The role of the Flb protein family in the life cycle of Aspergillus niger

Genes flbA-E are involved in sporulation and vegetative growth in Aspergillus nidulans. Inactivation of either of these genes results in a fluffy phenotype with delayed or even abolished sporulation. Previously, a non-sporulating phenotype was obtained by inactivating flbA in Aspergillus niger, which was accompanied by lysis, thinner cell walls, and an increased secretome complexity. Here, we further studied the role of the flb genes of A. niger. Strains ΔflbA, ΔflbB and ΔflbE showed increased biomass formation, while inactivation of flbA-D reduced, or even abolished, formation of conidia. Strain ΔflbA was more sensitive to H2O2, DTT, and the cell wall integrity stress compounds SDS and Congo Red (CR). Also, ΔflbC was more sensitive to SDS, while ΔflbB, ΔflbD, and ΔflbE were more sensitive to CR. On the other hand, inactivation of flbE increased resistance to H2O2. Enzyme secretion was impacted when the Δflb strains were grown on xylose. Strain ΔflbE showed reduced xylanase, cellulase and amylase secretion. On the other hand, amylase secretion at the periphery of the ΔflbA colony was reduced but not in its center, while secretion of this enzyme was increased in the center of the ΔflbB colony but not at its periphery. Inactivation of flbC and flbD also impacted zonal cellulase and amylase activity. Together, the Flb protein family of A. niger function in biomass formation, sporulation, stress response, and protein secretion. Supplementary Information The online version contains supplementary material available at 10.1007/s10482-024-01957-x.


Introduction
The FlbA-E proteins of Aspergillus nidulans play a role in asexual sporulation.Inactivation of their encoding genes results in fluffy colonies due to the production of high numbers of aerial hyphae combined with the delay or even abolished production of spore producing conidiophores (Wieser et al. 1994).
FlbA is a RGS domain protein, which negatively regulates vegetative growth signaling and thereby stimulates asexual development.It does so by stimulating the intrinsic GTPase activity of the Gα subunit FadA (Yu et al. 1996).Overexpression of flbA in A. nidulans inhibits hyphal growth and stimulates conidiophore development (Lee and Adams 1994a).By contrast, inactivation of flbA results in abolished asexual development (Wieser et al. 1994) and in autolysis of hyphae when colonies mature (Lee and Adams 1994a;Wieser et al. 1994).
Genes flbB, flbC, and flbD encode transcription factors.FlbB is a fungal specific bZIP-type transcription factor (Etxebeste et al. 2008).Deletion of its encoding gene results in defective branching patterns, in susceptibility to autolysis under high sorbitol or sucrose concentrations and in delayed conidiation with a fluffy appearance.Moreover, mis-scheduled flbB upregulation reduces the diameter of the vesicle of the conidiophore and reduces the number of metulae (Etxebeste et al. 2009).FlbC is a transcription factor containing two C 2 H 2 zinc finger DNA binding domains (Kwon et al. 2010a).Inactivation of its encoding gene results in delayed and reduced conidiation and in enhanced sexual fruiting body formation, while its overexpression restricts hyphal growth and delays conidiation.FlbC acts as a transcriptional regulator in a pathway parallel to that involving FlbA and the FlbB/FlbE and FlbB/FlbD complexes (Garzia et al. 2010).Double mutants cause additive effects, resulting in a prolonged delay in conidiation (Kwon et al. 2010b).Deletion of the c-Myb transcription factor gene flbD results in delayed conidiation and a fluffy phenotype (Wieser et al. 1994;Wieser and Adams 1995), while its overexpression causes sporulation in liquid submerged cultures.FlbD functions by interacting with FlbB (Garzia et al. 2009(Garzia et al. , 2010;;Etxebeste et al. 2010).
FlbE does not have any known conserved domain (Garzia et al. 2009).Both inactivation and overexpression of its encoding gene results in the absence of conidiophore formation, accelerated vegetative growth, and accelerated autolysis and cell death (Kwon et al. 2010b).FlbE is involved in FlbB stability and may thus protect FlbB from proteolytic degradation, possibly due to their physical interaction (Garzia et al. 2009).The FlbB/FlbE complex is a prerequisite for flbD expression in the wild-type (Garzia et al. 2010).
The Flb proteins are conserved in A. nidulans, Aspergillus fumigatus, Aspergillus oryzae, and Aspergillus niger (Pel et al. 2007;Ogawa et al. 2010;Kwon et al. 2010a).The phenotypic changes in the Δflb strains of A. oryzae are similar to those in A. nidulans.Conservation of function of the Flb proteins is also implied by the finding that inactivation of flbB in A. fumigatus results in delayed and reduced sporulation and precocious cell death (Xiao et al. 2010).Also, the A. fumigatus flbE gene is involved in conidiation (Kwon et al. 2010b).However, its inactivation does not result in increased vegetative proliferation, accelerated autolysis, or cell death.
Like in A. nidulans, inactivation of flbA in A. niger results in a fluffy phenotype with abolished sporulation and an increased lysis incidence (Krijgsheld et al. 2013).The latter is probably caused by a reduced thickness (Krijgsheld et al. 2013) and reduced integrity (van Munster et al. 2015) of the cell wall.The ΔflbA strain secretes a higher diversity and amount of proteins in the culture medium.From this and the fact that A. niger does not secrete proteins in zones that sporulate it was concluded that sporulation inhibits protein secretion in A. niger.The fact that inactivation of flbC in A. oryzae results in reduced expression of the glucoamylase gene glaB and the acid protease pepA indicates that this sporulation gene also has a positive impact on secretion (Tanaka et al. 2016).
We here assessed the role of the FlbA-E proteins of A. niger in sporulation, vegetative growth, secretion, and stress responses.To this end, flbA (ATCC64974_61450), flbB (ATCC64974_29960), flbC (ATCC64974_58210), flbD (ATCC64974_19410), and flbE (ATCC64974_100850) were inactivated by CRISPR Cas9.All Flb proteins were shown to impact stress resistance and protein secretion.FlbA-D also play a role in formation of conidia, while FlbA, FlbB and FlbE suppress biomass formation.

