C-terminus of serine–arginine protein kinase-like protein, SrpkF, is involved in conidiophore formation and hyphal growth under salt stress in Aspergillus aculeatus

The serine–arginine protein kinase-like protein, SrpkF, was identi�ed as a regulator for the cellulose-responsive induction of cellulase genes in Aspergillus aculeatus. To analyze various aspects of SrpkF function, we examined the growth of the control strain (MR12); C-terminus deletion mutant, which produced SrpkF 1–327 (DCsrpkF); whole gene-deletion mutant of srpkF (DsrpkF), srpkF overexpressing strain (OEsprkF), and the complemented strain (srpkF + ) under various stress conditions. All test strains grew normally on minimal medium under control, high salt (1.5 M KCl), and high osmolality (2 M sorbitol and 1.0 M sucrose). However, only DCsrpkF showed reduced conidiation and hyperbranched hyphal growth on 1.0 M NaCl media. Conidiation of DCsrpkF on 1.0 M NaCl media was reduced to 12% compared with that of srpkF + . By contrast, deletion of srpkF did not affect hyphal growth and conidiation under the same conditions. We then quanti�ed the transcripts of the regulators involved in the central asexual conidiation pathway in A. aculeatus. The �ndings revealed that the expression of brlA, abaA, wetA, and vosA was reduced in DCsrpkF under salt stress. These data suggest that in A. aculeatus, SrpkF regulates conidiophore development. The C-terminus of SrpkF seems to be important for regulating SrpkF function in response to culture conditions.


Introduction
Aspergillus aculeatus no.F-50, which was isolated from soil, produces cellulolytic enzymes that cooperatively hydrolyze pulp together with enzymes from Trichoderma reesei (Murao et al. 1979).This coordinated hydrolysis of pulp is attributed to the secretion of enzymes by A. aculeatus, such as βglucosidase 1 (Murao and Sakamoto 1979), which has a different substrate speci city from the enzymes of T. reesei (Baba et al. 2015).A. aculeatus belongs to Aspergillus section Nigri, which is used in the fermentation industry to produce enzymes and organic acids (Gams et al. 1986;Hong et al. 2013;Meijer et al. 2011).Therefore, we believe that A. aculeatus can be a useful source of enzymes.We aim to evaluate how carbohydrate-active enzymes (CAZymes) are produced in A. aculeatus and construct a mass production system for the enzymes.
In A. aculeatus, CAZyme production, in response to the inducer, is mainly regulated at the transcriptional level (Tani et al. 2014).To identify factors involved in this regulation, we established an Agrobacterium tumefaciens-mediated transformation system that enabled us to identify the locus of the inserted transfer DNA (T-DNA) by PCR, followed by sequencing of ampli ed DNA fragments (Kunitake et al. 2011).We have identi ed various proteins as regulators: a putative transcription factor (cellobiose response regulator) (Kunitake et al. 2013); dipeptidyl peptidase IV (Tani et al. 2017); SepM, a component of the septation initiation network (Tsumura et al. 2021); and the serine-arginine protein kinase F (SrpkF) (Katayama et al. 2022).Each factor participates in the early induction stage of CAZyme expression; however, the mechanisms of regulation are unknown.
The SRPK subfamily of serine-threonine protein kinases is widely conserved in metazoans, such as fungi (de Souza et al. 2013) and humans (Giannakouros et al. 2011).SRPK is involved in the phosphorylation of serine residues in serine-arginine (SR) dipeptide repeats of SR proteins, which are essential for pre-mRNA splicing (Zahler et al. 1992).Saccharomyces cerevisiae has only one Srpk protein, namely Sky1p, which is homologous to Srpk1 in humans.Because both proteins are involved in the phosphorylation of several SR proteins, they participate in the regulation of various biological processes (Rodriguez Gallo et al. 2022).
The number of SRPK paralogs varies among organisms.S. cerevisiae has only one SRPK, possibly because the yeast genome has few introns (Siebel et al. 1999).By contrast, lamentous fungi have many

Strains and culture conditions
All A. aculeatus strains used in this study were derived from wild-type A. aculeatus no.F-50 [NBRC 108796].A. aculeatus strains were propagated at 30°C in minimal medium (MM) or complete medium (CM) (Adachi et al. 2009;Kunitake et al. 2015).Four mM uridine was complemented to grow the uridine auxotroph.The strains used in this study are summarized in Table 1.

