Stage-specific RNA regulomes of Trichophyton mentagrophytes: mRNA-lncRNA-miRNA interplay in spore-hypha transition

1
S age-speci ic RNA egulomes o T ichophy on men ag ophy es:
mRNA-lncRNA-miRNA in e play in spo e-hypha ansi ion
Wudian Xiao1* , Zhaodan Wu2*, Jia Zhang1*, Jun Wan1, Ruihuan Zhang1, Xinyi Xiang1, Yang Yu1, Lu Fu1,
Kui Yang3, Yang Chen4, Ziyao Xiao1, Ziyu Wang1, L qin He1, Jingcan You1, Chunxiang Zhang1
1 BasicMedicineResea chInno a ionCen e  o ca diome abolicdiseases,Minis yo Educa ion,Depa men o Ca diology,TheA ilia edHospi alo Sou hwes 
Medical Uni e si y, Key Labo a o y o Medical Elec ophysiology, Minis y o Educa ion, Ins i u e o Ca dio ascula Resea ch, Nucleic Acid Medicine o Luzhou
KeyLabo a o y,ModelAnimalandHumanDiseaseResea cho LuzhouKeyLabo a o y,Labo a o yAnimalCen e ,Schoolo BasicMedicalSciences,Schoolo 
ClinicalMedicine,Schoolo PublicHeal h,Sou hwes MedicalUni e si y,Luzhou,646000,China
2 Depa men o O hodon ics,S a eKeyLabo a o yo O alDiseases,Na ionalClinicalResea chCen e  o O alDiseases,Wes ChinaHospi alo S oma ology,
SichuanUni e si y,Chengdu,China
3 Collegeo In elligen Manu ac u ing,ChangchunSci-TechUni e si y,Changchun,130600,China
4 Depa men o Respi a o yandC i icalCa eMedicine,LuzhouPeople'sHospi al,Luzhou,China
Co espondingau ho s:WudianXiao([email p o ec ed]);L qinHe([email p o ec ed]);
JingcanYou([email p o ec ed]);ChunxiangZhang([email p o ec ed])
Copy igh : © Wudian Xiao e al.
This is an open access a icle dis ibu ed unde
e ms o he C ea i e Commons A ibu ion
License (A ibu ion 4.0 In e na ional – CC BY 4.0).
Resea ch A icle
Abs ac
Backg ound: As a globally dis ibu ed de ma ophy e, T ichophy on men ag ophy es
(T. men ag ophy es) causes di e se de ma ophy oses in humans and animals. Long
non-coding RNAs (lncRNAs) and mic oRNAs (miRNAs), which se e as c i ical egula-
o s o di e se biological p ocesses, ha e been ex ensi ely cha ac e ized in nume ous
ungal species. Howe e , he ole o mRNAs, lncRNAs, and miRNAs du ing T. men ag o-
phy es ge mina ion emains unexplo ed.
Objec i es: In his s udy, he molecula mechanisms in ol ed in he ge mina ion o
T. men ag ophy es we e sys ema ically in es iga ed.
Me hods: RNA-sequencing echnology, small RNA-sequencing echnology, ela ed bio-
in o ma ics me hods, and qRT-PCR we e used o sys ema ically cha ac e ize he ex-
p ession p o iles o mRNAs, miRNAs, and lncRNAs in T. men ag ophy es spo es and hy-
phae, and analyze he egula o y mechanisms o mRNAs, miRNAs, and lncRNAs du ing
T. men ag ophy es ge mina ion.
Resul s: In ou s udy, RNA-sequencing was pe o med o iden i y mRNAs, lncRNAs,
and miRNAs in spo es and hyphae o T. men ag ophy es. A o al o 3,193 di e en-
ially exp essed mRNAs, 409 di e en ially exp essed lncRNAs, and 119 di e en ially
exp essed miRNAs we e iden i ied, wi h qRT-PCR subsequen ly used o e i y he de-
pendabili y o he sequencing da a. In addi ion, an mRNA-lncRNA-miRNA egula o y
ne wo k con aining 2,672 mRNAs, 107 miRNAs, and 329 lncRNAs was cons uc ed.
Gene On ology, Kyo o Encyclopedia o Genes and Genomes, and Gene Se En ichmen
Analysis sugges ed ha mRNAs, lncRNAs, and miRNAs may play impo an oles
du ing spo e ge mina ion, po en ially pa icipa ing in undamen al biosyn he ic, cell
wall emodelling, cell cycle egula ion, cy oskele al eo ganiza ion, epigene ic egula-
ion, and me abolic p ocesses.
Academic edi o : Adéla Wenn ich
Recei ed:
27 July 2025
Accep ed:
6 Oc obe 2025
Published:
5 No embe 2025
Ci a ion: Xiao W, Wu Z, Zhang J,
Wan J, Zhang R, Xiang X, Yu Y, Fu
L, Yang K, Chen Y, Xiao Z, Wang
Z, He L, You J, Zhang C (2025)
S age-speci ic RNA egulomes
o T ichophy on men ag ophy es:
mRNA-lncRNA-miRNA in e play in
spo e-hypha ansi ion. IMA Fungus
16: e166433. h ps://doi.o g/10.3897/
ima ungus.16.166433
IMA Fungus 16: e166433 (2025)
DOI: 10.3897/ima ungus.16.166433
* These au ho s con ibu ed equally o his wo k.
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IMA Fungus 16: e166433 (2025), DOI: 10.3897/ima ungus.16.166433
Wudian Xiao e al.: mRNA, lncRNA, and miRNA in T ichophy on men ag ophy es spo es and hyphae
Conclusion: Ou s udy e ealed he cha ac e is ics o mRNAs, lncRNAs, and miRNAs in
T. men ag ophy es using ansc ip omic me hods, and se he s age o u u e pa hoge-
nici y s udies and an i ungal d ug de elopmen o T. men ag ophy es.
Key wo ds: Hyphae, lncRNAs, miRNAs, mRNAs, RNA-sequencing, Spo es, T ichophy on
men ag ophy es
In oduc ion
T ichophy on men ag ophy es (T. men ag ophy es) is a globally dis ibu ed
zoono ic de ma ophy e ha in ec s ke a inized issues (skin, nails, hai olli-
cles), causing pe sis en de ma ophy oses in humans and di e se mamma-
lian hos s. Clinical mani es a ions include inea manuum, onychomycosis,
inea capi is, and inea pedis (Wei zman and Summe bell 1995; Che me e
e al. 2008). The ecalci ance o hese in ec ions cha ac e ized by high e-
cu ence a es and escala ing an i ungal esis ance due o he apeu ic o e -
use, es ablishes T. men ag ophy es as a c i ical public heal h h ea (Ebe
e al. 2020).
The spo e- o-hypha ansi ion cons i u es a pi o al i ulence de e minan .
Du ing ge mina ion, spo es adhe e o he s a um co neum ia ex acellula
ligands, de elop ge m ubes, and sec e e ke a inoly ic enzymes (ke a inases,
lipases, phospholipases) ha acili a e issue in asion (Duek e al. 2004; Ela-
a ashi e al. 2017). No ably, while scanning elec on mic oscopy has delin-
ea ed mo phological e en s, he ansc ip omic egula o y a chi ec u e—pa -
icula ly dynamic in e ac ions among mRNAs, lncRNAs, and miRNAs, emains
unexplo ed in T. men ag ophy es (Duek e al. 2004).
