| Mycology | Resea ch A icle
Iden i ica ion and unc ional cha ac e iza ion o he pu a i e
membe s o he CTDK-1 kinase complex as egula o s
o g ow h and de elopmen in Aspe gillus nidulans and
Aspe gillus umiga us
Z. Agi ezabala,1 X. Gu uceaga,2 A. Ma in-Vicen e,2 A. O amendi,1 A. Fagoaga,1 J. R. Fo wendel,2 E. A. Espeso,3 O. E xebes e1
AUTHOR AFFILIATIONS See a ilia ion lis on p. 26.
ABSTRACT Asexual spo es a e he main ehicle used by ungi o dispe se o new
niches. The Eu o iomyce e Aspe gillus nidulans is he main e e ence o he s udy o
he gene ic/molecula con ol o asexual de elopmen . In his species, Flb p o eins
con ol he exp ession o he mas e gene b lA, and hus, loss-o - unc ion mu a ions
in lb (ups eam de elopmen al ac i a ion [UDA]) genes block b lA ansc ip ion and,
consequen ly, he p oduc ion o conidiopho es, he s uc u es bea ing asexual spo es
known as conidia. Howe e , he aconidial pheno ype o speci ic lb mu an s, such as
ha o he Δ lbB s ain, is e e ed unde sal -s ess condi ions. P e iously, we gene a ed
a collec ion o second-si e mu an s o Δ lbB unable o conidia e on cul u e medium
supplemen ed wi h NaH2PO4 (0.65 M). He e, we iden i ied a Gly347S op mu a ion wi hin
lpA as esponsible o he FLIP57 pheno ype and cha ac e ized he ole o he pu a i e
cyclin FlpA and he emaining pu a i e componen s o he C- e minal domain kinase-1
(CTDK-1) complex in A. nidulans and Aspe gillus umiga us. FlpA, S k47, and FlpB a e
necessa y (i) o imely ge mina ion, (ii) in he ansi ion om me ulae o phialides ( he
cells gene a ing conidia) du ing conidiopho e de elopmen , and (iii) o he de elop
men o sexual s uc u es (cleis o hecia) in A. nidulans. The h ee p o eins a e nuclea ,
and he nucleoplasmic localiza ion o S k47 depends on he ac i i y o FlpA, which
co ela es wi h he e en ion o S k47 by FlpA in pull-down assays. O e all, his wo k
links he pu a i e CTDK-1 complex o aspe gilli wi h g ow h and de elopmen al con ol.
Iden i ica ion o a mu a ion in lpA as inhibi o o conidia ion in A. nidulans and unc ional
cha ac e iza ion o FlpA, S k47 and FlpB as pu a i e membe s o he C- e minal domain
kinase complex CTDK-1 in A. nidulans and A. umiga us.
IMPORTANCE Aspe gillus umiga us has been included by he Wo ld Heal h O ganiza
ion in he p io i y lis o ungal pa hogens because (i) i causes 90% o in asi e
aspe gillosis cases, wi h a high mo ali y a e, and (ii) in ec ions a e becoming inc eas
ingly esis an o azole an i ungals. A. nidulans is an oppo unis ic pa hogen and
a sap o oph which has se ed du ing he las 80 yea s as a e e ence sys em o
ilamen ous ungi. He e, we cha ac e ized he ole in mo phogenesis and de elop
men o he pu a i e ansc ip ional cyclin/kinase complex CTDK-1 in bo h aspe gilli.
The null mu an s o he co esponding genes showed delayed ge mina ion, abe an
conidiopho e de elopmen , and inhibi ion o cleis o hecia p oduc ion. While in highe
euka yo es his complex is o med only by a cyclin and a kinase, he ungal complex
would inco po a e a ungal-speci ic hi d componen , FlpB, which would enable he
in e ac ion be ween he kinase (S k47) and he cyclin (FlpA) and may be used as a a ge
o an i ungals.
No embe /Decembe 2023 Volume 14 Issue 6 10.1128/mbio.02452-23 1
Edi o Gus a o H. Goldman, Uni e sidade de Sao
Paulo, Sao Paulo, B azil
Add ess co espondence o O. E xebes e,
oie [email p o ec ed].
Z. Agi ezabala and X. Gu uceaga con ibu ed
equally o his a icle. The o de o hese wo
au ho s was de e mined alphabe ically and wi h
hei consen .
The au ho s decla e no con lic o in e es .
See he unding able on p. 27.
Recei ed 12 Sep embe 2023
Accep ed 3 Oc obe 2023
Published 9 No embe 2023
Copy igh © 2023 Agi ezabala e al. This is an
open-access a icle dis ibu ed unde he e ms o
he C ea i e Commons A ibu ion 4.0 In e na ional
license.
Downloaded om h ps://jou nals.asm.o g/jou nal/mbio on 03 May 2024 by 158.227.89.37.
KEYWORDS ilamen ous ungi, Aspe gillus nidulans, Aspe gillus umiga us, ege a i e
g ow h, asexual de elopmen , conidia ion, sexual de elopmen , s ess esponse, cyclin,
kinase, CTDK-1, RNA polyme ase II
Aspe gilli cons i u e an impo an genus o ilamen ous ungi. Taxonomically, he
genus is loca ed wi hin he phylum Ascomyco a, subphylum Pezizomyco ina, and he
class o Eu o iomyce es. I is composed o hund eds (~350) o species (1). Some o hem
a e pa hogens o ui s, seeds, animals, and humans. Fo example, Aspe gillus umiga us is
he main agen causing in asi e aspe gillosis (2). O he aspe gilli a e used in indus y as a
sou ce o aluable p oduc s (3, 4). Aspe gillus species can ep oduce sexually o asexually
(5). Sexual de elopmen has been desc ibed in a subse o species o his genus, bu
sequencing o Aspe gillus genomes has unco e ed ha he p esence o genes coding
main egula o s o sex is a gene al end (1). Sexual ep oduc ion is based on meio ic cell
di isions, gene a ing spo es wi h combina ions o he gene ic con en o he pa en als (6,
7). Asexual ep oduc ion is based on mi osis, p oducing spo es wi h an iden ical gene ic
con en . This explains why sexual ep oduc ion is mainly di ec ed o he in e change o
gene ic ma e ial, while he main aim o asexual de elopmen is dispe sal.
Spo es ge mina e unde a o able en i onmen al condi ions. Pola g ow h and
b anching o ge mlings gene a e ma u e hyphae, while usion o hyphae h ough
anas omosis gene a es he mycelium, he s uc u e specialized in subs a e coloniza ion
(8). Depending on he s imulus (O2 o CO2, ligh /da kness, nu ien a ailabili y/s a a
ion, sal o osmo ic s ess, p esence/absence o speci ic me aboli es) (9), asexual o
sexual de elopmen al p og ams will be ac i a ed o ep essed. Those spo es dispe se
again o new niches, ini ia ing new li e cycles.
One o he main model o ganisms wi hin he genus Aspe gillus is Aspe gillus nidulans.
Se e al ea u es make his species a sui able e e ence o ganism: as g ow h a es a
labo a o y condi ions, he ac ha i is no a pa hogen, he possibili y o ca ying ou
sexual c osses in sho pe iods o ime, he amenabili y o gene ic manipula ion, and
he a ailabili y o a as a ay o s anda dized cellula and molecula echniques (10).
Toge he wi h he So da iomyce e Neu ospo a c assa, A. nidulans is he main e e ence
species in he s udy o de elopmen al p og ams (3, 10, 11), since mos o he known
egula o s o sexual and asexual de elopmen we e iden i ied and cha ac e ized o
he i s ime in A. nidulans (2). Asexual spo es o A. nidulans a e known as conidia
since hey a e p oduced by localized budding and subsequen cons ic ion om an
ex e nal spo ogenous cell, called he phialide (12). Phialides and conidia a e he las
cell ypes p oduced in he asexual de elopmen al cycle, gi ing ise o mul i-cellula
s uc u es called conidiopho es. Each A. nidulans conidiopho e, and he same holds ue
o A. umiga us, p oduces housands o conidia, and each colony gene a es millions o
conidiopho es on solid cul u e (9, 10, 13–15).
In his seminal wo ks, Timbe lake es ima ed ha mo e han a housand mRNAs
inc eased hei concen a ion a e he induc ion o conidiopho e de elopmen (16).
To da e, he numbe o iden i ied and unc ionally cha ac e ized p o eins wi h a ole in
asexual de elopmen is a om his es ima ion. These egula o s ha e been classi ied
in o signal ansduce s, cen al egula o s, ep esso s, and balance s (9, 17). The e a e
complex unc ional and gene ic ela ionships among hese g oups o egula o s, as well
as be ween hem and egula o s o o he cellula p ocesses such as pola g ow h, sexual
de elopmen , seconda y me abolism, o cell dea h. A simpli ied model desc ibes he
ac i i y o wo main pa hways (18). UDA pa hways a e signal ansduc ion pa hways
in ol ed in he inhibi ion o pola g ow h o hyphae and he decision o whe he o
induce o no , depending on ex acellula and in acellula s imuli, asexual de elopmen .
The e a e a leas h ee UDA subpa hways, which a e de ined by lbA, lbB/ lbD/ lbE, and
lbC, espec i ely. FlbB, FlbC, and FlbD a e ansc ip ion ac o s and play an impo an
ole in he induc ion o he exp ession o b lA. In ac , b lA is he cen al gene in his
model, since i s exp ession is con olled by UDA-s, and hen, B lA con ols he exp ession
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o cen al de elopmen al pa hway (CDP) genes, which egula e he o ma ion o mos
o he cell ypes o he conidiopho e (9, 17, 19). As men ioned abo e, he e a e known
ep esso s o b lA exp ession, which ac a wo le els: ep esso s ha also ac i a e sexual
de elopmen and hose ha ep ess b lA exp ession once asexual de elopmen has
been comple ed (20–24).