Strains and culture conditions
Escherichia coli TOP10 was used for cloning.Static and liquid cultures of A. niger MA234.1, its derived strains ΔflbA, ΔflbB, ΔflbC, ΔflbD, ΔflbE, and the strains in which the flb genes had been reintroduced (Table 1) were inoculated with spores and grown at 30 ℃. Spores were isolated from 3-day-old cultures that had been grown on potato dextrose agar (PDA) after confluent inoculation with 10 6 spores.The spores were harvested with 0.9% NaCl using a cotton swab.Spore suspensions were filtered through a syringe with cotton to remove hyphae and counted using a hemocytometer.
Colonies were grown in between two perforated polycarbonate membranes (pores of 0.1 µm, diameter 76 mm; Profiltra, Almere, The Netherlands) (Wösten et al. 1991) on MMA-G for biomass assessment of static cultures.The upper polycarbonate membrane was placed 24 h after inoculation.To monitor spatial protein secretion, 7-day-old colonies that had been grown on a single PC membrane were transferred for 24 h to a ring plate (Levin et al. 2007).The five concentric wells of this plate were filled with MM with 25 mM xylose (MM-X).

Enzyme activity assays
Cellulase activity was measured using the filter paper activity assay (Fpase) (Xiao et al. 2004).To this end, 7 mm diameter circles of Whatman No.1 filter paper were placed in 96 well plates with 60 µl culture medium for 24 h at 50 °C, followed by a 5 min incubation at 95 °C after adding 120 µl DNS (10 g L −1 3,5-dinitrosalicylic acid, 400 g L −1 KNa-tartrate and 16 g L −1 NaOH).Samples (100 µl) were transferred to the wells of a 96 wells flat-bottom plate (Cellstar, Greiner Bio-one, www.gbo.com) and the A 540 was determined using a Synergy HTX Microplate Reader (BioTek, www.agile nt.com).Activity was determined using a glucose standard curve.
A unit of cellulase activity was defined as 1 µmol glucose released in 1 min.Amylase activity was determined in a similar way as cellulase activity but the Whatman filter paper was replaced by 60 μl 1% starch.Xylanase activity was determined using the Xylanase Assay Kit (XylX6 Method)(Megazyme, www.megaz yme.com).In this case, one unit of activity was defined as the amount of enzyme required to release 1 µmole of 4-nitrophenol from the XylX6 substrate in one minute under the defined assay conditions.