Growth Assays
The effect of salt or osmotic stress on A. aculeatus strains was tested on MM, including 1.0 M NaCl, 1.5 M KCl, 2.0 M sorbitol, or 1.0 M sucrose.Then, 2 µL of 1 × 10 6 /mL conidia was spotted on prepared solid media and cultured for 5 d at 30°C.Then, the colonies were examined.
Conidia from A. aculeatus strains were collected in a solution of 0.01% Tween 80 and 0.8% NaCl and counted using a hemocytometer.The number of conidia was normalized by colony area.Conidial germination was tested by using a conidium suspension prepared from the strains grown on MM with or without 1 M NaCl.Then 50 µL of 1 × 10 6 /mL conidia was inoculated into 10 mL MM liquid media with or without 1 M NaCl in 90-mm dishes.Germination rate indicates the number of germinated conidia divided by the total number of conidia.

Gene Expression Analysis By Qrt-pcr
The conidium suspension (1 × 10 6 conidia) was prepared from strains pregrown on MM with or without 1 M NaCl.The collected conidia were inoculated into 200 mL of MM supplemented with 0.1% Bacto Tryptone (Thermo Fisher Scienti c, Tokyo, Japan) and incubated at 160 rpm for 24 h at 30°C.Mycelia were harvested with Miracloth (Merck Millipore, Tokyo, Japan), placed on MM plus 0.1% Bacto Tryptone supplemented with or without 1.0 M NaCl, and incubated for the indicated periods.RNA was prepared from the mycelia and qRT-PCR was used to quantify the expression of genes, as described (Tani et al. 2017).Primers used for qRT-PCR are listed in Supplementary Table S1.The speci city of PCR ampli cation was con rmed by melting curve analysis.The expression pro le of each gene was analyzed with the delta-deltaC T method.The expression level of each gene was normalized to the reference gene, glyceraldehyde-3-phosphate dehydrogenase A (gpdA).Relative expression levels are means of at least three independent experiments and error bars indicate standard deviations.More than three biological replicates were performed for each experiment, and each replicate was evaluated in triplicate.

Additional Methods
Primers for qRT-PCR were designed by using a sequence in an in-house A. aculeatus draft genome database.The genes and their accession numbers are listed in Supplementary Table S2.A scanning electron microscope (SEM) SU-1510 (Hitachi, Tokyo, Japan) was used to observe conidium formation.

Hyphal growth and conidium formation defects in ΔC srpkF
We investigated whether SrpkF is involved in morphogenesis in A. aculeatus because some regulators involved in the expression of cellulase and hemicellulase genes also affect stress response or conidiation (He et  Microscopic analysis of the tips of the colony of each strain revealed increased hyphal branching frequency in ΔCsrpkF compared with other strains on MM with 1 M NaCl (Fig. 1c).The hyper branching and reduced conidiation phenotypes of ΔCsrpkF were not observed on CM containing 1 M NaCl (Fig. 1b  and c) and MM without salt stress (data not shown).
Germination in ΔC sprkF was stimulated under salt stress We next evaluated conidial germination in strains MR12, ΔsrpkF, ΔCsrpkF, and OEsrpkF in submerged MM with or without 1.0 M NaCl.Because the phenotypes of MR12 and srpkF + were the same under all test conditions, we used MR12 as control.The four strains were grown on MM plates with or without 1.0 M NaCl for 5 d.Conidia of each strain were harvested, inoculated into submerged MM with or without 1.0 M NaCl, and incubated at 30°C for 24 h.Conidial germination of each strain was examined microscopically.Conidia of strains pregrown on salt-free MM, 60-70% of conidia of all test strains germinated in salt-free submerged culture (Fig. 2).In all strains, conidial germination was drastically reduced in submerged culture with 1.0 M NaCl.In the case of conidia collected from MM with 1.0 M NaCl, 60-80% of conidia in all test strains germinated under salt-free conditions.Interestingly, preincubation under salt stress signi cantly stimulated conidial germination in ΔCsrpkF and OEsrpkF even under salt stress (Fig. 2, n = 3, p < 0.05, one-way ANOVA).