Due o hei ole in he con ol o b lA exp ession, dele ion o loss-o - unc ion
mu a ions in UDA genes inhibi o delay conidia ion. Mu an s show an aconidial
pheno ype known as lu y (14). An example is he Δ lbB mu an , in which he CDP
pa hway is blocked. Ne e heless, he lu y pheno ype o he null lbB mu an is
e e ed unde speci ic s ess condi ions, such as supplemen a ion o he s anda d solid
Aspe gillus minimal medium (AMM) wi h high concen a ions (abo e 0.5 M) o NaH2PO4
(25–27). In a p e ious wo k, we ook ad an age o his pheno ype and mu agenized
h ough UV ligh conidia o a Δ lbB s ain wi h he aim o isola ing second-si e mu an s
unable o conidia e when cul u ed on AMM supplemen ed wi h 0.65 M NaH2PO4 (27).
These mu an s we e named as FLIP ( lu y in phospha e mu an s).
In his wo k, mu an s FLIP57 and FLIP76 ha e been cha ac e ized. Iden i ica ion o
he mu a ions causing hei pheno ypes led o he cha ac e iza ion o AN10640/FlpA
( lu y in phospha e A) as a pu a i e cyclin equi ed in he ansi ion om me ulae o
phialides du ing conidiopho e de elopmen . Fluo escence mic oscopy sugges ed ha i s
localiza ion is cell cycle dependen , being loca ed in he nucleoplasm in he in e phase
bu no du ing mi osis. The same pheno ype and subcellula localiza ion we e obse ed
o i s pu a i e in e ac ion pa ne s AN8190/S k47, a cyclin-dependen kinase (CDK), and
AN6312/FlpB, a homolog o Schizosaccha omyces pombe C k3. Speci ic dependencies
we e desc ibed among hese h ee p o eins o nuclea accumula ion and immunode ec
ion pa e ns. Besides de elopmen , ge mina ion and adial g ow h we e also ema kably
a ec ed in he null mu an s o lpA, s k47, o lpB bo h in A. nidulans and A. umiga us.
O e all, esul s sugges ha he ac i i y o he pu a i e C- e minal domain kinase-1
(CTDK-1) complex in he genus Aspe gillus is equi ed in mul iple cellula p ocesses,
pa icipa ing in he coo dina ion o g ow h and de elopmen al p og ams.
RESULTS
Sequencing and analysis o FLIP57 and FLIP76 genomes
In a p e ious wo k, 80 second-si e mu an s o Δ lbB unable o conidia e on AMM
supplemen ed wi h 0.65 M NaH2PO4 (FLIP mu an s) we e g ouped in o se en pheno ypic
g oups (27). We de e mined ha he mu an pheno ype o FLIP166, which belonged o
he g oup o FLIP mu an s wi h a o ally aconidial pheno ype unde phospha e s ess
condi ions, was caused by a mu a ion in AN1459/pm C (27, 28). Eigh addi ional FLIP
mu an s we e selec ed (Fig. S1), hei genomic DNA ex ac ed and pm C sequenced.
None o he FLIP mu an s analyzed bo e a mu a ion in his gene, showing ha hei
pheno ypes we e no caused by mu an pm C alleles. Genomic DNA samples o FLIP57
and FLIP76 (Fig. 1A) we e sequenced and compa ed o he e e ence FGSC4 genome,
he FLIP166 genome, and A. nidulans ansc ip omes (see Ma e ials and Me hods; see
File S1) (23, 27, 29). Fou and h ee exonic candida e mu a ions we e iden i ied in FLIP57
and FLIP76, espec i ely (Fig. 1B). Howe e , in FLIP76, an exonic mu a ion was iden i ied
in AN5717/kapI, leading o a p ema u e s op codon (R557E+8-S op; KapI is 1,095 amino
acids long; File S1; Fig. 1B). Since we p e iously desc ibed ha he double ΔkapI;Δ lbB
mu an was comple ely aconidial unde s ess condi ions induced by he addi ion o
0.5 M NaH2PO4 (30), we concluded ha he FLIP76 pheno ype was caused by he
addi i e e ec o lbB dele ion and a loss-o - unc ion mu a ion in kapI. Consequen ly, we
disca ded FLIP76 and con inued he analysis wi h FLIP57.
In his second mu an , ou o ou exonic mu a ions iden i ied, AN6932 and AN7856
(File S1) we e disca ded because he co esponding null mu an did no show a de ec
in conidia ion (31) o because he gene showed e y low exp ession le els (AN7856).
AN7842 was also disca ded because, despi e he low exp ession le els, RNA-seq eads
sugges ed ha i is no co ec ly anno a ed. Thus, he analysis was ocused on he
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mu a ion ound in AN10640 because (i) RNA-seq da a showed ha he anno a ion o
exons and in ons was co ec ; (ii) i showed mode a e exp ession le els (see below);
and (iii) he gene is p edic ed o encode a cyclin. Tha would link he con ol o asexual
de elopmen wi h he con ol o he cell cycle, ansc ip ion, and/o addi ional cellula
p ocesses.
A poin mu a ion co esponding o a Gly347S op unca ion in AN10640/
FlpA caused he FLIP57 pheno ype
AN10640 is loca ed in ch omosome V (coo dina es 1,323,461–1,325,516, coding s and)
and is composed o i e exons and ou in ons. The G-T mu a ion o FLIP57 is loca ed
FIG 1 Mu a ions ound in FLIP57 and FLIP76. (A) Pheno ypes o mu an s FLIP57 and FLIP76 a e 72 hou s o cul u e a 37°C in AMM ( ow 1) o AMM
supplemen ed wi h 0.65 M NaH2PO4 ( ow 2). A pa en al Δ lbB and he mu an FLIP166 (27) we e used as con ols. (B) Mu a ions ound in he genomes o FLIP57
and FLIP76, which could be esponsible o he co esponding FLIP pheno ypes.
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in exon 5 and modi ies he codon in he posi ion 347 (GGA, Gly) by a s op signal (TGA,
Gly347S op) (Fig. 2A). Since he co esponding p o ein is p edic ed o be 392 amino acids
long, he FLIP57 mu a ion would cause he unca ion o he las 45 amino acids (Fig. 2A).
RNA-seq da a showed ha , in he cul u e condi ions and gene ic backg ounds analyzed
by o he s in A. nidulans ( e iewed in e e ence 32), he posi ion and ex ension o in ons
o AN10640 ma ched he FungiDB anno a ions. Fu he mo e, RNA-seq da a, including
FPKM alues o wild ype and he null lbB backg ounds be o e (VG) and a e (AD,
5 hou s) he induc ion o asexual de elopmen (23, 33), showed mode a e exp ession
le els in all condi ions/backg ounds analyzed, wi h no ema kable induc ion/inhibi ion
o AN10640 in any o he samples (Fig. 2B). The In e p o se e p edic ed he p esence
o a cyclin-like domain (IPR013763, amino acids 59–263; Fig. 2A), which is desc ibed
o be ound in cyclins bu also in ansc ip ion ac o IIB and in he e inoblas oma
umo supp esso (34–36). The FungiDB da abase desc ibed ha o hologs o AN10640
ha e oles in posi i e egula ion o sep a ion ini ia ion signaling, egula ion o phospho
yla ion o RNA polyme ase II C- e minal domain, and ime ic posi i e ansc ip ion
elonga ion ac o complex b localiza ion. The FLIP57 mu a ion is loca ed ou side o his
pu a i e cyclin-like domain.
To con i m ha he mu a ion Gly347S op in AN10640 ( he gene was named as lpA,
lu y in phospha e A) was he cause o he FLIP57 pheno ype, p o oplas s o his
s ain and hose o i s pa en al Δ lbB s ain we e ans o med wi h lpA::ha3x::py GA um,
lpA(Gly347S op)::ha3x::py GA um, lpA::g p::py GA um, o lpA(Gly347S op)::g p::py GA um cons uc s (Fig.
2C). The inse ion o he mu an cons uc s in he null lbB pa en al caused an
inhibi ion o conidia ion in AMM supplemen ed wi h 0.5 M NaH2PO4, while he
wild- ype cons uc s did no al e he Δ lbB pheno ype. In e sely, he wild- ype
cons uc s e e ed he FLIP57 pheno ype unde phospha e s ess. No di e ence was
obse ed be ween lpA::ha3x and lpA::g p coun e pa s, sugges ing ha he size o
he 3′- ag is no de imen al o FlpA ac i i y. On he con a y, he mu an cons uc
lpA(Gly347S op)::g p::py GA um did no e e he FLIP57 pheno ype unde phospha e s ess
(Fig. 2C). These esul s con i med he a o emen ioned hypo hesis.
FlpA and i s pu a i e in e ac o s S k47/AN8190 and FlpB/AN6312 a e widely
conse ed in he kingdom Fungi
Paolillo and colleagues ca ied ou a deep bioin o ma ics and phylogene ic analysis o
A. nidulans cyclins (34). They classi ied AN10640 wi hin g oup III cyclins, as a T/K-like
cyclin, as was AN4981/PchA. To de e mine he conse a ion pa e n o FlpA in he ungal
kingdom, we ca ied ou BLAST analyses o iden i y pu a i e o holog sequences. Table
S1 shows he axonomy o he species co esponding o he FlpA hi s analyzed, he
leng h o he hi , sco e and e alues, he que y co e age, and he esul ( i s sequence)
o he con i ma o y e e se e ie al o each hi sequence a he FungiDB da abase. Only
hi s gi ing AN10640/FlpA as he i s sequence in his con i ma o y e e se e ie al we e
conside ed. O e all, he alues gi en in Table S1, oge he wi h he phylogene ic ee in
Fig. 3A, show ha FlpA is widely conse ed in he kingdom Fungi.
Xie and colleagues ha e ecen ly de e mined he s uc u e o he Schizosaccha omy
ces pombe o holog o FlpA, C k2, as pa o he ime ic CTDK-1 (C- e minal domain
[CTD] kinase I) complex (37). CTDK-1 ac s as he p ima y RNA polyme ase II CTD Se 2
kinase complex. The Swiss-Model websi e modeled FlpA ( om His26 o Lys374; Model
7j 7.1.B was aken as he e e ence; sequence iden i y o 18.67%) (37), wi h highe
con idence, as expec ed, in he cyclin-like domain han in he case o he C- e minus (Fig.