Statistics
Experiments were performed using biological triplicates.Data were subjected to One-way Anova analysis of variance.Mean value was analysed with a confidence P ≤ 0.05.

Inactivation and reintroduction of flbA-flbE
FlbA-E from A. niger show 62.0-79.7% and 66.2-82.6%identity to their homologues of A. fumigatus and A. nidulans, respectively (NCBI, https:// www.ncbi.nlm.nih.gov/).Genes flbA, flbB, flbC, flbD, and flbE were inactivated in A. niger.Their inactivation was confirmed by PCR (see Material and Methods) and Sanger sequencing.In addition, wild-type phenotypes were obtained after reintroducing the inactivated genes in the deletion strains (data not shown).

Growth and sporulation
Radial growth of ∆flbA, ∆flbB, ∆flbC, ∆flbD, and ∆flbE colonies was similar to the reference strain To monitor spatial protein secretion, 7-day-old colonies that had grown on a single PC membrane were transferred for 24 h to a ring plate (Levin et al. 2007).The five concentric wells of this plate were filled with MM with 25 mM xylose (MM-X).Ring 2 is a central zone, ring 4 is just behind the colony periphery.Statistical analysis was done with One-way ANOVA with different letters indicating statistical differences ▸ Page 7 of 11 58 Vol.: (0123456789) when grown on PDA or on defined MMA with glucose, sucrose, xylose, sorbitol, maltose, starch, xylan or pectin as a carbon source (Fig. 1A).However, strains ΔflbA, ΔflbB, and ΔflbE produced 10.4-20.3%more biomass on MMA-G (Fig. 1B) compared to the reference strain.Spores were counted from cultures grown on MMA-G.The ΔflbA strain did not form spores at all, while ΔflbB, ΔflbC, and ΔflbD showed a reduced formation of conidia of 27.4%-62.0%(Fig. 1C, D).Spore formation of ΔflbE was similar to that of the reference strain.

Stress resistance
The reference and the Δflb strains were exposed to the cell wall stressors sodium dodecyl sulfate (SDS) and Congo Red (CR) and the endoplasmic reticulum stressor dithiothreitol (DTT) as well as to H 2 O 2 induced oxidative stress (Fig. 2).Strain ΔflbA showed increased sensitivity to all stressors.Strain ΔflbC was more sensitive to SDS, while ΔflbB, ΔflbD and ΔflbE were more sensitive to CR.On the other hand, ΔflbE showed higher resistance to H 2 O 2 .

