Fungal-fungal cocultivation leads to widespread secondary metabolite alteration requiring the partial loss-of-function VeA1 protein

Abstract

Microbial communication has attracted notable attention as an indicator of microbial interactions that lead to marked alterations of secondary metabolites (SMs) in varied environments. However, the mechanisms responsible for SM regulation are not fully understood, especially in fungal-fungal interactions. Here, cocultivation of an endophytic fungus Epicoccum dendrobii with the model fungus Aspergillus nidulans and several other filamentous fungi triggered widespread alteration of SMs. Multiple silent biosynthetic gene clusters in A. nidulans were activated by transcriptome and metabolome analysis. Unprecedentedly, gene deletion and replacement proved that a partial loss-of-function VeA1 protein, but not VeA, was associated with the widespread SM changes in both A. nidulans and A. fumigatus during cocultivation. VeA1 regulation required the transcription factor SclB and the velvet complex members LaeA and VelB for producing aspernidines as representative formation of SMs in A. nidulans. This study provides new insights into the mechanism that trigger metabolic changes during fungal-fungal interactions.

A partial loss-of-function VeA1 protein regulatory network mediates secondary metabolism during fungal-fungal interaction.

INTRODUCTION

Microbial secondary metabolites (SMs) are diverse low–molecular weight compounds produced by microorganisms to ensure their formidable adaptive capacity for variable environments. Driven by the discovery of penicillin (1, 2) and its development into an antibiotic, great progress has been achieved for chemical characterization of a multitude of natural products from microorganisms in the past decades. To date, approximately 61.5% of microbial SMs are reported from fungi (The Natural Products Atlas database, www.npatlas.org/), underlying their significance as natural product sources (3). Among them, about 13.5% SMs are contributed by Aspergillus species, which are one of the largest number of fungi, owning about 378 species as reported by the World Data Centre for Microorganisms (4). Nonetheless, the large number of predictive gene clusters from sequenced fungal genomes suggests their potential to produce more SMs. One obstacle to characterizing these unknown SMs is that most biosynthetic gene clusters (BGCs) are not transcriptionally expressed or only low-expressed under standard laboratory cultivation conditions.

In nature, microorganisms never live in isolation but constantly coinhabit all environments. They generally exchange and share chemical signals including SMs with each other. There is a consensual perspective that the interaction among microorganisms constitutes a driving force for the production of SMs (5). However, standard laboratory cultivation normally omits the complex microbial community, possibly leading to less SM production (6). To exploit the hidden SM treasure, cocultivation becomes a prospective strategy to stimulate SM synthesis and has been successfully applied to find metabolites with bioactivity or new chemical scaffolds (7). One famous example is the unanticipated discovery of penicillin in a culture of Staphylococcus aureus contaminated by Penicillium notatum (2). Since then, hundreds of cocultivation studies have been reported for the mining of natural products, especially new molecules previously not detected in monocultivation. For example, bacterial-fungal cocultures resulted in the identification of a polyketide pestalone (8), a large macrolactone ibomycin (9), four diterpenoid libertellenones (10), N-formyl alkaloids (11), and hybrid polyketide synthase–nonribosomal peptide synthetase (PKS-NRPS)–derived tetramic acid analogs (12). Likewise, fungal-fungal cocultivation also activated silent gene clusters and induced the corresponding natural product formation. Citrifelins, which have a unique tetracyclic framework, were characterized from a coculture of Penicillium citrinum with Beauveria felina (13). Cocultivation of two marine fungi activated a rare class of 2-alkenyl-tetrahydropyran and deactivated the antifungal metabolite pyridoxatin yielding methyl-pyridoxatin, implying a complex offensive and counteroffensive fungal-fungal interaction (14).

Although some interesting structures have been found during coculture, the mechanism of SM activation in fungus-containing coculture has been rarely studied in detail. In previous studies, Streptomyces hygroscopicus was shown to trigger the activation of polyketide biosynthesis in Aspergillus nidulans through physical interactions, leading to the formation of orsellinic acid and its anti-osteoporosis derivatives F-9775 A and B (15). Gene deletion and RNA sequencing (RNA-seq) experiments indicated that a member of the histone acetyltransferase Spt-Ada-Gcn5-acetyltransferase/ADA (SAGA/ADA) complex, GcnE, induced the activation of orsellinic acid BGC during the bacterial-fungal cocultivation (16, 17). The Myb-like transcription factor BasR was shown to be the central regulatory node for integrating bacterial signals to regulate the fungal metabolism (18). Additional study on monocultivation of A. nidulans connected the velvet complex member VeA to SAGA-ADA activity in suppression of the silent orsellinic acid BGC (19). These studies thus tied bacterial-fungal communication to the epigenetic machinery and BasR mediating signal transduction. However, the mechanism of pathway activation during fungal-fungal interactions remains unclear.

To address this issue, we carried out fungal-fungal cocultivation of the endophytic fungus Epicoccum dendrobii with the model fungus A. nidulans or other filamentous fungi. SM identification and genetic characterization led to the discovery that partial loss-of-function mutation of VeA is a prerequisite for mediating coculture widespread alteration of SMs in A. nidulans. High-performance liquid chromatography (HPLC) analysis and subsequent isolation allowed us to identify 14 aspernidine derivatives (1 to 14) including 8 new ones, which were encoded by the activated pkf gene cluster. Comprehensive data from transcriptome and following genetic mutations revealed that the transcription factor SclB regulated SM production under the control of VeA1. This genetic evidence indicates an apparent positive impact of a VeA1-containing velvet complex mediating SclB activation of silent gene clusters in the fungal-fungal system. Our study provides a new example for the promising cocultivation approach to altering secondary metabolism and opens up the opportunity to understand the regulation mechanism involved in the fungal-fungal interaction.