Srpkf Is Involved In The Regulation Of The Central Asexual Conidiation Pathway
SEM analysis revealed that conidiophores were formed normally in MR12, ΔCsrpkF, ΔsrpkF, srpkF + , and OEsrpkF that were grown on MM for 5 d (Fig. 3).Moreover, vesicles and conidia were normally formed in MR12, ΔsprkF, and OEsrpkF cultured for 5 d on MM supplemented with 1.0 M NaCl.Interestingly, distorted vesicles were formed in ΔCsrpkF under the same condition, which entailed reduced conidial chains (Fig. 3).These results suggest that SrpkF 1-327 compromised vesicle and conidium formation under salt stress.The C-terminus region, including 89 amino acids of SrpkF, could play an important role in the development of A. aculeatus.
Because asexual conidiation and normal shape of vesicles were reduced in ΔCsrpkF under salt stress, we quanti ed the expression of regulator genes involved in asexual sporulation in A. aculeatus, namely brlA, abaA, wetA, vosA, bB, bC, bD, bE, and uG.The functions of their products in A. nidulans are summarized in Table S2 and Fig. 4a (Ojeda-Lopez et al. 2018).After conidium germination, mycelia of MR12, ΔsrpkF, and ΔCsrpkF were scraped off from MM plates with or without 1.0 M NaCl, and RNA was extracted.qRT-PCR con rmed that srpkF was expressed equally in MR12 and ΔCsrpkF (Fig. 4b).The expression of srpkF in OEsrpkF was ~ 50-fold more than that in MR12 and ΔCsrpkF (data not shown).
The expression of brlA in ΔCsrpkF was signi cantly reduced at 3 h under the control condition, but reached the same expression levels as the control strain at 6, 9, and 12 h.In all test strains, brlA expression was lower under salt stress than under salt-free conditions at all time points; however, in ΔCsrpkF, brlA expression was signi cantly reduced under salt stress at all time points (p < 0.05, Student's t-test).There were no critical differences in the expression of abaA, wetA, and vosA in all test strains under the control condition.However, the expression of their genes under salt stress was signi cantly reduced at 9 and 12 h only in ΔCsrpkF (p < 0.05, Student's t-test).The expression of bB, bC, bD, bE, and uG was not affected by the C-terminus deletion of SrpkF even under salt stress (Fig. 4c).The expression of bD under normal conditions increased in ΔCsrpkF (p < 0.05, Student's t-test).However, this increase did not stimulate the expression of brlA, abaA, wetA, and vosA.These data demonstrate that the reduced expression of brlA, abaA, and wetA in ΔCsrpkF under salt stress resulted in distorted vesicle formation and reduced conidium formation.