3D; Fig. S2A). Since i co esponded o a s op signal, Dynamu (38) was unable o p edic
he hypo he ic e ec o he FLIP57 mu a ion in he s uc u e o AN10640.
The emaining wo componen s o he CTDK-1 complex in S. pombe a e C k1, a
cyclin-dependen kinase, and C k3, he la e being a C k1 ac i a o and con ibu ing
o he assembly o he complex by in e ac ing wi h C k1 and C k2 (37). The A. nidulans
o holog o C k1 is AN8190/S k47 (39), while ha o C k3 is AN6312/FlpB. Exons and
in ons o bo h genes we e anno a ed co ec ly in he FungiDB da abase (no shown).
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FIG 2 A mu a ion wi hin AN10640/ lpA causes he FLIP57 pheno ype. (A) Anno a ion o AN10640/ lpA and analysis o he mu a ion causing he FLIP57
pheno ype. The locus lpA was compa ed in FLIP57, FLIP76, and FLIP166 gene ic backg ounds and wi h ansc ip omes o a null lbB s ain (RNA-seq1) and a null
sl A s ain (RNA-seq2). FLIP57 bo e a mu a ion in he las exon o AN10640, which led o he subs i u ion o he codon o Gly347 by a s op
(Con inued on nex page)
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Table S1 includes mo e han 400 ungal species con aining FlpA, S k47, and FlpB (see
below) o hologs, ep esen ing 22 classes wi hin he phyla Ascomyco a, Basidiomyco a,
and Muco omyco a (Fig. 3B and C). The Swiss-Model websi e modeled bo h p o eins
aking, as in he case o FlpA, PDB s uc u e 7j 7.1 as he e e ence (subuni s α and γ,
espec i ely, o S k47 and FlpB; much lowe co e age in he case o S k47, om Se 746
o Leu1052; om Glu7 o Asn234 in he case o FlpB) (Fig. 3D; Fig S2B and C). ColabFold
p edic ed he s uc u e o a pu a i e complex o med by he h ee A. nidulans p o eins
( ull-leng h e sions o FlpA and FlpB we e used as que ies and he agmen om P o735
o Leu1067 in he case o S k47, based in his la e case on he Swiss-Model p edic ion).
The model in Fig. 3E shows ha FlpB would in e ac wi h bo h FlpA and S k47, enabling
he assembly o he complex, as has been desc ibed in S. pombe (see he s uc u e in
Fig. S2D) (37). O e all, bioin o ma ics esul s s ongly sugges ha CTDK-1 is a widely
conse ed ime ic complex in he kingdom Fungi, while in Homo sapiens, he e is no
C k3 o holog (37) (see Discussion).
Dele ion o lpA causes a dec ease in adial g ow h and conidial yield, while
unca ion o he las 45 amino acids o FlpA has negligible e ec s in a
wild- ype backg ound
To unc ionally cha ac e ize he ole o FlpA in g ow h and de elopmen o A. nidulans,
a null lpA mu an was gene a ed, bo h in wild- ype and Δ lbB gene ic backg ounds.
Wild- ype p o oplas s we e also ans o med wi h wild- ype and mu an (Gly347S op)
lpA::ha3x::py GA um cons uc s and also a wild- ype lpA::g p::py GA um cons uc (see
Ma e ials and Me hods). Pheno ypes o selec ed s ains we e analyzed in solid AMM and
AMM supplemen ed wi h 0.5 M NaH2PO4 (Fig. 4A). Fi s , we obse ed ha , a e 72 hou s
o cul u e a 37°C, dele ion o lpA caused a signi ican inhibi ion in adial ex ension, bo h
in wild- ype and null lbB backg ounds, and mainly in AMM supplemen ed wi h 0.5 M
NaH2PO4 (Fig. 4A h ough C). The a e age g ow h a es om 48 o 96 hou s o cul u e
we e 0.027 ± 0.004 and 0.025 ± 0.003 cm/hou o wild- ype and Δ lbB pa en als in
medium supplemen ed wi h sodium dihyd ogen phospha e. The alues o he null lpA
and double-null Δ lpA;Δ lbB s ains we e 0.012 ± 0.003 cm/hou and 0.012 ± 0.001 cm/
hou , espec i ely, in he same cul u e medium (n = 3 o each s ain, P = 0.006 and 0.004
o each null mu an compa ed o he pa en al s ain). Fu he mo e, compa ed o he null
lbB pa en al, he s ain ha in eg a ed he lpA(Gly347S op)::ha3x::py GA um cons uc in his
backg ound showed only a mino dec ease in he g ow h a e (0.023 ± 0.004 cm/h, n = 3,
P = 0.629).
The s ain ha in eg a ed he lpA(Gly347S op)::ha3x::py GA um cons uc (wild- ype
backg ound) showed no inhibi ion o conidia p oduc ion compa ed o he e e ence
s ain (6.17 × 107 ± 1.45 × 107 and 3.62 × 107 ± 1.55 × 107 conidia/cm2, o mu an and
wild- ype s ains, espec i ely; n = 3 o each s ain; mu an no shown in he g aphs;
P = 0.035) in AMM cul u e medium. In he null lpA s ain, howe e , he inhibi ion was
s a is ically signi ican (1.86 × 106 ± 3.23 × 105 conidia/cm2 in he null lpA s ain, n = 3,
P = 0.003) (Fig. 4A and D). The esul s ob ained in he Δ lbB backg ound con i med he
ends desc ibed o he wild- ype backg ound, wi h he di e ence ha bo h dele ion o
unca ion o he las 45 codons comple ely inhibi ed conidia p oduc ion, p obably due
o he addi i e e ec on conidia ion caused by he absence o lbB (26, 30, 40). Howe e ,
he C- e minus o FlpA could play a mino ole in hese p ocesses, as obse ed in he
wild- ype backg ound.
FIG 2 (Con inued)
codon (loss o he las 45 amino acids). The iden i ied mu a ion is ou side he cyclin-like domain (IPR013763, amino acids 59–263). (B) FPKM o TPM alues
o AN10640/ lpA in he mul iple RNA-seq expe imen s a ailable o A. nidulans (see e e ences wi hin e e ence 32). (C) Con i ma ion o he Gly347S op
unca ion wi hin AN10640/ lpA as he mu a ion causing he FLIP57 pheno ype. (Le ) Pheno ypes o FLIP57 and i s pa en al Δ lbB s ain on AMM and AMM
supplemen ed wi h 0.5 M NaH2PO4. (Middle) T ans o man s o Δ lbB p o oplas s wi h he syn he ic DNA cons uc s lpA::ha3x::py GA um, lpA(Gly347S op)::ha3x::py GA um,
lpA::g p::py GA um o lpA(Gly347S op)::g p::py GA um. (Righ ) T ans o man s o FLIP57 p o oplas s wi h he syn he ic DNA cons uc s lpA::ha3x::py GA um, lpA::g p::py GA um
o lpA(Gly347S op)::g p::py GA um.
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FIG 3 E olu iona y analysis o FlpA/AN10640, S k47/AN8190, and FlpB/AN6312. Phylogene ic ees (maximum-likelihood me hod and he JTT ma ix, wi h 50
eplica es in each case) co esponding o he pu a i e o hologs o FlpA (A), S k47 (B) and FlpB (C) included in Table S3. Co e age (ba g aphs in he ees) and
sco e (shape plo s) alues o each hi compa ed o A. nidulans que ies a e included. The colo key indica es which ungal class each o holog belongs o.
(Con inued on nex page)
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Null mu an s o s k47 and lpB show he same pheno ype as he null lpA
s ain
Since FlpA, S k47, and FlpB may be he componen s o he CTDK-1 complex in A.
nidulans, single-null mu an s o s k47 and lpB we e also gene a ed, and hei pheno ypes
we e compa ed o ha o he Δ lpA s ain a e 72 hou s o cul u e a 37°C in solid
AMM o AMM supplemen ed wi h 0.5 M NaH2PO4 (Fig. 4E and F). Di e en combina ions
o double-null mu an s we e also gene a ed by ans o ma ion o p o oplas s o he
single-null mu an s wi h he co esponding dele ion casse es. The h ee single-null
mu an s showed a highly simila pheno ype. We obse ed a sligh inc ease in colony
diame e and p oduc ion o conidia only in he case o he null lpB s ain. While he
e e ence wild- ype s ain p oduced 2.67 × 107 ± 0.82 × 107 conidia/cm2, Δ lpA, Δs k47,
and Δ lpB s ains p oduced, espec i ely, 5.94 × 105 ± 3.80 × 105, 5.63 × 105 ± 2.11 ×
105, and 1.02 × 106 ± 2.26 × 105 conidia/cm2 (P = 0.032 in he compa ison be ween he
wild- ype and he null lpA s ains, P = 0.908 and 0.172 when Δ lpA was compa ed o
Δs k47 and Δ lpB s ains, P = 0.063 when null s k47 and null lpB s ains we e compa ed; n
= 3 o each s ain). O no e is he pheno ypic di e ence be ween null s k47 s ains when
A. umiga us py G o iboB genes we e used as selec ion ma ke s (Fig. 4E and F). Conidia
p oduc ion inc eased om 5.63 × 105 ± 2.11 × 105 conidia/cm2 in he Δs k47::py G s ain
o 1.38 × 107 ± 2.02 × 106 conidia/cm2 in he Δs k47:: iboB s ain (P = 0.008, n = 3 o each
s ain).