Ad ancemen s in RNA sequencing (RNA-seq) enable genome-scale p o iling
o ansc ip ional ne wo ks (H dlicko a e al. 2017), as demons a ed by an i-
ungal a ge iden i ica ion in T ichophy on ub um (Gal ão-Rocha e al. 2023).
Non-coding RNAs ha a e ansc ibed om non-coding egions o he genome,
o ches a e key ungal adap a ions. Long non-coding RNAs (lncRNAs; >200 n )
modula e cell wall biogenesis, ansc ip ional egula ion (e.g., GAL-media ed
R-loop o ma ion in yeas ), and s ess esponses (Clou ie e al. 2016; Hom-
bach and K e z 2016; No ačić e al. 2020; Shuman 2020). Mic oRNAs (miRNAs;
19–25 n ) guide RNA-induced silencing complexes (RISCs) o a ge mRNAs ia
3’/5’ UTR binding, pos - ansc ip ionally egula ing de elopmen al ansi ions
(Iwakawa and Toma i 2022), as e idenced by s age-speci ic miRNAs in T icho-
phy on ub um and i on adap a ion in Pa acoccidioides b asiliensis (Wang e al.
2018; de Cu cio e al. 2021). Despi e documen ed oles o lncRNAs and miR-
NAs in ungal mo phogenesis, no in eg a ed analysis o he ipa i e RNA eg-
ulome (mRNA-lncRNA-miRNA) exis s o T. men ag ophy es ge mina ion (Lau
e al. 2020; Lai e al. 2023).
Recen ly, we epo ed a ansc ip ome analysis o ci cRNAs in T. men ag o-
phy es du ing spo e ge mina ion (Zhang e al. 2023). Tha wo k p o ided he
i s insigh s in o RNA-based egula ion o ungal mo phogenesis in de ma o-
phy es, bu was inhe en ly es ic ed o a single class o non-coding RNAs. To
achie e a mo e comp ehensi e unde s anding, he p esen s udy in eg a es
exp ession p o iles o mRNAs, lncRNAs, and miRNAs in o a uni ied egula o y
amewo k. This in eg a ed app oach allows us o no only ca alogue di e se
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IMA Fungus 16: e166433 (2025), DOI: 10.3897/ima ungus.16.166433
Wudian Xiao e al.: mRNA, lncRNA, and miRNA in T ichophy on men ag ophy es spo es and hyphae
RNA species bu also o cons uc candida e compe ing endogenous RNA
(ceRNA) ne wo ks, he eby explo ing he mul ilaye ed con ol unde lying he
c i ical spo e- o-hypha ansi ion.
Ma e ials and me hods
Fungal cul u e and sample collec ion
The T. men ag ophy es wild- ype s ain ATCC MYA-4439 was p o ided by BeNa
Cul u e Collec ion (BNCC, Beijing, China). T. men ag ophy es s ain was cul-
u ed and main ained on po a o dex ose aga medium (BD, Spa ks, MD, USA)
o 14 days a 28 °C o ha es spo es (Wang e al. 2022; Zang e al. 2024).
Spo es on he mycelium su ace we e insed using 5 mL o s e ile dis illed wa-
e and passed sequen ially h ough h ee cell il e s wi h po e sizes o 0.05 mm,
0.026 mm, and 0.01 mm (Zhang e al. 2023). In o de o collec hyphae, spo es
we e cul u ed and main ained in yeas ex ac pep one dex ose medium solu-
ion shaken on a THZ-300 shake (Yiheng Scien i ic Ins umen Co., Shanghai,
China) a 200 pm o 5 days a 28 °C. The hyphae we e hen il e ed h ough
gauze, ollowed by washing wi h s e ile dis illed wa e o collec pu e hyphae.
Ge mina ion analysis o T. men ag ophy es spo es
Fo ge mina ion analysis, 20 mL o 1 × 105 spo es/mL T. men ag ophy es spo e
suspension was inocula ed in o each o a se ies o 250 mL e lenmeye lasks
con aining 80–100 mL o yeas ex ac pep one dex ose medium, and immedi-
a ely incuba ed a 28 °C wi h cons an shaking (200 pm) (Yiheng Scien i ic Ins u-
men Co., Shanghai, China). Samples a he ime-poin s 0, 6, 12, and 18 h pos -in-
ocula ion we e aken di ec ly om he medium and subjec ed o mo phological
obse a ion using a mo ic AE2000 mic oscope (Mo ic, Fujian, China).
mRNA-lncRNA isola ion, ibosomal RNA-deple ed s and-speci ic
lib a y cons uc ion, and sequencing
A e o al RNA was ex ac ed om he h ee spo e (S, n = 3) and hyphae (H,
n = 3) samples using TRIzol® eagen (In i ogen, Ca lsbad, CA, USA) acco d-
ing o he manu ac u e ’s ins uc ions, ibosomal RNA was emo ed o e ain
bo h coding and non-coding RNAs. The emaining RNA was en iched using
SPRI beads, agmen ed a high empe a u e, and e e se- ansc ibed in o dou-
ble-s anded cDNA. The cDNA was hen end- epai ed, A- ailed, and liga ed o
adap e s, ollowed by PCR ampli ica ion. Ampli ied p oduc s we e pu i ied wi h
VAHTS DNA Clean Beads and sequenced on an Illumina No aSeq X Plus by
Gene Deno o Bio echnology Co. (Guangzhou, China).
miRNA isola ion, lib a y cons uc ion, and sequencing
To al RNA o spo es (S, n = 3) and hyphae (H, n = 3) was ex ac ed wi h he
TRIzol® eagen , ollowed by en ichmen o RNA molecules in he 18–30n size
ange by polyac ylamide gel elec opho esis (PAGE). The lib a y cons uc ion
in ol ed sequen ial liga ion o adap e s. A e he 3’ adap e s we e liga ed, a
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IMA Fungus 16: e166433 (2025), DOI: 10.3897/ima ungus.16.166433
Wudian Xiao e al.: mRNA, lncRNA, and miRNA in T ichophy on men ag ophy es spo es and hyphae
size-selec ion s ep was pe o med o en ich RNAs be ween 36–44 nucleo-
ides. The 5’ adap e s we e hen liga ed o hese size-selec ed molecules. The
liga ion p oduc s we e hen e e se ansc ibed by PCR ampli ica ion, and he
140–160 bp size PCR p oduc s we e en iched o gene a e a cDNA lib a y and
sequenced using Illumina No aSeq X Plus by Gene Deno o Bio echnology Co.
(Guangzhou, China).