RESULTS

Coculture of E. dendrobii with other filamentous fungi leads to the widespread alteration of SMs and activation of aspernidine biosynthesis in A. nidulans

The model fungus A. nidulans was cultivated by exposing to air to screen microbes that would affect A. nidulans morphology and SM production. One fungal strain, YL001, stimulated the spore color of A. nidulans becoming yellow and caused our attention (Fig. 1A). The strain was subsequently identified as E. dendrobii by alignment analysis of the sequences of internal transcribed spacer regions and intervening 5.8S nuclear ribosomal DNA (nrDNA) (ITS), partial 28S large subunit nrDNA (LSU), partial regions of RNA polymerase II second largest subunit (RPB2), and β-tubulin (fig. S1). To systematically study the fungal-fungal interactions, we cocultivated E. dendrobii with the filamentous fungi from different genera including Aspergillus, Penicillium, Trichoderma, and Fusarium (fig. S2A); among them, the cocultures with each A. nidulans, Aspergillus oryzae, Aspergillus terreus, Penicillium chrysogenum, Fusarium proliferatum, or Trichoderma hypoxylon showed widespread alterations of SMs (fig. S2B).

Fig. 1. Cocultivation of A. nidulans with E. dendrobii demonstrated a widespread alteration of secondary metabolism.

(A) Growth of A. nidulans (veA1) and E. dendrobii strains on PDA plates under monocultivation and cocultivation conditions. (B) HPLC analysis of crude extracts from monocultivated and cocultivated strains. Ultraviolet (UV) absorptions at 210 nm are illustrated. AN, A. nidulans; ED, E. dendrobii. (C) Structures of the identified aspernidine derivatives 1 to 14. New ones are shown in red; compounds 13 and 14 are instable intermediates with the proposed structures.

To reveal the mechanism of how E. dendrobii could affect secondary metabolism on a broad range of filamentous fungi, different VeA variants of A. nidulans were chosen for coculture due to the differential roles of VeA and VeA1 in fungal secondary metabolism (20). Specific yellow spore pigmentation was only observed on the cocultures for veA1-genotype strains, i.e., RDIT2.3 (21), LO4389 (22), and LO8030 (23), but not for veA-genotype strains RDIT9.32 (21) and RJMP1.49 (Fig. 1A and fig. S3A) (24). This indicated that either the gene yA, which encodes the laccase required to convert the yellow pigment to the final green pigment (25), or its gene product was partially inhibited in the cocultures. Moreover, in contrast to the monocultures of A. nidulans and E. dendrobii, a broad accumulation of new SMs was detected during cocultivation with veA1-genotype strains based on HPLC analysis (Fig. 1B), whereas there was no significant chemical difference for veA-genotype strains (fig. S3B).

To identify the up-regulated metabolites, a scale-up coculture of A. nidulans RDIT2.3 with E. dendrobii was performed in 20 liters of potato dextrose broth (PDB) medium for 6 days. Fourteen compounds were isolated from the crude extracts and characterized as aspernidine derivatives (1 to 14, Fig. 1C) by comprehensive mass spectrometry (MS) and nuclear magnetic resonance (NMR) analysis (tables S5 to S16 and figs. S15 to S67). Compounds 1 and 2 were derivatives of farnesyl pyrophosphate, which was one of the aspernidine precursors (26, 27). Compounds 3 to 14 were identified as meroterpenoids, sharing the same skeleton of trihydroxyphthalaldehyde but with varying modifications on the side chain or the benzoic aldehyde. Compound 5 was elucidated as aspernidine F (28), and compound 8 was elucidated as asperugin B (29), referring to the published data. Compounds 13 and 14 were instable during isolation and converted to compound 8 (fig. S4). Compounds 3, 4, 6, 7, and 9 to 12 are novel molecules.

Previously, aspernidine A was reported to be biosynthesized by the aspernidine biosynthesis gene cluster (pkf) composed of six genes (pkfA to pkfF) in A. nidulans (Fig. 2A) (30). The nonreducing PKS PkfA and prenyltransferase PkfE were responsible for ortho-orsellinaldehyde formation and prenylation, respectively. To prove the involvement of pkf BGC in the biosynthesis of 1 to 14, the PKS gene was deleted in A. nidulans carrying the veA1 allele to yield a ΔpkfA-mutant, which was subsequently cocultivated with E. dendrobii. HPLC analysis of the coculture extract revealed the complete abolishment of 1 to 14 (Fig. 2B). Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that five genes, i.e., pkfA, pkfB, pkfD, pkfE, and pkfF, were transcriptionally silent in monocultivation of A. nidulans, whereas they showed a high expression level when cocultivated with E. dendrobii (Fig. 2C). These results confirmed that cocultivation of E. dendrobii with veA1-genotype A. nidulans significantly activated the pkf BGC for the formation of aspernidines. Biosynthesis of 1 to 14 was proposed according to the reported pkf pathway (fig. S5).

Fig. 2. Biosynthetic pathway of aspernidine derivatives was activated during coculture of A. nidulans with E. dendrobii.

