Negative regulation and developmental competence in Aspergillus

Lee, Mi-Kyung; Kwon, Nak-Jung; Lee, Im-Soon; Jung, Seunho; Kim, Sun-Chang; Yu, Jae-Hyuk

doi:10.1038/srep28874

Download PDF

Article
Open access
Published: 01 July 2016

Negative regulation and developmental competence in Aspergillus

Mi-Kyung Lee^1,2,
Nak-Jung Kwon¹^nAff5,
Im-Soon Lee²,
Seunho Jung³,
Sun-Chang Kim⁴ &
…
Jae-Hyuk Yu¹

Scientific Reports volume 6, Article number: 28874 (2016) Cite this article

4161 Accesses
60 Citations
Metrics details

Subjects

Abstract

Asexual development (conidiation) in the filamentous fungus Aspergillus nidulans is governed by orchestrated gene expression. The three key negative regulators of conidiation SfgA, VosA, and NsdD act at different control point in the developmental genetic cascade. Here, we have revealed that NsdD is a key repressor affecting the quantity of asexual spores in Aspergillus. Moreover, nullifying both nsdD and vosA results in abundant formation of the development specific structure conidiophores even at 12 h of liquid culture, and near constitutive activation of conidiation, indicating that acquisition of developmental competence involves the removal of negative regulation exerted by both NsdD and VosA. NsdD’s role in repressing conidiation is conserved in other aspergilli, as deleting nsdD causes enhanced and precocious activation of conidiation in Aspergillus fumigatus or Aspergillus flavus. In vivo NsdD-DNA interaction analyses identify three NsdD binding regions in the promoter of the essential activator of conidiation brlA, indicating a direct repressive role of NsdD in conidiation. Importantly, loss of flbC or flbD encoding upstream activators of brlA in the absence of nsdD results in delayed activation of brlA, suggesting distinct positive roles of FlbC and FlbD in conidiation. A genetic model depicting regulation of conidiation in A. nidulans is presented.

The function of a conidia specific transcription factor CsgA in Aspergillus nidulans

Article Open access 16 September 2022

Velvet activated McrA plays a key role in cellular and metabolic development in Aspergillus nidulans

Article Open access 15 September 2020

srdA mutations suppress the rseA/cpsA deletion mutant conidiation defect in Aspergillus nidulans

Article Open access 15 March 2023

Introduction

Asexual development (conidiation) in the fungal class Ascomycetes results in the formation of mitotically derived conidiospores, or conidia¹. Despite a great variety in conidial form and function, all conidia represent non-motile asexual propagules that are usually made from the side or tip of specialized sporogenous cells, i.e., phialides in Aspergillus, via asymmetric mitotic cell division¹.

The genetic mechanisms of conidiation have been extensively studied in the model fungus Aspergillus nidulans. In a simple way, the A. nidulans asexual reproductive cycle can be divided into four distinct stages, beginning with a growth phase, proceeding through initiation of the developmental pathway, execution of the developmentally regulated events leading to sporogenesis, and concluding with switching off conidiation by feed-back control². The growth phase involves germination of a conidium and formation of an undifferentiated network of interconnected hyphal cells that form the mycelium. After a certain period of vegetative growth, under appropriate conditions, some of the hyphal cells stop normal growth and begin conidiation by forming complex structures called conidiophores that bear multiple chains of conidia (Fig. 1A; reviewed in ref. 1).

**Figure 1: Background information and quantitative analyses of conidiation.**

Conidiation does not usually occur in A. nidulans until cells have gone through a defined period of vegetative growth necessary for cells to acquire the ability to respond to development signals, which is defined as a competence³. Under normal media conditions, A. nidulans can be maintained in the vegetative stage of its life cycle by growing hyphae submerged in liquid medium. In liquid submerged culture, conidiation hardly takes place and sexual fruiting never occurs unless vegetative cells are exposed to air. Previous studies have revealed that A. nidulans cells require approximately 18 h of growth before they are competent to respond to the inductive signal provided by exposure to air^3,4.

A key event responding to the developmental inductive signal is activation of brlA, which encodes a C₂H₂ zinc finger transcription factor (TF) (Fig. 1B)⁵. Further genetic and biochemical studies have identified the abaA and wetA genes as necessary regulators of conidiation. The abaA gene encodes a putative TF that is activated by brlA during the middle stages of conidiophore development after differentiation of metulae^6,7. The wetA gene, activated by AbaA, functions in late phase of conidiation for the synthesis of crucial cell wall components and conidial metabolic remodeling^8,9. These three genes have been proposed to define a central regulatory pathway that acts in concert with other genes to control conidiation-specific gene expression and determine the sequence of gene activation during conidiophore development and spore maturation^10,11,12 (reviewed in ref. 1).

Subsequent studies have identified various upstream developmental activators (UDAs), fluG, flbA, flbB, flbC, flbD, and flbE that influence brlA expression (Fig. 1B)^13,14,15. Mutations in any of these genes result in “fluffy” colonies that are characterized by undifferentiated cotton-like masses of vegetative cells (reviewed in ref. 1). Each of the FlbB, FlbC and FlbD proteins contains a DNA binding domain and they are shown to be direct activators of brlA expression^16,17. The two genetic cascades composed of fluG →→ flbE/flbB→ flbD → brlA, and fluG →→ flbC → brlA were proposed, in which fluG functions upstream¹⁸.

Our studies to further understand the developmental control mechanisms have identified three key negative regulators of conidiation, SfgA, VosA, and NsdD^19,20,21. The fluG suppressor sfgA is predicted to encode a Zn(II)₂Cys₆ domain protein, and positioned between FluG and FLBs (Fig. 1B)^16,22. The velvet domain TF VosA and the GATA-type TF NsdD were isolated via gain-of-function genetic screens as repressors of conidiation^19,21. VosA, which is activated by AbaA, governs spore maturation and exerts negative feedback regulation of brlA by binding to the 11 nucleotide VosA responsive element (VRE) in the brlAβ promoter^19,23. NsdD, initially identified as a key activator of sexual fruiting²⁴, was found to be also a key repressor of conidiation²¹. The deletion of nsdD bypasses the needs for FluG and all UDAs, but not brlA, for conidiation, indicating that NsdD acts downstream of UDAs and upstream or at the same level of brlA²⁵.

