Co-option of an extracellular protease for transcriptional control of nutrient degradation in the fungus aspergillus nidulans

ABSTRACT Nutrient acquisition is essential for all organisms. Fungi regulate their metabolism according to environmental nutrient availability through elaborate transcription regulatory

programs. In filamentous fungi, a highly conserved GATA transcription factor AreA and its co-repressor NmrA govern expression of genes involved in extracellular breakdown, uptake, and

metabolism of nitrogen nutrients. Here, we show that the _Aspergillus nidulans_ PnmB protease is a moonlighting protein with extracellular and intracellular functions for nitrogen

acquisition and metabolism. PnmB serves not only as a secreted protease to degrade extracellular nutrients, but also as an intracellular protease to control the turnover of the co-repressor

NmrA, accelerating AreA transcriptional activation upon nitrogen starvation. PnmB expression is controlled by AreA, which activates a positive feedback regulatory loop. Hence, we uncover a

regulatory mechanism in the well-established controls determining the response to nitrogen starvation, revealing functional evolution of a protease gene for transcriptional regulation and

extracellular nutrient breakdown. SIMILAR CONTENT BEING VIEWED BY OTHERS A MULTILAYERED REGULATORY NETWORK MEDIATED BY PROTEIN PHOSPHATASE 4 CONTROLS CARBON CATABOLITE REPRESSION AND

DE-REPRESSION IN _MAGNAPORTHE ORYZAE_ Article Open access 28 January 2025 REGULATION OF EXTRACELLULAR MATRIX COMPONENTS BY AMRZ IS MEDIATED BY C-DI-GMP IN _PSEUDOMONAS OGARAE_ F113 Article

Open access 13 July 2022 THE DEOR-LIKE PLEIOTROPIC REGULATOR SCO1897 CONTROLS SPECIALISED METABOLISM, SPORULATION, SPORE GERMINATION, AND PHOSPHORUS ACCUMULATION IN _STREPTOMYCES COELICOLOR_

Article Open access 07 November 2024 INTRODUCTION Fungi have evolved remarkable metabolic versatility for utilizing diverse nutrient substrates for growth in their saprophytic, symbiotic,

and pathogenic lifestyles. Evolution of fungal lifestyle correlates with their secretome, and specific classes of secreted proteases are associated with distinct environmental niches1,2,3,4.

Secreted proteases are important in nutrient recycling via autolysis5 and function to promote extracellular nutrient breakdown and acquisition, a process known as lysotrophy3,6,7. The large

repertoire of proteases expressed by fungi allows them to break down complex organic matter, usually not available to many other organisms. The extracellular release of oligopeptides and

amino acids makes them available for the individual providing the extracellular protease as well as other cohabiting osmotrophic organisms, which obtain their nutrients from the

environment6,7. This unique decomposing ability has rendered fungi an indispensable part of the ecosystem and popular partners for establishing symbiotic relationships. Moreover, secreted

proteases have been implicated in the virulence of many fungal pathogens and they are employed for evasion of host recognition, colonization, and invasion, in addition to nutrient

acquisition8,9. Therefore, proteases could offer significant advantages to fungi. The ability to reprogram cellular metabolism according to the availability and quality of nutrients in the

environment is fundamental for fungi to survive and thrive in diverse habitats10,11. In environments where multiple nitrogen nutrients are available, fungi first metabolize the most

preferred ones, and as they become depleted, fungi switch their metabolism to utilize less preferred ones12,13. This preferential utilization of nutrients is a critical energy-efficient

strategy for survival. In _Aspergillus nidulans_, control of nitrogen source utilization is mediated by an elaborate regulatory mechanism in which the expression of hundreds of nitrogen

metabolic genes is orchestrated by the conserved wide-domain GATA transcription factor AreA13. AreA modulates target gene expression according to nitrogen availability and quality. In the

presence of a preferred nitrogen source, most nitrogen catabolic genes are not expressed. When the preferred nitrogen source is limiting or in the presence of an alternative nitrogen source,

AreA, often together with pathway-specific transcription factors, activates the expression of specific sets of transporter and nitrogen catabolic genes for utilization of the available

nutrient13. Moreover, in response to complete nitrogen starvation, AreA-mediated expression of certain nitrogen scavenging genes increases—a transcription response known as the nitrogen

starvation response14,15. Multiple control mechanisms underlie the differential regulation pattern12,13. First, AreA expression is controlled according to nitrogen conditions. With a

preferred nitrogen source, transcription of _areA_ is low, while _areA_ transcripts are also unstable due to polyA tail deadenylation and CU modification16,17,18. Consequently, little AreA

protein is present in the cell19. Transcription of _areA_ is elevated through autogenous regulation under conditions when an alternative nitrogen source is available or when nitrogen

nutrients are limiting20. At the same time, _areA_ transcripts are stabilized due to lack of deadenylation and polyA tail modification17,21, leading to an overall increase in AreA protein

levels19. The AreA protein is shuttled between the nucleus and cytoplasm through nuclear import and export14,22. During nitrogen starvation, AreA levels are further increased due to

