Effects of MrwetA on Sexual Reproduction and Secondary Metabolism of Monascus ruber M7 Based on Transcriptome Analysis

wetA, one of the conidiation center regulatory genes in many filamentous fungi, plays an important role in promoting asexual spores (conidia) maturation. Our recent research has found that knocking out or overexpressing MrwetA (a homolog of wetA) in Monascus ruber M7 does not affect the development of its asexual spores like other fungi, but both repress the development of its sexual spores (ascospores). However, the mechanism remains unclear. In this study, the function of MrwetA on sexual reproduction and secondary metabolism in M. ruber M7 was confirmed by a complementary experiment. Moreover, the regulatory roles of MrwetA in modulating the expression of genes involved in sexual reproduction, meiosis, and biosynthesis of Monascus pigment and citrinin were analyzed based on the transcriptional data. These results not only contribute to clarifying the regulation of the reproduction and secondary metabolism of Monascus spp., but also to enriching the regulation molecular mechanism of reproduction in filamentous fungi.


Introduction
Monascus spp., a sort of filamentous fungi used to produce fermented foods and medicines, possess a history of application for nearly 2000 years in China and other Asian countries [1,2].Monascus spp.are highly valued due to their ability to produce a diverse range of beneficial secondary metabolites, such as Monascus pigments (MPs) used as food colorants [3,4], and monacolin K (MK), a functional ingredient for lowering blood pressure [5].Although some Monascus strains may also produce citrinin (CIT) [6], the CIT issue in Monascus-relative products has been well controlled through the unremitting efforts of scientists [7][8][9][10].Over the past decades, numerous studies have investigated the biosynthesis of MPs, CIT, and MK in Monascus spp.And their gene clusters and biosynthesis pathways have been well-established [1,[11][12][13].However, due to the long fermentation time of Monascus-relative products, their production cost is often high and they are prone to contamination by miscellaneous microorganisms, resulting in food safety issues.Therefore, how to promote the growth of Monascus strains, and shorten their fermentation time is a major challenge at present [14].
Monascus spp.can produce asexual (conidia) and sexual (ascospores) spores, and both spores are very important for Monascus reproduction and growth [15].The mechanisms by which the Monascus genus produces asexual and sexual spores were previously believed to be similar to the Aspergillus genus due to their close phylogenesis [16][17][18].However, our recent research has found that in the Monascus genomes, there is no homologous of abaA, one of the core asexual development regulatory genes [19].Additionally, our findings indicate that MrwetA, homologous of another central asexual development regulatory gene Potato dextrose agar (PDA), malt extract agar (MA), Czapek yeast extract agar (CYA), and 25% glycerol nitrate agar (G25N) were utilized for morphological characteristics.PDA medium was used for the analyses of MPs and CIT production.Inducing medium (IM) was used to activate Agrobacterium tumefaciens before co-culture, and coculture-inducing medium (Co-IM) was used to generate mutants for the co-culture process.To select mutants, PDA supplied with 15 µg/mL G418 sulfate (Sigma-Aldrich, Shanghai, China) was used to isolate neomycin-resistant transformants.All strains were maintained on PDA slants at 28 • C.

Construction of the MrwetA Complemented Strain
In our recent research, the mutants of knocking out and overexpressing MrwetA were prepared [20].In this study, the MrwetA complementation strain was constructed, according to the strategy of homologous recombination [36,37].The genomic DNA of M7 was used to amplify the complete MrwetA gene sequence (1659 bp), 5 ′ flanking region (1052 bp), and 3 ′ flanking region (915 bp).neo (neomycin phosphotransferase resistance gene, 1221 bp) as the selected maker was amplified from plasmid pKNI.hph (hygromycin-B resistance marker gene, 2137 bp) as a maker for screening ∆MrwetA.The relevant primer pairs are shown in Table S1.
The construction steps of the complementation strain are as follows.Firstly, these cloned DNA fragments were ligated to plasmid pCAMBIA3300 (digested with Sal I and Hind III) by using pEASY ® -Basic Seamless Cloning and Assembly Kit (TransGen Biotech, Beijing, China) to form plasmid pCCMrwetA.The construction procedure is shown in Figure S1.Secondly, the plasmid pCCMrwetA was transformed into A. tumefaciens EHA105 using a freeze-thaw method.Finally, the A. tumefaciens EHA105 clones containing pCCMr-wetA were incubated for transformation with the ∆MrwetA strain to generate the MrwetA complementation strains (∆MrwetA::MrwetA).