In the present study, we further investigate negative regulation of conidiation and developmental competence. Through combinatorial genetic studies, we have found that VosA and NsdD are the major factors repressing brlA expression, and thereby influencing the acquisition of developmental competence in A. nidulans. We also report that the repressive role of NsdD in conidiation is conserved in other aspergilli. In A. nidulans conidia, NsdD directly binds to the brlAβ promoter region, which contains a GATAA sequence potentially interacting with NsdD. We also demonstrate that FlbC and FlbD are necessary for full activation of brlA even in the absence of nsdD. A working genetic model depicting the positive and negative regulations of brlA expression and conidiation in A. nidulans is presented.

Results

NsdD is a key factor determining the number of conidia

Previously, we showed that vosA and nsdD play an additive role in repressing conidiation and brlA expression in vegetative cells²¹. To further expand our understanding on the genetic interactions of the three negative regulators, we generated double mutants: ∆nsdD ∆vosA, ∆sfgA ∆nsdD and ∆sfgA ∆vosA. We then quantified the conidiation levels of FGSC4 (wild type; WT), ∆sfgA, ∆nsdD, ∆vosA, ∆nsdD ∆vosA, ∆sfgA ∆nsdD and ∆sfgA ∆vosA strains by spreading conidia onto solid MMG and incubating for 2 days. As shown in Fig. 1C, the ∆nsdD mutant produced ~2.3 fold more conidia than WT and other mutant strains (p < 0.001). The ∆sfgA ∆nsdD double mutant produced less number of conidia than the ∆nsdD single mutant, but more than WT (p < 0.001). On the contrary, the ∆sfgA ∆vosA mutant produced a highly reduced number of conidia, ~6 fold less than WT and the ∆vosA mutant. The ∆nsdD ∆vosA double mutant produced a similar number of conidia to WT. These results suggest that NsdD is a major determinant of the number of conidia being produced on solid culture condition. The ∆sfgA ∆vosA mutant exhibited a highly reduced number of conidia than each single mutant, suggesting that the ∆sfgA and ∆vosA mutations have synthetic negative effects on conidiogenesis.

NsdD and VosA cooperatively repress brlA expression and conidiation

One approach to investigate elevated or hyper activation of conidiation is to grow the strains in liquid shake culture and check conidiophore development and mRNA levels of brlA. Under this condition, WT hardly ever produces asexual developmental structure. When WT, ∆nsdD, ∆vosA, ∆sfgA, ∆sfgA ∆vosA, ∆sfgA ∆nsdD and ∆nsdD ∆vosA strains were examined at 16 h liquid shake culture, only the ∆nsdD ∆vosA double mutant formed a high number of conidiophores (Fig. 2A). We then examined the mRNA levels of brlA, abaA and wetA in WT and various mutant strains at 16 h of vegetative growth, and found that only the ΔnsdD ΔvosA double mutant showed a high level accumulation of brlA mRNA (Fig. 2B). Accumulation of abaA and wetA mRNA was consistent with the brlA mRNA expression pattern in the ΔnsdD ΔvosA mutant. These led us to determine the levels of brlA mRNA in WT, ΔnsdD, ΔvosA, and ΔnsdD ΔvosA strains in conidia and very early phases of growth (4~16 h of liquid culture). As shown in Fig. 2C, the ΔvosA mutant displayed a high level of brlA mRNA in conidia and somewhat reduced levels of brlA mRNA in vegetative cells, lacking further activation of brlA expression. On the contrary, the ΔnsdD ΔvosA mutant exhibited a high level of brlA mRNA in conidia, and began to show induced activation of brlA expression even at 6 h of liquid culture, and a sudden strong activation of brlA expression at 10 h and thereafter. In fact, the ΔnsdD ΔvosA mutant formed conidiophores as early as 12 h of liquid culture (data now shown). These findings indicate that NsdD and VosA are major negative regulators of brlA expression and conidiation, and that the removal of the repressive effects imposed by NsdD and VosA might be a key factor determining the developmental competence.

**Figure 2: Accelerated conidiation by the lack of *vosA* and *nsdD*.**

We also check timing of conidiation on solid air-exposed culture condition. Somewhat consistent with the above findings, the time required for the first conidiophore formation in a colony derived from a single conidium on solid medium was about 25 h in the ΔnsdD ΔvosA mutant (Fig. 2D). The ΔsfgA ΔnsdD and ΔnsdD mutants showed initial conidiophore development at 27 h. The ΔsfgA ΔvosA and ΔsfgA mutants formed the first conidiophore at 29 h. The ΔvosA showed elaboration of conidiophore at ~31 h, whereas WT formed the first conidiophore at 33 h. These results suggest collectively that negative regulation of brlA by both NsdD and VosA is a key attribute determining the developmental competence.

NsdD represses conidiation in A. flavus and A. fumigatus

All Aspergillus species appear to have an ortholog of NsdD (AspGD; http://www.aspgd.org/). The predicted NsdD polypeptide, especially the GATA domain in the C-terminus, is highly conserved in A. flavus and A. fumigatus (Fig. 3A). We hypothesized that NsdD might play a similar repressive role in conidiation in these aspergilli.

**Figure 3: Characterization of *nsdD* in *A. flavus*.**

In A. flavus, nsdD mRNA levels are high in conidia, and undulate during the lifecycle (Fig. 3B). The deletion of nsdD in A. flavus by replacing its coding region with the pyrG⁺ marker from A. fumigatus, caused restricted colony growth coupled with abnormal conidiophores compared to WT (NRRL3357; Fig. 3C). The size of the ∆nsdD conidiophores was averaged 45.81 μm, whereas WT conidiophore size was averaged 125.6 μm (P < 0.005; data not shown). This is consistent with the previous report demonstrating that NsdD is a major determinant of developmental morphogenesis²⁶. We then examined levels of conidiation in varying ways, and found that the absence of nsdD resulted in hyper-active conidiation evidenced by the elaboration of a high number of conidiophores at 28 h of liquid shake culture (Fig. 3D), the formation of abundant conidiophores imbedded in agar at 28 h of solid culture (Fig. 3E), as well as enhanced production of conidia per plate (Fig. 3F). These results indicate that NsdD is a key repressor of conidiation in A. flavus.