upregulation of _areA_ transcription, and nuclear export of AreA is blocked, leading to high levels of AreA accumulation in the nucleus14. AreA transcriptional activity also depends on

control by its co-repressor NmrA. NmrA physically interacts with the AreA DNA binding domain23,24 and mutants with the _nmrA_ gene deleted or with a C-terminal truncation of AreA also lead

to partial derepression18,25,26. Overexpression of NmrA suppresses the nitrogen starvation response, and full suppression requires the AreA C-terminal region19. These previous findings

highlight the importance of the levels of NmrA and its interactions with AreA for determining AreA transcriptional activation capacity. Relatively little is known about how NmrA levels are

controlled. In wild type, NmrA expression is regulated in an opposite pattern to AreA under different nitrogen conditions19; e.g. NmrA levels decrease with decreasing nitrogen availability

and quality. The expression pattern is independent of AreA function, but is mediated by a bZIP transcription factor MeaB that binds to the _nmrA_ promoter19. Deletion of _meaB_ abolishes

differential regulation of _nmrA_ with only a very low basal level of NmrA present in the cell irrespective of nitrogen conditions, and as a result, _meaB_∆ mutants have a derepression

phenotype similar to _nmrA_∆ mutants19. While MeaB is critical during favorable nitrogen conditions, transcriptional control of NmrA may be somewhat slow for reducing NmrA levels when

preferred nitrogen sources become exhausted as rapid AreA-mediated transcriptional responses activating expression of nitrogen metabolism genes are required. NmrA protein levels are thought

to be rapidly degraded during the transition to nitrogen starvation27. Three proteolytic activities against bacterial-expressed NmrA have been detected in total protein extracts of

nitrogen-starved _A. nidulans_ mycelia and one of them, PnmB, was purified to homogeneity based on its protease activity and cleavage of NmrA27. Mass spectrometry and peptide sequence

analysis revealed the corresponding gene as _pnmB_ (_AN2366_). The protein sequence of PnmB suggests it is a member of the trypsin-like serine protease family, which is supported by its

sensitivity to the serine protease inhibitor benzamidine and its in vitro cleavage site on NmrA27. However, the in vivo role of _pnmB_ towards NmrA degradation has not been examined. Here,

we demonstrate that PnmB degrades NmrA in vivo. PnmB expression and proteolytic activity against NmrA are dramatically induced upon nitrogen starvation and they are absolutely dependent on

activation of _pnmB_ transcription by AreA. Expression of PnmB causes rapid NmrA turnover and swift AreA transcriptional activation in response to nitrogen starvation. We also discover

another role of PnmB as a secreted protease for extracellular protein breakdown. Finally, we uncover an interesting expansion of _pnmB_-like genes in insect-associated fungi. Overall, this

work reveals a positive regulatory feedback mechanism for establishing prompt AreA activation, and also reports a protease that serves two distinct functions for the same ultimate goal of

scavenging nitrogen. RESULTS DELETION OF _PNMB_ HAS NO DETECTABLE EFFECT ON GROWTH The PnmB protease cleaves bacterial-expressed NmrA in vitro27. However, the actual in vivo function in _A.

nidulans_ of the _pnmB_ gene has not been established. We noticed a 5 bp intron containing an in-frame TAA stop codon was likely incorrectly assigned near the 3’ end of the _pnmB_ annotation

(Supplementary Fig. 1a). As the size of the intron is too small to be a typical intron, we reannotated the _pnmB_ gene removing this proposed intron (Supplementary Fig. 1b) and confirmed

our reannotation by analysis of published RNAseq data (Supplementary Fig. 1c) and introduction of a hemagglutinin (HA) epitope-tag in front of both the originally annotated and reannotated

stop codons. PnmBHA was only detected by western blot for the reannotated stop codon (Supplementary Fig. 1d and e). Interestingly, two distinct bands of similar mobility to the predicted

molecular weight of PnmB were observed, suggesting different PnmB forms are expressed (see below). These findings demonstrate that the _pnmB_ gene lacks an intron and encodes a protein of

249 amino acids (Supplementary Fig. 2). A deletion construct to replace the entire _pnmB_ coding region with the glufosinate resistance gene _Bar_ from _Streptomyces hygroscopicus_28 was

transformed into a wild-type strain (MH11036—_nkuA_∆) and glufosinate-resistant transformants were obtained. Growth of the _pnmB_∆ mutant on solid media was indistinguishable from wild type

on all sole nitrogen sources analyzed (ammonium, glutamine, alanine, proline, nitrate, uric acid, acetamide, GABA, L-histidine, 2-pyrrolidinone), and on various sources of proteins (BSA,

tryptone, peptone, skim milk, 25% honey) as the sole nitrogen source or sole nitrogen and carbon source (Supplementary Fig. 3a), suggesting that PnmB does not detectably affect nitrogen and

carbon metabolism under the conditions tested. We also did not observe any detectable growth phenotype for the _pnmB∆_ mutant under different temperature (25, 37, and 42 °C; Supplementary