Double-null mu an s displayed a highly simila pheno ype compa ed o hei
co esponding pa en al s ains, and no addi i e e ec s in conidia p oduc ion we e
obse ed a e dele ion o he second gene (Fig. 4E and F). The double-null Δ lpA;Δ lpB
(gene a ed a e ans o ma ion o Δ lpA p o oplas s wi h he cons uc o lpB dele ion)
p oduced 1.07 × 106 ± 2.75× 105 conidia/cm2 (P = 0.190 compa ed o i s pa en al s ain, n
= 3 o each s ain). Double-null mu an s Δs k47;Δ lpA and Δs k47;Δ lpB (bo h gene a ed
by ans o ma ion o p o oplas o he Δs k47:: iboB s ain) p oduced 1.36 × 107 ± 2.04 ×
106 and 2.08 × 107 ± 1.34 × 106 conidia/cm2 (P = 0.914 and 0.008 when compa ed o hei
pa en al s ain).
Finally, he abili y o gene a e cleis o hecia by he single-null lpA, s k47, and lpB
mu an s was assessed quali a i ely. Figu e 4G clea ly shows ha he mu an s do no
p oduce cleis o hecia in ou cul u e condi ions (7 days o cul u e a 37°C in solid AMM).
O e all, hese esul s s ongly sugges ha FlpA, S k47, and FlpB a e necessa y o
bo h mo phogenesis and sexual o asexual de elopmen al p ocesses. The pheno ypic
simila i ies among single-null and double-null mu an s also sugges ha he h ee
p o eins unc ion in he same pa hway, in co ela ion wi h hei hypo he ic pa icipa ion
in he o ma ion o he CTDK-1 complex.
Δ lpA, Δs k47, and Δ lpB s ains gene a e abe an conidiopho es wi h a
de icien me ula- o-phialide ansi ion
The null lbB mu an ails o induce conidiopho e de elopmen , while dele ion o bo h
lpA and lbB has an addi i e inhibi o y e ec on conidia p oduc ion. Thus, we hypo he
sized ha FlpA could ha e a ole di e en om ha ca ied ou by FlbB. While he
la e is necessa y o he induc ion o asexual de elopmen , FlpA may play a ole in
he co ec and imely gene a ion o he cell ypes ha o m conidiopho es ( oo -cell,
s alk, esicle, me ulae, phialides, and conidia) (13). To e i y ha , we ca ied ou a
pheno ypic cha ac e iza ion o he Δ lpA s ain in subme ged cul u e and unde ni ogen
FIG 3 (Con inued)
(D) P edic ed h ee-dimensional s uc u es o FlpA, S k47, and FlpB, modeled by Swiss-Model. The models co e he egions om His26 o Lys374 in FlpA, om
Se 746 o His1052 in S k47, and om Glu7 o Asn234 in FlpB, and a e based on PDB s uc u e 7j 7.1, which co esponds o he CTDK-1 complex o S. pombe and
is shown in Fig. S2D (37). The alignmen s be ween FlpA, S k47, o FlpB and he e e ence s uc u e can be seen in Fig. S2A–C. (E) P edic ed h ee-dimensional
s uc u e o a hypo he ic complex o med by FlpA, S k47, and FlpB, modeled by ColabFold. Full-leng h sequences o FlpA and FlpB we e used, oge he wi h an
N- e minally unca ed o m o S k47 co e ing esidues om P o735 o Leu1067.
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an i ungal compounds and o i ulence in a mouse model o in asi e aspe gillosis. The
h ee single-null mu an s showed a clea g ow h and de elopmen al pheno ype (Fig.
8A), wi h he di e ence ha he adius o Δ lpB colonies in medium supplemen ed wi h
0.5 M NaH2PO4 is sligh ly bigge han hose o Δ lpA and Δs k47 colonies (Fig. 8B). In all
cases, he dec ease in colony adii o single-null mu an s compa ed wi h he e e ence
wild- ype s ain was s a is ically signi ican (P < 0.05 in he h ee compa isons). We also
obse ed a delay in conidial ge mina ion in he h ee single-null mu an s (Fig. 8C), which
may pa ially explain he adial g ow h pheno ypes desc ibed in Fig. 8A and B.
To es o impac s on g ow h in hos -niche s ess, we nex examined he abili y o
he mu an s o g ow unde oxida i e, i on limi a ion, hypoxic, and cell wall s esses.
All s ains showed simila g ow h pa e ns in esponse o oxida i e s ess and unde
oxygen- o i on-limi ing condi ions (Fig. S6). Howe e , we ound ha when Δs k47,
Δ lpA, and Δ lpB conidia we e inocula ed on medium supplemen ed wi h Congo ed
(CR) o calco luo whi e (CFW), all mu an s showed educed adial g ow h compa ed
o he e e ence wild- ype s ain (Fig. 8D). To con i m ha he obse ed hype suscep
ibili y pheno ype o he mu an s o hese cell wall s esso s is no a esul o a ge
mina ion de ec , we decided o es he abili y o ma u e hyphae o de elop unde
hese condi ions. Thus, we g ew he same s ains in minimal medium o 36 hou s and
ans e ed mycelial pelle s o he same size o AMM aga pla es supplemen ed wi h
CR o CFW (80 µg/mL). A e 72 hou s o cul u e, CR caused a 48.48 ± 5.01 % g ow h
educ ion in he e e ence s ain, when compa ed o minimal medium (Fig. 8E and F).
G ow h educ ion was o 64.34 ± 4.33 %, 63.79 ± 3.69 %, and 69.42 ± 2.96% o Δs k47,
Δ lpA, and Δ lpB mu an s, espec i ely (P < 0.05 in all cases). The addi ion o CFW caused
a 28.66 ± 4.33% g ow h educ ion in he e e ence s ain, while i was much g ea e in he
single-null mu an s, mainly in Δ lpA (84.14 ± 2.13%) and Δ lpB (97.26 ± 1.25%) (P < 0.05 in
all cases).
To see i his suscep ibili y would also be e iden in esponse o cell wall- a ge ing
an i ungal d ugs, we nex es ed suscep ibili y o he mu an s o he echinocandin
caspo ungin, which inhibi s β-glucan syn hesis. Using a s ip di usion assay, we ound
he wild- ype and Δ lpA caspo ungin minimum e ec i e concen a ion (MEC) was
0.125 µg/mL, and he Δ lpB and Δs k47 displayed a caspo ungin MEC o 0.25 µg/mL
(Fig. S7). In addi ion, all s ains displayed simila suscep ibili ies o memb ane- a ge ing
an i ungals, wi h a o iconazole MIC ( ange o 0.125–0.25 µg/mL and posaconazole
MIC o 0.25 µg/mL. The e o e, loss o s k47, lpB o lpA does no impac an i ungal
suscep ibili y.
The hype suscep ibili y o he h ee mu an s ains o CR and CFW may sugges ha
he o ganiza ion o he cell wall, and subsequen ly PAMP exposu e, could be al e ed
by dele ion o lpB, lpA, o s k47. Al e ed PAMP exposu e could subsequen ly lead o
changes in hos ecogni ion du ing in ec ion. To check i dele ion o any gene al e s hos
ecogni ion, we nex challenged THP-1 monocy es wi h conidia o he ungal s ains and
assayed in lammasome ac i a ion using in e leukin (IL)-1β elease as an endpoin (44).
Howe e , only a sligh ly lowe elease o IL-1β in he single-null mu an s compa ed o
FIG 7 (Con inued)
he g een squa e, he ligh e band inc eases i s in ensi y in Δ lpA and Δ lpB backg ounds. (C) T ea men o c ude p o ein
ex ac s o S k47::HA3x wi h λ phospha ase (λPP) and λPP plus sodium o ho anada e, he la e as an inhibi o o phospha ase
ac i i y. Gels s ained wi h Bio-Sa e Coomassie (Bio-Rad) a e shown as loading con ols in panels A–C. (D) Pull-down assay
using he GFP-T ap esin o Ch omo ek. Resin samples we e sequen ially incuba ed (see Ma e ials and Me hods) wi h 3 mg
o a c ude p o ein ex ac o a s ain exp essing FlpA::GFP and a e washing o he non- e ained ac ion, wi h 3 mg o a
second c ude p o ein ex ac o a s ain exp essing S k47::HA3x. (E) Pull-down assays ca ied ou by sequen ially incuba ing he
GFP- ap esin wi h 3 mg o a c ude p o ein ex ac o a s ain exp essing FlpB::GFP (wild- ype o Δ lpA backg ound), and hen
wi h 3 mg o a second c ude p o ein ex ac o a s ain exp essing FlpA::HA3x o S k47::HA3x (wild- ype o Δ lpA backg ound).
S ain- ee gels (Bio-Rad) we e used o p o ein elec opho esis in panels D and E. The image in ow 1 was ob ained as loading
con ol be o e p o ein ans e ence o poly inylidene di luo ide (PVDF) memb anes. NR, non- e ained; R, e ained ac ion; TE,
o al ex ac .
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FIG 8 Pheno ype o single-null mu an s o lpA, s k47, and lpB in A. umiga us. (A) Pheno ypes o wild- ype and single-null lpA, s k47, and lpB s ains o A.
umiga us a e 72 hou s o cul u e a 37°C in AMM o AMM supplemen ed wi h 0.5 M NaH2PO4. Diame e o pla es is 5.5 cm. (B) Diame e o he colonies in panel
A a e 24, 48, and 72 hou s o cul u e unde he same condi ions (***P < 0.001, compa ed o he e e ence ΔakuB-py G + s ain; n = 3 o each s ain).
(Con inued on nex page)
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he e e ence wild- ype s ain was de ec ed, and hese di e ences we e no s a is ically
signi ican (P > 0.05 in he h ee compa isons; Fig. 8G). The e o e, we concluded ha
he e a e no signi ican di e ences in he abili y o he mu an s o igge he in lamma-
some.
Conside ing he g ow h, cell wall, and de elopmen al pheno ypes, we also analyzed
he i ulence o he A. umiga us single-null mu an s in a chemo he apeu ic model o
in asi e aspe gillosis. We obse ed ha , a e 12 days o in ec ion, 50% o hose animals
challenged wi h he knockou s we e ali e, compa ed o only a 20% o mice in ec ed wi h
he pa en al s ain. Howe e , due o he limi ed sample size, he mo ali y a es be ween
s ains we e no s a is ically signi ican (Fig. 8H). Taken oge he wi h he in i o s ess
assay da a, i is unlikely ha lpB, lpA, o s k47 play majo oles in suppo o A. umiga us
i ulence.