In A. fumigatus, nsdD is somewhat constitutively expressed, and its mRNA levels are high in conidia (Fig. 4A). Similar to A. nidulans and A. flavus, the deletion of nsdD in A. fumigatus caused restricted hyphal growth (Fig. 4B), and early and uncontrolled activation of conidiation leading to the formation of conidiophores at 19 h liquid shake culture (Fig. 4C), and elaboration of a high number of conidiophores imbedded in agar at 28 h of solid culture (Fig. 4D), and enhanced production of conidia per plate (Fig. 4E). As the mycotoxin gliotoxin (GT) biosynthesis is activated by BrlA^25,27, the deletion of nsdD resulted in elevated production of GT (Fig. 4F). These results indicate that NsdD functions as a negative regulator of conidiation and GT production in the opportunistic human pathogen A. fumigatus.

**Figure 4: Characterization of *nsdD* in *A. fumigatus*.**

NsdD directly binds to the brlAβ promoter region

We previously reported that, in the FluG-mediated conidiation pathway, NsdD functions downstream of FlbE/B/D/C and upstream of brlA²¹. In a simplistic interpretation, we hypothesized that NsdD directly binds to upstream of the brlA coding sequence. To test this hypothesis, we first scanned the brlA promoter region spanning 2 kb with JASPAR CORE database (http://jaspar.genereg.net) to search potential GATA-TF binding sites. NsdD contains a zinc finger GATA binding domain at 394^th–446^th amino acid at its C-terminus, which may interact with a core [A/T]GATA[A/G] consensus sequence. As shown in Fig. 5A, we identified five core GATA sequences located at the −1950 (+), −1013 (+), −894 (+), −668 (−), and −316 (−) nucleotide, where the transcription initiation site for brlAα is designated as +1 ⁵. Binding sites of VosA, FlbB, FlbC, and FlbD in the brlAβ promoter are also marked in Fig. 5A ^16,17,23.

**Figure 5: Regulatory elements of *brlA* and interaction of NsdD with the *brlA* upstream region.**

Our previous study revealed that nsdD encodes two distinct transcripts designated as nsdDβ and nsdDα²¹. The nsdDα transcript specifically accumulates in conidia, whereas the nsdDβ constitutively accumulates throughout the lifecycle. Specific expression of nsdDα in conidia requires activity of both VosA and VelB during the formation of spores²³. The nsdDβ and nsdDα transcripts contain 1,037 nt and 150 nt of 5′ untranslated region (UTR), respectively (Fig. 5B). Further analyses of cDNAs by RT-PCR indicate that these transcripts are predicted to encode the NsdDβ (461aa) and NsdDα (424aa) polypeptides, where NsdDα lacks the first 37 aa found in NsdDβ. To check the presence and expression levels of the predicted two NsdD proteins during the lifecycle, we carried out Western blot analysis employing a strain ectopically expressing NsdD::3XFLAG in ΔnsdD (TMK13, Table 1) and anti-FLAG antibody. We found that levels of both the NsdDβ and NsdDα proteins were very low in conidia, high in vegetative cells, then NsdDα became undetectable at 6 h post developmental induction (Fig. 5C). Employing the TMK13 strain and anti-FLAG antibody, we pulled-down the NsdD interacting DNAs in conidia, and PCR-amplified the five regions containing a core GATA site in the brlA promoter (ChIP-PCR). As shown in Fig. 5A,D, the three regions spanning −1,950, −1,013, and −894 containing the GATAA sequence in the + strand gave rise amplicons. However, the two regions containing GATAA in the − strand were not enriched by NsdD-ChIP. Taken together, while the precise NsdD binding sequence should be identified and validated, multiple NsdD might occupy the brlAβ promoter region. It can be further proposed that binding of NsdD and VosA to the brlA promoter results in the full repressive control of brlA expression, and the developmental competence might be determined by the removal of these key direct negative regulators of brlA (see Discussion).

Table 1 Aspergillus strains used in this study.

Full size table

The positive roles of FlbC and FlbD in conidiation

We previously showed that the deletion of nsdD suppressed the conidiation defects caused by the absence of FLBs²¹. We further asked whether the primary role of FLBs is to remove the NsdD-mediated negative regulation or they play distinct roles in activating conidiation. This was done by determining timing and levels of brlA in the ΔnsdD single and ∆flbC ∆nsdD and ∆flbD ∆nsdD double mutants. As shown in Fig. 6A, brlA accumulation was not observed in WT vegetative conditions, and was slightly increased 12 h post asexual developmental induction. The deletion of nsdD caused brlA mRNA accumulation at 36 h in submerged culture (vegetative), and at high levels at 12 and 24 h post induction. Importantly, we found that, even in the absence of nsdD, the deletion of flbC, or flbD resulted in significantly reduced and delayed accumulation of brlA mRNA at 24 and 48 h post induction. These results indicate that these positive regulators play distinct positive roles in activating brlA expression, and their activities are needed for full activation of brlA.

**Figure 6: The role FlbC and FlbD in activating *brlA*, and a model for regulation of the commencement of conidiation.**

Discussion

Conidiation in Aspergillus occurs as an integral part of the life cycle primarily controlled by the intrinsic genetic program rather than as a response to unfavorable environmental conditions¹. Neither the concentration of a limiting nutrient such as glucose or nitrogen source, nor continuous transfer to new medium modifies the timing with which cells become competent to develop^1,28.

Given that the timing of competence acquisition is endogenous and genetically determined, one can ask how the fungus keeps track of the time that has transpired following germination. Timberlake²⁹ came up with an explanation for this by proposing a repressor of conidiation, which becomes diluted during early growth. In this model, a fixed amount of repressor would be produced during the final stages of conidium differentiation and stored in the spore. During the (~18 h of) vegetative growth, such a repressor is diluted to a critical concentration before development can proceed. Timberlake further speculated that it could be a negatively acting TF that prevents expression of genes required for conidiation, e.g., brlA, and mutational inactivation of such a repressor would be expected to lead to precocious development.

Indeed, collectively, our studies have revealed that there are at least three negative regulators of conidiation, and that a key event for the acquisition of developmental competence is to remove the repressive effects imposed by NsdD and VosA. We further have found that the positive upstream regulators are needed for maximum level conidiation, but not for the commencement of development. This is based on the fact that the deletion of nsdD could bypass the need for fluG, flbB, flbE, flbD, and flbC, but not brlA, in conidiation²¹. Importantly, for the first time, we demonstrated that NsdD physically binds to three different regions in the brlAβ promoter, further supporting the idea that NsdD directly (rather than indirectly) represses the onset of brlAβ expression and conidiation.