Fig. 3b), osmotic stress (0.4 M and 1.0 M NaCl; Supplementary Fig. 3c), and pH (pH 5.0, 6.5, and 9.0; Supplementary Fig. 3d) conditions. OVEREXPRESSION OF PNMB PROMOTES NMRA DEGRADATION To

test whether PnmB can degrade NmrA in vivo, we overexpressed PnmB using the strong xylose-inducible promoter of the _Penicillium chrysogenum xylP_ gene29 under nitrogen-sufficient conditions

(i.e. ammonium as the sole nitrogen source) where NmrA levels are high, and tested whether NmrA degradation could be induced. Western blot analysis showed a distinct reduction of NmrA

levels when PnmB was overexpressed in a xylose-concentration-dependent manner (Fig. 1a). To rule out the possibility of non-specific degradation by the high level of PnmB protease in the

cell, we measured histone H3 levels by western blot analysis (Fig. 1a) and total proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with

Coomassie Blue (Supplementary Fig. 4) as controls and observed no difference with and without PnmB overexpression. Moreover, reciprocal co-immunoprecipitation of NmrAFLAG and PnmBHA was

observed in the presence of protease inhibitors including benzamidine known to inhibit PnmB proteolytic activity on NmrA27 (Fig. 1b), suggesting that the two proteins physically interact

with each other. Therefore, the observed reduction in NmrA level is a specific direct effect of PnmB function. PNMB ACCELERATES NMRA TURNOVER EARLY IN NITROGEN STARVATION NmrA is expressed

at high levels to antagonize AreA function under nitrogen sufficient conditions, but its levels decrease during nitrogen starvation thereby relieving repression of AreA for transcriptional

activation19. To test whether PnmB is involved in degrading NmrA upon nitrogen starvation, we compared NmrAFLAG protein levels of wild-type and _pnmB_∆ mutant strains during nitrogen

starvation in a time course experiment. Under nitrogen sufficient conditions, NmrAFLAG levels were similar between wild type and the _pnmB_∆ mutant (Fig. 1c). Consistent with previous

findings, NmrAFLAG levels decreased during nitrogen starvation in wild type. In contrast, NmrAFLAG levels were not reduced at the same rate and extent in the _pnmB_Δ strain, indicating that

NmrA turnover was slowed down in the absence of PnmB (Fig. 1c and Supplementary Fig. 5a). Therefore, this result indicates the importance of PnmB in controlling NmrA function (and therefore

AreA activation) upon nitrogen starvation. PNMB-MEDIATED NMRA TURNOVER IS REGULATED VIA _PNMB_ EXPRESSION We next determined the expression pattern of _pnmB_ under different nitrogen

conditions by measuring transcription levels using chromatin immuno-precipitation followed by next-generation sequencing (ChIPseq) against the elongating form of RNA polymerase II (PolII)30

(Fig. 1d). Under nitrogen sufficient conditions, _pnmB_ transcription levels were very low. A small increase was observed during growth on the alternative nitrogen source alanine. In stark

contrast, _pnmB_ transcription was induced nearly 20-fold after four hours of nitrogen starvation. Time course western blot analysis confirmed that PnmBHA protein levels were barely

detectable under nitrogen sufficient conditions but were substantially induced within 30 min of nitrogen starvation (Fig. 1e) and the levels continued to increase with time up to 4 h. This

observation suggests that PnmB levels are the key control for NmrA turnover. Loss of NmrA-mediated repression leads to derepression of nitrogen catabolic genes during nitrogen sufficient

conditions. This derepression phenotype can be assessed in plate tests using toxic nitrogen analogs; in wild type the genes for metabolism of toxic analogs are repressed during nitrogen

sufficiency and therefore normal growth occurs, whereas derepressed mutants express the genes to metabolize the analogs and show inhibited growth25,26. PnmB overexpression led to a

derepression phenotype (i.e. inhibited growth) on solid media containing the toxic analog of urea (thiourea (TU)) in the presence of ammonium (NH4) (Fig. 1f, g), which is similar to the

phenotype of the _nmrA_∆ mutant25. Taken together, the above results not only confirm the role of PnmB in NmrA turnover in vivo, but also indicate the importance of regulated PnmB expression

in modulating AreA transcriptional activation and nitrogen metabolic gene expression. AREA ACTIVATES _PNMB_ TO STIMULATE ITS OWN ACTIVATION CAPACITY AreA is the key regulator of the

nitrogen starvation response13,14,15,22. The nitrogen-starvation-induced expression pattern of _pnmB_ suggested a potential role of AreA in the regulation. The _pnmB_ promoter sequence

contains twelve GATA motifs (five belonging to the HGATAR consensus that was implicated as the AreA recognition sequence31) within the ~1.3 kilobase (kb) promoter region (Fig. 2a). To

determine whether AreA binds to the _pnmB_ promoter, ChIP followed by real-time PCR analysis (ChIP-qPCR) against AreAHA was carried out using six pairs of primers spanning the _pnmB_