(A) Schematic representation of aspernidine gene cluster (pkf) in A. nidulans. pkfA, polyketide synthase; pkfB, cytochrome P450; pkfC, short-chain dehydrogenase; pkfD, hypothetical protein; pkfE, prenyltransferase; pkfF, choline dehydrogenase. (B) HPLC analysis of crude extracts of A. nidulans carrying the veA1 allele with or without E. dendrobii and cocultivation of ΔpkfA-mutant in the veA1-genotype background with E. dendrobii. UV absorptions at 210 nm are illustrated. (C) Quantitative real-time polymerase chain reaction (RT-PCR) analysis of pkfA-pkfF genes in A. nidulans carrying the veA1 allele during (I) monocultivation and (II) cocultivation with E. dendrobii.

Cocultivation with E. dendrobii triggers the widespread alteration of transcription and secondary metabolism in A. nidulans

To probe how fungal-fungal cocultivation regulates fungal transcription and metabolism, RNA-seq analysis was performed for monocultivated or cocultivated A. nidulans RDIT 2.3 (veA1) strains in two biological replicates. The box plot analysis of two replicates showed similar gene expression level as displayed in fig. S6 to ensure data dependability. A principal components analysis for gene expression profiles in A. nidulans revealed a clear variance (96%) between monocultivation and cocultivation (fig. S7). Differential gene expression analysis was determined between these two conditions when the average reads of the corresponding transcripts differed. A total of 9561 unique transcripts were detected. Statistical analysis showed that 3267 genes (34.2%) of the differentially expressed genes (DEGs) were found between monocultivation and cocultivation with an adjusted P value of <0.01. Notably, 2047 genes (21.4%) of DEGs exhibited twofold or even higher expression level during cocultivation. Among them, 1128 genes were up-regulated and 919 genes were down-regulated (fig. S8). As shown in fig. S9, these DEGs were widely distributed on eight chromosomes of A. nidulans.

To find DEGs involved in the secondary metabolism, 64 backbone genes, which are responsible for the biosynthesis of chemical skeleton such as polyketides, nonribosomal peptides, and terpenes, were selected for deep analysis (31, 32). Consistent with HPLC and RT-PCR analysis, the PKS gene pkfA for aspernidine biosynthesis showed up-regulation under the cocultivation condition. Furthermore, 15 additional backbone genes were also significantly up-regulated (log₂ fold change ≥ 2.0), and 13 backbone genes were down-regulated (log₂ fold change ≤ −2.0) (adjusted P < 0.05) in A. nidulans (Fig. 3A). Similar gene expression levels were also found in 18 BGCs containing those backbone genes (Fig. 3B). These results revealed a widespread alteration of BGC gene expression levels in A. nidulans triggered by cocultivation. The corresponding SM analysis by LC-MS was well correlated to BGC gene expression during coculture (fig. S10). For example, AN2545 to AN2549, members of the emericellamide BGC, were activated in coculture (Fig. 3B), leading to the accumulation of nonribosomal peptides emericellamides A to F (15 to 19) (33). Monodictyphenone (20) and endocrocin (21), which were previously silent and discovered by deletion of the component of COMPASS complex cclA (34, 35), also showed increased production during cocultivation. The prenylated xanthone derivative arugosin A (22) identified on a strict Raulin-Thom oatmeal (RTO) medium (36) was easily produced in the coculture of A. nidulans with E. dendrobii. The production of austinol (23) and dehydroaustinol (24) was slightly increased under cocultivation (fig. S10). Biosynthetic genes of 20 to 22 (AN0146 to AN0150, AN10021 to AN10023, AN10035, AN10038, AN10044, and AN10049) were also correspondingly expressed to a higher level (Fig. 3B). However, the two common metabolites, sterigmatocystin and penicillin (37), were not detected in the coculture. Overall, transcriptome and secondary metabolism data demonstrated that cocultivation with E. dendrobii triggered the widespread alteration of SMs in A. nidulans with a veA1 allele. To address the intricate pathway activation mechanism, SM alteration was represented by detection of aspernidine derivatives (1 to 14) in the following investigations.

Fig. 3. Widespread alteration of transcriptome and metabolism triggered by cocultivation of A. nidulans carrying the veA1 allele with E. dendrobii.

(A) Expression levels of the 64 core biosynthetic genes in eight chromosomes of A. nidulans. Red, green, and gray color represent the genes that are up-regulated, down-regulated, and not regulated significantly under cocultivation condition, respectively. (B) The differential expressions of BGCs are indicated by heatmap during cocultivation. Their corresponding products referring to the published data are labeled on the right. Unknown: The biosynthetic pathway has not been elucidated yet. (C) Additional SMs in A. nidulans are activated under cocultivation condition.

VeA1 is crucial for the SM alteration in fungal-fungal cocultivation

Screening of different A. nidulans strains with E. dendrobii revealed that widespread SM alteration solely occurred in strains carrying a veA1 allele (fig. S3), suggesting the key role via VeA1 in fungal-fungal cocultivation. VeA is a light-dependent regulator governing development and secondary metabolism in A. nidulans, especially by coordination with LaeA and VelB in what is termed the trimeric velvet complex (38, 39). Site mutation at the start codon (ATG) of veA caused by ultraviolet (UV) radiation led to the veA1 allele, which was widely used in research laboratories as veA1 strains (40, 41). In contrast to VeA protein, VeA1 lacks the N-terminal 36 amino acids and fails to respond to light, in part due to a truncated bipartite nuclear localization signal (NLS) motif, which leads to a repressed impact on secondary metabolism (42).