NsdD is a GATA TF with a highly conserved DNA-binding domain consisting a Cys2-Cys2 type IV zinc finger in its C-terminal basic region²⁴. GATA TFs (GATA-1 ~ GATA-6) bind to a DNA sequence called a GATA motif [(A/T)GATA(A/G)] present in the regulatory regions of their target genes through two zinc finger domains³⁰. Our in vivo NsdD-DNA interaction analyses in conidia suggest that the NsdD responsive elements (NREs) might be composed of the GATAA core sequence. As shown in Fig. 5, the three regions containing GATAA in the + strand, but not the two regions with GATAA in the − strand, can be enriched by NsdD-ChIP. The GATAA sequence at −1950 is positioned between the predicted binding sites for the two key UDAs, FlbB and FlbD^13,17. The two sites at −1013 and −894 may overlap with the RNA polymerase binding region (the brlAβ transcript is marked by the arrow-head line in Fig. 5A). A revised VosA responsive element (VRE; TGGCTTGGGCTGG) is positioned at −1,496 between the two predicted FlbC binding sites^16,23. Thus, binding of multiple NsdD and one VosA-VelB in the promoter region of brlAβ may effectively inhibit the initiation of brlAβ transcription. In fact, as shown in Fig. 2A–D, the absence of both nsdD and vosA resulted in near constitutive expression of brlA throughout the life cycle, and abundant/precocious conidiophore development in liquid culture (as early as 12 h). However, the observation that about 25 h is required for the elaboration of the first conidiophore in the ΔnsdD ΔvosA mutant colony derived from a conidium (Fig. 2D) suggests that a single spore must undergo vegetative growth for a certain period even in the absence of the key negative regulators of conidiation.

The NsdD polypeptide(s) is highly conserved in most (if not all) Aspergillus species (Fig. 3A) and other fungi including Penicillium, Coccidioides, Ajellomyces, and Fusarium (not shown). Moreover, the A. fumigatus and A. flavus nsdD genes appear to encode two transcripts (and polypeptides; see the second Met position Fig. 3A,B and 4A). We demonstrated that the role of NsdD in negatively controlling brlA and conidiation is conserved in these two species. In A. fumigatus, there are four GATAA sequences at −2276, −1148, −976, and −932, where the BrlA ATG is +1. In A. flavus, two GATAA sequences are present at −1413 and −1389, where the BrlA ATG is +1. In both cases, the deletion of nsdD resulted in precocious and enhanced conidiation, which is consistent with a previous report³¹. Early and increased production of GT in the A. fumigatus nsdD mutant can be explained by precocious and enhanced expression of BrlA, which in turn directly activates gliotoxin biosynthesis^27,32.

Tight repression of brlA in a conidium and for a certain period of vegetative growth is important for the fitness of Aspergillus fungi. Adams and Timberlake³³ showed that overexpression of brlA in vegetative cells resulted in complete cessation of growth and generalized losses of protein and RNA. Collectively, we present a genetic model depicting the negative and positive regulations and the commencement of conidiation in A. nidulans (Fig. 6). During the formation of conidia, VosA and VelB are activated by AbaA³⁴, which in turn activate expression of the lower transcript of nsdD in conidia²³. VosA and multiple NsdD are bound to the upstream regulatory region of brlA, which confers full repression of brlA and conidiation. SfgA acts as an upstream negative regulator of conidiation functioning downstream of FluG²⁰. During early phase of vegetative growth, FluG accumulates to a certain level, which then removes the repressive effects of SfgA, thereby allowing UDAs (FlbB/D and FlbC) to function. Acquisition of the developmental competence might also involve the translocation of FlbB to the hyphal tip, became transcriptionally competent, then entering into the nucleus³⁵. In order for activated FlbB-FlbD¹⁷ and FlbC to trigger brlA expression and conidiation, both NsdD and VosA need to be removed from the brlA promoter. Currently, we do not know how NsdD and VosA are displaced from the brlA promoter. One possible explanation is degradation of the VosA and NsdD proteins. Upon removal of NsdD and VosA coupled with the cooperative activity of FLBs, brlAβ is expressed above threshold, which then fully activates itself and brlAα, triggering development of conidiophores²¹. While not shown in the model, activated BrlA leads to expression of AbaA, which in turn activates expression of VosA, VelB, and WetA in phialides and conidia (see Fig. 1B). The VosA-VelB heterodimer shuts off expression of brlA and β-glucan biosynthetic genes, and activates genes associated with trehalose biogenesis and nsdDα in conidia, allowing full repression of brlA for next generation^19,34,36.

Finally, while we presented a simplified single-path model for conidiation, it is important to note that that regulation of development is a complex multi-degree process involving both activation of the FluG-initiated conidiation pathway and inhibition of FadA-mediated G protein signaling pathway for vegetative growth^{37,38,39,40,41}. In A. nidulans, various G protein mutants displayed precocious activation of conidiation^42,43,44. Moreover, high level accumulation of brlA alone might not be sufficient to trigger conidiation as shown in our ricA study⁴⁵. In A. fumigatus, various developmental regulators including velvet proteins, G-proteins, and RAS proteins govern conidiation (reviewed in ref. 46 and 47). Additional studies integrating genome-wide and systems analyses are in progress to better address the developmental control mechanisms in Aspergillus.

Methods

Fungal strains and culture conditions

The Aspergillus strains used in this study are listed in Table 1. A. nidulans strains were grown on solid or in liquid minimal medium with 1% glucose (MMG) with supplements as described previously⁴⁸ at 7 °C. To determine the numbers of conidia in WT and mutant strains, approximately 10⁵ spores were spread onto solid MMG and incubated at 37 °C for 2 days. The conidia were collected from the entire plate and counted using a hemocytometer. To check elaboration of conidiophores in liquid submerged culture, conidia (10⁶/ml) of individual strains were inoculated in liquid MMG and incubated at 37 °C, 220 rpm. For Northern blot analyses, samples were collected as described⁴⁹. Briefly, for vegetative growth, conidia (10⁶/ml) of strains were inoculated in liquid MMG and cultured at 37 °C, 220 rpm. Samples of liquid submerged culture were collected at designated time points. Induction of asexual development or sexual development was done as described previously⁴⁹.