promoter region. Consistent with minimal PnmB expression, under nitrogen sufficiency (ammonium, NH4), there was no detectable AreAHA binding to any of these regions. In contrast, under

nitrogen starvation (-N), strong AreAHA ChIP signals were detected at the distal region (e.g. at ~−1300 to −900 bp), while albeit lower but significant binding signals can also be detected

in the proximal region (e.g. between −726 to −125 bp) (Fig. 2b). This result showed that AreAHA binds to the _pnmB_ promoter at multiple locations, in response to nitrogen starvation. We

next determined whether _pnmB_ transcription is dependent on AreA by comparing PolII binding at _pnmB_ in wild-type and _areA_∆ mutant strains. Strikingly, both the weak (~3-fold) and strong

(>150-fold) transcription induction of _pnmB_ observed during growth on the alternative nitrogen source and after nitrogen starvation in wild type, respectively, was completely abolished

in the _areA_∆ mutant (Fig. 2c), showing that AreA activates _pnmB_ expression. This result strongly indicates a model in which AreA establishes a positive feedback loop where AreA promotes

degradation of its co-repressor NmrA through activating expression of _pnmB_ to stimulate its own transcriptional activation during nitrogen limitation and starvation. Based on the model,

NmrA turnover is expected to slow down in the _areA_∆ mutant during nitrogen starvation, and this is indeed the case (Fig. 2d and Supplementary Fig. 5b). Moreover, NmrA turnover kinetics in

the _areA_∆ mutant is comparable to that observed in the _pnmB_∆ mutant (Fig. 1c and Supplementary Fig. 5a), consistent with the above finding that _pnmB_ activation during nitrogen

starvation is absolutely dependent on AreA. We further tested the model by assessing whether PnmB-mediated NmrA turnover is important for activation of AreA-dependent gene expression

immediately upon nitrogen starvation. To do this, we compared the transcription activity of AreA targets (_ureA_ and _mepA_) and housekeeping control genes (_actA_ and _benA_) between wild 

type and the _pnmB_∆ mutant in a time course of nitrogen starvation using PolII ChIP-qPCR analysis. In support of the proposed model, we found a consistent and significant delay for AreA

activation in the _pnmB∆_ mutant as compared to wild type (Fig. 2e and Supplementary Fig. 6). Taken together, the above results demonstrate that AreA kick-starts a regulatory feedback loop

to degrade its co-repressor NmrA in order to stimulate its own activation capacity during the early phase of nitrogen starvation. PNMB EXISTS AS TWO DISTINCT FORMS The intracellular protein

fraction of the wild-type PnmBHA strain contains two distinct PnmBHA protein bands of similar abundance in western blot analysis (Fig. 1e). The two bands were also observed in the

intracellular fraction of the total protein extract when PnmBHA was overexpressed (see below; Fig. 3a). There is no sign of transcript isoforms from RNAseq data. Therefore, the two PnmB

forms most likely result from a post-transcriptional event. The N-terminus of PnmB carries a signal peptide (Likelihood score of 0.9939 by SignalP-5.0)32 that may be proteolytically cleaved,

and the two forms could potentially represent the full-length and cleaved forms. Another possibility is that there could be two alternative translation initiation codons as a non-conserved

methionine codon at the 18th codon position of the _pnmB_ transcript may be used for internal translation initiation. At present, we cannot distinguish which of these or if other

post-translational events are responsible for the two PnmB forms observed. PNMB IS ALSO A SECRETED PROTEASE Protein sequence analysis of PnmB identified three Chymotrypsin Peptidase S1A

domains and an N-terminal signal peptide, which is responsible for sorting proteins for secretion33, suggesting that PnmB may also be a secreted protease in addition to its intracellular

role. To test this, we extracted secreted proteins from filtered growth media and assayed for the presence of PnmB. Since PnmB activity was previously found in wild type under nitrogen

starvation27, we first performed the assay on the wild-type PnmBHA strain after four hours of nitrogen starvation. A strain overexpressing PrtAHA, which is a known secreted protease34, from

the strong xylose inducible _xylP_ promoter29 was used as a positive control. While PrtAHA was found in both extracts of mycelia (intracellular) and growth media (secreted) (Fig. 3a), we

failed to detect PnmBHA in the growth media and suspected that PnmBHA, even if secreted, might be present at levels too low for detection by our assay. Indeed, PnmBHA, when overexpressed

from the _xylP_ promoter, was readily detected in the growth media (Fig. 3a). The detected PnmBHA and PrtAHA proteins are not due to intracellular proteins from contaminating mycelia or

remnants of lysed cells in the growth media, as the highly abundant histone H3 protein was not detected in the secreted fractions (Fig. 3a). It is interesting to note that only one band was

found in the secreted fraction, as compared to two in the intracellular extract, and close inspection of the size of the bands showed a slight difference between secreted PnmBHA and the two

intracellular PnmBHA forms (Supplementary Fig. 7). Secretion of the PnmB and PrtA proteases was independently confirmed using a milk-clearing assay, in which a halo indicative of protein

degradation was observed around the _xylP_(p)_pnmB_HA and _xylP_(p)_prtA_HA colonies when the proteins are overexpressed (Fig. 3b). Therefore, PnmB can be secreted and functions as an

extracellular protease, serving two distinct but related functions—direct nutrient breakdown and global nitrogen regulation of genes for nutrient acquisition. EXPANSIONS OF _PNMB_-LIKE GENES