To further prove a specific role for VeA1 in widespread SM alteration in fungal-fungal interactions, veA and veA1 genes were completely deleted in A. nidulans RJMP1.49 and LO4389, respectively (Fig. 4A). Cocultivation of ΔveA-mutant and ΔveA1-mutant with E. dendrobii both abolished the production of aspernidine derivatives 1 to 14, confirming that partial loss of function of VeA yielding the defective protein VeA1 still functions in the fungal-fungal interaction to alter SMs. Moreover, when veA1 was replaced with veA, the production of 1 to 14 was totally abolished during cocultivation, whereas veA replacement by veA1 led to their formation (Fig. 4A).

Fig. 4. Partial loss-of-function VeA1 protein leads to SM alteration in A. nidulans and A. fumigatus during cocultivation with E. dendrobii.

UV absorptions at 210 nm are illustrated in (A) and (C) and at 230 nm in (B) for crude extract analysis by HPLC. (A) Cocultivation of E. dendrobii with A. nidulans strains carrying ΔveA, ΔveA1, ΔveA::veA1, and ΔveA1::veA. (B) Cocultivation of E. dendrobii with A. fumigatus (AF) strains carrying veA and veA1 alleles. (C) Cocultivation of E. dendrobii with ΔvelB, ΔlaeA, and ΔvosA mutants in the veA1-genotype background. The structures of labeled peaks are shown in Fig. 1B (1 to 14) and fig. S11 (25 to 31), respectively.

As mentioned earlier, previous studies have shown that the veA1 allele normally reduces SM output in many cases, such as sterigmatocystin and penicillin (38, 43). Inspired by the unexpected positive regulatory setting in secondary metabolism of VeA1 protein in A. nidulans during cocultivation with E. dendrobii, we were curious about the generalizability of VeA1 activation mechanism. Taking Aspergillus fumigatus as an example, the veA1 strain was constructed by complementation of the veA1 gene in a veA deletion background. As expected, LC-MS analysis demonstrated that coculture of the veA1 mutant of A. fumigatus with E. dendrobii led to significant SM alteration compared with a veA strain (Fig. 4B). For example, the first three intermediates of the fumiquinazoline pathway fumiquinazolines A (27), B (28), and F (29), which were found at low production levels in all fungal tissues (44), were notably accumulated in the veA1-genotype strain. Furthermore, the intermediates and shunt products spirotryprostatin F (25), cyclotryprostatin D (30), and cyclotryprostatin B (31) derived from the fumitremorgin biosynthetic pathway (45) were overproduced in the A. fumigatus veA1 strain (fig. S11). These data proved that partial loss-of-function VeA1 protein potentially acts as a crucial role for the SM changes in Aspergillus genus.

Fungal-fungal cocultivation triggers a complex VeA1-mediated regulation network for SM pathway activation

Having identified the crucial role of partial loss-of-function VeA1 in the fungal-fungal interaction, we proceeded to investigate whether other velvet members, LaeA and VelB, and an additional velvet domain protein, VosA (38, 46), were involved in the SM pathway activation. Deletion mutants of velB, laeA, and vosA were constructed in A. nidulans LO4389, a veA1 strain. Removal of velB and laeA completely abolished the production of aspernidines, while the ΔvosA mutant still retained their production during cocultivation with E. dendrobii (Fig. 4C). These results demonstrated that velvet complex regulators VeA1, LaeA, and VelB, but not VeA or VosA, are required for positive regulation of aspernidine derivatives during cocultivation.

We then were interested in searching for the downstream regulators participating in aspernidine production. Because the pkf cluster does not contain a transcription factor, we hypothesized that a cis-acting transcription factor could be contained elsewhere in the genome for function (47). Thus, we analyzed the gene expression levels of 454 transcription factors and 91 epigenetic factors in A. nidulans genome, which are annotated as putative regulators in the Aspergillus Genome Database (www.aspgd.org/) (data files S1 and S2) (48). Of these, 12 candidate genes, including 7 genes encoding for transcription factors and 5 genes encoding for epigenetic regulators, showed significantly differential expressions in coculture (log₂ fold change ≥ 2.0 or ≤ −2.0) (Fig. 5A and table S3). Eleven of these genes were successfully deleted in the A. nidulans veA1 background, with the exception of spt10, which has already been correlated to normal cell division and growth retardation (fig. S68) (16, 49).

Fig. 5. The Zn(II)₂Cys₆ transcription factor SclB regulates SM production in A. nidulans and A. fumigatus.

(A) Transcriptome analysis of transcription factors and epigenetic regulators. (B) HPLC metabolic profiles of coculture extracts of E. dendrobii with ΔllmJ, ΔrmtB, ΔatrA, ΔcelA, ΔsclB, and ΔsclB^C mutants in the veA1-genotype background. UV absorptions at 210 nm are illustrated. (C) Total ion chromatogram of coculture extracts of A. fumigatus with wild type (WT) and ΔsclB mutant by LC-MS analysis. The structures of labeled peaks are shown in Fig. 1B (1 to 14) and fig. S14 (32 to 36).