Discussion
The ndings of this study revealed that hyphal branching and conidial germination were stimulated, but conidium formation was reduced, in ΔCsrpkF under salt stress.The C-terminus deletion of SrpkF resulted in the reduced expression of transcription factor genes involved in conidium formation, such as brlA, wetA, abaA, and vosA.However, no differences were observed in morphology and gene expression in the strain ΔsrpkF of A. aculeatus.Our study demonstrated that the C-terminus region of SrpkF-from 328 to 416 amino acids-could play critical roles in conidiation and hyphal growth under salt stress in A. aculeatus.
Our previous study revealed that the expression of cellulase genes was reduced in ΔCsprkF as well as ΔsprkF in A. aculeatus (Katayama et al. 2022), suggesting that SrpkF 1-327 is insu cient to stimulate the cellulose-responsive expression of cellulase genes.A. aculeatus srpkF encodes a 416-amino acid protein, which is predicted to have a conserved protein domain family of the serine-threonine kinase/serinearginine protein kinase STKc_SRPK (ID: cd14136), in 52-414 amino acid region.Although whether SrpkF 1-327 possesses kinase activity, needs to be biochemically assessed, SrpkF 1-327 seems to be involved in hyphal growth and conidium formation in A. aculeatus.
The expression of srpkF was 50-fold stimulated under carbon starvation than under 1% glucose in submerged MM (Katayama et al. 2022) and CM (data not shown) at 3 h post-incubation.Because the intact sprkF promoter controls the production of SrpkF 1-327 , the protein can only exist under shortage of energy sources.Reduced conidiation and hyper branching phenotypes under salt stress in ΔCsrpkF have been observed after 5-d incubation on MM supplemented with 1% glucose, but not on CM, suggesting that the energy source was depleted during the growth of ΔCsrpkF in 5 d and SrpkF 1-327 could exist in mycelia grown on MM.
The obvious question is what caused the variances and similarities in sporulation, hyperbranching, and conidium germination in strains ΔSrpkF, ΔCSrpkF, and OEsrpkF.Of these, only ΔCSrpkF showed a reduction of vesicle formation and conidiation under salt stress.When the three strains were pregrown under salt stress, conidium germination in ΔCSrpkF and OEsrpkF, but not ΔSrpkF, was stimulated under salt stress.These data suggest that under salt stress, active SprkF can disturb vesicle formation and conidiation, whereas active SrpkF stimulates mycelial growth from conidia.This regulation could be mediated through the C-terminal region (from 328 to 416 aa) of SrpkF.
Regulation of Srpk function has been well-studied in human Srpk1.In Homo sapiens, Srpk1 is constitutively active, and its function is regulated by controlling its localization.A diverse loop region between the N-and C-terminal lobes is involved in regulating its localization (Koutroumani et  The signaling properties of Src family kinases are regulated through Src homology domain (SH2 and SH3)-mediated interactions with its catalytic domain.SH2 and SH3 domains, located in the N-terminus of Src family kinases, positively regulate the activity of its interactors through heterocomplex formation.By contrast, intramolecular interaction between SH2 domains and their C-terminal phosphorylated tails negatively control their activities (Hirai and Varmus 1990;LaFevre-Bernt et al. 1998).Further, with-no-K(Lys) kinase-1 (WNK1) is rapidly activated and phosphorylated at multiple residues after exposure of cells to hyperosmotic conditions, which regulate the localization of WNK1 from the cytosol to vesicular structures (Zagorska et al. 2007).Phosphorylation sites on SrpkF were predicted by NetPhos3.1 using the default lters (http://www.cbs.dtu.dk/services/NetPhos/).Five serine and two tyrosine residues were identi ed in the 89 amino acids within SrpkF 328 − 416 , predicted to be phosphorylated with a score higher than 0.5 (Blom et al. 2004).Phosphorylation at multiple residues within SrpkF 328 − 416 might be stimulated under salt stress, which regulates the functional expression of SrpkF.

Statements & Declarations
Representative images of MR12, DCsrpkF, DsrpkF, OEsrpkF, and srpkF + examined by SEM, showing a de ciency in the formation of vesicle and conidium chain in DsrpkF on minimal medium supplemented with 1.0 M NaCl.The bar is 50 mM.
introns, and Aspergillus nidulans has seven SRPKs (de Souza et al. 2013).Only a few eurotiomycetes, to which A. nidulans belongs, have seven types of SRPKs (de Vries et al. 2017).SRPKs are classi ed by the homology of the catalytic domain and are not conserved, except for the catalytic domain.A study revealed that a few SRPKs are not involved in mRNA-splicing, and the biological processes that involve SRPK in metazoans are unknown (Gou et al. 2020; Hong et al. 2012; Wang et al. 2017).Therefore, the role of SRPK in the regulation of various signaling pathways must be investigated.In this study, we analyzed SrpkF function in morphological development in A. aculeatus.Microscopic analysis and quantitative real-time (qRT)-PCR demonstrated that the C-terminal region of SrpkF (amino acids 328-416) plays important roles in vesicle and conidium chain formation under salt stress.The reduction in conidium formation in ΔCsprkF correlated well with the reduced expression of key transcription factor genes involved in conidium formation.