IN MANY ENTOMO-FUNGAL SPECIES To study the evolutionary conservation of _pnmB_ in fungi, we performed BLASTP searches of PnmB against the 3375 publicly available fungal genomes in NCBI and

JGI databases, which represent major fungal lineages. PnmB BLASTP hits were not evenly distributed across fungal lineages (Fig. 4 and Supplementary Data 1). Notably, fungal species in the

_Zoopagomycota_ phylum generally carry multiple _pnmB-_like genes (Fig. 4 and Supplementary Data 2). Interestingly, those species carrying the highest number of _pnmB_ homologs are

associated with insects or arthropods as a pathogen, commensal or symbiont (Table 1). Therefore, the _pnmB_ gene family has expanded in many entomo-fungi. DISCUSSION This work shows that the

_A. nidulans_ PnmB protease has two functions in nitrogen metabolism (Fig. 5). First, it is secreted as an extracellular protease where it contributes to the degradation of proteins to

facilitate nutrient breakdown and nitrogen acquisition. The second role is to degrade the co-repressor NmrA to enhance the response to nitrogen starvation by increasing the turnover of NmrA

when its synthesis is reduced due to loss of activation by MeaB19. The dual functionality is clearly not essential in all fungi because ~20% of PnmB-like protein sequences do not carry a

signal peptide for secretion (Supplementary Data 1), but presumably provides a selective advantage in some environments such as an osmotrophic ecosystem7 where nitrogen liberated by

extracellular PnmB-mediated proteolysis may be competed for by other cohabiting microorganisms. It is tempting to speculate that those PnmB homologs without a signal peptide may also control

NmrA turnover and that this is the only role in the respective fungal species. The coupling of extracellular proteolysis with intracellular NmrA degradation (and hence, relief of repression

by NmrA of AreA-dependent activation of nutrient acquisition and metabolism genes) could ensure rapid nitrogen nutrient uptake in order to protect the released nutrients from competitors.

It appears that the _pnmB_ gene might offer a selective advantage in niches occupied by both fungi and insects or for fungi with insect hosts, based on the moderate to large expansions of

this gene in many entomo-fungi. How the protease gene evolved the two PnmB functions in _A. nidulans_ is a fascinating question. As most fungal PnmB-like proteins (~80%) carry a signal

peptide (Supplementary Data 1), the ancestral gene likely encodes an extracellular protease for protein breakdown, while the second transcription regulatory function was newly evolved. This

new function would require evolution to establish an intracellular population of PnmB in addition to the secreted form. One possibility is that secretion of the ancestral protease may not

have been very efficient and poor secretion may have led to an intracellular population. Acquisition of the transcriptional regulation role would have provided selection to maintain poor

secretion. Alternatively, if secretion of the ancestral protease was efficient, mutations affecting efficient recognition of the signal peptide sequence or establishing two forms differing

at the N-terminus with and without a signal peptide could lead to a switch from secretion to both secretion and intracellular retention. We observed two distinct intracellular isoforms of

PnmBHA protein. Our analysis suggests that they are not a result of alternative transcript isoforms. An internal methionine codon (codon 18) might act as an internal translation start site35

to generate the intracellular form of PnmB lacking a signal peptide. Another notable difference between the extracellular and intracellular forms of PnmB is their substrate specificity. Our

results showed that the secreted PnmB has broad-spectrum proteolytic activity able to degrade milk proteins, whereas intracellular PnmB is highly specific for NmrA with no sign of

non-specific degradation of histone and total proteins. A previous in vitro study had identified a specific PnmB cleavage site on NmrA27. It is currently unclear how the specificity for NmrA

(or the loss of broad-spectrum activity) is achieved by intracellular PnmB, but it is likely mediated by direct interaction between PnmB and NmrA. PnmB activities are controlled at the

level of its expression according to nitrogen availability (this study, ref. 27). The global nitrogen regulator AreA is absolutely required for _pnmB_ expression through direct binding to

the _pnmB_ promoter. We have further demonstrated that this positive regulation by AreA in turn creates a positive feedback regulatory loop through degrading the co-repressor NmrA to

accelerate AreA activation kinetics upon nitrogen starvation (Fig. 5). Consequently, genes required for nitrogen breakdown, uptake and metabolism are promptly induced such that _A. nidulans_

can quickly scavenge nitrogen nutrients in the environment. Therefore, our work has identified a mechanism for eliciting a rapid transcription response for nitrogen starvation. It is

noteworthy that two more unknown proteases that cleave NmrA were implicated in NmrA turnover27. It will be interesting to see whether they serve similar roles as PnmB. As _A. nidulans_ often

lives with many other microbes in the wild, the accelerated transcription response would provide a growth advantage in competitive and nutrient-limiting environments. Hence, our study

reveals a protease gene that plays a role in essential physiology in the cell to enhance its growth competitiveness in nature. METHODS STRAINS, MEDIA, AND PRIMERS USED IN THIS STUDY _A.