Observations of Sexual Reproduction
Referred to Jia et al. [20], WT, ∆MrwetA, and ∆MrwetA::MrwetA strains were cultured for 9 d at 28 • C to observe microscopic morphologies on PDA, CYA, G25N, and MA.To measure the number of cleistothecia and ascospores, freshly harvested spores (1 × 10 5 /mL) from each strain were inoculated in a PDA plate covered with sterile cellophane and incubated at 28 • C. Samples collected at every 4 d from the 3rd to the 11th d were scraped.The cleistothecia were counted using a five-point sampling method.Colonies with an area of 1 per cm 2 were collected from each point, washed with 1 mL of sterile water, and counted under a microscope.And the number of ascospores was counted after breaking the cleistothecia.

Detection of Monascus Pigments and Citrinin
To detect the yield of MPs and CIT, 200 µL freshly harvested spores (1 × 10 5 /mL) from each strain were inoculated onto Pa DA plate covered with sterile cellophane and incubated at 28 • C for 11 d.Hyphae and media were respectively collected at every 2 d from the 3rd to the 11th d, and freeze-dried.Then, the freeze-dried hyphae (20 mg) and media (20 mg) were separately ground into powder.Then, 1.5 mL of 80% (v/v) methanol (chromatographic grade) was added and mixed to extract under ultrasonic (KQ-250B, Kunshan, China) for 30 min at 30 • C.After centrifugation at 12,000× g for 10 min, the supernatants were used for measuring MPs and CIT.
The supernatant solution was diluted to an appropriate concentration with 80% methanol, and 80% (v/v) methanol was used as the control (CK).The yellow MPs, orange MPs, and red MPs were detected at 380 nm, 470 nm, and 520 nm using a UV-visible spectro-photometer (UV-1700, Shimadzu, Tokyo, Japan).Then, absorption values were multiplied by the dilution factor to obtain the yields of yellow, orange, and red MPs.In addition, the CIT content was determined by ultra-high performance liquid chromatography with the fluorescence detector (UPLC-FLR) following our previous paper [38].

RNA Extraction and RNA Sequencing
WT and ∆MrwetA were incubated at 28 • C on a cellophane-covered PDA plate.Two replicates were conducted for each strain.The fresh hyphae were obtained on the 3rd, 5th, and 7th d, respectively.The total RNA was extracted by using TransZol Up Plus RNA Kit (TransGen Biotech, Beijing, China).RNA sequencing was performed on DNBSEQ highthroughput sequencing platform (BGI, Wuhan, China, http://en.genomics.cn/(accessed on 8 January 2022)).The isolation of RNA, construction of libraries, sequencing, data filtering, and quality control were performed according to the platform's operation guides.The reference genome is the genome of M. ruber M7 sequenced by our lab [1].
Gene expression levels were calculated as RNA-Seq by Expectation-Maximization (RSEM) [39].The normalized value of fragments per kilobase transcript fragment per million map readings (FPKM) was used as a parameter to compare expression levels between WT and ∆MrwetA.The differentially expressed genes (DEGs) were detected by DEseq software package (DEseq 2) [40].DEGs were selected based on the standard of gene fold change (FC) ≥1.5 (|log 2 FC| ≥ 0.5849) and Q value (adjusted p-value) ≤ 0.05.The Kyoto Encyclopedia of Genes and Genomes (KEGG, https://www.kegg.jp/(accessed on 23 April 2022)) pathway is a database resource for understanding the high-level function and utility of biological systems, allowing analysis of the function of DEGs and the metabolic pathways involved.In addition, genes involved in fungal sexual reproduction and secondary metabolism were further analyzed to elucidate the role of MrwetA in sexual reproduction and secondary metabolism in M. ruber M7.

Statistical Analysis
p values were determined by using a one-way analysis of variance (ANOVA) test.A significance level of p < 0.05 was labeled with *, while p < 0.01 was denoted with **.Origin 2022 was utilized for graph plotting and statistical analysis.Error bars given in the figures represent the standard error.

Construction of the MrwetA Complemented Strain
Following the successful transformation of the constructed and verified recombinant vector (Figure S2) into ∆MrwetA, colonies with resistance to G418 sulfate were selected and subsequent PCR was conducted (primers in Table S2) to screen the complemented strains.MrwetA knockout strains were constructed in the previous study, and hph was used as the resistance screening marker [20].Putative target transformants were discerned by the amplification of MrwetA, neo, and hph fragments with lengths consistent with the expected sizes (Figure S3).In addition, PCR analysis confirmed the presence of MrwetA ORF and neo sequences as well as the absence of the hygromycin B sequence.

