{"paper_id":"32947b0e-c843-4657-a151-0bb45da29404","body_text":"The transcription factor UvCreA regulates ustilaginoidin synthesis and pathogenicity in Ustilaginoidea virens | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The transcription factor UvCreA regulates ustilaginoidin synthesis and pathogenicity in Ustilaginoidea virens xiaolong Bai, Zequn Pan, Ying Du, Muhammad Zulqar Nain Dara, Jing Wang, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9251727/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Ustilaginoidea virens generates several types of mycotoxins, including ustilaginoidins during infection. The transcription factor CreA plays a central role in carbon catabolite repression, coordinating carbon source utilization during fungal growth, development, and host infection. However, knowledge is limited regarding the specific functions and molecular mechanisms by which CreA regulates mycotoxin biosynthesis and pathogenicity. In this study, we found that the wild-type U. virens strain exhibited significantly different ustilaginoidin-producing capacities under distinct carbon sources. The UvCreA gene knockout mutants exhibited slowed mycelial growth, decreased pathogenicity and ustilaginoidin production, but produced significantly more conidiospores than the wild type. Consistently, the expression of ustilaginoidin biosynthetic genes was significantly down-regulated in the UvCreA mutant. Moreover, yeast one-hybrid and electrophoretic mobility shift assays confirmed that UvCreA binds to the promoter regions of polyketide synthase gene UvPKS1 and methyltransferase gene UgsJ . ChIP-qPCR showed significant enrichment in promoter regions of UvPKS1 and UgsJ . These findings indicate that UvCreA not only controls ustilaginoidin biosynthesis through directly binding the Ugs gene promoters, but also positively regulates mycelium growth and pathogenicity in U. virens . Therefore, this study provides theoretical insights into the mechanisms of carbon metabolism and mycotoxin biosynthesis in phytopathogenic fungi. Ustilaginoidea virens Rice false smut Transcription factor UvCreA Pathogenicity Ustilaginoidins Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Rice false smut (RFS) has become one of the most damaging fungal diseases of rice worldwide, which is caused by Ustilaginoidea virens . RFS epidemic is largely attributed to a combination of factors, including climate change, excessive use of chemical fertilizers, high-yield cultivation practices, and widespread adoption of hybrid rice varieties (Tang et al. 2013 ). U. virens infects rice florets by colonizing the stamen filaments (Li et al. 2013 ), and covers the rice grains with powdery chlamydospores, known as false smut balls (Chen et al. 2020 ). U. virens produces ustiloxins and ustilaginoidins, besides other multiple types of mycotoxins (Wen et al. 2023 ; Zhou et al. 2012 ), seriously affecting grain quality (Lin et al. 2018 ). Ustilaginoidins mycotoxins are part of the naphtho-γ-pyrone class, which are usually soluble in organic solvents (Meng et al. 2015 ; Wang et al. 2019 ). Specifically, Ustilaginoidin D has been demonstrated to induce a substantial delay in yolk sac absorption and hepatic injury in zebrafish (Wang et al. 2021 ). The biosynthesis of ustilaginoidins is controlled by the Ugs ( u stila g inoidin s ynthesis) gene cluster, which contains at least fourteen genes (Li et al. 2019 ). Among them, UvPKS1 encodes a polyketide synthase that catalyzes the initial step in ustilaginoidin biosynthesis. Knockout of UvPKS1 in U. virens results in complete cessation of ustilaginoidin production, including the intermediates. The gene UgsJ encodes a methyltransferase responsible for methylation at the C3-3' side chain of the pyranone ring. In comparison with the wild type, the UgsJ -knockout strain generates a greater quantity of ustilaginoidins F, G, and A, which are derivatives that lack the C3-3' methylation modification of the pyranone ring (Li et al. 2019 ). However, the regulatory mechanisms underlying ustilaginoidin biosynthesis remain largely unknown. Carbon catabolite repression (CCR) serves as a regulatory mechanism that controls carbon utilization, influencing growth, development, and secondary metabolism in fungi (Xu et al. 2022 ). The highly conserved transcription factors, catabolite-responsive elements, CreA/Cre1 have been considered as major regulatory factors in the CCR mechanism (Mogensen et al. 2006 ). When preferred carbon sources such as glucose are present, expression of genes encoding hydrolases for secondary carbon sources is suppressed (Yoav et al. 2018 ). CreA suppresses the transcription of secondary carbon source hydrolase genes through direct bindind with the conserved cis -acting