nidulans_ strains used and their genotypes are listed in Table 2. MH11726 was obtained by outcross from MH1162619. RT303 was obtained by outcross of MH964114. Strain constructions described

below used the gene-targeting _nkuA_∆ recipient strain MH1103636. _Aspergillus_ nitrogen-free medium (ANM) containing supplements and indicated carbon and nitrogen sources was used for

liquid and solid growth experiments37. Sequences of primers used in this study are listed in Table 3. DELETION OF _PNMB_ The _pnmB_ deletion strain (CWF579) was generated by replacing the

entire _pnmB_ coding region with the _Bar_ gene from _S. hygroscopicus_ that confers glufosinate resistance. The deletion construct was generated as described previously38 using primers

PnmB_delF1, PnmB_delR1, PnmB_delF2, and PnmB_delR2 and subsequently amplified using LongtineF and LongtineR primers. The resultant DNA construct was transformed into MH11036 (_pyroA4_,

_riboB2_, _nkuA_∆_::argB_), and glufosinate-resistant transformants were isolated. Deletion of _pnmB_ was confirmed in one transformant (CWF579) by Southern blot analysis (Supplementary Fig.

 8). GENERATION OF THE PNMBHA STRAIN The DNA construct inserting a HA epitope tag at the C terminus of PnmB was generated as described previously38 using the primers PnmB_tagF1, PnmB_tagR1,

PnmB_delF2, PnmB_delR2, LongtineF, and LongtineR. The DNA construct was transformed into MH11036, and glufosinate-resistant transformants were isolated. One transformant (CWF583) was

confirmed by Southern blot analysis for correct integration (Supplementary Fig. 8) and by western blot analysis for successful tagging. GENERATION OF _PNMB_ _HA_ AND _PRTA_ _HA_

OVEREXPRESSION STRAINS The coding region of _pnmB_ or _prtA_ was amplified by PCR using primers PnmB_OE_F, PnmB_HAOE_R, PrtA_OE_F and PrtA_HAOE_R specified in Table 3. The _pnmB_ and _prtA_

PCR products were introduced after the _xylP_ promoter in the pCWB588 plasmids, (which also contain a 5′ and 3′ truncated _yA_ gene fragment for targeting to the _yA_ locus) using the

Isothermal Assembly method39. The resultant _xylP_(p)::_pnmB_HA (pCWB288) and _xylP_(p)::_prtA_HA (pCWB290) constructs were verified by Sanger-sequencing and transformed into MH11036. The

_xylP_(p)::_pnmB_HA (CWF598) and _xylP_(p)::_prtA_HA (CWF664) transformants were tested for overexpression by western blot analysis. For _pnmB_, another overexpression strain (CWF666) was

created by targeting the _xylP_(p)-_pnmB_HA overexpression construct (pCWB287), which contains a 5′ and 3′ truncated _wA_ fragment, to the _wA_ locus in the CWF387 parent strain. GROWTH

TESTS Strains were point-inoculated and grown on solid ANM37 containing supplements and the indicated nitrogen source at 10 mM (except where indicated) under the specified temperature and pH

conditions and with or without 10 mM TU (Sigma) for 2 days. For quantification in the TU derepression assay, the average of three colony diameter measurements was calculated for the strains

in each growth assay. Three independent assays were carried out. The ratio was calculated by dividing the average colony diameter on the xylose condition by that under the glucose

condition. Statistical significance was calculated using _t_ test in GraphPad of Prism 5. For phenotypic assessment of _pnmB_ overexpression, 1% xylose was also added to the media to induce

overexpression. WESTERN BLOT ANALYSIS Strains were grown in 100 mL ANM liquid medium with supplements and 10 mM ammonium tartrate at 37 °C for 16 h. For nitrogen starvation, mycelia were

washed with and transferred to pre-warmed nitrogen-free ANM media for the indicated amount of time. Xylose was added to the media at a final concentration of 1% to induce PnmB and PrtA

overexpression in the respective overexpression strains. Mycelia were harvested on Miracloth, pressed dried, snap-frozen in liquid nitrogen, and kept at −80 °C until protein extraction.