Analysis o di e en ially exp essed mRNA
Ini ially, low-quali y eads we e il e ed om he aw da a using as p ( e sion
0.18.0) o ob ain high-quali y clean eads (Chen e al. 2018). Reads mapped
o he ibosome da abase we e hen emo ed using Bow ie2 ( e sion 2.2.8)
(Langmead and Salzbe g 2012). Pai ed-end clean eads we e mapped o he
T. men ag ophy es genome (GSA: CRA028745) using HISAT2 ( e sion 2.2.1),
and ansc ip s we e econs uc ed using S ing ie ( e sion 2.2.3) (T apnell e
al. 2010; Kim e al. 2015; Pe ea e al. 2015). All o he econs uc ed ansc ip s
we e aligned o e e ence genome and we e di ided in o wel e ca ego ies by
using g compa e. We de ined ansc ip s wi h one o he class codes ‘u, i, j,
x, c, e o o’ as no el ansc ip s. We used he ollowing pa ame e s o iden i y
eliable no el genes: ansc ip s longe han 200 bp, and had o ha e mo e han
wo exons. A FPKM ( agmen pe kilobase o ansc ip pe million mapped
eads) alue was calcula ed using he RSEM so wa e p ima ily o assessing
gene exp ession le els and acili a ing sample compa isons (Li and Dewey
2011). Co ela ion analysis was conduc ed using R o assess he eliabili y
and ope a ional s abili y o he expe imen al eplica es. The co ela ion coe i-
cien be ween wo samples was calcula ed, wi h alues close o 1 indica ing
s onge ep oducibili y. P incipal componen analysis (PCA) was pe o med
using he R package gmodels (h p://www. p ojec .o g/) o e eal ela ionships
be ween samples. To ensu e analy ical igo , di e en ial exp ession analysis
was pe o med using DESeq2, which u ilizes aw ead coun s and employs a
nega i e binomial model o iden i y signi ican ly di e en ially exp essed genes
(Lo e e al. 2014). Genes wi h a alse disco e y a e (FDR) < 0.05 and a old
change > 2 we e conside ed di e en ially exp essed.
Iden i ica ion and analysis o lncRNAs
All econs uc ed ansc ip s we e mapped o he T. men ag ophy es genome
and classi ied in o wel e ca ego ies using Cu compa e be o e selec ing an-
sc ip s longe han 200 bp and wi h an exon numbe g ea e han wo. By e-
qui ing mo e han one exon, ou pipeline sys ema ically excludes single-exon
lncRNAs. The p o ein coding po en ial o he new ansc ip s was assessed
wi h CNCI ( e sion 2), CPC ( e sion 0.9- 2) and FEELNC ( e sion 0.2) us-
ing he de aul pa ame e s (Kong e al. 2007; Sun e al. 2013; Wuche e al.
2017). T ansc ip s consis en ly p edic ed as non-p o ein-coding by all h ee
ools we e classi ied as lncRNAs. Po en ial lncRNAs we e classi ied in o i e
ca ego ies based on hei posi ion wi h espec o p o ein coding genes: in-
e genic lncRNAs, bidi ec ional lncRNAs, in onic lncRNAs, an isense lncRNAs,
and sense-o e lapping lncRNAs. The lncRNA a ge mRNAs we e iden i ied by
pe o ming an isense, ans- and cis- egula ion lncRNA analyses wi h RNAplex
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IMA Fungus 16: e166433 (2025), DOI: 10.3897/ima ungus.16.166433
Wudian Xiao e al.: mRNA, lncRNA, and miRNA in T ichophy on men ag ophy es spo es and hyphae
( e sion 0.2) (Ta e and Ho acke 2008). The FPKM alues, co ela ion coe i-
cien s, PCA, and di e en ially exp essed lncRNAs we e assessed as o mRNA
(see sec ion 2.5 abo e).
miRNA iden i ica ion and analysis
Reads con aining mo e han one low quali y (Q- alue ≤ 20) base o unknown
nucleo ides (N), eads wi hou 3’ adap e s, eads con aining 5’ adap e s, eads
con aining 3’ and 5’ adap e s bu no small RNA agmen be ween hem, eads
con aining poly (A) in he small RNA agmen , and eads sho e han 18 n
(no including adap e s) we e emo ed om he aw da a. The esul ing clean
ags we e aligned o he GenBank da abase (Release 209.0) and R am da a-
base (Release 14.10) o emo e RNA, scRNA, snoRNA, snRNA and RNA. All
clean ags we e also aligned o he e e ence genome (GSA: CRA028745) o
emo e agmen s and epe i i e sequences ha mapped o exons o in ons.
Clean ags we e aligned o he ungal miRBase da abase (Release 22) o iden-
i y known miRNAs using Bow ie ( e sion 1.1.2). Subsequen ly, he emaining
unaligned ags we e compa ed agains miRNA sequences om o he spe-
cies wi hin miRBase, applying a il e ha equi ed alignmen o p ecu so se-
quences while excluding ma ches beyond he 2 bp ex ensions o he ma u e
miRNA. The no el miRNA candida es we e iden i ied by mi Deep2 based on
hei genome posi ions and p edic ed hai pin s uc u es, a e excluding all
ags ma ching known miRNAs, miRNA edi ing a ian s, mRNA deg ada ion
agmen s, epe i i e egions, and o he non-coding RNAs (including RNA,
scRNA, snoRNA, snRNA, and RNA) (Mackowiak 2011). miRNA a ge mR-
NAs we e iden i ied by pa ma ch (Ve sion 1.2). The ansc ip s pe million
(TPM) alue was calcula ed wi h he ollowing o mula: TPM = Ac ual miRNA
coun s/To al coun s o clean ags × 106. Co ela ion coe icien s and PCA
we e assessed as o mRNA (see sec ion 2.5 abo e). We iden i ied miRNAs
wi h a old change > 2 and P < 0.05 in a compa ison as signi ican di e en-
ially exp essed miRNAs.
En ichmen analysis
Gene on ology (GO) en ichmen analysis and Kyo o Encyclopedia o Genes and
Genomes (KEGG) pa hway analysis we e pe o med on di e en ially exp essed
mRNAs, lncRNAs a ge mRNAs, miRNAs a ge mRNAs and ceRNA a ge
mRNAs. Fo GO en ichmen analysis, all di e en ially exp essed genes (DEGs)
we e mapped o GO e ms (h p://www.geneon ology.o g/). The numbe o
genes associa ed wi h each e m was calcula ed. GO e ms ha we e signi i-
can ly en iched in he DEGs compa ed o he exp essed gene uni e se we e
iden i ied using a hype geome ic es . Pa hway en ichmen analysis iden i ied
signi ican ly en iched me abolic pa hways o signal ansduc ion pa hways in
DEGs compa ing wi h he exp essed gene uni e se as backg ound gene se s.
GO e ms and KEGG pa hways wi h FDR ≤ 0.05 we e conside ed signi ican ly
en iched. Subsequen ly, GO en ichmen analysis and KEGG pa hway analysis
we e pe o med on a se o genes by using he Gene Se En ichmen Analysis
(GSEA) (Sub amanian e al. 2005). Gene se s wi h |NES| > 1, NOM p- al < 0.05,
and FDR q- al < 0.25 we e conside ed di e en ially exp essed.

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IMA Fungus 16: e166433 (2025), DOI: 10.3897/ima ungus.16.166433
Wudian Xiao e al.: mRNA, lncRNA, and miRNA in T ichophy on men ag ophy es spo es and hyphae
O hology-in e ed P o ein-P o ein in e ac ion (PPI)
PPI analysis was pe o med using he STRING 10 da abase and isualized
in Cy oscape ( 3.7.1) (Szkla czyk e al. 2015). Since STRING does no con ain
speci ic PPI da a o T. men ag ophy es, an o hology-based app oach was
employed. P o ein sequences o T. men ag ophy es we e mapped o he Sac-
cha omyces ce e isiae e e ence p o eome using ecip ocal bes BLAST hi o -
hology in e ence. The esul ing o hology-in e ed PPI ne wo k was analyzed
o iden i y pu a i e unc ional modules and hub p o eins. Pu a i e hub genes
we e iden i ied by in e sec ing he op- anked candida es om he cy oHubba
plugin (e alua ed by Deg ee, Edge Pe cola ed Componen (EPC), Eccen ici y,
and Maximum Neighbo hood Componen algo i hms (MNC)) wi h key modules
de i ed om Molecula Complex De ec ion (MCODE) analysis in Cy oscape.