DISCUSSION
Among he di e en ypes o kinases, which a e di e en ia ed based on he sequence
o he kinase domain, cyclin-dependen kinases, o CDKs, a e se ine/ h eonine kinases
whose ac i i y depends on he egula o y ole ca ied ou by a cyclin (45). Cyclins
we e named his way, i s , because hei p o ein le els luc ua ed in a cyclical ashion
du ing he cell cycle and, second, because hey we e de ined by he p esence o a
cyclin box domain (see e e ences wi hin e e ence 46). Cyclins can be di ided in o wo
main g oups, cell-cycle o canonical cyclins and ansc ip ional cyclins (46). The e m
ansc ip ional cyclins e e s o hei ole in he egula ion o RNA polyme ase ac i i y
du ing ansc ip ion ini ia ion and elonga ion. Thus, he ole o cyclins goes beyond he
con ol o e en s di ec ly linked o he cell cycle. Fu he mo e, membe s o bo h g oups
o cyclins play cellula unc ions independen ly o a pa ne CDK. Simila ly, CDKs also
con ol ansc ip ion, me abolism, and cell di e en ia ion (47).
Paolillo and cowo ke s iden i ied, in he genomes o A. nidulans and A. umiga us, 15
genes encoding cyclins ha ep esen he h ee phylogene ic g oups: g oup I o cell
cycle cyclins ( h ee in each genome), g oup II (se en in each genome) , and g oup III
( i e in each genome), which mainly include ansc ip ional cyclins (34). On he o he
hand, De Souza and Osmani cha ac e ized he pheno ypes o he single-null mu an s
o all he A. nidulans genes encoding kinases, o aling 128 (39). The null mu an o
AN8190/s k47 was desc ibed o show a s ong g ow h de ec and sensi i i y o s ess
condi ions igge ed by sodium chlo ide. Ou g oups ecen ly comple ed he sys ema ic
dis up ion o all pu a i e p o ein kinase encoding genes in A. umiga us (44). In e es
ingly, he A. umiga us s k47 dis up ion mu an displayed only wild- ype pheno ypes (ou
unpublished esul s), which is in con as o he A. umiga us Δs k47 esul s epo ed he e.
Howe e , he p e iously published wo k desc ibing hese esul s iden i ied addi ional
kinase dis up ion mu an s ha also displayed di e en ial esul s upon dis up ion o
dele ion (44). These inconsis encies a e p esumed o be caused by con inued ansc ip
ional ead h ough, and subsequen exp ession o unca ed kinase mu an s, in a leas
some o he dis up ion mu an s con ained wi hin he A. umiga us lib a y. In he analysis
o he se o lu y in phospha e mu an s o ou A. nidulans collec ion, we iden i ied a
Gly347S op subs i u ion in AN10640/ lpA as he mu a ion causing he FLIP57 pheno ype.
FIG 8 (Con inued)
(C) Ge mina ion a e o each s ain 8, 10, o 12 hou s a e inocula ion (n = 3 o each s ain a each cul u e ime). (D) Pheno ypes o he s ains a e 48 hou s
o cul u e on AMM supplemen ed wi h 40 o 80 µg/mL o CR o CFW. Conidia we e inocula ed a ou di e en concen a ions (104, 103, 102, o 10 conidia).
Diame e o pla es: 9 cm. (E) Pheno ypes (72 hou s a 37°C) o he same s ains a e he ans e ence o mycelial pelle s g own in liquid minimal medium o 36
hou s o pla es con aining minimal medium (con ol) o medium supplemen ed wi h 80 µg/mL o CR o CFW. Pic u es a e ep esen a i e o biological iplica es.
(F) Ba g aphs ep esen ing he pe cen age o g ow h educ ion o each s ain cul u ed in he p esence o CR o CFW compa ed o minimal medium. Analysis
was ca ied ou by one-way analysis o a iance wi h Dunne ’s mul iple compa ison es . (Top) P ≤ 0.0045; (Bo om) P ≤ 0.0006. (G) Quan i ica ion o elease o
in e leukin-1β by THP-1 monocy es a e co-cul u ing wi h conidia (mul iplici y o in ec ion: 10 conidia pe THP-1 cell). (H) Pe cen su i al o emale CD-1 (n = 10
mice pe g oup) o in ec ion by he wild- ype o he A. umiga us single-null mu an s o lpA, s k47, and lpB. Su i al was eco ded daily, and he Kaplan-Meie
cu es we e compa ed using log- ank es s in G aphPad P ism ( .9.2.0). CFW, calco luo whi e; CR, Congo ed. ** P < 0.01; *** P < 0.001; **** P < 0.0001
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Since FlpA is he o holog o S. pombe cyclin C k2/Lsc1, i s ac i i y was linked o ha o
he CDK S k47 and he hypo he ic o ma ion o an A. nidulans CTDK-1 complex (37, 43).
In humans, CDK12-Cyclin K and CDK13-Cyclin K complexes would be he coun e pa s o
he S. pombe C k1/C k2 complex (37). Ne e heless, he s uc u e o he S. pombe CTDK-1
complex di e s om he human coun e pa s in ha i adds a hi d membe , C k3, which
is no conse ed in Homo sapiens. C k3 enables he o ma ion o he S. pombe CTDK-1
complex by in e ac ing wi h bo h he cyclin and he kinase. This con igu a ion may be
conse ed in he kingdom Fungi based on (i) he wide conse a ion pa e ns o he
h ee componen s, (ii) he ColabFold p edic ion, (3) he highly simila pheno ypes o he
h ee single-null mu an s in A. nidulans and A. umiga us, and (4) he in e ac ion pa e ns
desc ibed in he pull-down assays ca ied ou in his wo k.
The NLS adamus algo i hm p edic ed he p esence o a NLS sequence in AnS k47
(1,119 amino acids) bu no in AnFlpA (392 amino acids) o AnFlpB (240 amino acids).
Howe e , ou esul s showed ha , in A. nidulans, nuclea accumula ion o AnS k47 was
dependen on AnFlpA ac i i y, while nuclea accumula ion o AnFlpA o AnFlpB was
no dependen on AnS k47. This beha io is opposi e o wha was desc ibed in S.
pombe. In ission yeas , Lsk1/C k1 was equi ed o nuclea localiza ion o Lsc1/C k2,
bu no in e sely (43). Rega ding he phospho yla ion pa e n o AnS k47, ou esul s a e
no conclusi e. The use o λ phospha ase did no modi y he immunode ec ion-band
pa e n o AnS k47, and hus, we we e no able o con i m ha he S k47 band pai
al e ed in he Δ lpA and Δ lpB backg ounds co esponds o a (poly)phospho yla ed
o m o he kinase. P e iously, p elimina y phosphopep ide-de ec ion analyses by ou
g oup (unpublished) de ec ed mul iple phospho yla ed pep ides o AnS k47, sugges
ing ha his kinase may be polyphospho yla ed a Se 173, Se 177, Se 221, Se 498,
Se 499, Se 505, Se 720, Th 1105, Se 1106, Se 1111, and Se 1112. In addi ion, he same
analysis iden i ied a phosphopep ide phospho yla ed bo h a Se 2 and Se 5 o a leas
he hep ad loca ed a posi ions 1684–1690 ( o al leng h: 1745 aa) o he la ge subuni
o RNA polyme ase complex II, AnRpb1. Phosphopep ides o AnFlpA o AnFlpB we e
no de ec ed. In he phosphopep ide-de ec ion expe imen s ca ied ou in his wo k,
we we e able o de ec jus a single phosphopep ide co esponding o he hypo he ic
phospho yla ion o Se 720 o AnS k47 (no phosphopep ides o AnRpb1, AnFlpA, o
AnFlpB we e de ec ed). In e es ingly, his phosphopep ide was no de ec ed in he null
lpA s ain, bu , un o una ely, we ha e no been able o ep oduce his esul . Dephou e
and colleagues desc ibed ha he p obabili y o obse ing si e-de e mining ions is
hampe ed by biases in pep ide agmen a ion ha a o b eakage a ce ain poin s and
dis a o i a o he s (48). The same au ho s also s a ed ha he ype o p o ease used in
sample p ocessing ( ypsin was used in his s udy; see Ma e ials and Me hods) may limi
phospho yla ion-si e iden i ica ion because no all phospho yla ed esidues a e loca ed
in egions ha will gene a e pep ides de ec able by mass spec ome y upon clea age
by a single p o ease (48). Fu u e expe imen s will ha e o de e mine i FlpA is he cyclin
o one o he cyclins o S k47, i bo h p o eins a e equi ed o phospho yla ion o Rpb1,
and i phospho yla ion o S k47 is a p e equisi e o i s nuclea impo and/o e en ion.
Dele ion o lpA, s k47, o lpB has pleio opic e ec s on colony o ma ion bo h in A.
nidulans and A. umiga us. Compa ed o he e e ence wild- ype s ain, (i) ge mina ion
is delayed; (ii) he adius o he colony is dec eased; (iii) abe an conidiopho es wi h a
misscheduled aisi ion om me ulae o phialides a e gene a ed; and (i ) cleis o hecia
a e no p oduced. I hese h ee genes encode he componen s o he CTDK-1 complex,
hen hese pheno ypic ai s a e p obably a consequence o an al e ed phospho yla ion
pa e n o Rpb1. I has been desc ibed ha phospho yla ion o se ine 2 o he C- e minal
hep ad epea s o Rpb1 is no an essen ial ea u e o gene al ansc ip ion in ission yeas
bu a he is c i ical o ce ain biological esponses, such as sexual de elopmen (49).
Fo example, Se 2 phospho yla ion is c i ical du ing sexual di e en ia ion o S. pombe o
he induc ion o s e11 ansc ip ion (49, 50), which encodes an HMG domain ma ing- ype
ansc ip ion ac o . In co ela ion wi h hese obse a ions, he single-null mu an s o
lpA, s k47 and lpB do no de elop cleis o hecia, in A. nidulans, in he condi ions es ed.