Reproduction Observations of M. ruber M 7 and Its MrwetA Modified Strains
Compared to M. ruber M7 (WT), ∆MrwetA, and ∆MrwetA::MrwetA exhibited no significant difference in conidial microscope morphologies across the four media (PDA, MA, CYA, and G25N), which are commonly used for morphological observation of Monascus spp.and cleistothecia could only be observed when cultivated on PDA and MA.As shown in Figure 1, the cleistothecia were similar in the WT and ∆MrwetA::MrwetA, whereas the sizes of cleistothecia were smaller in ∆MrwetA compared to them.change (FC) ≥1.5 (|log2 FC| ≥ 0.5849) and Q value (adjusted p-value) ≤ 0.05.The Kyoto Encyclopedia of Genes and Genomes (KEGG, https://www.kegg.jp/(accessed on 23 April 2022)) pathway is a database resource for understanding the high-level function and utility of biological systems, allowing analysis of the function of DEGs and the metabolic pathways involved.In addition, genes involved in fungal sexual reproduction and secondary metabolism were further analyzed to elucidate the role of MrwetA in sexual reproduction and secondary metabolism in M. ruber M7.

Statistical Analysis
p values were determined by using a one-way analysis of variance (ANOVA) test.A significance level of p < 0.05 was labeled with *, while p < 0.01 was denoted with **.Origin 2022 was utilized for graph plotting and statistical analysis.Error bars given in the figures represent the standard error.

Construction of the MrwetA Complemented Strain
Following the successful transformation of the constructed and verified recombinant vector (Figure S2) into ΔMrwetA, colonies with resistance to G418 sulfate were selected and subsequent PCR was conducted (primers in Table S2) to screen the complemented strains.MrwetA knockout strains were constructed in the previous study, and hph was used as the resistance screening marker [20].Putative target transformants were discerned by the amplification of MrwetA, neo, and hph fragments with lengths consistent with the expected sizes (Figure S3).In addition, PCR analysis confirmed the presence of MrwetA ORF and neo sequences as well as the absence of the hygromycin B sequence.

Reproduction Observations of M. ruber M 7 and Its MrwetA Modified Strains
Compared to M. ruber M7 (WT), ΔMrwetA, and ΔMrwetA::MrwetA exhibited no significant difference in conidial microscope morphologies across the four media (PDA, MA, CYA, and G25N), which are commonly used for morphological observation of Monascus spp.and cleistothecia could only be observed when cultivated on PDA and MA.As shown in Figure 1, the cleistothecia were similar in the WT and ΔMrwetA::MrwetA, whereas the sizes of cleistothecia were smaller in ΔMrwetA compared to them.The morphologies and yields of cleistothecia were analyzed from the 3rd d to the 11th d, to determine whether MrwetA delays or inhibits sexual development.The cleistothecia size in both WT and ∆MrwetA::MrwetA steadily increased from the 3rd to the 11th d.However, in ∆MrwetA, there was no significant change observed in cleistothecia size as time progressed, which remained smaller compared to that in the WT (Figure 2A).As shown in Figure 2B,C, the number of cleistothecia and ascospores was significantly reduced in ∆MrwetA compared to those in WT, whereas this defect could be restored in ∆MrwetA::MrwetA.Specifically, the number of cleistothecia in WT reached 36.93 ± 1.54/cm 2 , 104.67 ± 18.05/cm 2 , and 177.67 ± 2.38/cm 2 on the 3rd, 7th, and 11th d, respectively, which was 1.33, 1.58, and 2.43 times higher than its yield in ∆MrwetA.Correspondingly, the WT ascospore numbers reached (2.21 ± 0.57) × 10 5 (per cm 2 ) and (4.14 ± 0.58) × 10 5 (per cm 2 ) on the 7th and 11th d, respectively, which were 96.22 and 129.50 times of that in ∆MrwetA.The morphologies and yields of cleistothecia were analyzed from the 3rd d to the 11th d, to determine whether MrwetA delays or inhibits sexual development.The cleistothecia size in both WT and ΔMrwetA::MrwetA steadily increased from the 3rd to the 11th d.However, in ΔMrwetA, there was no significant change observed in cleistothecia size as time progressed, which remained smaller compared to that in the WT (Figure 2A).As shown in Figure 2B,C, the number of cleistothecia and ascospores was significantly reduced in ΔMrwetA compared to those in WT, whereas this defect could be restored in ΔMrwetA::MrwetA.Specifically, the number of cleistothecia in WT reached 36.93 ± 1.54/cm 2 , 104.67 ± 18.05/cm 2 , and 177.67 ± 2.38/cm 2 on the 3rd, 7th, and 11th d, respectively, which was 1.33, 1.58, and 2.43 times higher than its yield in ΔMrwetA.Correspondingly, the WT ascospore numbers reached (2.21 ± 0.57) × 10 5 (per cm 2 ) and (4.14 ± 0.58) × 10 5 (per cm 2 ) on the 7th and 11th d, respectively, which were 96.22 and 129.50 times of that in ΔMrwetA.