The esul ing p o ein-p o ein in e ac ion ne wo k was cons uc ed based on
hese high-con idence hub genes (Shannon e al. 2003).
Cons uc ion and analysis o candida e lncRNA – miRNA – mRNA
ne wo k (ceRNA)
CeRNA e e s o he pool o ansc ip s (such as mRNA and lncRNA) ha can
compe i i ely seques e miRNA ac i i y. Based on he same miRNA esponse
elemen s (MREs), candida e ceRNA ne wo ks be ween lncRNAs, mRNAs, and
miRNA we e cons uc ed by assembling di e en ially exp essed mRNAs, ln-
cRNAs and miRNAs. Depending on di e en ially exp essed mRNAs, lncRNAs,
and miRNAs, exp ession co ela ion be ween mRNA-miRNA o lncRNA-miRNA
wi h a Spea man Rank co ela ion coe icien (SCC) ≤ -0.7 we e iden i ied as
nega i ely co-exp essed lncRNA-miRNA pai s o mRNA-miRNA pai s. Exp es-
sion co ela ion be ween lncRNA-mRNA wi h a Pea son co ela ion coe icien
(PCC) > 0.9 we e selec ed as co-exp essed lncRNA-mRNA pai s. The po en ial
lncRNA-mRNA-miRNA pai s wi h p- alues less han 0.05 (wi hou mul iple- es -
ing co ec ion) in he abo e analysis we e sc eened by he hype geome ic dis-
ibu ion es o ob ain he inal ceRNA pai s. All co-exp essed compe ing ip-
le s we e hen assembled o cons uc ceRNA and isualized wi h Cy oscape
so wa e ( 3.6.0). The numbe o co-exp essed a ge ed miRNAs was de ined
as he RNA connec i i y o iden i y hub genes.
qRT-PCR alida ion
To alida e he RNA-seq esul s, quan i a i e eal- ime polyme ase chain e-
ac ion (qRT-PCR) was conduc ed o ou andomly selec ed di e en ially ex-
p essed mRNAs, ou di e en ially exp essed lncRNAs, and six di e en ially ex-
p essed miRNAs. To al RNA was ex ac ed om spo es and hyphae, ollowed by
e e se ansc ip ion o cDNA wi h Re e R a Ace qPCR RT Mas e Mix (Toyobo,
Osaka, Japan). Nex , qRT-PCR was conduc ed wi h Powe UpTM SYBRTM G een
Mas e Mix (Applied Biosys ems, The mo Fishe Scien i ic, Wal ham, MA, USA).
B ie ly, he eac ion olume con ained 5 μL o SYBR G een Mas e Mix, 1 μL o
10 μM o wa d and e e se p ime s, 1 μL o empla e cDNA, and 3 μL o dH2O in
a inal olume o 10 μL. The eac ions we e pe o med on a Quan S udio 5 PCR
Sys em (Applied Biosys ems, The mo Fishe Scien i ic) as ollows: 50 °C o
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IMA Fungus 16: e166433 (2025), DOI: 10.3897/ima ungus.16.166433
Wudian Xiao e al.: mRNA, lncRNA, and miRNA in T ichophy on men ag ophy es spo es and hyphae
2 min and 95 °C o 2 min, ollowed by 40 cycles o 95 °C o 1 s and 60 °C o
30 s. A mel cu e analysis was pe o med a 95 °C o 1 s, 60 °C o 20 s, and
95 °C o 1 s. No- empla e con ols and no- e e se ansc ip ion con ols we e
included in e e y qPCR un o moni o con amina ion. None o hese con ols
showed speci ic ampli ica ion, con i ming he speci ici y and eliabili y o he
expe imen al esul s. The ampli ica ion e iciency o all qPCR p ime s was al-
ida ed ia s anda d cu es cons uc ed om se ially dilu ed cDNA empla es
( ypically 2- old o 10- old dilu ions). Ampli ica ion e iciency (E) was calcula ed
using he o mula E = (10^(-1/slope) - 1)*100%. The e iciency o all p ime s
used ell wi hin he ange o 90%–110%, wi h R² alues o he s anda d cu es
g ea e han 0.98, mee ing he ecommended c i e ia o he MIQE guidelines.
All eac ions p oduced a single sha p peak in he mel cu e analysis, con i m-
ing ampli ica ion speci ici y wi hou nonspeci ic ampli ica ion o p ime -dime
o ma ion. Th ee echnical eplica es we e pe o med o each biological epli-
ca e. The C alues o he h ee echnical eplica es o each biological eplica e
we e i s a e aged o ob ain a ep esen a i e C alue o ha biological epli-
ca e. Subsequen ly, all g oup-based s a is ical analyses and da a p esen a ions
we e pe o med using hese consolida ed C alues, each ep esen ing one bi-
ological eplica e. The ela i e quan i ica ion o mRNAs, lncRNAs, and miRNAs
was calcula ed by he 2-ΔΔC me hod using chs and U6 as he in e nal e e ence
(Li ak and Schmi gen 2001). The s abili y o he e e ence genes chs and U6
was e alua ed using he geNo m and No mFinde so wa e. Thei exp ession
emained s able ac oss all expe imen al condi ions, wi h an M alue less han
0.5, con i ming hei sui abili y as e e ences o da a no maliza ion.
S a is ical analyses
S a is ical analyses we e pe o med using SPSS S a is ics 20.0 (SPSS Inc., Chica-
go, IL, USA). Unless o he wise speci ied, alues a e exp essed as he mean ± s an-
da d e o o he mean (SEM). P < 0.05 was conside ed s a is ically signi ican .
Abb e ia ions
lncRNAs Long non-coding RNAs
miRNAs Mic o RNAs
ceRNA Compe ing endogenous RNA
cDNA Complemen a y DNA
scRNA Small cy oplasmic RNA
RNA Ribosomal RNA
snoRNA Small nucleola RNA
snRNA Small nuclea RNA
RNA T ans e RNA
mRNA Messenge RNA
T. men ag ophy es T ichophy on men ag ophy es
qRT-PCR Quan i a i e eal- ime polyme ase chain eac ion
RNA-seq RNA sequencing
RISC RNA-induced Silencing Complex
UTR Un ansla ed egion
PAGE Polyac ylamide gel elec opho esis
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FPKM F agmen pe kilobase o ansc ip pe million mapped eads
PCA P incipal componen analysis
FDR False disco e y a e
TPM T ansc ip s pe million
GO Gene on ology
KEGG Kyo o Encyclopedia o Genes and Genomes
GSEA Gene Se En ichmen Analysis
PPI P o ein-P o ein in e ac io
EPC Edge Pe cola ed Componen
MNC Maximum Neighbo hood Componen
MCODE Molecula Complex De ec ion
MREs MiRNA esponse elemen s
SCC Spea man Rank co ela ion coe icien
PCC Pea son co ela ion coe icien
SEM S anda d e o o he mea
MAPK Mi ogen-ac i a ed p o ein kinase
Resul s
Ge mina ion o T. men ag ophy es spo es
Ge mina ion o T. men ag ophy es spo es p og essed h ough wo dis inc
phases: swelling and ge m ube o ma ion. Do man spo es displayed a ound-
ed o o al mo phology (Fig. 1A). Wi hin 6 hou s o incuba ion, spo e swelling
commenced, esul ing in an app oxima e doubling o cellula diame e . Concu -
en ly, pola ized g ow h was ini ia ed a one si e on he spo e su ace (Fig. 1B).