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In he case o asexual de elopmen , he abe an conidiopho e s uc u es desc ibed
in his wo k a e p obably ela ed o ge mina ion and g ow h de ec s, a he han (i)
misscheduled o incomple e sep a ion p ocesses, as was desc ibed in S. pombe (43), o
(ii) a di ec ole o hese h ee p o eins in he con ol o he exp ession pa e ns o UDA
o CDP genes. The in ol emen o genes encoding egula o s o ge mina ion and hyphal
pola i y in he o ma ion o conidiopho es has been p e iously desc ibed [see, e.g., he
cases o cdc42 and ac1 o hologs o A. nidulans (51)].
The ole o ansc ip ional cyclins and kinases in A. nidulans conidia ion was
p e iously desc ibed by he g oup o Fische e al. (52, 53). P kA, a Cdk9 kinase, in e ac s
wi h cyclins PchA, PclA and PclB, and also wi h he kinase PipA, modula ing i s in e ac ion
pa ne s and ac i i y o con ol mo phogenesis and de elopmen . Cdk9 is supposed
o phospho yla e Se 2 esidues in he CTD o Rpb1 and enable ansc ip elonga ion
(54). Thus, he phospho yla ion s a us o he CTD domain o he la ge subuni o RNA
polyme ase II is egula ed by mul iple cyclin-kinase complexes, which would modi y
hei con igu a ion, depending on he mo phogene ic/de elopmen al s age and he cell
ype. I con i med in he u u e o FlpA and S k47, such a mechanis ic lexibili y would
enable, on he one hand, coo dina ion o mul iple pa hways and accu a e in eg a ion o
he co esponding signals and, on he o he hand, modi ica ion o he a ini y o RNA
polyme ase II complex o a ge p omo e s.
MATERIALS AND METHODS
DNA sequencing
Genomic DNA o mu an s FLIP57 and FLIP76 (Table S3) was ex ac ed ollowing s anda d
p ocedu es and using a phenol:chlo o o m:isoamyl alcohol mix u e (25:24:1 ol/ ol,
PanReac Applichem) (55). An1459/pm C was ampli ied om hese genomic samples
using oligonucleo ides Pm C-seq1 and Pm C-GSP2 and sequenced using oligonucleo i
des Pm C-seq1, Pm C-seq4, Pm C-Up, and Pm C-GSP2 (Table S4) o disca d ha he
pheno ypes o FLIP57 o FLIP76 we e caused by mu a ions in pm C, as occu ed wi h
FLIP166 (27). A e in eg i y, pu i y and quan i y checks (1.5% aga ose elec opho esis
and Qubi luo ime e analyses), DNA samples we e sequenced a S ab ida (Capa ica,
Po ugal) using an Illumina No aseq pla o m and 150-bp pai ed-end sequencing eads.
The samples gene a ed 16,607,684 (2,507 Mbp) and 16,279,736 sequence eads (2,458
Mbp), espec i ely, esul ing in a heo e ical a e age dep h co e age o 83× and 82×,
assuming a genome size o 30 Mpb.
Bioin o ma ics
Reads we e mapped using BWA-MEM (56) a he Galaxy pla o m (h ps://usegalaxy.o g/),
and he SAM iles gene a ed we e con e ed o BAM coun e pa s (SAM- o-BAM). SNPs
compa ed o he e e ence A. nidulans genome (A_nidulans_FGSC_A4_ e sion_s10-m02-
03_ch omosomes. as a) we e iden i ied using Snippy. Resul s o s ains FLIP57 and
FLIP76 we e compa ed also wi h hose ob ained o he mu an FLIP166 (27). Exonic
mu a ions p esen in FLIP57 o FLIP76 bu absen in he genomes o FLIP166 and
he e e ence genome we e selec ed as candida es. The In eg a i e Genomics Viewe
so wa e (57) was used o isualize and analyze DNA-seq and RNA-seq eads and o
compa e hem wi h e e ence genomes.
Gene and p o ein sequence analyses we e ca ied ou in he FungiDB da abase
(58). The p esence o pu a i e unc ional domains in p o ein sequences was p edic ed
wi h In e P o (59), while he Na ional Cen e o Bio echnology In o ma ion websi e
(h ps://blas .ncbi.nlm.nih.go /Blas .cgi) was used o BLAST que y sequences. Clus al
Omega was used o align p o ein sequences (60). The .ms ile gene a ed was used o
isualize he alignmen s wi h Genedoc, and he clus al ile gene a ed was impo ed in o
MEGAX (61) in o de o gene a e phylogene ic ees, which we e edi ed using iTOL (62).
S uc u e homology models o FlpA, S k47, and FlpB we e buil wi h Swiss Model (63)
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and ColabFold (64). The p esence o nuclea localiza ion signals was p edic ed using
NLS adamus and SeqNLS algo i hms (65, 66). Finally, GO analyses we e ca ied ou a he
ShinyGO websi e ( .0.61) (67).
S ains, oligonucleo ides, and cul u e condi ions
Aspe gillus nidulans and A. umiga us s ains used in his s udy a e lis ed in Table S3,
while oligonucleo ides can be seen in Table S4. Pa en al s ains FLIP57 and FLIP76 we e
p e iously gene a ed in a mu agenesis sc eening o a null lbB (BD177) s ain (27). A.
nidulans s ains we e cul i a ed in adequa ely supplemen ed liquid o solid AMM using
glucose (2%) and ammonium a a e (5 mM) as he sou ces o ca bon and ni ogen,
espec i ely (68, 69). A medium con aining 25 g/L co n s eep liquo (Sigma-Ald ich) and
suc ose (0.09 M) as he ca bon sou ce was used as he e men a ion medium (Aspe gillus
e men a ion medium [AFM]) o cul u e samples o p o ein ex ac ion (70). A. umiga us
s ains we e ou inely cul u ed on AMM aga pla es and NaH2PO4 (0.5–0.65 M) was
added o AMM o induce sal -s ess condi ions.
To assess sensi i i y o A. umiga us s ains o CR o CFW, 10 µL o 10- old se ial
dilu ions o conidia om each s ain (104, 103, 102, and 10 conidia) we e spo ed on Pe i
pla es illed wi h AMM supplemen ed wi h 40 o 80 µg/mL o each compound (71).
Pla es we e incuba ed o 48 and 72 hou s a 37°C. Colony g ow h o he mu an s was
compa ed o ha o he e e ence s ain o e he inoculum dilu ion ange. To e alua e
he suscep ibili y o CR and CFW using ma u e hyphae, 100 mL o AMM b o h was
inocula ed wi h 5 × 107 conidia and incuba ed o 36 hou s a 37°C and 250 pm. Then,
mycelial pelle s o each s ain we e isola ed and placed on he cen e o Pe i pla es
con aining minimal medium supplemen ed wi h 80 µg/mL o CR o CFW. Con ol pla es
con ained no d ug (minimal medium). The pla es we e incuba ed a 37°C and diame e s
we e measu ed a e 72 hou s.
The abili y o he A. umiga us s ains o espond o hos -s ess condi ions was
e alua ed wi h he ollowing assays: o de e mine he abili y o g ow in condi ions o
i on s a a ion, 103 conidia we e poin inocula ed in he cen e o AMM pla es o AMM
lacking i on, and colony mo phologies we e obse ed a e 48 hou s a 37°C. To e alua e
he g ow h in low oxygen en i onmen s, AMM pla es con aining 103 conidia o each
s ain we e incuba ed in a hypoxia chambe a 37°C o 48 hou s. The suscep ibili y o
oxida i e s ess was e alua ed as p e iously desc ibed (72), and g ow h inhibi ion halos
we e measu ed a e 48 hou s o incuba ion. Suscep ibili y o he s ains o posaconazole,
o iconazole, and caspo ungin was de e mined using g adien di usion s ips (Lio il-
chem) in RPMI aga pla es. Resul s we e ead a e 24 o 48 hou s o g ow h a 37°C. All
he expe imen s we e pe o med in biological iplica es.
The p ocedu es de eloped by Tilbu n and colleagues, o Szewczyk and colleagues,
we e used o ob ain and ans o m p o oplas s o A. nidulans, and selec ans o man s
on selec i e (lacking u idine and u acil, on he one hand, o py idoxine, on he o he
hand) egene a ion medium ( egene a ion minimal medium [RMM]: AMM supplemen
ed wi h 1 M suc ose) (73, 74). P o oplas s o A. umiga us s ain ΔakuB-py G+ we e
ob ained and ans o med using he p ocedu e desc ibed by Yel on and colleagues
(75). A lpA (AFUB_007390, A u1g07020), A s k47 (AFUB_051670; A u5g03160), and A lpB
(AFUB_027820, A u2g12130) we e dele ed using a CRISPR-Cas9 gene edi ing p ocedu e
p e iously desc ibed by us (see below how DNA casse es we e gene a ed) (76).
Selec ion o ans o man s was ca ied ou based on hyg omycin esis ance and co ec
in eg a ion o he DNA cons uc s was con i med by diagnos ic-PCR using he oligo
nucleo ide pai s shown in Table S4. The con ol ΔakuB-py G + s ain was cons uc ed
p e iously by eplacing he mu an py G locus o he A. umiga us KU80Δpy G s ain wi h
he unc ional A. pa asi icus py G homolog (71).
Fo luo escence mic oscopy analyses, conidiospo es o he s ains o in e es we e
incuba ed o app oxima ely 18 hou s a 25°C in Ibidi µ-Dishes o Falcon mul i-well pla es
con aining, espec i ely, 2.0 o 1.5 mL o supplemen ed wa ch minimal medium (WMM)
(77). Nuclei we e isualized by adding o he cul u es wo d ops o NucBlueTM li e cell
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s ain eadyP obesTM eagen (DAPI, In i ogen) plus i on X100 (PanReac AppliChem,
0.05%) and by incuba ing 15–20 minu es a oom empe a u e (RT) be o e luo escence
mic oscopy analysis.