MPs and CIT Analysis of WT and Its MrwetA Modified Strains
The effects of MrwetA deletion on secondary metabolites were investigated by assessing the production of MPs and CIT, as illustrated in Figure 3. Compared to the WT, ΔMrwetA exhibited no significant difference in yellow, orange, and red MP yields during the early stage (3 d-5 d), but showed an increase on the 7th and 11th d, while the MPs content of ΔMrwetA::MrwetA closely resembled that of WT across different time points (Figure 3A-C).In addition, the yield of CIT in ΔMrwetA was significantly increased at all

MPs and CIT Analysis of WT and Its MrwetA Modified Strains
The effects of MrwetA deletion on secondary metabolites were investigated by assessing the production of MPs and CIT, as illustrated in Figure 3. Compared to the WT, ∆MrwetA exhibited no significant difference in yellow, orange, and red MP yields during the early stage (3 d-5 d), but showed an increase on the 7th and 11th d, while the MPs content of ∆MrwetA::MrwetA closely resembled that of WT across different time points (Figure 3A-C).In addition, the yield of CIT in ∆MrwetA was significantly increased at all time points compared to those in the WT, with no significant difference observed in ∆MrwetA::MrwetA (Figure 3D).The yield of total MPs and citrinin in the MrwetA overexpressed strain was higher on the 7th d compared to WT, similar to the ∆MrwetA [20].In summary, the deletion of MrwetA resulted in enhanced MP production in the later period and CIT production in all observed time points.
time points compared to those in the WT, with no significant difference observed in ΔMrwetA::MrwetA (Figure 3D).The yield of total MPs and citrinin in the MrwetA overexpressed strain was higher on the 7th d compared to WT, similar to the ΔMrwetA [20].In summary, the deletion of MrwetA resulted in enhanced MP production in the later period and CIT production in all observed time points.

The Transcriptomic Analysis of WT and ΔMrwetA
High-throughput sequencing technology was used to elucidate the mechanism of MrwetA in M7 on the transcription level.Based on the previous findings [20] and the results in this study, the deletion of MrwetA disturbed the sexual development and secondary metabolites, while complementation of MrwetA restored these disturbances, verifying the important role of MrwetA in the strain.As described by Yang et al. [41], when cultivated on PDA, M7 was at a logarithmic phase on the 3rd d, followed by stable growth from the 4th to the 7th d, and subsequently entering the decline phase after the 7th d.Consequently, samples were collected and analyzed on the 3rd, 5th, and 7th d in this study.

The Transcriptomic Analysis of WT and ∆MrwetA
High-throughput sequencing technology was used to elucidate the mechanism of MrwetA in M7 on the transcription level.Based on the previous findings [20] and the results in this study, the deletion of MrwetA disturbed the sexual development and secondary metabolites, while complementation of MrwetA restored these disturbances, verifying the important role of MrwetA in the strain.As described by Yang et al. [41], when cultivated on PDA, M7 was at a logarithmic phase on the 3rd d, followed by stable growth from the 4th to the 7th d, and subsequently entering the decline phase after the 7th d.Consequently, samples were collected and analyzed on the 3rd, 5th, and 7th d in this study.

Analysis of Differentially Expressed Genes
The screened differentially expressed genes (DEGs) were analyzed as shown in Figure 4, compared to WT, the number of up-regulated and down-regulated DEGs in ∆MrwetA were 285 and 386 on the 3rd d (Figure 4A), 616 and 844 on the 5th d (Figure 4B), 690 and 1013 on the 7th d (Figure 4C), respectively.In addition, the number of common DEGs was 163 at all time points (Figure 4D).In summary, the number of DEGs increased along with cultivation, and the number of down-regulated DEGs was more than that of up-regulated DEGs at all time points.This trend indicates a potential stronger involvement of MrwetA in the up-regulation of gene expression.
gi 2024, 10, x FOR PEER REVIEW 7 of

Analysis of Differentially Expressed Genes
The screened differentially expressed genes (DEGs) were analyzed as shown in Fi ure 4, compared to WT, the number of up-regulated and down-regulated DEGs ΔMrwetA were 285 and 386 on the 3rd d (Figure 4A), 616 and 844 on the 5th d (Figure 4B 690 and 1013 on the 7th d (Figure 4C), respectively.In addition, the number of commo DEGs was 163 at all time points (Figure 4D).In summary, the number of DEGs increase along with cultivation, and the number of down-regulated DEGs was more than that up-regulated DEGs at all time points.This trend indicates a potential stronger involv ment of MrwetA in the up-regulation of gene expression.