By 12 hou s, he ge m ube eme gence phase was e iden , cha ac e ized by he
p o usion o a single ge m ube (Fig. 1C). Ex ensi e g ow h o mul iple ge m
ubes, culmina ing in hyphal o ma ion, was obse ed by 18 hou s (Fig. 1D;
Suppl. ma e ial 1: ig. S1).
O e iew o RNA-sequencing
To cha ac e ize he exp ession p o iles and co-exp ession ne wo ks o mRNAs,
lncRNAs, and miRNAs du ing T. men ag ophy es ge mina ion, cDNA and small
RNA lib a ies we e cons uc ed and subjec ed o high- h oughpu sequencing.
Fo he lncRNA-mRNA ansc ip ome, 1,567,771,788 high-quali y clean eads
(Q30 > 93%) we e e ained a e il e ing aw da a (1,570,717,414 eads; Suppl.
ma e ial 2: able S1). Base composi ion and co e age analyses indica ed uni-
o m genomic dis ibu ion wi hou 3′/5′ bias, wi h > 77% o eads exhibi ing
≥ 80% gene co e age (Fig. 2A, B). Sequencing sa u a ion analysis con i med
adequa e dep h o cap u ing exp essed genes (Suppl. ma e ial 1: ig. S2).
Small RNA sequencing yielded 136,978,282 clean eads (Suppl. ma e ial
2: able S1), wi h dis inc size dis ibu ions: 22-n RNAs domina ed in spo es,
whe eas 23-n RNAs we e p edominan in hyphae (Fig. 2C, D). A e emo ing
known non-coding RNAs in GeneBank ( RNA, scRNA, snoRNA, snRNA, and
RNA), 80,548,874 unmapped ags we e e ained (Suppl. ma e ial 1: ig. S3A).
Subsequen genome alignmen iden i ied 102,779,029 mapped ags, including
7,548,666 exon-ma ching ags (Suppl. ma e ial 1: ig. S3B, C), wi h no epea
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sequence ma ches de ec ed (Suppl. ma e ial 1: ig. S3D). Collec i ely, hese
esul s demons a e high-quali y, ep oducible sequencing da a sui able o
downs eam bioin o ma ic analyses.
Analysis o mRNAs
Iden i ica ion and cha ac e iza ion o mRNAs
To iden i y unanno a ed ansc ip s, we pe o med de no o ansc ip ome as-
sembly using S ingTie. Alignmen o econs uc ed ansc ip s o he T. men-
ag ophy es genome e ealed 8,059 mRNAs, comp ising 7,554 known and 505
no el ansc ip s (Suppl. ma e ial 2: able S2). Exp ession le els we e quan i-
ied using FPKM (Fig. 3A), and iolin plo s demons a ed dis inc mRNA exp es-
sion pa e ns be ween spo es and hyphae (Fig. 3B). P incipal componen anal-
ysis (PCA) and co ela ion analysis u he con i med clea sepa a ion be ween
spo e and hyphal g oups (Fig. 3C, D).
Di e en ial exp ession analysis (FDR < 0.05, |log2FC| > 1) iden i ied 3,193 sig-
ni ican ly di e en ially exp essed mRNAs, including 692 up- egula ed and 2,501
down- egula ed ansc ip s in hyphae compa ed o spo es (Suppl. ma e ial 2:
able S2). No ably, he mos highly up- egula ed mRNAs included SRY1 (g3940),
YIL010W (g3025), and alg2 (g3846), while he mos s ongly down- egula ed
A
B
C
D
Figu e 1. T. men ag ophy es obse ed by op ical mic oscopy du ing ge mina ion. A. 0 h, B. 6 h, C. 12 h, D. 18 h. Scale ba :
10 μm.
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AMP-ac i a ed p o ein kinase ac i i y, and glu aminase ac i i y) (Fig. 9B; Suppl.
ma e ial 2: able S7), and 105 biological p ocess e ms (pa icula ly posi i e
egula ion o G2/M ansi ion, es ablishmen o cell pola i y, and di e en ia-
ion- ela ed mo phogenesis) (Fig. 9C; Suppl. ma e ial 2: able S7). These ind-
ings s ongly sugges an isense lncRNAs play c ucial oles in mi o ic egula ion
and spo e ac i a ion. KEGG pa hway analysis u he suppo ed hese obse a-
ions, wi h en ichmen in: Yeas MAPK signaling pa hway, Euka yo ic ibosome
biogenesis, Yeas cell cycle egula ion, Oxida i e phospho yla ion, Amino acid
biosyn hesis, and Seconda y me aboli e biosyn hesis (Fig. 9D; Suppl. ma e ial
2: able S7). The coo dina ed en ichmen o hese pa hways highligh s he po-
en ial in ol emen o an isense lncRNAs in undamen al cellula p ocesses
du ing ungal de elopmen and mo phogenesis.
Func ional en ichmen analysis o cis-lncRNAs
P e ious s udies ha e es ablished ha lncRNAs can cis- egula e adjacen
genes on he same allele (Yan e al. 2017). Applying a 10 kb genomic win-
dow (ups eam o downs eam) o iden i y po en ial cis- egula o y lncRNAs,
we iden i ied 1,158 signi ican cis-in e ac ions in ol ing 389 di e en ially ex-
p essed lncRNAs and 950 a ge mRNAs (Suppl. ma e ial 2: able S8).
Figu e 7. Iden i ica ion and cha ac e iza ion o lncRNAs. A. Iden i ica ion o no el lncRNAs. B. lncRNA ype. C. FPKM
dis ibu ion. D. lncRNA exp ession iolin plo . p  =  6.17E-147***.
AB
CD

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Func ional en ichmen analysis o hese cis- egula ed a ge mRNAs e ealed
signi ican en ichmen in 160 unc ional ca ego ies (P < 0.05), comp ising: 28
cellula componen e ms (e.g., ungal- ype acuole lumen, cell wall compo-
nen s), 50 molecula unc ion e ms (including uc ose-bisphospha e aldolase
ac i i y, G p o ein-coupled pep ide ecep o ac i i y, and ATP ci a e syn hase
ac i i y), and 82 biological p ocess e ms (no ably ungal- ype cell wall disas-
sembly du ing cellula usion, uc ose 1,6-bisphospha e me abolism, and cell
cycle egula ion) (Fig. 10A–C; Suppl. ma e ial 2: able S8). These en iched e ms
s ongly implica e cis-ac ing lncRNAs in c i ical de elopmen al p ocesses, pa -
icula ly hyphal o ma ion and spo e ac i a ion. KEGG pa hway analysis u he
demons a ed en ichmen in key me abolic pa hways (P < 0.05), including: Ami-
no acid me abolism ( yp ophan, aline/leucine/isoleucine biosyn hesis), Ca -
bohyd a e me abolism ( uc ose and mannose), Ene gy me abolism (oxida i e
phospho yla ion) (Fig. 10D; Suppl. ma e ial 2: able S8). Collec i ely, hese ind-
ings demons a e ha cis-ac ing lncRNAs pa icipa e in ungal de elopmen by
locally egula ing genes in ol ed in cell wall emodeling, me abolic ep og am-
ming, and cellula di e en ia ion du ing hyphae o ma ion and spo e ac i a ion.
Figu e 8. Analysis o di e en ially exp essed lncRNAs. A. Volcano plo o lncRNAs. B. P incipal componen analysis.