To quan i y conidia p oduc ion in A. nidulans, asexual spo es we e poin inocula ed
and cul u ed o 72 hou s a 37°C. Conidia we e collec ed in Tween 20 (0.02%), dilu ed (i
necessa y), and he o al amoun o conidia was de e mined using a Thoma cell coun e .
The numbe o conidia was di ided by he a ea o he colony. Mic oso Excel was used o
de e mine s a is ically signi ican di e ences in conidia p oduc ion among s ains and o
d aw he co esponding column ba g aphs. Radii o A. nidulans colonies and hei adial
g ow h a es we e de e mined by measu ing and by compa ing he diame e o he
colonies a e 48, 72, and 96 hou s o cul u e in adequa ely supplemen ed AMM media.
Conidia o A. umiga us we e ha es ed om 5-day-old cul u es g own on AMM.
Colony mo phologies o A. umiga us mu an s we e assessed by spo ing 5 µL con aining
1,000 conidia on o he cen e o AMM pla es (5.5 cm) and incuba ion o 72 hou s a 37°C.
Diame e o colonies we e measu ed a e 24, 48, and 72 hou s o cul u e a 37°C.
The pheno ypes o Δ lpA, Δs k47, and Δ lpB mu an s o A. nidulans unde ni ogen
s a a ion condi ions we e assessed as ollows (25, 26, 41, 78). Fi s , 106 conidia pe mL
o he mu an s and hei pa en al wild- ype s ain we e inocula ed in E lenmeye lasks
illed wi h 25 mL o adequa ely supplemen ed liquid AMM. A e 18 hou s o cul u e
a 37°C and 200 pm, mycelia we e il e ed using Mi aclo h pape and inocula ed in
liquid AMM lacking a ni ogen sou ce. A e addi ional 20 hou s o cul u e, pheno ypes
we e analyzed using an Imaging Sou ce DFK23UP031 digi al came a coupled o a Nikon
Eclipse E100 mic oscope.
Hypo he ic ge mina ion de ec s o single-null mu an s we e assessed by inocula ing
5 × 104 (A. nidulans) conidia pe mL in E lenmeye lasks illed wi h adequa ely supple
men ed liquid AMM, and by cul u ing hem a 37°C and 200 pm. Fo he e alua ion o
ge mina ion a es in A. umiga us, 10,000 conidia om each s ain we e inocula ed in o
AMM b o h, pou ed o e s e ile co e slips, and incuba ed a 37°C in s a ic condi ions.
A e 6 o 8 (A. nidulans), o 8, 10, o 12 (A. umiga us) hou s o cul u e, he ge mina ed
conidia we e coun ed om a minimum numbe o 100 conidia o each s ain and ime
poin . A leas wo eplica es we e analyzed pe s ain. The co esponding g aphs we e
d awn wi h Mic oso Excel o G aphPad P ism ( .9.2.0).
Dis ibu ion o sep a in A. nidulans hyphae was analyzed using luo escence
b igh ene 28 (CFW). B ie ly, conidia o he s ains o in e es we e inocula ed in
Falcon mul i-well pla es con aining adequa ely supplemen ed WMM and incuba ed o
app oxima ely 18 hou s a oom empe a u e. Then, he cul u e medium was eplaced
by esh WMM con aining 0.01% CFW. Samples we e incuba ed a oom empe a u e o
5 minu es. Finally, he cul u e medium was eplaced wice (plus addi ional 5 minu es o
incuba ion) be o e obse a ion using a Nikon Eclipse Ci manual up igh mic oscope (see
below; phase con as and GFP images we e p ocessed). Sep um- o-sep um dis ances
(a minimum o 90 o each s ain in h ee biological eplica es) we e measu ed using
ImageJ (h ps://imagej.nih.go /ij/), and he co esponding sca e plo was gene a ed
using G aphPad P ism ( .5.01).
Gene a ion o DNA casse es o ans o ma ion
The usion-PCR echnique was used o gene a e DNA cons uc s o ans o ma ion o A.
nidulans p o oplas s (79). Fo 3′-end gene agging (wild- ype o mu an e sions), h ee
DNA agmen s we e ampli ied and used: i s , 1.5 kb o he 3′-end o he coding egion
was ampli ied using oligonucleo ides GSP1 and GSP2 (Table S4); second, g p, mChe y, o
ha3x ags plus he selec ion ma ke (py GA um o py oAA um o A. umiga us) we e ampli ied
using oligonucleo ides GFP1 and GFP2 (2.6 o 1.9 kb, espec i ely, when g p::py GA um,
g p::py oAA um, o mChe y::py oAA um, and ha3x::py GA um we e ampli ied); hi d, 1.5 kb o he
3′-UTR egion o he gene (oligonucleo ides GSP3 and GSP4). The usion-PCR eac ion
was ca ied ou wi h an aliquo o each agmen and oligonucleo ides GSP1 and GSP4.
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To gene a e he cons uc s o A. nidulans gene dele ion, he ollowing h ee
agmen s we e ampli ied and used. Fi s , 1.5 kb o he p omo e egion o he gene was
ampli ied using oligonucleo ides PP1 and PP2. Second, he selec ion ma ke s py GA um
o py oAA um we e ampli ied wi h oligonucleo ides SMP1 and GFP2. Thi d, 1.5 kb o he
3′-UTR egion o he gene was ampli ied wi h oligonucleo ides GSP3 and GSP4. The
usion-PCR eac ion was ca ied ou wi h oligonucleo ides PP1 and GSP4. To cons i u
i ely exp ess FlpA::GFP o FlpA::HA3x chime as unde he con ol o he “mini” e sion
o he p omo e gpdAp (42), i e agmen s we e ampli ied and used (27, 80): i s ,
1.5 kb o he p omo e using oligonucleo ides PP1 and PP2′-ATG; second, he gpdAp
p omo e wi h oligonucleo ides gpdAUp and gpdADw; hi d, he coding egion o lpA
was ampli ied wi h oligonucleo ides geneSP and GSP2; ou h, he ag plus he selec ion
ma ke (GFP1 and GFP2); and inally, 1.5 kb o he 3′-UTR egion wi h oligonucleo ides
GSP3 and GSP4. The usion o all hose i e agmen s was done using oligonucleo i
des PP1 and GSP4. A e ans o ma ion o A. nidulans p o oplas s and genomic DNA
ex ac ion, co ec ecombina ion o he cons uc s was con i med by diagnos ic PCR
using oligonucleo ides sPP1 and sGSP4.
Fo A. umiga us gene ic enginee ing, hyg omycin esis ance epai empla es,
con aining 40-bp mic o-homology egions a bo h 5′- and 3′-ends o A s k47, A lpA o
A lpB, we e ampli ied om plasmid pJMR2 (81) and u ilized in a CRISPR/Cas9-media ed
ans o ma ion o gene a e comple e gene dele ions (82).
Analysis o cy okine elease in THP-1 cells
Measu emen s o IL1-β eleased by THP-1 cells was ca ied ou ollowing p ocedu es
desc ibed p e iously (44, 83, 84). THP-1 cells we e cul u ed in RPMI-1640 medium
con aining HEPES (25 mM) supplemen ed wi h hea -inac i a ed e al bo ine se um
(10%), penicillin and s ep omycin (100 U/mL), and 100 µg/mL no mocin, and assayed
o iabili y by exclusiona y ypan blue s aining. Pla ing was ca ied ou a a densi y
o 5 × 104 cells pe well (96-well pla es) in he same medium bu lacking no mocin.
Pho bol 12-my is a e 13-ace a e a a inal concen a ion o 100 nM was added, and cells
we e incuba ed o 24 hou s o di e en ia e o a mac ophage pheno ype. Supe na an s
we e disca ded and eplaced wi h 180 µL o esh RPMI (wi hou phenol ed) and 20 µL
o dis illed wa e con aining 5 × 105 A. umiga us conidia (mul iplici y o in ec ion, 10:1)
o he single nulls o he e e ence s ain. Cells we e co-cul u ed o 16 hou s a 37°C
and 95% humidi y. A e incuba ion ime, we collec ed he supe na an s and measu ed
he IL-1β concen a ions using IL-1β enzyme-linked immunoso ben assay (ELISA) ki s
(In i ogen) and ollowing manu ac u e ins uc ions.
Vi ulence assays in A. umiga us
All animal expe imen s we e ca ied ou acco ding o he p o ocols app o ed by he
Uni e si y o Tennessee Ins i u ional Animal Ca e and Use Commi ee. G oups o 10,
6-week-old emale CD-1 mice (Cha les Ri e s Labo a o ies) we e immunosupp essed
wi h 150 mg/kg o cyclophosphamide (neu openic model) on day −3 and subsequen ly
e e y hi d day s a ing again on day +1 (85) and an addi ional subcu aneous injec ion
o iamcinolone ace onide on day −1. On he day o he in ec ion, mice we e sligh ly
anes he ized wi h 3.5% iso lu ane and inocula ed in anasally wi h 106 eshly ha es ed
conidia con ained in 35 µL o py ogen- ee saline solu ion. Mice we e moni o ed a leas
wice a day and we e gi en ood and wa e ad libi um, and hose animals showing
se e e signs o in ec ion we e humanely eu hanized by anoxia wi h CO2. Mo ali y was
moni o ed o 12 days. G aphPad P ism .9.2.0 was used o plo su i al da a on a
Kaplan-Meie cu e and o ca y ou log- ank (Man el-Cox) es s.
Fluo escence mic oscopy
Subcellula localiza ion o FlpA, S k47, and FlpB was analyzed using a Zeiss Axio
Obse e Z1 in e ed mic oscope as p e iously desc ibed (27, 86) o a Nikon Eclipse
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Ci manual up igh mic oscope. The o me was equipped wi h a ×63 Plan Apoch oma
1.4 oil imme sion lens, and il e s 38 (exci a ion a 470 nm and emission a 525 nm)
and 43 (exci a ion a 545 nm and emission a 605 nm). The la e included 40× and
100× (oil) Nikon CFI Plan Fluo lenses, and Sem ock il e s DAPI-5060C (exci a ion a
377 nm and emission a 447 nm), GFP-4050B (exci a ion a 466 nm and emission a
525 nm), and mChe y-C (exci a ion a 562 nm and emission a 641 nm). ImageJ (h ps://
imagej.nih.go /ij/) (U.S. Na ional Ins i u es o Heal h, Be hesda, MA, USA) was used o
p ocess luo escence mic oscopy and phase con as images.