Total proteins were extracted as previously described19. In all, 50 μg of total proteins were separated in 10% SDS-PAGE gel and transferred to PVDF membrane for immuno-detection. PnmBHA,

NmrAFLAG, and histone H3 were detected using HA-probe (Santa Cruz sc-7392), anti-FLAG® M2 (Sigma F1804), and anti-Histone H3 (Abcam ab1791) antibodies, respectively, at the concentration of

0.1 µg/mL. Goat anti-mouse (Millipore AP124P) or goat anti-rabbit horseradish peroxidase (HRP)-conjugated (Millipore AP132P) antibodies were used as the secondary antibody at the

concentration of 0.1 µg/mL for Chemiluminescence detection using Clarity™ Western ECL Substrate (Bio-rad 1705060) kit. CO-IP OF NMRAFLAG AND PNMBHA Strains expressing NmrAFLAG (MH11626)19,

PnmBHA (CWF664), or both NmrAFLAG and PnmBHA (CWF666) were grown in 100 mL ANM media containing supplements and 10 mM ammonium tartrate at 37 °C for 16 h. Overexpression of PnmBHA was

induced with 1% xylose for 4 h at 37 °C. Mycelia were collected by filtering using a Mira-cloth, immediately frozen in liquid nitrogen and then ground into fine powder in liquid nitrogen

using a mortar and pestle. Ground mycelia powder was then transferred into a fresh tube containing zirconium beads (~100 μL in volume) and subjected to 5 cycles of 3-min lysis at the maximum

speed using a Bullet Blender in 1 mL IP buffer (250 mM NaCl, 100 mM Tris-HCl pH 7.5, 10% glycerol, 1 mM EDTA, and 0.1% NP-40). Protease inhibitors (1 mM PMSF, 1x Roche EDTA-free

cOmplete-mini Protease Inhibitor Cocktail containing benzamidine), phosphatase inhibitors (100 mM NaF, 50 mM Na vanadate, 80 mM β-glycerol phosphate), and 1.5 mM dithiothreitol were added

immediately before lysis. For IP, 1 mL of total protein lysate was incubated with 2 µg mouse anti-FLAG (Sigma F3165) or mouse anti-HA (Santa Cruz SC-7392) antibody with gentle rotation for 2

 h at 4 °C. 10 µL of pre-washed protein A beads (GE Healthcare 17-0963-03) was added and the mixture was incubated for additional 2 h with gentle rotation. The protein A beads were washed 5

times with 1 mL of IP buffer. In the final round, beads were resuspended in 50 µL of 4× Laemmli protein loading buffer and incubated at 95 °C for 10 min for elution. The immuno-precipitated

samples were separated on a 10% SDS-PAGE gel for western blot analysis using mouse anti-FLAG HRP-conjugated antibody (Abcam ab49763) for NmrAFLAG and rabbit anti-HA (Abcam ab9110) primary

antibody followed by goat anti-rabbit HRP-conjugated secondary antibody (Merck Millipore, AP132P) for PnmBHA at the concentration of 0.1 µg/mL. PROTEIN SECRETION ANALYSIS Strains were first

grown in ANM media containing supplements and 10 mM ammonium tartrate at 37 °C for 16 h, and then transferred to 10 ml nitrogen-free ANM media for an additional 4 h. For PnmB and PrtA

overexpression, 1% xylose was added to the media. Mycelia were collected by filter with miracloth, and mycelia were subjected to total protein extraction as the intracellular fraction the

TCA extraction method described above, while the conditioned growth media was further filtered using a 0.2 μm filter to remove all mycelia before protein extraction by a modified TCA method.

Briefly, 10 μg BSA and 1 mL of ice-cold 100% TCA was added to 5 mL of filtered medium, and then 2 ml of the mixture was transferred to a microfuge tube and centrifuged at 4 °C 15000 rpm for

30 min. The protein pellet was washed with 1 mL ice-cold acetone and air-dried, followed by re-suspension in 50 μL protein loading buffer with incubation at 95 °C for 10 min. 50 μL secreted

protein and 10 μg intracellular total protein was separated in 10% SDS-PAGE and subjected to western blot analysis using HA-probe (Santa Cruz sc-7392) and anti-Histone H3 (Abcam ab1791)

antibodies at the concentration of 0.1 µg/mL. CHIP AND CHIP-SEQ ANALYSIS Strains were cultured as described in the “Western blot analysis” section. Crosslinking and chromatin preparation

were carried out according to a previous study10. Briefly, formaldehyde was added to the growth media at a final concentration of 1%, followed by gentle shaking for 20 min at room

temperature. Subsequently, 25 mL 2.5 M glycine was added to terminate crosslinking and the mixture was left at room temperature for 10 min with gentle shaking. Mycelia were washed with

ice-cold water and harvest by filtering on Miracloth. Harvested mycelia were press-dried, snap-frozen in liquid nitrogen, and then lyophilized for 1–2 h before storing at −80 °C for

chromatin preparation. Lyophilized mycelia were lysed with zirconium beads in ice-cold FA lysis buffer for five times of 3 min in a Bullet Blender. Extracts were recovered and centrifuged to

remove the supernatant. Pellets were re-suspended in cold FA lysis buffer and subjected to sonication at 100% amplitude with 10 s on and off cycles for a total sonication time of 20 min

using the Qsonica Q800R machine. For IP, 2 µg of anti-RNA polymerase II antibody (Millipore 04-1572) was used for ChIP as described previously40. Quantitative real-time PCR was performed

using primers listed in Table 3. IP efficiency was calculated for each gene by comparing the amount between IP product and input DNA. Open reading frame-free region was used as background

control to compare the IP efficiency between different genes and samples. Library preparation for PolII ChIP-seq analysis was carried out as described previously41. Sequencing was performed

on Illumina HiSeq2500. Raw reads were mapped to _A. nidulans_ genome version _A_nidulans__FGSC_A4_version_s10-m04-r03 using Bowtie242,43. PolII ChIPseq signals were calculated by first

summing the total number of reads overlapping at each base pair across the coding region of genes and then normalizing to gene length and total number of mapped reads number. The Integrated