C. Hea map o di e en ially exp essed lncRNAs.
A
B
C
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Func ional en ichmen analysis o ans-lncRNAs
In addi ion o cis- egula ion, lncRNAs can modula e gene exp ession h ough
ans-ac ing mechanisms (Yan e al. 2017). Ou analysis iden i ied 247,586
signi ican co-exp ession ela ionships be ween 409 di e en ially exp essed
lncRNAs and 3,138 a ge mRNAs (Suppl. ma e ial 2: able S9). Func ional
en ichmen o hese ans- egula ed a ge s e ealed signi ican associa ions
(P < 0.05) wi h key biological p ocesses, including: 30 cellula componen
e ms (e.g., ungal- ype cell wall, acuola lumen, hyphal cell wall), 53 molec-
ula unc ion e ms (such as L-amino acid ansmemb ane anspo e ac i -
i y and DNA-binding ansc ip ion ac o ac i i y), and 192 biological p ocess
e ms (including alpha-amino acid me abolic p ocess, pigmen biosyn he ic
Figu e 9. GO and KEGG classi ica ion o an isense lncRNAs – a ge mRNAs. A. Top 20 GO e ms en iched by cellula
componen s. B. Top 20 GO e ms en iched by molecula unc ion. C. Top 20 GO e ms en iched by biological p ocess.
D. Top 20 pa hways o KEGG en ichmen analysis.
A B
C D
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p ocess, and adhesion o symbion o hos ) (Fig. 11A–C; Suppl. ma e ial 2:
able S9). KEGG pa hway analysis u he iden i ied 17 signi ican ly en iched
me abolic pa hways (P < 0.05), no ably encompassing amino acid biosyn hesis
( yp ophan, a ginine/p oline, and b anched-chain amino acids), ca bohyd a e
me abolism ( uc ose and mannose), lipid me abolism (glyce olipids me abo-
lism), and co ac o syn hesis (pan o hena e and CoA biosyn hesis) (Fig. 11D;
Suppl. ma e ial 2: able S9). These comp ehensi e indings demons a e ha
ans-ac ing lncRNAs likely o ches a e ungal de elopmen by globally egu-
la ing genes in ol ed in cell wall o ganiza ion, me abolic ep og amming, and
hos -mic obe in e ac ions.
Figu e 10. GO and KEGG classi ica ion o cis-lncRNAs a ge mRNAs. A. The op 20 en iched GO e ms o he cellula
componen . B. The op 20 en iched GO e ms o he molecula unc ion. C. The op 20 en iched GO e ms o he biological
p ocess. D. Top 20 o KEGG en ichmen analysis.
A B
CD
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Analysis o miRNAs
Iden i ica ion and cha ac e iza ion o miRNAs
Ou small RNA sequencing analysis iden i ied 733 known miRNAs (miR-
Base-ma ching sequences) h ough alignmen wi h he miRBase da abase
(Suppl. ma e ial 2: able S10) and p edic ed 52 no el miRNAs based on ge-
nomic posi ioning and hai pin s uc u e analysis (Suppl. ma e ial 2: able S10).
Bo h known and no el miRNAs exhibi ed a cha ac e is ic 5’ u acil bias in he
18–25 n size ange ac oss spo e and hyphal s ages (Suppl. ma e ial 1: ig.
S4A–D), wi h posi ion-speci ic nucleo ide p e e ences consis en wi h canoni-
cal miRNA ea u es (Suppl. ma e ial 1: ig. S4E–H). Comp ehensi e anno a ion
o 129,717,858 small RNA ags e ealed di e se RNA species, including 44.5
Figu e 11. GO and KEGG classi ica ion o ans-lncRNAs a ge mRNAs. A. The op 20 en iched GO e ms o he cellula
componen . B. The op 20 en iched GO e ms o he molecula unc ion. C. The op 20 en iched GO e ms o he biological
p ocess. D. Top 20 o KEGG en ichmen analysis.
A B
C
D
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million RNAs, 7.5 million exon-mapping sequences, 574,139 known miRNAs,
and 38,066 no el miRNAs (Suppl. ma e ial 1: ig. S5A). No ably, TPM-based
exp ession analysis e ealed signi ican di e ences in miRNA abundance be-
ween spo es and hyphae (Suppl. ma e ial 1: ig. S5B), sugges ing s age-speci -
ic egula o y oles du ing ungal de elopmen .
Iden i ica ion and analysis o di e en ially exp essed miRNAs
PCA e ealed dis inc clus e ing pa e ns be ween spo e and hyphal g oups
o bo h known and no el miRNAs (Fig. 12A, B). Di e en ial exp ession anal-
ysis iden i ied 119 signi ican ly di e en ially exp essed miRNAs (P < 0.05,
|log2FC| > 1), comp ising 56 up egula ed and 63 down egula ed species
(Suppl. ma e ial 2: able S10). No ably, no el-m0011-5p, miR2109-z, and no el-
m0019-5p eme ged as he mos s ongly up egula ed miRNAs, while miR319-y,
miR-12295-x, and miR6149-x showed he mos p onounced down egula ion
(Fig. 12C). Hie a chical clus e ing o hese di e en ially exp essed miRNAs
clea ly dis inguished hyphal om spo e samples (Fig. 12D), demons a ing o-
bus s age-speci ic miRNA exp ession p o iles du ing ungal de elopmen .
Func ional en ichmen analysis o miRNAs
Th ough comp ehensi e a ge p edic ion, we iden i ied 8,662 mRNAs po en ial-
ly egula ed by 785 miRNAs, encompassing 626,128 a ge si es (Suppl. ma e-
ial 2: able S11). Func ional en ichmen analysis o hese miRNA- a ge ed mR-
NAs e ealed signi ican associa ions (P < 0.05) wi h 19 unc ional ca ego ies,
including plasma memb ane (cellula componen ), ans e ase/oxido educ ase
ac i i y (molecula unc ion), and key biological p ocesses such as mul i-o gan-
ism ep oduc ion, g ow h, and mi o ic cell cycle egula ion (Fig. 13A–C; Suppl.
ma e ial 2: able S11). KEGG pa hway analysis u he demons a ed en ich-
men in 140 me abolic and signaling pa hways, mos no ably biosyn hesis- e-
la ed p ocesses, including seconda y me aboli es, amino acids, and co ac o s,
as well as ibosome biogenesis and yeas MAPK signaling (Fig. 13D; Suppl.
ma e ial 2: able S11). These indings s ongly sugges ha miRNAs play a pi -
o al ole in coo dina ing ungal g ow h, de elopmen , and me abolic adap a ion
h ough widesp ead pos - ansc ip ional egula ion.