P o ein ex ac ion and immunode ec ion
Fo p o ein ex ac ion, conidia o he s ains o in e es we e collec ed in Tween 20 (4
mL, 0.02%) and washed wice by cen i uga ion a 4,000 pm o 10 minu es. By de aul ,
inocula (106 conidia/mL) we e cul u ed in AFM (o AMM o phosphopep ide en ichmen
analyses) o 15 hou s a 37°C and 200 pm (87). Mycelia we e il e ed, ozen, lyophilized,
homogenized, and esuspended in 1 mL pe sample o A-40 bu e (25 mM HEPES, pH
= 7.5, 50 mM KCl, 5 mM MgCl2, 0.1 M EDTA, 10% glyce ol, and 0.5 mM di hio h ei ol
(DTT), supplemen ed wi h a p o ease inhibi o cock ail om Roche) o T ap bu e (10
mM HEPES, pH = 7.5, 1 mM EDTA, 0.1% NP-40, 150 mM NaCl, 0.5 mM DTT, plus a p o ease
inhibi o cock ail om Roche; used in pull-down expe imen s wi h he GFP-T ap esin o
P o ein ech). A e incuba ion a 4°C du ing 90 minu es wi h gen le end-o e -end mixing,
samples we e cen i uged a 4°C and 13,000 pm o 30 minu es. Supe na an s we e
ans e ed o new 1.5 mL ubes, and p o ein concen a ions we e de e mined by he
B ad o d assay. Two hund ed mic og ams o p o ein was p ecipi a ed pe sample wi h
ichlo oace ic acid (TCA) and pu i ied wi h e hanol/e he 1:1 and 1:3 mixes, espec i ely.
Finally, p ecipi a es we e esuspended in 80 µL o SDS-PAGE loading bu e (62.5 mM
T is-HCl, pH = 6.8, 2% SDS, 5% β-me cap oe hanol, 6 M u ea, and 0.05% b omophenol
blue), and he in eg i y o he samples was assessed by polyac ylamide gel elec opho e
sis.
Al e na i ely, he p ocedu e desc ibed by He ás-Aguila and Peñal a was ollowed
(88). Mycelial samples (app oxima ely 6 mg o lyophilized powde ) we e lysed using 1 mL
o alkaline lysis bu e (0.2 M NaOH and 0.2% β-me cap oe hanol), and p o eins we e
p ecipi a ed by adding 7.5% TCA. Samples we e cen i uged and he supe na an s we e
disca ded, esuspending he pelle s in 100 µL T is-base (1 M) and 200 µL o SDS-PAGE
loading bu e . Aliquo s o 10 µL we e hen loaded and sepa a ed in 10% polyac ylamide
gels.
Samples o a hund ed mic og ams o p o ein om he c ude ex ac s o he s ains
o in e es we e incuba ed wi h λPP ( o al olume o 100 µL pe eac ion; New England
Biolabs) o 20 minu es a 30°C (89). Sodium o ho anada e (10 mM) was used as a
λPP inhibi o when necessa y. A e incuba ion, p o eins we e p ecipi a ed as desc ibed
abo e and esuspended in 50 µL o SDS-PAGE loading bu e . The in eg i y o samples
was checked by polyac ylamide gel elec opho esis.
Fo immunode ec ion, p o ein samples we e sepa a ed in Any kD Mini-PROTEAN TGX
p ecas p o ein gels (Bio-Rad; s ain- ee gels we e also used in speci ic expe imen s)
be o e elec o- ans e ence (T ans-blo Tu bo ans e sys em, Bio-Rad) o ni ocellulose
il e s. GFP- o HA3x- agged p o eins we e de ec ed using α-GFP (1:1,000; Roche) o
α-HA3x (1:1,000; San a C uz Bio echnology) mouse monoclonal an ibody cock ails.
Pe oxidase-conjuga ed α-mouse (1:2,500; Jackson Immuno-Resea ch Labo a o ies) was
used as seconda y an ibody. Pe oxidase ac i i y was induced using he Cla i y Wes e n
ECL subs a e (Bio-Rad). Chemiluminescence was de ec ed using a Chemidoc + XRS
sys em (Bio-Rad).
Phosphopep ide en ichmen
Aliquo s o app oxima ely 300 µg o he c ude p o ein ex ac s we e p ecipi a ed wi h
ace one and esuspended in a mix u e o u ea (8 M) and ammonium bica bona e
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(50 mM). P o eins we e educed wi h di hio ei ol (10 mM) o 30 minu es a RT, alkyla ed
wi h iodoace amide (50 mM) in he da k o 30 minu es (RT), and diges ed wi h 15 µg
o ypsin o e nigh a 37°C. C18 SEP-PAK columns we e used o desal ing o pep ide
mix u es. Samples we e d ied a RT using a speed ac concen a o .
Fo TiO2 phosphopep ide en ichmen , slu y o Ti ansphe e TiO (5 µm) beads (ca . no.:
5020–75,000) was p epa ed in bu e C (300 mg/mL lac ic acid; 53% ace oni ile, ACN;
0.07% i luo oace ic acid, TFA) a 25 mg/mL.
Pep ides pelle s we e esuspended in 600 µL o bu e C, and 72 µL o he i anium
slu y was added o each sample. A e incuba ion o 15–30 minu es a RT wi h
end-o e -end o a ion, samples we e cen i uged o spinning down he beads. Pelle s
we e esuspended in 150 µL o bu e C and ans e ed o op o he C8 disks s age
ips (90). Bu e C was emo ed a mode a e speed (10–30 µL/min) using a sy inge, and
ano he 150 µL o bu e C was added on op o he C8/TiO2 s age ip and passed i
h ough a he same speed o washing. Addi ional washing was ca ied ou wi h 100 µL
o bu e B (80% ACN, 0.1% TFA) a he same condi ions. Fo sample elu ion, 2 × 50 µL o
bu e D (0.5% NH4OH) was applied, collec ing each elua e in 100 µL o TFA (2%). Samples
we e d ied in a speed ac concen a o and econs i u ed in TFA (0.1%) o desal ing using
C 18 Zip-Tip columns.
Fo mass spec ome y analysis, ac ions o 1/10 om each phosphopep ide-
en iched sample we e analyzed by nano-LC-MS/MS. Pep ides we e apped on o a
AcclaimPepMap 100 C18 (2 cm) p ecolumn (The moScien i ic), elu ed on o a Acclaim
PepMap 100 C18 column (inne diame e 75 µm, 50 cm long, 2-µm pa icle size;
The moScien i ic) and sepa a ed using a 180-minu e g adien (0%–21% bu e B o 60
minu es, 21%–35% bu e B o 100 minu es, 95% bu e B o 10 minu es, and 0% bu e
B o 10 minu es; bu e A: 0.1% o mic acid, 2% ACN; bu e B: 0.1% o mic acid in ACN) a
a low- a e o 250 nL/min on a nanoEasy HPLC (P oxeon) coupled o a nanoelec osp ay
ion sou ce (The moScien i ic).
A Q-Exac i e mass spec ome e (The moScien i ic) in he posi i e ion mode was
used o mass spec a acquisi ion. Full-scan MS spec a (m/z 400–1,500) we e acqui ed
in he O bi ap a a esolu ion o 70,000 a m/z 200, and he 10 mos in ense ions we e
selec ed o anden mass spec ome y (MS/MS). F agmen a ion was ca ied ou wi h a
no malized collision ene gy o 27 eV. MS/MS scans we e acqui ed wi h a s a ing mass o
m/z 200; au oma ic gain con ol (AGC) a ge was 2e5, esolu ion o 17,500 (a m/z 200),
in ensi y h eshold o 8e3, isola ion window o 2.0 m/z uni s, and maximum IT was 100
ms. Cha ge s a e sc eening was enabled o ejec unassigned, singly cha ged, and equal
o mo e han se en p o ona ed ions. A dynamic exclusion ime o 20 seconds was used
o disc imina e agains p e iously selec ed ions.
Mass spec a *. aw iles we e sea ched agains he A. nidulans as a da abase (10,720
p o ein en ies) using Seques sea ch engine h ough P o eome Disco e e ( .1.4.0.288,
The moScien i ic). Sea ch pa ame e s included a maximum o wo missed clea ages
allowed, ca bamidome hyla ion o Cys esidues as a ixed modi ica ion and N- e minal
ace yla ion, C- e minal oxida ion, and Se , Th , and Ty phospho yla ion as a iable
modi ica ions. P ecu so and agmen mass ole ance was se o 10 ppm and 0.02 Da,
espec i ely. Node 3 phosphoRS was used as sco ing algo i hm. This node e alua es
s a is ical con idence o loca ion o phospho yla ion si es. Iden i ied pep ides we e
alida ed using Pe cola o algo i hm wi h a q- alue h eshold o ≤0.01 (91).
Pull-down assays
Pie ce An i-HA aga ose beads (50 µL pe sample, The moScien i ic) we e cen i uged
5–10 seconds a 12,000 g and washed wi h a 1:1 ol o T is bu e ed saline (TBS,
The moScien i ic) by cen i uga ion a he same condi ions. A e disca ding he liquid,
2 mg o c ude p o ein lysa e was added. Samples we e incuba ed o 90 minu es a
4°C wi h gen le end-o e -end mixing. A e pelle ing he esin wi h a 5- o 10-second
pulse a 12,000 g, supe na an s we e collec ed, and 200 mg o p o ein was p ecipi a ed
wi h TCA. This ac ion was sa ed o analysis o binding e iciency (non- e ained, NR,
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