Unexpectedly, deletion of sclB solely suppressed pkf gene cluster expression and subsequent production of aspernidine derivatives in the coculture with E. dendrobii (Fig. 5B and fig. S12). This suggested that SclB is a possible downstream regulator of VeA1. SclB was previously reported as a Zn(II)₂Cys₆ transcription factor containing a unique CX₂-CX₆-CX₅-CX₂-CX₈-C domain architecture and connected to secondary metabolism in standard monocultivation of A. nidulans (50). To further concretely assess its function under cocultivation conditions, we constructed the sclB complementary (ΔsclB^C) strain in the ΔsclB background. HPLC analysis revealed that the ΔsclB^C strain restored aspernidine production in comparison with the ΔsclB strain, suggesting that SclB functions as a positive regulator of the pkf cluster and is induced in the VeA1 background under cocultivation condition (Fig. 5B). Furthermore, blast analysis indicated that SclB is highly conserved throughout the genus Aspergillus, including species such as A. mulundensis, A. versicolor, A. puulaauensis, and A. oryzae (table S4). Putative orthologs of the backbone enzyme PkfA required for aspernidine biosynthesis were also found in these strains, possibly establishing the correlation of SclB homologies to pkf BGC expression throughout Aspergillus species. In addition to aspernidines, SclB was reported to activate the expression of emericellamides (15 to 19), austinol (23), and dehydroaustinol (24) BGCs under monocultivation of A. nidulans, and disruption of sclB led to the elimination of 15 to 19, 23, and 24 (50). By contrast, under cocultivation of E. dendrobii and A. nidulans, deletion of sclB maintained the capability for the production of 15 to 19, 23, and 24, although in decreased yields of 23 and 24 (fig. S13). The production of monodictyphenones (20 to 22) was vanished in coculture extract in the absence of SclB. The complementation of sclB restored the accumulation of 20 to 24. To confirm the role of SclB in another fungal system, gene AFUB_077130 from A. fumigatus sharing a 42.2% identity with A. nidulans sclB was knocked out and incubated in potato dextrose agar (PDA) plates for product detection. As demonstrated by LC-MS analysis of the culture extracts, several SM biosynthesis pathways were regulated in ΔsclB mutant compared to wild type (Fig. 5C). The production of sesquiterpenoids fumagillenes A (32) and B (33) (51) was increased in sclB deletion strain, while the alkaloids pseurotins A (34), A₁ (35), and A₂ (36) (52) showed significantly reduced production (fig. S14). These results demonstrate the widespread regulatory role of SclB in fungal secondary metabolism.

DISCUSSION

Cross-species cocultivation of bacteria-bacteria, fungi-fungi, or bacteria-fungi represents a prospective approach mimicking ecological and physiological conditions to uncover cryptic SMs (6). Various mechanisms might be triggered by the interacting partners in response to their survival, colonization, and pathogenesis. One of them is direct physical interaction between bacteria and fungi. This results in a fungus as a scaffold bound by bacterium externally or internally, leading to the formation of mixed-species biofilms (53). A set of surface proteins and saccharides has been proved to be involved in the attachment of bacterial cells to fungal hyphae (54). In the specific interaction between A. nidulans and Streptomyces rapamycinicus, activation of the silent orsellinic acid gene cluster was mediated by manipulating the chromatin-based epigenetic factor gcnE in the eukaryotic partner by bacterial signal transduction (15, 16, 18). Cryptic SM stimulations were also observed in fungal-fungal cocultivation of P. citrinum with B. felina, Trichoderma hamatum with Chaunopycnis sp., Trametes robiniophila with Pleurotus ostreatus, and Aspergillus fischeri with Xylaria cubensis (13, 14, 55, 56). Despite increasing research on fungal-fungal interactions for activation of silent BGCs, the complex regulation involved in fungal metabolism triggered by cocultivation still remains uncovered.

To address this question, we asked whether different veA alleles (veA and veA1) were differentially involved in fungal-fungal interactions based on previous studies showing a differential impact of the two alleles in monoculture (20). Unexpectedly, A. nidulans with veA1-genotype, when cocultivated with E. dendrobii, triggered widespread alteration of transcriptome and metabolism in A. nidulans but not veA alleles. Numerous silent biosynthetic genes were activated and subsequently led to the formation of their products (Fig. 3), such as pkf gene cluster activation for the accumulation of aspernidine derivatives (Figs. 1 and 2). In previous studies, the synthesis of aspernidines was activated by genetic inactivation of a mitogen-activated protein kinase gene mpkA in A. nidulans (30, 57). In contrast, cocultivation with E. dendrobii triggered aspernidine formation in the presence of mpkA, indicating cocultivation as a promising approach to activating silent BGCs; however, this required the presence of a mutated veA allele, veA1. Deletion of veA1 (ΔveA) failed to induce the biosynthesis of aspernidines, and replacement of veA by veA1 in A. nidulans RJMP1.49 led to the accumulation of aspernidines (Fig. 4A). Our results concretely established the connection of VeA1, but not the null veA mutant or the WT veA allele, to pkf gene activation and widespread changes in secondary metabolism in A. nidulans. The marked SM alteration was also found when E. dendrobii was cocultivated with A. fumigatus carrying the veA1-genotype strain but not the veA-genotype strain (Fig. 4B). Therefore, an unexpected and overlooked function of VeA1 was highlighted under the fungal-fungal cocultivation conditions in this work. Previously, in vitro interaction analysis established the expression of VeA1 using an VeA1::GFP protein and supported the formation of the VelB-VeA1-LaeA velvet complex with the assistance of the importin α protein KapA (42). Cocultivation of E. dendrobii with ΔlaeA and ΔvelB mutants in the veA1 background completely eliminated the production of aspernidine derivatives, confirming a synergistic regulation of VeA1, LaeA, and VelB on the activation of the pkf gene cluster (Fig. 4C). This result demonstrated that the widespread regulation of secondary metabolism by VeA1 in fungal-fungal interactions relied on the integrity of a parallel velvet complex. Thus, our study provides a new perspective on the regulation of fungal metabolism depending on which the velvet complex (VelB-VeA1-LaeA or VelB-VeA-LaeA) is present.