KEGG Pathway Enrichment Analysis of DEGs
The functions of DEGs at three time points (3rd, 5th, and 7th d) were investigate from five categories by KEGG pathway, including cellular processes, environmental i formation processing, genetic information processing, metabolism, and organismal sy tems.Transport and catabolism, signal transduction, folding, sorting and degradatio carbohydrate metabolism, amino acid metabolism, lipid metabolism, and global and ove view maps were the main KEGG subcategories in which DEGs were involved (Figure S4 On the 3rd d, key pathways enriched with DEGs included glycolysis/gluconeogenes

KEGG Pathway Enrichment Analysis of DEGs
The functions of DEGs at three time points (3rd, 5th, and 7th d) were investigated from five categories by KEGG pathway, including cellular processes, environmental information processing, genetic information processing, metabolism, and organismal systems.Transport and catabolism, signal transduction, folding, sorting and degradation, carbohydrate metabolism, amino acid metabolism, lipid metabolism, and global and overview maps were the main KEGG subcategories in which DEGs were involved (Figure S4).On the 3rd d, key pathways enriched with DEGs included glycolysis/gluconeogenesis, riboflavin metabolism, and thiamine metabolism (Figure 5A).Subsequently, on the 5th and 7th d, major pathways enriched with DEGs were similar, including biosynthesis of antibiotics, biosynthesis of amino acids, amino sugar and nucleotide sugar metabolism, and glycine, serine and threonine metabolism (Figure 5B,C).

Effects of MrwetA on Sexual Reproduction
As evidenced in Figure 2, the deletion of MrwetA inhibited the development of sexual structure cleistothecia.Both M7 and the model fungus A. nidulans belong to Aspergillaceae family and share a similar sexual reproductive process.Sexual reproduction is a complex biological process that requires the participation of many genes.To elucidate the molecular groundwork, BLASTP was used to identify homologues of sexual development-related genes, verified in A. nidulans, in the M7 genome (Table S3).Furthermore, the DEGs involved in M7 sexual reproduction were analyzed in six subgroups (Mating processes, signal transduction, perception of environmental signals, transcription factors, regulatory proteins, and fruiting body maturation) as shown in Figure 6.