Genome Browser program was used to visualize the data44. PROTEIN SEQUENCE AND BLASTP ANALYSIS PnmB motifs information was acquired from AspGD42. Signal peptide prediction was performed via

SignalP5.032. BLASTP analysis of PnmB was performed at NCBI45 against the non-redundant Protein Sequence (fungi) database with default parameters except for the maximum target sequences set

at 5,000 and at the JGI MycoCosm database46. The BLASTP output hits from NCBI and JGI were combined and assigned to the 6 phyla shown in Fig. 4 and Supplementary Data 1. Taxonomy information

of BLAST hits was assigned using the taxonomy database from NCBI (https://www.ncbi.nlm.nih.gov/taxonomy). STATISTICS AND REPRODUCIBILITY Statistics of colony diameter measurements was

performed by one-tailed unpaired _t_ test using the GraphPad Prism 5 software. REPORTING SUMMARY Further information on research design is available in the Nature Research Reporting Summary

linked to this article. DATA AVAILABILITY The PolII ChIPseq data are available from NCBI SRA database under the accession number PRJNA560791. The full blot images for all western blot

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metabolism. _Appl. Environ. Microbiol_. 64, 3232–3237 (1998). Article  CAS  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS We thank members of Wong laboratory

for their constructive suggestions to the work, Professor Michael Hynes for his critical and editorial comments, Dr Jason Slot for expert advice, Lakhansing Pardeshi for Next Generation

Sequencing data processing, and Jacky Chan from Information and Communication Technology Office (ICTO) of the University of Macau for his support on the High Performance Computing Cluster

(HPCC). This work was performed in part at the HPCC supported by ICTO of the University of Macau and was funded by the Research Services and Knowledge Transfer Office (RSKTO) of the

University of Macau (grant number: MYRG2019-00099-FHS) and the Science and Technology Development Fund, Macao S.A.R (FDCT) (project reference number: 0106/2020/A and 0033/2021/A1) (to

K.H.W.), and was supported by the Plant Biotechnology Center of Kansas State University (R.B.T.) as Contribution no. 20-038-J from the Kansas Agricultural Experiment Station. We also

acknowledge the support from the Collaborative Research Fund Equipment Grant (C5012-15E) from the Research Grant Council, Hong Kong Government. AUTHOR INFORMATION Author notes * Ang Li

Present address: Department of Otolaryngology-Head and Neck Surgery, First Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510120, China * Chirag Parsania Present address:

Gene & Stem Cell Therapy Program, Centenary Institute, Camperdown, NSW, 2050, China AUTHORS AND AFFILIATIONS * Faculty of Health Sciences, University of Macau, Avenida da Universidade,

Taipa, Macau SAR, China Ang Li, Chirag Parsania, Kaeling Tan & Koon Ho Wong * Gene Expression, Genomics and Bioinformatics Core, Faculty of Health Sciences, University of Macau, Avenida

da Universidade, Taipa, Macau SAR, China Kaeling Tan * Department of Plant Pathology, Kansas State University, 1712 Claflin Road, 4024 Throckmorton Plant Sciences Center, Manhattan, KS,

66506, USA Richard B. Todd * Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Avenida da Universidade, Taipa, Macau SAR, China Koon Ho Wong * MoE

Frontiers Science Center for Precision Oncology, University of Macau, Avenida da Universidade, Taipa, Macau SAR, China Koon Ho Wong Authors * Ang Li View author publications You can also

search for this author inPubMed Google Scholar * Chirag Parsania View author publications You can also search for this author inPubMed Google Scholar * Kaeling Tan View author publications

You can also search for this author inPubMed Google Scholar * Richard B. Todd View author publications You can also search for this author inPubMed Google Scholar * Koon Ho Wong View author

publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS A.L. and K.H.W. conceived the work. A.L. performed the experiments and sequence analysis. A.L., C.P.,

K.T., R.B.T., and K.H.W. designed the experiments and analyzed and interpreted the data. A.L., R.B.T., and K.H.W. wrote the paper. CORRESPONDING AUTHORS Correspondence to Richard B. Todd or

Koon Ho Wong. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. PEER REVIEW INFORMATION _Communications Biology_ thanks Jason Stajich and the other

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transcriptional control of nutrient degradation in the fungus _Aspergillus nidulans_. _Commun Biol_ 4, 1409 (2021). https://doi.org/10.1038/s42003-021-02925-1 Download citation * Received:

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