In addition to widespread regulation of SM by the velvet complex, a series of transcriptional elements are also in cooperation with or mediated by the VelB-VeA1-LaeA complex, together composing a complicated regulatory network (58, 59). Using transcriptome analysis and gene deletions of transcription factors, we targeted the sole Zn(II)₂Cys₆ transcription factor SclB affecting SM production in A. nidulans under cocultivation conditions (Fig. 5 and fig. S13). Aspernidine formation was significantly activated, and production of austinols and monodictyphenones was increased by SclB in the VeA1 background in our case. This differs from SM production by SclB regulation under monocultivation, which is repressed by VosA. As aspernidine synthesis was retained in both vosA-veA1-sclB and ΔvosA-veA1-sclB strains but abolished in ΔsclB-vosA-veA1 mutant during cocultivation with E. dendrobii, it appeared that this repression impact of VosA on SclB was disturbed during the fungal-fungal interaction in the presence of the VelB-VeA1-LaeA complex. In the routine axenic situation, VosA repressed the activation of SclB to BrlA, which encodes a master regulator for conidia formation (60), by specifically binding 9 base pairs (bp) in the promoter of SclB. Meanwhile, SclB regulated the biosynthetic pathway by direct activation or through downstream factors RsmA and RcoA. SclB functions downstream of the VosA-VosA homodimer, acting as an antagonist to VosA and linking fungal development and secondary metabolism by controlling the process of spore viability (50). In fungal regulation network, VeA competes with VosA for VelB to form VeA-VelB or VosA-VelB heterodimer. The existence of VeA1 with a shortened NLS region in our case reduced protein interaction with VelB and decreased nuclear import of both proteins (61). This might lead to an accumulation of VosA-VelB heterodimer and cutback of VosA-VosA homodimers, weakening the suppression of SclB. Furthermore, blast analysis showed SclB with high conservation in Aspergillus species; for example, in Aspergillus niger, the ortholog of SclB was proved to repress the sclerotial formation and related to the production of indoloterpenes and aurasperones (62). Our data demonstrated that SclB in A. fumigatus might not be involved in fungal development, but still regulated secondary metabolism (Fig. 5C). Together, we present a newly discovered coordinated regulatory network comprising a VeA1 velvet complex and its downstream components for pathway activation in fungal-fungal coculture (Fig. 6).

Fig. 6. A model to show partial loss-of-function mutation of VeA-mediated secondary metabolism alterations during cocultivation of A. nidulans with E. dendrobii.

In summary, cocultivation of E. dendrobii with A. nidulans carrying the veA1 allele led to widespread alteration of secondary metabolism, representing a new example for pathway activation. Partial loss-of-function of VeA (VeA1) is crucial for fungal chemical signal transduction triggered by cocultivation. Therefore, cocultivation with E. dendrobii stimulated the VelB-VeA1-LaeA complex in A. nidulans, which subsequently induced the expression of SclB for eventually activating the silent pkf BGC and leading to the discovery of cryptic SMs. Our study presented an undescribed regulatory network mediated by partial loss-of-function mutation of VeA in a fungal-fungal interaction and provided insights into understanding the mechanism of microbial communication.

MATERIALS AND METHODS

Strains, media, and growth conditions

The fungal strains used in this study are summarized in table S1. All A. nidulans and A. fumigatus strains were grown at 37°C on glucose minimum medium [1.0% (w/v) glucose, salt solution (50 ml/liter), trace element solution (1 ml/liter), and 1.6% (w/v) agar] with appropriate supplements corresponding to the auxotrophic markers (63). The salt solution comprises (w/v) 12% NaNO₃, 1.04% KCl, 1.04% MgSO₄∙7H₂O, and 3.04% KH₂PO₄. The trace element solution contains (w/v) 2.2% ZnSO₄∙7H₂O, 1.1% H₃BO₃, 0.5% MnCl₂∙4H₂O, 0.16% FeSO₄∙7H₂O, 0.16% CoCl₂∙5H₂O, 0.16% CuSO₄∙5H₂O, 0.11% (NH₄)₆Mo₇O₂₄∙4H₂O, and 5% Na₄EDTA. E. dendrobii was grown at 28°C on PDA (BD) medium. For cocultivation of E. dendrobii with fungi from different genera, four Aspergillus species (i.e., A. nidulans, A. fumigatus, A. oryzae, and A. terreus), three Penicillium species (i.e., P. chrysogenum, P. crustosum, P. previcompactum), two Trichoderma species (i.e., T. hypoxylon and T. reesei), and one Fusarium species (i.e., F. proliferatum) were cultivated on PDA plates with or without the inoculation of E. dendrobii. For SM production, cocultivation was carried out on PDA plates, or in the liquid PDB (BD), shaking medium (180 rpm) was used at 28°C for 6 days.