Effects of MrwetA on Sexual Reproduction
As evidenced in Figure 2, the deletion of MrwetA inhibited the development of sexual structure cleistothecia.Both M7 and the model fungus A. nidulans belong to Aspergillaceae family and share a similar sexual reproductive process.Sexual reproduction is a complex biological process that requires the participation of many genes.To elucidate the molecular groundwork, BLASTP was used to identify homologues of sexual development-related genes, verified in A. nidulans, in the M7 genome (Table S3).Furthermore, the DEGs involved in M7 sexual reproduction were analyzed in six subgroups (Mating processes, signal transduction, perception of environmental signals, transcription factors, regulatory proteins, and fruiting body maturation) as shown in Figure 6.Mating-type (MAT) genes and genes related to signal transduction pathways play a crucial role in regulating fungal sexual reproduction.In comparison with the M7, MAT1-2 expression levels were down-regulated on the 5th and 7th d, and pheromone receptor preA (regulated by MAT1-2) expression levels were down-regulated on the 3rd and 5th d in the deletant (Figure 6A).G protein-coupled receptors (GPCRs), possessing seven transmembrane structures, are activated upon binding to pheromones and then bind to heterotrimeric G proteins.In G protein signaling pathway, the expression levels of gprD-1 (encoding GPCR) were elevated on the 3rd and 5th d.In addition, the expression levels of flbA (regulator of G protein signaling) and gprD-2 (encoding GPCR) were also elevated in ΔMrwetA on the 5th d, while the opposite was observed for rasA (GTPase) (Figure 6B).Moreover, in the MAPK (mitogen-activated protein kinase) cascade signaling pathway, the expression levels of rsp5 (E3 ubiquitin-protein ligase) and ste11 (mitogen-activated protein kinase) were down-regulated at different time points, while the other genes were up-regulated on the 5th and 7th d (Figure 6C).Among DEGs related to fruiting body maturation as shown in Figure 6D, only the expression level of vosA (trehalose production) decreased on the 5th and 7th d.
Light and nutrient factors in the environment serve as important signals for the fungal sexual reproduction process.As shown in Figure 6E, the expression levels of veA and velB related to light response increased on the 5th and 7th d, while the opposite was observed for silG.Changes in the expression levels of lsdA, phoA, An-pho80, and fhbA related to nutrient perception varied across time points.In addition, the expression levels of esdC, which regulates early sexual development, increased on the 5th and 7th d.On the other hand, DEGs related to various physiological processes (Figure 6F) demonstrated decreased expression levels of noxA (NADPH oxidase), mutA (α-1,3 glucanase/mutanase), and hxtA (high-affinity hexose transporter) on the 3rd d compared with WT.Moreover, the expression of noxA was increased on the 5th and 7th d.Mating-type (MAT) genes and genes related to signal transduction pathways play a crucial role in regulating fungal sexual reproduction.In comparison with the M7, MAT1-2 expression levels were down-regulated on the 5th and 7th d, and pheromone receptor preA (regulated by MAT1-2) expression levels were down-regulated on the 3rd and 5th d in the deletant (Figure 6A).G protein-coupled receptors (GPCRs), possessing seven transmembrane structures, are activated upon binding to pheromones and then bind to heterotrimeric G proteins.In G protein signaling pathway, the expression levels of gprD-1 (encoding GPCR) were elevated on the 3rd and 5th d.In addition, the expression levels of flbA (regulator of G protein signaling) and gprD-2 (encoding GPCR) were also elevated in ∆MrwetA on the 5th d, while the opposite was observed for rasA (GTPase) (Figure 6B).Moreover, in the MAPK (mitogen-activated protein kinase) cascade signaling pathway, the expression levels of rsp5 (E3 ubiquitin-protein ligase) and ste11 (mitogen-activated protein kinase) were down-regulated at different time points, while the other genes were up-regulated on the 5th and 7th d (Figure 6C).Among DEGs related to fruiting body maturation as shown in Figure 6D, only the expression level of vosA (trehalose production) decreased on the 5th and 7th d.
Light and nutrient factors in the environment serve as important signals for the fungal sexual reproduction process.As shown in Figure 6E, the expression levels of veA and velB related to light response increased on the 5th and 7th d, while the opposite was observed for silG.Changes in the expression levels of lsdA, phoA, An-pho80, and fhbA related to nutrient perception varied across time points.In addition, the expression levels of esdC, which regulates early sexual development, increased on the 5th and 7th d.On the other hand, DEGs related to various physiological processes (Figure 6F) demonstrated decreased expression levels of noxA (NADPH oxidase), mutA (α-1,3 glucanase/mutanase), and hxtA (high-affinity hexose transporter) on the 3rd d compared with WT.Moreover, the expression of noxA was increased on the 5th and 7th d.
Transcription factors and genes related to fruiting body maturation are indispensable from the mating process to the maturation of cleistothecia.In transcription factors, compared with WT, the expression levels of nsdD and fhpA decreased on the 3rd d; On the 5th d, the expression levels of nsdC, nosA, and flbC increased, while medA decreased; on the 7th d, both nosA expression levels increased, in contrast to fhpA (Figure 6G).
In summary, MrwetA regulated various genes associated with the sexual reproduction of M7, consistent with the findings presented in Figure 2.

Effects of MrwetA on Meiosis
During sexual reproduction in fungi, the formation of haploid spores requires meiosis.As depicted in Figure 7, five DEGs (cdc28, snf1, ume6, dbf4, and sok2) were detected during the Premeiotic S phase and prophase, three DEGs (hop1, cdc28, and ndt80) during meiosis I, and three DEGs (cdc20, ama1, and cdc15) during meiosis II.Additionally, five DEGs (clb3/4, slk19, rad24, rad9, and cdc14) were detected between meiosis I and meiosis II.Compared with WT, the expression levels of cdc28, ndt80, and cdc15 were decreased on the 3rd d.Furthermore, the number of DEGs on the 5th and 7th d significantly exceeded that of the 3rd d, with a consistent expression trend observed at both time points, predominantly characterized by decreased expression levels among the majority of DEGs.These changes in DEGs involved in meiosis align with the morphology and yield changes in cleistothecia and ascospores.
Transcription factors and genes related to fruiting body maturation are indispensable from the mating process to the maturation of cleistothecia.In transcription factors, compared with WT, the expression levels of nsdD and fhpA decreased on the 3rd d; On the 5th d, the expression levels of nsdC, nosA, and flbC increased, while medA decreased; on the 7th d, both nosA expression levels increased, in contrast to fhpA (Figure 6G).
In summary, MrwetA regulated various genes associated with the sexual reproduction of M7, consistent with the findings presented in Figure 2.