A. flavus and A. fumigatus strains were grown on solid or in liquid MMG with 0.1% yeast extract (YE, v/v) and supplements as described^48,50, at 30 °C and 37 °C, respectively. To check elaboration of conidiophores in liquid submerged culture, conidia (2 × 10⁵/ml) of individual strains were inoculated in liquid MMG with 0.5% YE and incubated, 220 rpm. For developmental induction, vegetative cells were collected and transferred to solid medium, and the culture plates were air exposed for asexual developmental induction or tightly sealed induction in dark condition as described⁴⁹.

Construction of A. nidulans strains

The oligonucleotides used in this study are listed in Table S1. The double joint PCR (DJ-PCR) method⁵¹ was used to generate the ∆sfgA ∆vosA, ∆nsdD ∆vosA and ∆sfgA ∆nsdD mutants. Both 5′ and 3′ flanking regions of the sfgA and nsdD genes were amplified from genomic DNA of FGSC4 using OMK556;OMK557 and OM558;OMK559 (for sfgA), and OMK562;OMK563 and OMK564;OMK565 (for nsdD). The A. nidulans pyroA⁺ marker was amplified with the primer pair ONK395;ONK396. The final DJ-PCR sfgA deletion construct was amplified with OMK560;OMK561, and the nsdD deletion construct was amplified with OMK566;OMK567. The sfgA deletion amplicon was introduced into THS15.1 to generate the ∆sfgA ∆vosA mutant. The nsdD deletion amplicon was introduced into THS15.1 and TNJ57 to generate the ∆nsdD ∆vosA and ∆sfgA ∆nsdD mutants, respectively. Protoplasts were generated using the Vinoflow FCE lysing enzyme (Novozymes)⁵². At least three independent deletion mutant strains were isolated. To complement ∆nsdD and epitope-tag NsdD, the FGSC4 nsdD fragment including its 2kb 5′ and coding regions was amplified with the primer pair OMK574;OMK575, digested with PstI and NotI, and cloned into the pHS13 vector³⁴, which contains ^3/4pyroA⁵³, a 3xFLAG tag, and the trpC terminator. The resulting plasmid pMK20 was then introduced into the recipient ∆nsdD strain TNJ108, and several TMK13 class transformants expressing the WT NsdD fused with the 3XFLAG tag under its native promoter have been isolated and confirmed.

Construction of A. flavus and A. fumigatus strains

The nsdD gene was deleted in A. flavus NRRL3357.5 (pyrG⁻)⁵⁴ and A. fumigatus AFU293.1 (pyrG⁻)⁵⁵ employing DJ-PCR⁵¹. In A. flavus strain, the 5′ and 3′ flanking regions of the nsdD gene were amplified using A. flavus WT (NRRL3357) genomic DNA with the primer pairs ONK1037;ONK1038 and ONK1039;ONK1040. The A. fumigatus pyrG⁺ marker was amplified from A. fumigatus WT (AFU293) genomic DNA with the primer pair OMK589;OMK590. The 5′ and 3′ flanking regions of nsdD were fused to the marker, and the resulting fusion product was further amplified by the nested primer pair ONK1041;ONK1042. The final deletion construct was introduced into A. flavus NRRL3357.5, and the ∆nsdD mutant (LNJ11) was isolated and confirmed by PCR followed by restriction enzyme digestion⁵¹.

In A. fumigatus strain, the 5′ and 3′ flanking regions of nsdD were amplified from A. fumigatus WT (AFU293) with the primer pairs ONK1043;ONK1044 and ONK1045;ONK1046. The A. nidulans pyrG⁺ marker was amplified from FGSC4 genomic DNA with the primer pair OHS696;OHS697. The 5′ and 3′ flanking regions of nsdD were fused to the marker, and the fusion product was further amplified by the nested primer pair ONK1047;ONK1048. The final deletion construct was introduced into A. fumigatus AFU293.1, and the ∆nsdD mutant (LNJ12) was isolated and confirmed by PCR followed by restriction enzyme digestion⁵¹. At least three independent deletion strains were isolated and confirmed.

Nucleic acid isolation and manipulation

Genomic DNA and total RNA isolation was carried out as previously described^18,56. In Northern blot analyses, DNA probes were prepared by PCR amplification of the coding region of individual genes with appropriate oligonucleotide pairs using FGSC4 genomic DNA as template (Table S1). Probes were labelled with ³²P-dCTP (PerkinElmer) using Random Primer DNA Labeling Kit (Takara) and purified by illustra MicroSpin G-25 columns (GE Healthcare).

Protein extraction and Western blot analysis

Western blot analysis of NsdD was performed using conidia, vegetative cells, and developing cells of TMK13. Individual samples including 2 d old conidia (2 × 10⁸) were collected and resuspended in the lysis buffer (50 mM Tris-HCl [pH7.2], 150 mM NaCl, 1% Triton X-100, 1 mM EDTA), and homogenized by using a Mini Bead beater and 0.2 ml of silica-zirconium beads (Biospec)⁵⁷. Protein concentration was colorimetrically determined using a Protein Assay System (BioRad). Approximately 10 μg of total proteins per a lane were separated on 10% SDS-PAGE gel and transferred onto immobilon-P membrane (Millipore). The membrane was incubated with the mouse monoclonal Anti-FLAG antibody (M2 clone, Sigma-Aldrich), and then subsequently incubated with a secondary antibody conjugated with horseradish peroxidase, HRP-Goat anti-mouse IgG (Millipore). The membrane was developed using enhanced chemilluminescence reagents (Amersham Biosciences).