Figure 1 Comparison
Figure 1

Table 1
(Katayama et al. 2022;Tani et al. 2013)he functions of SprkF, we examined the phenotypes of previously generated strains: control (MR12) strain; srpkF overexpressing strain (OEsprkF); C-terminus deletion mutant, which produced SrpkF 1-327 (ΔCsrpkF); whole gene-deletion mutant of srpkF (ΔsrpkF), and the complemented strain (srpkF + ) (Table1)(Katayama et al. 2022;Tani et al. 2013).srpkF in OEsrpkF was expressed under the control of the promoter of translation elongation of 1α gene, which is constitutively expressed in lamentous fungi (Fig.1a)(Katayama et al. 2022).SrpkF 1-327 was produced under the intact promoter to investigate the function of its protein-produced as a result of the T-DNA insertion-which entails the reduction of cellulose-responsive expression of cellulase genes(Katayama et al. 2022).Strains MR12, ΔCsrpkF, ΔsrpkF, OEsrpkF, and srpkF + were grown under salt stress or osmotic stress.Hyphal growth and conidium formation of all test strains appear to be the same in control, 1.5 M KCl, 2.0 M sorbitol, or 1.0 M sucrose (Fig.1b).However, ΔCsrpkF displayed a relatively compact colony morphology, with severe defects in conidium formation only on MM supplemented with 1.0 M NaCl.The number of conidia in ΔCsrpkF (average 1.6 × 10 6 conidia/cm 2 ) signi cantly reduced to 12% compared with the control strain (average 1.3 × 10 7 conidia/cm 2 ) (n = 3, p < 0.05, Student's t-test) under salt stress.
(Jumper et al. 2021;Mirdita et al. 2022oteins are evolutionarily conserved and are involved in mRNA splicing in various organisms(Nikolakaki et al. 2001).SrpkA (AN7185), an Srpk1 (UniProt, Q96SB4) homolog in A. nidulans, was predicted to have a loop region between N-and C-terminal lobes in the AlphaFold Protein Structure Database, although the identity between SrpkA and SrpkF in A. aculeatus was just 40% and a model of SrpkF from the AlphaFold CoLab notebook was predicted not to have this loop region (data not shown)(Jumper et al. 2021;Mirdita et al. 2022).
(Baltussen et al. 2020)an and Prade 2002)d by a central regulatory pathway composed of transcription factors BrlA, AbaA, and WetA(Baltussen et al. 2020).When brlA expression was increased in the mpkB deletion mutant, which is the S. cerevisiae mitogen-activated protein (MAP) kinase Fus3 homolog, conidiation was increased even in submerged culture.Moreover, transcripts of vosA, a repressor of brlA expression, were not detected in the mpkB deletion strain(Kang et al. 2013).BrlA is one of the key regulators for conidiation in Aspergillus.Our data showed that the expression of brlA, abaA, wetA, and vosA was only reduced under salt stress in ΔCsrpkF, implying that SrpkF induces brlA expression, which regulates conidiation.Conidial germination under salt stress was less inhibited in ΔCsrpkF that were precultured under salt stress.Typically, conidia remain dormant until favorable conditions occur.However, mRNAs can be synthesized in the conidia of A. nidulans, Aspergillus fumigatus, and Talaromyces marneffei, thereby affecting the tness of fungal cells after germination(Wang et al. 2021).Recognition of osmotic stress and its signal transduction are relayed through a MAP kinase network, known as the HOG pathway, in the budding yeast and lamentous fungi(Brewster et al. 1993;Han and Prade 2002).Although hyperbranched hyphal tips were observed under salt stress in the hogA deletion mutant of A. nidulans, conidiation in the strain was normal (Han and Prade 2002).To our knowledge, no factors are involved in the Hog pathway causing both hyphal hyperbranching and abnormal sporulation under salt stress, suggesting that SrpkF acts on a different pathway.Hyperbranching and reduced sporulation occur in A. fumigatus Af293, producing a dihydroxyl oxylipin(Niu et al. 2020).The expression of various genes uctuates during mycelial elongation from spores(Baltussen et al. 2020).The ndings of this study indicate that investigating the contribution of SrpkF in gene expression and metabolite production is important to elucidate the regulatory mechanism of morphogenesis through the C-terminal of SrpkF.