Protein secretion
Colonies of the reference and the Δflb strains that had been grown for 7 days on a perforated PC membrane (see Material and Methods) were transferred for 24 h to a ring plate with 5 concentric wells (Levin et al. 2007) filled with MM-X.This medium contains xylose, which induces a wide range of xylanolytic and cellulolytic enzymes (van Peij et al. 1998).Also, it inhibits but not abolishes glucoamylase secretion (Nunberg et al. 1984;Wösten et al. 1991).SDS-PAGE showed that protein profiles of the central zone 2 of ΔflbA and ΔflbC colonies was different from that of the reference strain, while those of the other flb strains were not affected (Fig. 3A).Strains ΔflbA, ΔflbC, and ΔflbD indicated higher protein intensity in the subperiphery zone 4 of the colony when compared to the reference strain, while protein profiles were not affected in ΔflbB and ΔflbE (Fig. 3A, B).Activity of cellulase, xylanase, and amylase was determined in the culture medium of the reference and the Δflb strains in zones 2 (centre) and 4 (subperiphery) of the ring plate (Fig. 3C-E).Xylanase activity was not affected in the Δflb strains except for ΔflbE that showed a 2.5-fold (zone 2) and 2.6-fold (zone 4) lower activity (Fig. 3D).Cellulase and amylase activities were also lower in the culture medium of ΔflbE.Cellulase activity was 3.1-fold (zone 2) and 1.8-fold (zone 4) lower (Fig. 3C), while amylase activity was 1.9-fold (zone 2) and 1.7-fold (zone 4) lower (Fig. 3E).A decreased amylase activity (8.2fold) was also found in zone 4 of ΔflbC, zone 2 (6.7fold) and zone 4 (13.1-fold) of ΔflbD, and zone 4 (2.5-fold) of ΔflbA.On the other hand, amylase was 1.4 fold and 1.6 fold higher in zone 2 of ΔflbC and ΔflbB.Also, cellulase activity was 2.1-fold and 3.8fold higher in zone 4 of the ΔflbC and ΔflbD strains, respectively, when compared to the reference strain (Fig. 3C).Together, the Flb proteins can play both a stimulatory as well as a repressing role in release of enzymes that are involved in substrate degradation.