Chromatin immunoprecipitation assay

Briefly, two days-old conidia (1 × 10⁹ conidia) of TMK13 were crosslinked with fresh 1% formaldehyde for 30 min at RT. Then, 125 mM of glycine buffer was added to stop the cross-linking reaction. The conidia were resuspended in spore lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 5 mM EDTA, 15 mM EGTA, 0.5% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 1x protease inhibitor cocktail). Resuspended samples were mixed with silica beads and broken by a mini-bead beater for 3 min. Subsequently, the samples were sonicated for five cycles (30 s on, 60 s off) with a sonifier microtip at 70% amplitude and level 5 of output control⁵⁷. The lysates were finally diluted in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris/HCl, 167 mM NaCl, pH 8.0), and then the lysates were applied for ChIP assays according to the manufacturer’ instructions with a slight modification (MAGnify Chromatin Immunoprecipitaion System, Invitogen). The lysate was reacted with 1 μg of the mouse monoclonal Anti-FLAG antibody (Sigma-Aldrich). As a negative control, the chromatin extract was incubated with 1 μg of Anti-rabbit IgG in this assay. Individual input DNA samples before immune-precipitation (IP) were used as positive controls. Finally, the enriched DNA was purified and used as a template for PCR reactions with the GO Taq DNA polymerase (Promega). The primer sets used for PCR are shown in Supplementary Table S1.

Gliotoxin analysis

Gliotoxin (GT) production was analyzed by thin-layer chromatography (TLC) as described⁵⁵. Briefly, spores (10⁵/ml) of A. fumigatus strains were inoculated into 2 ml liquid MMG with 0.5% YE and stationary cultured at 37 °C up to 3 days. GT was extracted by CHCl_3. Each sample was loaded onto a TLC silica plate including a fluorescence indicator (Kiesel gel 60, 0.25 mm; Merck). As a positive control, ~1 μg of GT standard (Sigma-Aldrich, St. Louis, MO) was applied. The plate was then developed with toluene-ethyl acetate-formic acid (5:4:1, v/v/v) as mobile phase, where the R(f ) value of GT was ~0.61. Photographs of TLC plates were taken following exposure to UV (365 nm) using a Sony DSC-T70 digital camera. This analysis was performed in triplicates.

Microscopy

The colony photographs were taken by using a Sony digital camera (DSC-F28). Photomicrographs were taken using a Zeiss M² Bio microscope equipped with AxioCam and AxioVision (Rel. 4.8) digital imaging software.

Additional Information

How to cite this article: Lee, M.-K. et al. Negative regulation and developmental competence in Aspergillus. Sci. Rep. 6, 28874; doi: 10.1038/srep28874 (2016).