Analysis o ceRNAs
Cons uc ion o a candida e ceRNA ne wo k
Th ough comp ehensi e a ge p edic ion, we iden i ied 34,680 miRNA-mR-
NA and 3,227 miRNA-lncRNA in e ac ion pai s in ol ing 119 di e en ially ex-
p essed miRNAs, 3,177 mRNAs, and 403 lncRNAs. Using s ingen co ela ion
h esholds (SCC ≤ -0.7 o nega i e co-exp ession and PCC > 0.9 o ceRNA
pai s), we de i ed 229,789 po en ial ceRNA in e ac ions be ween 2,911 mRNAs
and 338 lncRNAs. Fo s a is ical sc eening o high-con idence pai s, we pe -
o med hype geome ic es ing and e ained in e ac ions wi h an unco ec ed
P- alue < 0.05 (no e: mul iple- es ing co ec ion such as FDR adjus men was
no applied in his s ep), ul ima ely yielding a candida e ne wo k comp ising

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14,360 signi ican ceRNA pai s, in eg a ing 2,627 mRNAs, 107 miRNAs, and
329 lncRNAs (Suppl. ma e ial 2: able S12). Topological analysis e ealed 636
hub nodes in he p ima y ne wo k ie , wi h he highes connec i i y obse ed
o i e mRNAs (gl 1, d xS1, g3795, SPBC20F10.07, and MSTRG.440) and i e
lncRNAs (MSTRG.9453.1, MSTRG.2311.1, MSTRG.2710.1, MSTRG.3534.1 and
MSTRG.8002.1), along wi h hei miRNA pa ne s (Fig. 14), highligh ing cen al
egula o s in he ungal ceRNA ne wo k.
Explo a o y en ichmen analysis o ceRNAs
Func ional anno a ion o ceRNA- ela ed mRNAs e ealed signi ican en ichmen
(unco ec ed P- alues < 0.05) in 286 ca ego ies (32 cellula componen s, 68
molecula unc ions, and 186 biological p ocesses), including ungal- ype ac-
uole lumen, ungal- ype cell wall, hyphal cell wall, L-amino acid ansmemb ane
Figu e 12. Iden i ica ion and cha ac e iza ion o miRNAs. A. PCA o known miRNAs. B. PCA o no el miRNAs. C. Volcano
plo o miRNAs. D. Hea map o di e en ially exp essed miRNAs.
A B
CD
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anspo e ac i i y, DNA-binding ansc ip ion ac o ac i i y, L-amino acid ans-
po , me hionine me abolic p ocess, and cellula amino acid biosyn he ic p o-
cess (Fig. 15A–C; Suppl. ma e ial 2: able S12). KEGG pa hway analysis u he
iden i ied 18 signi ican ly en iched me abolic pa hways (unco ec ed P- alues <
0.05), wi h p edominan ep esen a ion in amino acid biosyn hesis ( yp ophan),
seconda y me aboli e p oduc ion, and cen al ca bon me abolism (glyce olipid
and py u a e me abolism) (Fig. 15D; Suppl. ma e ial 2: able S12). These ind-
ings p o ide p elimina y clues ha lncRNAs modula e ungal de elopmen and
me abolism h ough miRNA-media ed ceRNA ne wo ks, compe i i ely egula -
ing mRNAs in ol ed in cell wall dynamics and me abolic ep og amming.
Figu e 13. GO and KEGG classi ica ion o miRNAs a ge mRNAs. A. Top 20 GO e ms en iched by cellula componen s.
B. Top 20 GO e ms en iched by molecula unc ion. C. Top 20 GO e ms en iched by biological p ocess. D. Top 20 pa h-
ways o KEGG en ichmen analysis.
A B
C D
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qRT-PCR e i ica ion o he RNA-Seq esul s
To alida e he RNA sequencing da a, qRT-PCR was pe o med on a andom-
ly selec ed panel o di e en ially exp essed mRNAs, lncRNAs, and miRNAs
using p ime s designed ia P ime -BLAST (Suppl. ma e ial 2: able S13). Ex-
p ession ends o mRNAs and lncRNAs exhibi ed signi ican conco dance
wi h RNA-seq p o iles (Fig. 16; Suppl. ma e ial 2: able S13). Among miRNAs,
miR-150-x showed signi ican up egula ion du ing ge mina ion, whe eas
h ee o he s we e down- egula ed (Fig. 16). C i ically, he posi i e co ela ion
be ween up- egula ed lncRNA-MSTRG.10182.6 and up- egula ed a ge gene
o-4 aligned wi h he p edic ed ceRNA ne wo k in e ac ion. These esul s col-
lec i ely con i m he eliabili y o ou ansc ip omic da a and he accu acy o
iden i ied exp ession dynamics.
Figu e 14. The highes connec i i y o 5 mRNAs and 5 lncRNAs. Red ep esen ed mRNAs, yellow ep esen ed lncRNAs,
and blue ep esen ed miRNAs.
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Discussion
The ansi ion om do man spo es o in asi e hyphae in de ma ophy es (e.g.,
T ichophy on ub um, T. men ag ophy es) is o ches a ed by mul ilaye ed mo-
lecula con ols, including ansc ip ional, pos - ansc ip ional, and epigene ic
egula ion. This p ocess in ol es dynamic in e ac ions be ween mRNAs, miR-
NAs, and lncRNAs, which collec i ely ine- une ungal de elopmen and pa ho-
genici y (Liu e al. 2007; Wang e al. 2018; Zhang e al. 2023). In he p esen
s udy, we p esen a comp ehensi e ansc ip omic landscape o he spo e- o-
Figu e 15. GO and KEGG classi ica ion o ceRNA- ela ed mRNAs. A. Top 20 GO e ms en iched by cellula componen s.
B. Top 20 GO e ms en iched by molecula unc ion. C. Top 20 GO e ms en iched by biological p ocess. D. Top 20 pa h-
ways o KEGG en ichmen analysis.
A B
C
D
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dis up ing lncRNA-p o ein in e ac ions could selec i ely inhibi spo e ge mina-
ion wi hou a ec ing hos cells.
In summa y, his s udy sys ema ically deciphe ed he lncRNA-mRNA-miR-
NA egula o y ne wo k du ing T. men ag ophy es ge mina ion by in eg a ing
mul i-omics da a, e ealing how lncRNAs coo dina e de elopmen al ansi ions
h ough cis-, ans-, and ceRNA mechanisms. These indings p o ide no el in-
sigh s in o he egula o y amewo k o ilamen ous ungal mo phogenesis and
lay a heo e ical ounda ion o RNA in e e ence-based an i ungal s a egies.
Conclusion
This s udy es ablishes a comp ehensi e egula o y a las o T. men ag ophy es
ge mina ion, e ealing ha : (1) T ansc ip ional ep og amming supp esses
anabolism while ac i a ing cell di ision machine y. (2) lncRNAs ope a e h ough
spa ially dis inc mechanisms (an isense, cis, and ans) o con ol mo phogen-
esis. (3) miRNAs ine- une de elopmen al ansi ions by a ge ing cell cycle and
me abolic egula o s. (4) ceRNA ne wo ks in eg a e lncRNA-miRNA-mRNA in-
e ac ions o coo dina e hyphal g ow h. These indings p o ide no el insigh s
in o he egula o y amewo k o ilamen ous ungal mo phogenesis and lay a
heo e ical ounda ion o RNA in e e ence-based an i ungal s a egies.
Acknowledgemen s
We app ecia e Gene Deno o Bio echnology Co. (Guangzhou, China) o assis-
ance in sequencing and bioin o ma ics analysis.
Addi ional in o ma ion
Con lic o in e es
The au ho s ha e decla ed ha no compe ing in e es s exis .
E hical s a emen
No e hical s a emen was epo ed.
Use o AI
No use o AI was epo ed.
Adhe ence o na ional and in e na ional egula ions
All necessa y pe mi s we e ob ained.
Funding
This s udy was suppo ed by Na ional Na u al Science Founda ion o China (G an Nos.
