fungi. Here, the orthologs of PdeH and PdeL were characterized in a typical nematode-trapping fungus Arthrobotrys oligospora by gene disruption and phenotypic comparison. Deletion of AopdeH caused serious defects in mycelial growth, conidiation, stress response, trap formation, and nematicidal efficiency compared to the wild-type strain. In contrast, these phenotypes have no significant difference in the absence of AopdeL. In addition, deletion of AopdeH and AopdeL resulted in a remarkable increase in cAMP level during vegetative growth and trap formation, and the number of autophagosomes was decreased in ΔAopdeH and ΔAopdeL mutants, whereas their volumes considerably increased. Moreover, metabolomic analyses revealed that many metabolites were downregulated in ΔAopdeH mutant compared to their expression in the wild-type strain. Our results indicate that AoPdeH plays a crucial role in mycelial growth, conidiation, stress response, secondary metabolism, and trap formation. In contrast, AoPdeL only plays a minor role in hyphal and conidial morphology, autophagy, and trap formation in A. oligospora. This work expands the roles of phosphodiesterases and deepens the understanding of the regulation of trap formation in nematode-trapping fungi.

Phospholipase C (AoPLC2) regulates mycelial development, trap morphogenesis, and pathogenicity of the nematode-trapping fungus Arthrobotrys oligospora.

Phospholipase C (PLC) is a hydrolase involved in signal transduction in eukaryotic cells. This study aimed to understand the function of PLC in the nematode-trapping fungus Arthrobotrys oligospora. Orthologous PLC (AoPLC2) of A. oligospora was functionally analysed using gene disruption and multi-phenotypic analysis. Disrupting Aoplc2 caused a deformation of partial hyphal cells (about 10%) and conidia (about 50%), decreased the number of nuclei in both conidia and hyphal cells, and increased the accumulation of lipid droplets. Meanwhile, the sporulation-related genes fluG and abaA were downregulated in ΔAoplc2mutants than in the wild-type strain. Moreover, ΔAoplc2mutants were more sensitive to osmotic stressors. Importantly, the number of traps, electron-dense bodies in traps, and nematicidal activity of ΔAoplc2mutants were reduced, and the shape of the traps was deformed. In addition, AoPLC2 was involved in the biosynthesis of secondary metabolites in A. oligospora. AoPLC2 plays an important role in the development of hyphae, spores, and cell nuclei, responses to stress, formation of traps, and predation of nematodes in A. oligospora. This study reveals the various functions of phospholipase C and elucidates the regulation of trap morphogenesis in nematode-trapping fungi.

Aolatg1 and Aolatg13 Regulate Autophagy and Play Different Roles in Conidiation, Trap Formation, and Pathogenicity in the Nematode-Trapping Fungus Arthrobotrys oligospora.

Autophagy is a conserved cellular recycling and trafficking pathway in eukaryotes that plays an important role in cell growth, development, and pathogenicity. Atg1 and Atg13 form the Atg1–Atg13 complex, which is essential for autophagy in yeast. Here, we characterized the roles of the Aolatg1 and Aolatg13 genes encoding these autophagy-related proteins in the nematode-trapping fungus Arthrobotrys oligospora. Investigation of the autophagy process by using the AoAtg8-GFP fusion protein showed that autophagosomes accumulated inside vacuoles in the wild-type (WT) A. oligospora strain, whereas in the two mutant strains with deletions of Aolatg1 or Aolatg13, GFP signals were observed outside vacuoles. Similar results were observed by using transmission electron microscopy. Furthermore, deletion of Aolatg1 caused severe defects in mycelial growth, conidiation, conidial germination, trap formation, and nematode predation. In addition, transcripts of several sporulation-related genes were significantly downregulated in the ΔAolatg1 mutant. In contrast, except for the altered resistance to several chemical stressors, no obvious differences were observed in phenotypic traits between the WT and ΔAolatg13 mutant strains. The gene ontology analysis of the transcription profiles of the WT and ΔAolatg1 mutant strains showed that the set of differentially expressed genes was highly enriched in genes relevant to membrane and cellular components. The Kyoto Encyclopedia of Genes and Genomes analysis indicated that differentially expressed genes were highly enriched in those related to metabolic pathways, autophagy and autophagy-related processes, including ubiquitin-mediated proteolysis and SNARE interaction in vesicular transport, which were enriched during trap formation. These results indicate that Aolatg1 and Aolatg13 play crucial roles in the autophagy process in A. oligospora. Aolatg1 is also involved in the regulation of asexual growth, trap formation, and pathogenicity. Our results highlight the importance of Aolatg1 in the growth and development of A. oligospora, and provide a basis for elucidating the role of autophagy in the trap formation and pathogenicity of nematode-trapping fungi.