References

Adams, T. H., Wieser, J. K. & Yu, J.-H. Asexual sporulation in Aspergillus nidulans . Microbiol Mol Biol Rev 62, 35–54 (1998).
CAS Google Scholar
Ni, M., Gao, N., Kwon, N.-J., Shin, K.-S. & Yu, J.-H. Regulation of Aspergillus conidiation. In Cellular and Molecular Biology of Filamentous Fungi, Borkovich, K. A. & Ebbole, D. J. (eds). Washington, D.C., ASM Press 559–576 (2010).
Axelrod, D. E., Gealt, M. & Pastushok, M. Gene control of developmental competence in Aspergillus nidulans . Dev Biol 34, 9–15 (1973).
CAS Google Scholar
Yager, L. N., Kurtz, M. B. & Champe, S. P. Temperature-shift analysis of conidial development in Aspergillus nidulans . Dev Biol 93, 92–103 (1982).
CAS Google Scholar
Adams, T. H., Boylan, M. T. & Timberlake, W. E. brlA is necessary and sufficient to direct conidiophore development in Aspergillus nidulans . Cell 54, 353–62 (1988).
CAS Google Scholar
Andrianopoulos, A. & Timberlake, W. E. The Aspergillus nidulans abaA gene encodes a transcriptional activator that acts as a genetic switch to control development. Mol Cell Biol 14, 2503–15 (1994).
CAS Google Scholar
Andrianopoulos, A. & Timberlake, W. E. ATTS, a new and conserved DNA binding domain. Plant Cell 3, 747–8 (1991).
CAS Google Scholar
Sewall, T. C., Mims, C. W. & Timberlake, W. E. Conidium differentiation in Aspergillus nidulans wild-type and wet-white (wetA) mutant strains. Dev Biol 138, 499–508 (1990).
CAS Google Scholar
Marshall, M. A. & Timberlake, W. E. Aspergillus nidulans wetA activates spore-specific gene expression. Mol Cell Biol 11, 55–62 (1991).
CAS Google Scholar
Mirabito, P. M., Adams, T. H. & Timberlake, W. E. Interactions of three sequentially expressed genes control temporal and spatial specificity in Aspergillus development. Cell 57, 859–68 (1989).
CAS Google Scholar
Park, H.-S. & Yu, J.-H. Genetic control of asexual sporulation in filamentous fungi. Curr Opin Microbiol 15, 669–77 (2012).
CAS Google Scholar
Etxebeste, O., Garzia, A., Espeso, E. A. & Ugalde, U. Aspergillus nidulans asexual development: making the most of cellular modules. Trends in Microbiology 18, 569–576 (2010).
CAS Google Scholar
Etxebeste, O. et al. Basic-zipper-type transcription factor FlbB controls asexual development in Aspergillus nidulans . Eukaryot Cell 7, 38–48 (2008).
CAS Google Scholar
Oiartzabal-Arano, E., Perez-de-Nanclares-Arregi, E., Espeso, E. A. & Etxebeste, O. Apical control of conidiation in Aspergillus nidulans . Curr Genet 62, 371–7 (2016).
CAS Google Scholar
D’Souza, C. A., Lee, B. N. & Adams, T. H. Characterization of the role of the FluG protein in asexual development of Aspergillus nidulans . Genetics 158, 1027–36 (2001).
Google Scholar
Kwon, N.-J., Garzia, A., Espeso, E. A., Ugalde, U. & Yu, J.-H. FlbC is a putative nuclear C2H2 transcription factor regulating development in Aspergillus nidulans . Mol Microbiol 77, 1203–19 (2010).
CAS Google Scholar
Garzia, A., Etxebeste, O., Herrero-Garcia, E., Ugalde, U. & Espeso, E. A. The concerted action of bZip and cMyb transcription factors FlbB and FlbD induces brlA expression and asexual development in Aspergillus nidulans . Mol Microbiol 75, 1314–24 (2010).
CAS Google Scholar
Seo, J.-A., Guan, Y. & Yu, J.-H. Suppressor mutations bypass the requirement of fluG for asexual sporulation and sterigmatocystin production in Aspergillus nidulans . Genetics 165, 1083–93 (2003).
CAS Google Scholar
Ni, M. & Yu, J.-H. A novel regulator couples sporogenesis and trehalose biogenesis in Aspergillus nidulans . PLoS One 2, e970 (2007).
Google Scholar
Seo, J.-A., Guan, Y. & Yu, J.-H. FluG-dependent asexual development in Aspergillus nidulans occurs via derepression. Genetics 172, 1535–44 (2006).
CAS Google Scholar
Lee, M.-K. et al. NsdD is a key repressor of asexual development in Aspergillus nidulans . Genetics 197, 159–73 (2014).
Google Scholar
Kwon, N.-J., Shin, K.-S. & Yu, J.-H. Characterization of the developmental regulator FlbE in Aspergillus fumigatus and Aspergillus nidulans . Fungal Genet Biol 47, 981–93 (2010).
CAS Google Scholar
Ahmed, Y. L. et al. The velvet family of fungal regulators contains a DNA-binding domain structurally similar to NF-kappaB. PLoS Biol 11, e1001750 (2013).
Google Scholar
Han, K.-H. et al. The nsdD gene encodes a putative GATA-type transcription factor necessary for sexual development of Aspergillus nidulans . Mol Microbiol 41, 299–309 (2001).
CAS Google Scholar
Xiao, P., Shin, K.-S., Wang, T. & Yu, J.-H. Aspergillus fumigatus flbB encodes two basic leucine zipper domain (bZIP) proteins required for proper asexual development and gliotoxin production. Eukaryot Cell 9, 1711–23 (2010).
CAS Google Scholar
Cary, J. W. et al. NsdC and NsdD affect Aspergillus flavus morphogenesis and aflatoxin production. Eukaryot Cell 11, 1104–11 (2012).
CAS Google Scholar
Shin, K.-S., Kim, Y.-H. & Yu, J.-H. Proteomic analyses reveal the key roles of BrlA and AbaA in biogenesis of gliotoxin in Aspergillus fumigatus . Biochem Biophys Res Commun 463, 428–33 (2015).
CAS Google Scholar
Pastushok, M. & Axelrod, D. E. Effect of glucose, ammonium and media maintenance on the time of conidiophore initiation by surface colonies of Aspergillus nidulans . J Gen Microbiol 94, 221–4 (1976).
CAS Google Scholar
Timberlake, W. E. Molecular genetics of Aspergillus development. Annu Rev Genet 24, 5–36 (1990).
CAS Google Scholar
Urnov, F. D. A feel for the template: zinc finger protein transcription factors and chromatin. Biochem Cell Biol 80, 321–33 (2002).
CAS Google Scholar
Szewczyk, E. & Krappmann, S. Conserved regulators of mating are essential for Aspergillus fumigatus cleistothecium formation. Eukaryot Cell 9, 774–83 (2010).
CAS Google Scholar
Scharf, D. H. et al. Biosynthesis and function of gliotoxin in Aspergillus fumigatus . Appl Microbiol Biotechnol 93, 467–72 (2012).
CAS Google Scholar
Adams, T. H. & Timberlake, W. E. Developmental repression of growth and gene expression in Aspergillus . Proc Natl Acad Sci USA 87, 5405–9 (1990).
CAS Google Scholar
Park, H.-S., Ni, M., Jeong, K.-C., Kim, Y.-H. & Yu, J.-H. The role, interaction and regulation of the velvet regulator VelB in Aspergillus nidulans . PLoS One 7, e45935 (2012).
CAS Google Scholar
Herrero-Garcia, E. et al. Tip-to-nucleus migration dynamics of the asexual development regulator FlbB in vegetative cells. Mol Microbiol 98, 607–24 (2015).
CAS Google Scholar
Park, H.-S. et al. Velvet-mediated repression of beta-glucan synthesis in Aspergillus nidulans spores. Sci Rep 5, 10199 (2015).
CAS Google Scholar
Alkahyyat, F., Ni, M., Kim, S.-C. & Yu, J.-H. The WOPR Domain Protein OsaA Orchestrates Development in Aspergillus nidulans . PLoS One 10, e0137554 (2015).
Google Scholar
Yu, J.-H., Mah, J.-H. & Seo, J.-A. Growth and developmental control in the model and pathogenic aspergilli. Eukaryot Cell 5, 1577–84 (2006).
CAS Google Scholar
Yu, J.-H. Heterotrimeric G protein signaling and RGSs in Aspergillus nidulans . J Microbiol 44, 145–54 (2006).
CAS Google Scholar
Krijgsheld, P. et al. Development in Aspergillus . Stud Mycol 74, 1–29 (2013).
CAS Google Scholar
Kong, Q. et al. Gbeta-like CpcB plays a crucial role for growth and development of Aspergillus nidulans and Aspergillus fumigatus . PLoS One 8, e70355 (2013).
CAS Google Scholar
Seo, J.-A., Han, K.-H. & Yu, J.-H. Multiple roles of a heterotrimeric G-protein gamma-subunit in governing growth and development of Aspergillus nidulans . Genetics 171, 81–9 (2005).
CAS Google Scholar
Chang, M.-H., Chae, K.-S., Han, D.-M. & Jahng, K.-Y. The GanB Galpha-protein negatively regulates asexual sporulation and plays a positive role in conidial germination in Aspergillus nidulans . Genetics 167, 1305–15 (2004).
CAS Google Scholar
Rosen, S., Yu, J.-H. & Adams, T. H. The Aspergillus nidulans sfaD gene encodes a G protein beta subunit that is required for normal growth and repression of sporulation. EMBO J 18, 5592–600 (1999).
CAS Google Scholar
Kwon, N.-J., Park, H.-S., Jung, S., Kim, S.-C. & Yu, J.-H. The putative guanine nucleotide exchange factor RicA mediates upstream signaling for growth and development in Aspergillus . Eukaryot Cell 11, 1399–412 (2012).
CAS Google Scholar
Park, H.-S. & Yu, J.-H. Developmental regulators in Aspergillus fumigatus . J Microbiol 54, 223–31 (2016).
CAS Google Scholar
Yu, J.-H. Regulation of Development in Aspergillus nidulans and Aspergillus fumigatus . Mycobiology 38, 229–37 (2010).
CAS Google Scholar
Pontecorvo, G., Roper, J. A., Hemmons, L. M., Macdonald, K. D. & Bufton, A. W. The genetics of Aspergillus nidulans . Adv Genet 5, 141–238 (1953).
CAS Google Scholar
Seo, J.-A., Han, K.-H. & Yu, J.-H. The gprA and gprB genes encode putative G protein-coupled receptors required for self-fertilization in Aspergillus nidulans . Mol Microbiol 53, 1611–23 (2004).
CAS Google Scholar
Kafer, E. Meiotic and mitotic recombination in Aspergillus and its chromosomal aberrations. Adv Genet 19, 33–131 (1977).
CAS Google Scholar
Yu, J.-H. et al. Double-joint PCR: a PCR-based molecular tool for gene manipulations in filamentous fungi. Fungal Genet Biol 41, 973–81 (2004).
CAS Google Scholar
Szewczyk, E. et al. Fusion PCR and gene targeting in Aspergillus nidulans . Nat Protoc 1, 3111–20 (2006).
CAS Google Scholar
Osmani, A. H., May, G. S. & Osmani, S. A. The extremely conserved pyroA gene of Aspergillus nidulans is required for pyridoxine synthesis and is required indirectly for resistance to photosensitizers. J Biol Chem 274, 23565–9 (1999).
CAS Google Scholar
He, Z. M., Price, M. S., Obrian, G. R., Georgianna, D. R. & Payne, G. A. Improved protocols for functional analysis in the pathogenic fungus Aspergillus flavus . BMC Microbiol 7, 104 (2007).
Google Scholar
Xue, T., Nguyen, C. K., Romans, A., Kontoyiannis, D. P. & May, G. S. Isogenic auxotrophic mutant strains in the Aspergillus fumigatus genome reference strain AF293. Arch Microbiol 182, 346–53 (2004).
CAS Google Scholar
Park, H.-S. & Yu, J.-H. Multi-copy genetic screen in Aspergillus nidulans . Methods Mol Biol 944, 183–90 (2012).
CAS Google Scholar
Jeong, K.-C. & Yu, J.-H. Investigation of in vivo protein interactions in Aspergillus spores., Methods Mol Biol 944, 251–257 (2012).
CAS Google Scholar
Shaaban, M. I., Bok, J. W., Lauer, C. & Keller, N. P. Suppressor mutagenesis identifies a velvet complex remediator of Aspergillus nidulans secondary metabolism. Eukaryot Cell 9, 1816–24 (2010).
CAS Google Scholar
Brookman, J. L. & Denning, D. W. Molecular genetics in Aspergillus fumigatus . Curr Opin Microbiol 3, 468–74 (2000).
CAS Google Scholar