Isolation and phylogenetic analysis of E. dendrobii

Symbiotic microorganisms were isolated from contaminated plates and further cultivated on PDA medium. Type specimen was deposited in the China General Microbiological Culture Collection Center.

The mycelia of E. dendrobii were collected from a 6-day-old PDB culture and subsequently used for genomic DNA extraction as the described method (64). Phylogenetic analyses were conducted on the basis of ITS, LSU, RPB2, and β-tubulin as actin genes to construct a robust backbone tree. The four actin genes were amplified using the primer pairs listed in table S2. PCR amplification was carried out by using Phusion High-Fidelity DNA polymerase from New England Biolabs on a T100 Thermal cycler from Bio-Rad. PCR mixtures and thermal profiles were set as recommended by the manufacturer’s instruction. Consensus sequences were assembled in MEGA 5.0 (65), and the 55 additional reference sequences were obtained from GenBank (www.ncbi.nlm.nih.gov/genbank/). The concatenated aligned dataset and each locus were analyzed separately using Maximum Likelihood (66).

RNA isolation

For RNA extraction, A. nidulans was cultivated with or without E. dendrobii in 15 ml of liquid PDB medium for 7 days at 37°C, and the cells were collected by centrifugation. Total RNA was isolated using TRIzol (TransGen Biotech, China) reagent as described previously. Reverse transcription of 3 mg of RNA was performed with the TIANScript II RT Kit (TIANGEN, China). β-Actin gene (AN6542) from A. nidulans served as an internal standard for calculation of expression levels. The oligonucleotide sequences for PCR primers are given in table S2.

Transcriptional analysis by RNA-seq and qRT-PCR

For RNA-seq analysis, RNA examples were sequenced on Illumina HiSeq X Ten platform using (2 × 150 bp) paired-end module after disposal at Vazyme Biotech Co. Ltd. Proteins that refer to the genome sequence of A. nidulans FGSC A4 from the National Center for Biotechnology Information (NCBI) were annotated. The level of gene expression was calculated as normalized FPKM (fragments per kilobase of transcript per million mapped reads) using RSEM (RNA-seq by expectation-maximization). Differential expression analysis was performed with a cutoff of P < 0.01. DEGs were calculated by DESeq2 and identified with P value and log₂ fold change/ratio between monocultivation and cocultivation samples.

Meanwhile, qRT-PCR was carried out by using the CFX96 Real-Time System (Bio-Rad) to analyze the expression level of pkf genes. The KAPA SYBR FAST qPCR Kit was used for the reactions [2× KAPA SYBR FAST qRCP Master Mix, 0.2 μM forward/reverse primer, A. nidulans complementary DNA (cDNA) template ~5 ng, and ddH₂O to a total volume of 20 μl]. Reactions were performed as follows: 95°C for 3 min followed by 40 cycles of (95°C for 3 s, 60°C for 20 s, and 72°C for 20 s) and then equilibrated at 65°C for 5 s before being slowly reheated (dissociated) back to 95°C. Each cDNA sample was performed in triplicate, and the average threshold cycle was calculated. Relative expression levels were calculated using the 2^−∆∆Ct method. qRT-PCR primers were listed in table S2.

Genetic cassette construction

The cassettes for gene deletion, integration, replacement, and replenishment were constructed as described in fig. S68. Complementary overhangs of 30 to 35 bp were designed for sequence assembly. Double-joint PCR amplifications were carried out for homologous recombination to create corresponding cassettes (67). The oligonucleotide sequences for PCR primers are given in table S2.

To identify key genes involved in the induction between fungal-fungal interactions, we deleted veA, veA1, laeA, velB, and sclB genes. Upstream and downstream regions of the respective genes with a length of 1.0 kb were amplified from the genomic DNA of A. nidulans using the designed primers listed in table S2. Two fragments were combined with a suitable marker gene by double-joint PCR strategy to yield gene deletion cassettes. To replace veA by veA1 in A. nidulans, partial veA gene (encoding amino acids 37 to 573) including the mutation site was amplified from A. nidulans LO4389 and combined with veA upstream region. The cassette was constructed as mentioned above and transformed into RJMP1.49 to yield TYWG40.1 carrying the veA1 allele. The replacement of veA1 by veA in LO4389 was generated using the same method. To obtain veA1 mutation of A. fumigatus, the veA gene (AFUB_011960) was knocked out from A. fumigatus cea 17-2 with a hygromycin marker to construct strain TYRHM 1.2. Subsequently, the veA1 gene (encoding amino acids 35 to 571) was amplified from A. fumigatus cea17-1 and combined with upstream region including the promoter and terminator of veA as well as the downstream region. The cassette was constructed as mentioned above and transformed into TYRHM1.2 to generate veA1-genotype A. fumigatus strain TYRHM2.3.