Fungistatic Mechanism of Ammonia against Nematode-Trapping Fungus Arthrobotrys oligospora, and Strategy for This Fungus To Survive Ammonia.

ABSTRACTSoil fungistasis is a phenomenon in which the germination and growth of fungal propagules is widely inhibited in soils. Although fungistatic compounds are known to play important roles in the formation of soil fungistasis, how such compounds act on soil fungi is little studied. In this study, it was found that ammonia (NH3) induced global protein misfolding marked by increased ubiquitination levels of proteins (ubiquitylome data and Western blot verification). The misfolded proteins should trigger the endoplasmic reticulum (ER) stress, which was indicated by electron microscope image and proteome data. Results from the mutants of BiP and proteasome subunit alpha 7 suggested that ER stress played a mechanistic role in inhibiting conidial germination. Results from proteome data indicated that, to survive ammonia fungistasis, conidia first activated the unfolded protein response (UPR) to decrease ER stress and restore ER protein homeostasis, and the function of UPR in surviving ammonia was confirmed by using mutant strains. Second, ammonia toxicity could be reduced by upregulating carbon metabolism-related proteins, which benefited ammonia fixation. The results that metabolites (especially glutamate) could relieve the ammonia fungistasis confirmed this indirectly. Finally, results from gene knockout mutants also suggested that the fungistatic mechanism of ammonia is common for soil fungistasis. This study increased our knowledge regarding the mechanism of soil fungistasis and provided potential new strategies for manipulating soil fungistasis.IMPORTANCE Soil fungistasis is a phenomenon in which the germination and growth of fungal propagules is widely inhibited in soil. Although fungistatic compounds are known to play important roles in the formation of soil fungistasis, how such compounds act on soil fungi remains little studied. This study revealed an endoplasmic reticulum stress-related fungistatic mechanism with which ammonia acts on Arthrobotrys oligospora and a survival strategy of conidia under ammonia inhibition. Our study provides the first mechanistic explanation of how ammonia impacts fungal spore germination, and the mechanism may be common for soil fungistasis. This study increases our knowledge regarding the mechanism of soil fungistasis in fungal spores and provides potential new strategies for manipulating soil fungistasis.

Historical Differentiation and Recent Hybridization in Natural Populations of the Nematode-Trapping Fungus Arthrobotrys oligospora in China.

Maintaining the effects of nematode-trapping fungi (NTF) agents in order to control plant-parasitic nematodes (PPNs) in different ecological environments has been a major challenge in biological control applications. To achieve such an objective, it is important to understand how populations of the biocontrol agent NTF are geographically and ecologically structured. A previous study reported evidence for ecological adaptation in the model NTF species Arthrobotrys oligospora. However, their large-scale geographic structure, patterns of gene flow, their potential phenotypic diversification, and host specialization remain largely unknown. In this study, we developed a new panel of 20 polymorphic short tandem repeat (STR) markers and analyzed 239 isolates of A. oligospora from 19 geographic populations in China. In addition, DNA sequences at six nuclear gene loci and strain mating types (MAT) were obtained for these strains. Our analyses suggest historical divergence within the A. oligospora population in China. The genetically differentiated populations also showed phenotypic differences that may be related to their ecological adaptations. Interestingly, our analyses identified evidence for recent dispersion and hybridization among the historically subdivided geographic populations in nature. Together, our results indicate a changing population structure of A. oligospora in China and that care must be taken in selecting the appropriate strains as biocontrol agents that can effectively reproduce in agriculture soil while maintaining their nematode-trapping ability.