U23A20398, 82030007, and 32200165), Noncommunicable Ch onic Diseases-Na ional
Science and Technology Majo P ojec (G an Nos. 2024ZD0537707), Na u al Science
Founda ion o Sichuan, China (G an Nos., 2023NSFSC1553), Luzhou People’s Go e n-
men -Sou hwes Medical Uni e si y Science and Technology s a egic coope a ion p ojec
(G an Nos. 2024LZXNYDJ034), Science and Technology S a egic Coope a ion P og ams
o Sichuan Uni e si y and Luzhou Municipal People’s Go e nmen (G an Nos. 2021CDLZ-
7), and School-le el p ojec o Sou hwes Medical Uni e si y (G an Nos. 2018-ZRQN-121).

33
IMA Fungus 16: e166433 (2025), DOI: 10.3897/ima ungus.16.166433
Wudian Xiao e al.: mRNA, lncRNA, and miRNA in T ichophy on men ag ophy es spo es and hyphae
Au ho con ibu ions
Concep ualiza ion, Chunxiang Zhang, Wudian Xiao, and Jingcan You, Da a cu a ion, Zha-
odan Wu, Jia Zhang, Jun Wan, and Xinyi Xiang; Fo mal analysis, L qin He and Wudian
Xiao; Funding acquisi ion, Chunxiang Zhang and Wudian Xiao; In es iga ion, Zhaodan
Wu, Jia Zhang, Yang Chen, Ziyu Wang, and Wudian Xiao; Me hodology, L qin He, Lu Fu,
Yang Yu, and Wudian Xiao; So wa e, Zhaodan Wu, Jingcan You, Kui Yang, and Ziyao
Xiao; Visualiza ion, Zhaodan Wu, Ruihuan Zhang, and Wudian Xiao; W i ing – o iginal
d a , Zhaodan Wu, Jia Zhang, Wudian Xiao; W i ing – e iew & edi ing, Chunxiang Zhang
and Wudian Xiao.
Au ho ORCIDs
Wudian Xiao h ps://o cid.o g/0000-0002-2718-4298
Da a a ailabili y
Da a om his s udy ha e been deposi ed in public eposi o ies as ollows: Genomic aw
eads and sca olding da a ha e been deposi ed o he Genome Sequence A chi e (GSA)
o he China Na ional Cen e o Bioin o ma ion (CNCB) unde accession CRA028745.
The inal assembled and anno a ed genome is a ailable om he CNCB Genome Wa e-
house (GWH) unde accession GWHGQOF00000000.1. All ansc ip omic sequencing
da a ha e been submi ed o he NCBI Sequence Read A chi e (SRA) unde accessions
SRR26663347–SRR26663352 (mRNA/lncRNA) and SRR26416206–SRR26416211
(miRNA).
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Supplemen a y ma e ial 1
Supplemen a y igu es S1–S5
Au ho s: Wudian Xiao, Zhaodan Wu, Jia Zhang, Jun Wan, Ruihuan Zhang, Xinyi Xiang,
Yang Yu, Lu Fu, Kui Yang, Yang Chen, Ziyao Xiao, Ziyu Wang, L qin He, Jingcan You,
Chunxiang Zhang
Da a ype: zip
Explana ion no e: igu e S1. T. men ag ophy es obse ed by op ical mic oscopy du ing
ge mina ion. (A) 0 h, (B) 18 h. Scale ba : 20 μm. igu e S2. Assessmen o Sequenc-
ing Sa u a ion ac oss all samples. (A) Sequencing sa u a ion dis ibu ion o sample
S1. (B) Sequencing sa u a ion dis ibu ion o sample S2. (C) Sequencing sa u a ion
dis ibu ion o sample S3. (D) Sequencing sa u a ion dis ibu ion o sample H1. (E)
Sequencing sa u a ion dis ibu ion o sample H2. (F) Sequencing sa u a ion dis i-
bu ion o sample H3. igu e S3. Classi ica ion o clean eads based on hie a chi-
cal genomic mapping. The mapping s a us o clean eads hie a chically aligned o
non-coding RNA in GenBank (A), genome (B), exon egions (C), and epea sequence
(D). igu e S4. Nucleo ide bias analysis o known and no el miRNAs in hyphae and
spo e samples. Fi s nuleo ide bias o known miRNAs o hyphe (A) and spo e (B).
Fi s nuleo ide bias o no el miRNAs o hyphe (C) and spo e (D). Nucleo ide bias a
each posi ion o known miRNAs o hyphe (E) and spo e (F). Nucleo ide bias a each
posi ion o no el miRNAs o hyphe (G) and spo e (H). igu e S5. Analysis o small
RNA composi ion and miRNA exp ession p o ile. (A) Comp ehensi e anno a ion o
small RNA ags. (B) miRNA exp ession iolin plo . p  =  0.0416*.
Copy igh no ice: This da ase is made a ailable unde he Open Da abase License
(h p://openda acommons.o g/licenses/odbl/1.0/). The Open Da abase License
(ODbL) is a license ag eemen in ended o allow use s o eely sha e, modi y, and
use his Da ase while main aining his same eedom o o he s, p o ided ha he
o iginal sou ce and au ho (s) a e c edi ed.
Link: h ps://doi.o g/10.3897/ima ungus.16.166433.suppl1
40
IMA Fungus 16: e166433 (2025), DOI: 10.3897/ima ungus.16.166433
Wudian Xiao e al.: mRNA, lncRNA, and miRNA in T ichophy on men ag ophy es spo es and hyphae
Supplemen a y ma e ial 2
Supplemen a y ables S1–S13
Au ho s: Wudian Xiao, Zhaodan Wu, Jia Zhang, Jun Wan, Ruihuan Zhang, Xinyi Xiang,
Yang Yu, Lu Fu, Kui Yang, Yang Chen, Ziyao Xiao, Ziyu Wang, L qin He, Jingcan You,
Chunxiang Zhang
Da a ype: zip
Explana ion no e: able S1. Summa y o sequencing lib a ies o T. men ag ophy es
spo e and hyphal samples. able S2. Iden i ica ion and di e en ial exp ession analy-
sis o mRNAs in T. men ag ophy es spo e and hyphal samples. able S3. Func ional
en ichmen analysis o di e en ially exp essed mRNAs. able S4. Gene se en ich-
men analysis o di e en ially exp essed mRNAs. able S5. Analysis o he p o-
ein-p o ein in e ac ion ne wo k. able S6. Iden i ica ion and di e en ial exp ession
analysis o lncRNAs. able S7. Lis o an isense lncRNA-mRNA egula o y pai s and
hei unc ional en ichmen . able S8. Lis o cis lncRNA-mRNA egula o y pai s and
hei unc ional en ichmen . able S9. Lis o ans lncRNA-mRNA egula o y pai s and
hei unc ional en ichmen . able S10. Iden i ica ion and di e en ial exp ession anal-
ysis o miRNAs. able S11. Func ional en ichmen analysis o miRNA a ge genes.
able S12. CeRNA ne wo k pai s and unc ional en ichmen analysis o ceRNA- ela -
ed mRNAs. able S13. P ime sequences and qRT-PCR alida ion esul s.
Copy igh no ice: This da ase is made a ailable unde he Open Da abase License
(h p://openda acommons.o g/licenses/odbl/1.0/). The Open Da abase License
(ODbL) is a license ag eemen in ended o allow use s o eely sha e, modi y, and
use his Da ase while main aining his same eedom o o he s, p o ided ha he
o iginal sou ce and au ho (s) a e c edi ed.
Link: h ps://doi.o g/10.3897/ima ungus.16.166433.suppl2