Genetic manipulation in A. nidulans and A. fumigatus

Fresh spores of A. nidulans were inoculated into 30 ml of liquid minimal medium (LMM) [1.0% glucose, salt solution (50 ml/liter), trace element solution (1 ml/liter), and 0.5% yeast extract] in 100-ml flask and incubated at 37°C and 230 rpm for germination. Mycelia were harvested after 6 hours by centrifugation at 5000 rpm for 10 min and washed with distilled H₂O. The mycelia were then transferred into a 50-ml flask with 10 ml of osmotic buffer [1.2 M MgSO₄ in 10 mM sodium phosphate (pH 5.8)] containing 50 mg of lysing enzyme from Trichoderma harzianum (Sigma-Aldrich) and 20 mg of yatalase from Corynebacterium sp. OZ-21 (OZEKI Co. Ltd.). After shaking at 30°C and 100 rpm for 2.5 hours, the cells were transferred into a 50-ml falcon tube and overlaid gently with 10 ml of trapping buffer [0.6 M sorbitol in 0.1 M tris-HCl (pH 7.0)]. After centrifugation at 4°C and 5000 rpm for 10 min, the protoplasts were collected from the interface of the two buffer systems. The protoplasts were then transferred to a sterile 15-ml falcon tube and resuspended in 200 μl of STC buffer [1.2 M sorbitol, 10 mM CaCl₂, and 10 mM tris-HCl (pH 7.5)] for transformation. The constructed gene cassettes via PCR mentioned above were transformed into corresponding A. nidulans by polyethylene glycol–mediated protoplast transformation according to the method described previously. After incubation for 3 days, transformants were transferred onto fresh supplemented minimal medium (SMM) plates [1.0% glucose, salt solution (50 ml/liter), trace element solution (1 ml/liter), 1.2 M sorbitol, and 1.6% agar] for second selection. Protoplast generation and transformation for A. fumigatus were carried out as previously described (68). Screening of auxotrophic transformants was carried out by PCR amplification (fig. S68). After cultivation in PDB liquid medium at 25°C for 3 days, SMs were extracted with a solvent mixture (ethyl acetate:methanol:acetic acid, 89:10:1), dissolved in methanol (MeOH), and analyzed on HPLC or LC-MS.

Large-scale fermentation, extraction, and isolation of SMs

To isolate 1 to 14, A. nidulans RDIT2.3 was inoculated into 60× 250-ml flask containing 80 ml of PDB liquid medium and incubated on a rotary shaker at 180 rpm and 28°C for 6 days. The supernatant and mycelia were separated by filtration. The supernatant was extracted with equal volume of a solvent mixture (ethyl acetate:methanol:acetic acid, 89:10:1) for three times and evaporated under reduced pressure to give a crude extract (4.87 g). Subsequently, the crude extract was subjected to silica gel column chromatography and eluted with dichloromethane (DCM):MeOH (100:0 to 0:100 in gradient, v/v) to give seven fractions. Fraction 1, eluted with DCM:MeOH (100:0), was further purified on semipreparative HPLC [MeOH:H₂O (70:30 to 100:0)] to yield 8 (2.5 mg), 9 (2.1 mg), 10 (2.0 mg), 11 (0.8 mg), and 12 (3.1 mg). Compounds 3 to 7 were obtained from fraction 2 (DCM:MeOH, 100:1) by using a Sephadex LH-20 column eluting with methanol (MeOH) and subsequent semipreparative HPLC (MeOH:H₂O, 85:15) to give 3 (2.3 mg), 4 (1.9 mg), 5 (3.1 mg), 6 (2.4 mg), and 7 (2.2 mg). Fraction 3 (DCM:MeOH, 100:3) was separated on semipreparative HPLC (acetonitrile:H₂O, 70:30) to yield 1 (0.9 mg) and 2 (1 mg).

HPLC and LC-MS analysis of SMs

HPLC analysis was conducted with a Waters HPLC system (Waters e2695, Waters 2998, Photodiode Array Detector) using an XTerra MS C18 column (250 by 4.6 mm, 5 μm, Waters). Water with 0.1% (v/v) formic acid (A) and MeOH (B) was used as the solvent at a flow rate of 1 ml/min. For analysis of the crude extracts, substances were eluted with a linear gradient from 60 to 100% B in 20 min, washed with 100% (v/v) solvent B for 5 min, and equilibrated with 60% (v/v) solvent B for 5 min. UV absorptions at 210 nm were illustrated. Semipreparative purification on HPLC was performed on an SSI HPLC system (Teledyne SSI Lab Alliance Series III pump system and Series 1500 Photodiode Array Detector) with an ODS column (C18, 10.0 by 250 mm, 5 μm, YMC) and a flow rate of 2.5 ml/min.

LC-MS analysis was performed on an Agilent HPLC 1200 series system equipped with a single-quadrupole mass-selective detector and an Agilent 1100LC MSD model G1946D mass spectrometer by using a Venusil XBP C18 column (3.0 by 50 mm, 3 μm, Bonna-Agela Technologies, China). Water (A) with 0.1% (v/v) formic acid and acetonitrile (B) was used as the solvent at a flow rate of 0.5 ml/min. The substances were eluted with a linear gradient from 5 to 100% B in 30 min, then washed with 100% (v/v) solvent B for 5 min, and equilibrated with 5% (v/v) solvent B for 10 min. The mass spectrometer was set in electrospray positive ion mode for ionization.

NMR analysis

NMR spectra were recorded on a Bruker Avance 500-MHz spectrometer at room temperature (Bruker Corporation, Karlsruhe, Germany). All spectra were processed with MestReNova 12.0 (Metrelab). Chemical shifts are referenced to those of the solvent signals. NMR data are given in tables S5 to S16, and spectra are given in figs. S15 to S67.

Supplementary Materials

This PDF file includes:

Figs. S1 to S68

Tables S1 to S16

References

Other Supplementary Material for this manuscript includes the following:

Dataset S1 and S2

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