Discussion
The Flb proteins of A. niger were shown to have roles in biomass formation, sporulation, stress resistance, and secretion.All Flb family proteins play a role in protein secretion and in resistance to cell wall stress.FlbA also functions in resistance to oxidative and ER stress, while FlbE has a negative effect on oxidative stress resistance.In addition, FlbA, FlbB and FlbE have repressive effects on biomass formation, while FlbA-D function in production of conidia.Together, Flb proteins of A. niger have pleiotropic phenotypes (Fig. 4).FlbA, FlbB and FlbE seem to have a conserved role in biomass formation in aspergilli.Overexpression of flbA in A. nidulans inhibits hyphal growth (Lee and Adams 1994a), while its inactivation results in autolysis of hyphae (Lee and Adams 1994a;Wieser et al. 1994).Deletion of flbB in A. nidulans results in defective branching patterns and in susceptibility to autolysis when exposed to high osmotic media (Etxebeste et al. 2009), while inactivation of flbB in A. fumigatus results in precocious cell death (Xiao et al. 2010).Notably, both inactivation and overexpression of flbE in A. nidulans results in accelerated vegetative growth, autolysis and cell death (Kwon et al. 2010b).This indicates that the level of FlbE within hyphae of A. nidulans is important for its function.Whether this is also the case for other aspergilli needs to be established.A role in vegetative growth was also shown for FlbC of A. nidulans (Kwon et al. 2010a).This was not observed in our study in A. niger but this may be due to the fact that the phenotype in A. nidulans was found upon overexpression of flbC, while we performed a flbC deletion.Similarly, a role of FlbD in hyphal growth has been described in A. oryzae (Ogawa et al. 2010) but we did not find it in A. niger.
The flb genes of A. nidulans were originally found by the isolation of fluffy colonies with a delayed or even abolished sporulation (Wieser et al. 1994).Similar sporulation phenotypes were found for all flb genes of A. oryzae (Ogawa et al. 2010) and for flbB and flbE of A. fumigatus (Xiao et al. 2010;Kwon et al. 2010b).We here showed that flbA-D of A. niger also play a role in sporulation but this was not the case for flbE.Thus, although the flb genes seem to have conserved functions in biomass formation and sporulation there are differences between the aspergilli.This was previously also shown for fluG of A. niger (Wang et al. 2015).FluG is involved in sporulation in A. nidulans (Lee andAdams 1994b, 1996) and A. oryzae (Ogawa et al. 2010) but not in A. niger (Wang et al. 2015) and in airexposed cultures of A. fumigatus (Mah and Yu 2006).
Previously, a relation between sporulation and repression of secretion was found in A. niger (Levin et al. 2007;Krijgsheld et al. 2013).Secretion of proteins was only observed in non-sporulating colonies of this fungus (Levin et al. 2007).Secretion was observed throughout the colony after inactivating flbA, which is explained by the non-sporulating phenotype (Krijgsheld et al. 2013).By contrast, the ΔbrlA strain did not show altered secretion.This strain that lacks the central regulator of sporulation initiates but does not complete sporulation.This indicates that the regulatory link between sporulation and secretion occurs upstream of BrlA.The secretome of xylose-grown ΔflbA colonies contained 18 proteins with a signal sequence for secretion that had never been reported to be part of the secretome of A. niger, while 101 proteins had previously not been identified in the culture medium of xylose-grown wild type colonies (Krijgsheld et al. 2013).From these data it was concluded that inactivation of flbA results in spatial changes in secretion and in a more complex secretome.SDS PAGE and enzyme activity assays showed that the other Flb proteins also impact secretion of enzymes by xylose-grown colonies.Inactivation of flbE resulted in a reduced xylanase, cellulase and amylase activity in the culture medium underlying the outer and central zones of the colony.Decreased amylase activity was also found in the culture medium underlying the inner and outer zone of ΔflbD, as well as in the outer zone of ΔflbC.On the other hand, amylase activity was higher in the culture medium underlying the inner zone of ΔflbC and ΔflbB and cellulase activity was higher in the outer zone of the ΔflbC and ΔflbD strains.Together, these data show that the Flb proteins of A. niger can both stimulate and repress protein activity in the medium.Repression of protein secretion into the culture medium makes sense when Page 9 of 11 58 Vol.: (0123456789) a colony initiates sporulation and thereby "decides" to invest in reproduction and not in vegetative growth.
The stimulatory role of Flb proteins in this respect is not clear yet.Apart from the Flb proteins, also FluG of A. niger seems to play a role in regulation of secretion (Wang et al. 2015).Thus, FluG of A. niger has lost its role in sporulation but would still be functional in regulation of secretion.The Flb proteins of A. niger also function in stress resistance.FlbE has a negative effect on resistance to H 2 O 2 .On the other hand, FlbA has a stimulatory role in resistance to the cell wall stressors SDS and Congo Red, as well as to the endoplasmic reticulum stressor DTT and to H 2 O 2 .The reduced resistance of ΔflbA to cell wall stressors is explained by a reduced thickness (Krijgsheld et al. 2013), andreduced integrity (van Munster et al. 2015) of its cell wall.The role of FlbA in resistance to H 2 O 2 and to DTT is less easily explained.Previously it was shown that FlbA downregulates rpnR (Aerts et al. 2019).RpnR promotes resistance to H 2 O 2 and to DTT.The fact that FlbA promotes resistance to these stressors and at the same time represses RpnR is contradictory and may be explained by the complex regulatory pathways of stress resistance.The fact that FlbB-D also protect against one of the cell wall stressors suggests that such a protection may be particular relevant during sporulation.
Together, we here showed that Flb proteins of A. niger are not only involved in regulation of vegetative growth and sporulation as was previously shown in A. nidulans, but also in regulation of secretion and in stress resistance.The fact that FlbB of A. nidulans represses production of the secondary metabolite 2,4-dihydroxy-3-methyl-6-(2-oxopropyl) benzaldehyde (DHMBA) (Oiartzabal-Arano et al. 2015), while FlbC of A. oryzae stimulates expression of the glucoamylase gene glaB and the acid protease pepA (Tanaka et al. 2016) suggests that the Flb proteins of other aspergilli also have pleiotropic phenotypes.

Fig. 3
Fig. 3 Protein profiles (A, B) and cellulase (C), xylanase (D), and amylase (E) activity of the reference and the Δflb strains.To monitor spatial protein secretion, 7-day-old colonies that had grown on a single PC membrane were transferred for 24 h to a ring plate(Levin et al. 2007).The five concentric wells of this plate were filled with MM with 25 mM xylose (MM-X).Ring 2 is a central zone, ring 4 is just behind the colony periphery.Statistical analysis was done with One-way ANOVA with different letters indicating statistical differences

Fig. 4
Fig. 4 Pleiotropic roles of the Flb proteins of A. niger.Red and green lines indicate repression and stimulation, respectively

Table 1
Strains used in this work