Effects of MrwetA on Meiosis
During sexual reproduction in fungi, the formation of haploid spores requires meiosis.As depicted in Figure 7, five DEGs (cdc28, snf1, ume6, dbf4, and sok2) were detected during the Premeiotic S phase and prophase, three DEGs (hop1, cdc28, and ndt80) during meiosis I, and three DEGs (cdc20, ama1, and cdc15) during meiosis II.Additionally, five DEGs (clb3/4, slk19, rad24, rad9, and cdc14) were detected between meiosis Ⅰ and meiosis Ⅱ.Compared with WT, the expression levels of cdc28, ndt80, and cdc15 were decreased on the 3rd d.Furthermore, the number of DEGs on the 5th and 7th d significantly exceeded that of the 3rd d, with a consistent expression trend observed at both time points, predominantly characterized by decreased expression levels among the majority of DEGs.These changes in DEGs involved in meiosis align with the morphology and yield changes in cleistothecia and ascospores.

Effects of MrwetA on Secondary Metabolites
MPs and CIT are the main secondary metabolites in M7 and deletion of MrwetA disturbed the yield of these products.To elucidate the underlying mechanism, the genes involved in the biosynthesis of MPs and CIT [11][12][13] were analyzed (Figure 8).Compared with WT, there was no significant difference in the expression level of genes related to

Effects of MrwetA on Secondary Metabolites
MPs and CIT are the main secondary metabolites in M7 and deletion of MrwetA disturbed the yield of these products.To elucidate the underlying mechanism, the genes involved in the biosynthesis of MPs and CIT [11][12][13] were analyzed (Figure 8).Compared with WT, there was no significant difference in the expression level of genes related to MPs biosynthesis on the 3rd d in ∆MrwetA (Figure 8A).However, the expression levels of MrPigA-G and MrPigJ-P were significantly higher on the 5th and 7th d, while the expression level of MrPigI decreased only on the 7th d.According to He and Cox.[11], key genes in the CIT major biosynthetic pathway include pksCT, mrl1, mrl2, mrl4, mrl6, and mrl7.As depicted in Figure 8B, the expression levels of mrl1, mrl2, mrl5, and mrl7 increased on the 3rd d, mrl3 and mrl5-7 on the 5th d, pksCT, mrl1-3, and mrl7 on the 7th d in ΔMrwetA compared with WT.These changes in MPs and CIT biosynthesis-related genes were consistent with the increased yield of MPs and CIT, indicating that MrwetA regulated the production of MPs and CIT through the biosynthesis pathway.

Discussion and Conclusions
Based on the previous findings [20], deletion of MrwetA hindered sexual development and affected the yield of secondary metabolites, while complementation of MrwetA totally restored these observed disorders, verifying its role in sexual development and biosynthesis of secondary metabolites.On the other hand, MrwetA deletants and overexpressed mutants both exhibited defects in asexual development [20].This pattern of According to He and Cox.[11], key genes in the CIT major biosynthetic pathway include pksCT, mrl1, mrl2, mrl4, mrl6, and mrl7.As depicted in Figure 8B, the expression levels of mrl1, mrl2, mrl5, and mrl7 increased on the 3rd d, mrl3 and mrl5-7 on the 5th d, pksCT, mrl1-3, and mrl7 on the 7th d in ∆MrwetA compared with WT.These changes in MPs and CIT biosynthesis-related genes were consistent with the increased yield of MPs and CIT, indicating that MrwetA regulated the production of MPs and CIT through the biosynthesis pathway.