Download references

Acknowledgements

This work was supported by the Intelligent Synthetic Biology Center of Global Frontier Project (2011-0031955) funded by the Ministry of Education, Science and Technology grants, and a National Research Foundation of Korea grant funded by the Korean Government (NRF-2011-619-E0002).

Author information

Nak-Jung Kwon
Present address: Present address: Macrogen Co, Seoul, Korea.,

Authors and Affiliations

Department of Bacteriology, The University of Wisconsin-Madison, Madison, Wisconsin, 53706, USA
Mi-Kyung Lee, Nak-Jung Kwon & Jae-Hyuk Yu
Department of Biological Sciences and Center for Biotechnology Research, Institute for Ubiquitous Information Technology and Applications (UBITA), Konkuk University, Seoul 143-701, Republic of Korea,
Mi-Kyung Lee & Im-Soon Lee
Department of Bioscience and Biotechnology and Center for Biotechnology Research, Institute for Ubiquitous Information Technology and Applications (UBITA), Konkuk University, Seoul, 143-701, Republic of Korea
Seunho Jung
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Dae-Jeon, Republic of Korea
Sun-Chang Kim

Authors

Mi-Kyung Lee
View author publications
You can also search for this author in PubMed Google Scholar
Nak-Jung Kwon
View author publications
You can also search for this author in PubMed Google Scholar
Im-Soon Lee
View author publications
You can also search for this author in PubMed Google Scholar
Seunho Jung
View author publications
You can also search for this author in PubMed Google Scholar
Sun-Chang Kim
View author publications
You can also search for this author in PubMed Google Scholar
Jae-Hyuk Yu
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

M.-K.L. and N.-J.K. performed the experiments. M.-K.L., I.-S.L., S.J., S.-C.K. and J.-H.Y. designed the experiments and analyzed the data. M.-K.L. and J.-H.Y. wrote the main manuscript text. M.-K.L. prepared all Figures and Tables. All authors have reviewed the manuscript.

Corresponding author

Correspondence to Jae-Hyuk Yu.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Table S1 (PDF 54 kb)

Rights and permissions

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

Reprints and permissions

About this article

Cite this article

Lee, MK., Kwon, NJ., Lee, IS. et al. Negative regulation and developmental competence in Aspergillus. Sci Rep 6, 28874 (2016). https://doi.org/10.1038/srep28874

Download citation

Received: 21 March 2016
Accepted: 16 May 2016
Published: 01 July 2016
DOI: https://doi.org/10.1038/srep28874

This article is cited by

srdA mutations suppress the rseA/cpsA deletion mutant conidiation defect in Aspergillus nidulans
- Masahiro Ogawa
- Ryouichi Fukuda
- Hiroyuki Horiuchi
Scientific Reports (2023)
Evaluating the potential of semi-continuous itaconic acid fermentation by Aspergillus terreus: operational profile and experiences
- Éva Hülber-Beyer
- Katalin Bélafi-Bakó
- Nándor Nemestóthy
World Journal of Microbiology and Biotechnology (2023)
The function of a conidia specific transcription factor CsgA in Aspergillus nidulans
- He-Jin Cho
- Hee-Soo Park
Scientific Reports (2022)
A methanolic extract of Zanthoxylum bungeanum modulates secondary metabolism regulator genes in Aspergillus flavus and shuts down aflatoxin production
- Asmaa Abbas
- Colin W. Wright
- Anssi M. Malinen
Scientific Reports (2022)
Genome-wide identification of the GATA transcription factor family and their expression patterns under temperature and salt stress in Aspergillus oryzae
- Chunmiao Jiang
- Gongbo Lv
- Bin Zeng
AMB Express (2021)

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.