AoBck1 and AoMkk1 Are Necessary to Maintain Cell Wall Integrity, Vegetative Growth, Conidiation, Stress Resistance, and Pathogenicity in the Nematode-Trapping Fungus Arthrobotrys oligospora.

The cell wall integrity (CWI) pathway is composed of three mitogen-activated protein kinases (MAPKs), Bck1, Mkk1/2, and Slt2, and is one of the main signaling pathways for fungal pathogenesis, cell wall synthesis, and integrity maintenance. In this study, we characterized orthologs of Saccharomyces cerevisiae Bck1 and Mkk1 in the nematode-trapping (NT) fungus Arthrobotrys oligospora by multiple phenotypic comparison, and the regulation of conidiation and cell wall synthesis was analyzed using real-time PCR (RT-PCR). Both ΔAoBck1 and ΔAoMkk1 mutants showed severe defects in vegetative growth, cell nucleus number, and stress resistance. Both the mutants were unable to produce spores, and the transcription of several genes associated with sporulation and cell wall biosynthesis was markedly downregulated during the conidiation stage. Further, cell walls of the ΔAoBck1 and ΔAoMkk1 mutants were severely damaged, and the Woronin body failed to respond to cellular damage. In particular, the mutants lost the ability to produce mycelial traps for nematode predation. Taken together, AoBck1 and AoMkk1 play a conserved role in mycelial growth and development, CWI, conidiation, multi-stress tolerance, trap formation, and pathogenicity. We highlighted the role of AoBck1 and AoMkk1 in regulating the Woronin body response to cellular damage and cell nucleus development in A. oligospora.

The High Osmolarity Glycerol (HOG) Pathway Functions in Osmosensing, Trap Morphogenesis and Conidiation of the Nematode-Trapping Fungus Arthrobotrys oligospora.

Hog1, a mitogen-activated protein kinase (MAPK), has been identified in diverse fungal species, and it regulates various cellular processes, such as osmoadaptation, nutrient-sensing, and pathogenesis. However, the roles that Hog1 plays in nematode-trapping fungi were previously unclear. Here, we characterized orthologs of Saccharomyces cerevisiae Hog1 and membrane mucin Msb2 in the nematode-trapping fungus Arthrobotrys oligospora. We generated gene deletion mutants of HOG1 and MSB2 in A. oligospora, and characterized their roles in osmosensing, growth, and trap morphogenesis. We found that both hog1 and msb2 mutants were highly sensitive to high osmolarity. Predation analyses further revealed that hog1 and msb2 deletion caused a reduction in trap formation and predation efficiency. Furthermore, HOG1 is required for conidiation in A. oligospora, demonstrating its critical role in this developmental pathway. In summary, this study demonstrated that the conserved Hog1 and Msb2 govern physiology, growth and development in the nematode-trapping fungus A. oligospora.

Novel Polyketide-Terpenoid Hybrid Metabolites from a Potent Nematicidal Arthrobotrys oligospora Mutant ΔAOL_s00215g278.

Here, we reported that detailed investigation on trace targeted metabolites from nematode-trapping fungus Arthrobotrys oligospora mutant with deletion of P450 gene AOL_s00215g278 led to isolation of 9 new polyketide-terpenoid hybrid derivatives, including four new glycosides of the key precursor farnesyl hydrotoluquinol (1) and, surprisingly, four new sesquiterpenyl epoxy-cyclohexenoids (SECs) analogues. Among them, two major target metabolites 1 and 14 displayed moderate nematode inhibitory ability. Moreover, the mutant lacking AOL_s00215g278 could form far more nematode-capturing traps within 6 h in contact with nematodes and show rapid potent nematicidal activity with killing 93.7% preys, though deletion of the P450 gene resulted in dramatic decrease in fungal colony growth and failure to produce fungal conidia. The results unequivocally revealed that gene AOL_s00215g278 should be involved in not only the SEC biosynthetic pathway in the nematode-trapping fungus A. oligospora but also fungal conidiation and nematicidal activity.