Discussion and Conclusions
Based on the previous findings [20], deletion of MrwetA hindered sexual development and affected the yield of secondary metabolites, while complementation of MrwetA totally restored these observed disorders, verifying its role in sexual development and biosynthesis of secondary metabolites.On the other hand, MrwetA deletants and overexpressed mutants both exhibited defects in asexual development [20].This pattern of consistent phenotypes resulting from both deletion and overexpression is also observed in genes in other fungi, where the strict expression levels of these genes are crucial either for their own functionality [43,44] or for their involvement in the formation of protein complexes [45] essential for an organism to form protein complexes for organisms.Although this study confirmed the important role of M7, especially for the cleistothecia, further investigation is needed to determine whether it works through complex or solely.Furthermore, in this study, 1703 DEGs (|log2 FC| ≥ 0.5849, Q value ≤ 0.05) were detected on the 7th d, accounting for 22.2% of all genes (9% up-regulated and 13.2% down-regulated) of all genes.However, the RNA-seq data showed that after wetA deletion, DEGs (|log2 FC| ≥ 1, Q value ≤ 0.05) accounted for 52% (28% up-regulated and 24% down-regulated), 57% (30% up-regulated and 27% down-regulated), 43% (21.6% up-regulated and 21.4% down-regulated) of all genes in A. nidulans, A. fumigatus, and A. flavus, respectively [34].The percentage of wetA-regulated genes in M7 was significantly lower than those in the aforementioned species.Additionally, a larger proportion of down-regulated genes was observed, suggesting a unique regulatory network of wetA in M7.
wetA has been reported to regulate asexual reproduction in various filamentous fungi such as A. nidulans, A. fumigatus, F. graminearum, and B. bassiana [25,30,46,47].However, disruption of it in M7 hindered sexual reproduction and showed no effect on asexual reproduction [20].The underlying mechanism remains elusive, because of the complexity of sexual reproduction involving numerous genes.Referred to Dyer and O'Gorman [17], the genes related to sexual reproduction in M7 also can be categorized into six groups (Mating processes, signal transduction, perception of environmental signals, transcription factors, regulatory proteins, and fruiting body maturation).
MAT genes play a pivotal role in fungal sexual reproduction, orchestrating the expression of pheromone-related genes and GPCRs, which are pivotal for downstream signaling cascades, including the MAPK pathway, essential for sexual development [48][49][50].In this study, there have been alterations in the expression levels of MAT1-2, gprD, rasA, and ste11, which are involved in MAT processes, GPCRs, and the MAPK pathway, respectively.Furthermore, environmental signal reception, transcription factors, endogenous physiological processes and genes related to fruiting body maturation also participate in the process of sexual reproduction.In our study, expression changes in genes related to these processes were identified, suggesting their involvement in sexual reproduction regulation.For instance, changes in the expression of vosA and noxA genes imply alterations in trehalose production and redox reactions, respectively, influencing cleistothecia development.
Meiosis is another necessary process for the sexual reproduction of haploid spores in filamentous fungi.In budding yeast, specific transcription factors such as ime1 initiate the meiosis process, subsequently regulating the expression of genes including ime2 and cdc28 to promote meiosis [51,52].Based on RNA-Seq data, the expression levels of cdc28, ndt80, and cdc15 were significantly decreased on the 3rd d, while the number of down-regulated genes increased on the 5th and 7th d.This suggests a delayed onset of meiosis-related gene expression in ∆MrwetA, potentially contributing to the observed reduction in cleistothecia and ascospores.
In summary, our findings underscore the role of MrwetA in the sexual development and secondary metabolism of M7.By affecting the expression of genes involved in sexual reproduction, meiosis, and secondary metabolite biosynthesis, MrwetA contributes to the regulatory network governing these processes.These insights advanced our understanding of reproduction and metabolism in M7, while also broadening our knowledge of wetA function and sexual reproduction regulation in filamentous fungi.

Supplementary Materials:
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/jof10050338/s1, Figure S1.Schematic diagram of constructing transformation plasmid pCCMrwetA.Figure S2.Verification of PCR products and vector on MrwetA complementation cassette.Figure S3.PCR verification of MrwetA complementation strain.Figure S4.KEGG Pathway classification of DGEs.Table S1.Primers used in the construction of MrwetA complementation strain.Table S2.Primer combinations and expected sizes for validation of ∆MrwetA::MrwetA strains.Table S3.Homologues of sexual reproduction related genes of Aspergillus nidulans in Monascus ruber M7.
Author Contributions: Y.H.: formal analysis, writing-original draft.L.J.: methodology, writing-draft and editing.F.C.: project administration, supervision, writing-review and editing.All authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by National Natural Science Foundation of China (Nos.31730068 and 31330059).

Figure 4 .
Figure 4.The number of DEGs.(A) Number of DEGs on the 3rd d (B) Number of DEGs on the 5 d (C) Number of DEGs on the 7th d (D) Venn diagram of DEGs.DEGs: |log2 FC| ≥ 0.5849, Q val ≤ 0.05.

Figure 4 .
Figure 4.The number of DEGs.(A) Number of DEGs on the 3rd d (B) Number of DEGs on the 5th d (C) Number of DEGs on the 7th d (D) Venn diagram of DEGs.DEGs: |log2 FC| ≥ 0.5849, Q value ≤ 0.05.

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. Fungi 2024, 10, x FOR PEER REVIEW 8 of 15 antibiotics, biosynthesis of amino acids, amino sugar and nucleotide sugar metabolism, and glycine, serine and threonine metabolism (Figure5B,C).

Figure 5 .
Figure 5. KEGG enrichment analysis of DEGs in WT vs. ΔMrwetA.The top 20 KEGG pathways enriched by DEGs on the 3rd d (A), 5th d (B), and 7th d (C).The size of the dot indicates the number of DEGs, and a smaller p-value indicates a more significant enrichment.

Figure 5 .
Figure 5. KEGG enrichment analysis of DEGs in WT vs. ∆MrwetA.The top 20 KEGG pathways enriched by DEGs on the 3rd d (A), 5th d (B), and 7th d (C).The size of the dot indicates the number of DEGs, and a smaller p-value indicates a more significant enrichment.

Table 1 .
Strains used in this study.