element 5'-SYGGRG-3' in the promoter region of the target gene (Benocci et al. 2017 ). This conserved mechanism enables efficient utilization of carbon sources and represents a crucial transcriptional control system for carbon nutrient selectivity in microorganisms (New et al. 2014 ). CreA is also involved in growth and development, secondary metabolite synthesis, and pathogenicity in phytopathogenic fungi. Aspergillus ochraceus contaminates a wide range of foods and feeds by producing ochratoxin A (OTA). AoCreA plays a crucial role in modulating OTA biosynthesis and influences morphological development in response to different carbon sources (Wang et al. 2020 ). In A. flavus , CreA regulates the biosynthesis of aflatoxin and sclerotium production (Fasoyin et al. 2018 ). A recent study showed that UvCreA plays an important role in virulence and carbon source utilization through transcriptional regulation in U. virens , and is a key regulatory factor in CCR (Xie et al. 2024 ). Additionally, CreA responds to carbon source signals and controls the expression of secondary metabolic genes and the production of secondary metabolites across various species. (Yu et al. 2023 ). Therefore, CreA is implicated not only in the regulation of CCR but also in the biosynthesis of secondary metabolites and fungal pathogenicity. However, limited knowledge exists regarding the molecular mechanisms through which the transcription factor regulates mycotoxin biosynthesis. In this study, we demonstrate that different carbon sources significantly influence ustilaginoidin accumulation. We identify UvCreA as a global regulator affecting fungal growth, conidial production and germination, secondary metabolism, stress response, and pathogenicity. More importantly, UvCreA directly binds to the promoters of UvPKS1 and UgsJ within the Ugs gene cluster, thereby regulating ustilaginoidin biosynthesis. These findings not only reveal the role of UvCreA in U. virens development and pathogenicity but also offer new insights into the molecular mechanisms regulating ustilaginoidin biosynthesis, suggesting possible molecular drug targets for managing rice false smut. 2. Materials and methods Both nucleotide and amino-acid sequences were retrieved from the NCBI database ( https://www.ncbi.nlm.nih.gov/ ). The alignment analyses were conducted using DNAMAN 6 (v 6.0) and MEGA Ⅹ (v 10.0). Primer sequences employed in this work are provided in Supplementary Table S1 . The assays conducted in this study were independently repeated three times, unless noted. 2.1 Strains and culture media The strain was cultivated on PSA plates consisting of 200 g/L potato, 20 g/L sucrose, and 20 g/L agar. The U. virens strain P1FZ was used as the wild type (WT). Furthermore, strains were cultured on PSA plates (Jiang et al. 2026 ) or complete medium (CM, containing 1 g of yeast extract, 6 g of NaNO₃, 2 g of peptone, 1 g of acid hydrolyzed casein, 1 g of MgSO₄·7H₂O, 0.5 g of NaCl, and 1.5 g of KH₂PO₄ per liter) supplemented with different carbon sources (10g glucose, sucrose, D-fructose, or trehalose), subsequently incubated at 28°C, as described earlier (Momany et al. 1999 ). 2.2 Construction of UvCreA deletion mutants, complemented, and overexpression strains Deletion of the UvCreA gene was accomplished by the CRISPR/Cas9 system and homologous recombination (Jiang et al. 2026 ). The flanking sequences of the target gene and the hygromycin resistance gene ( Hyg ) from plasmid pGKO2- Hyg were independently amplified. Subsequently, these UvCreA sequences were fused to the 5’ and 3’ termini of the Hyg gene via fusion PCR. Primer pairs specific for the Cas9 target site and sgRNA were designed using an online tool ( https://portals.broadinstitute.org/gppx/crispick/public ). Following the annealing process, the sgRNA primers were incorporated into the pCAS9-tRp vector, which had been pre-digested with BsmB I (Li et al. 2019 ). The PCR products and construct were transformed into U. virens protoplasts using the polyethylene glycol method (Fu et al. 2018 ). The mutants were first screened by PCR and then confirmed by Southern blot analysis as described earlier (Meshram et al. 2010). For complementation and overexpression purposes, the coding sequence of UvCreA and the robust HSP70 promoter (OE8266) were cloned into the pY2P102-FLAG vector (Li et al. 2019 ). The constructed plasmid was introduced into the wild-type and Δ UvcreA -64 strains. An anti-FLAG antibody (Sigma-Aldrich, 12352203, St. Louis, MO) was used to confirm protein expression by immunoblot analysis. 2.3 Determination of growth rate, conidiogenesis, and conidial germination rate Mycelial plugs were inoculated onto PSA plates and subsequently cultured at 28°C for 14 days. The colony morphology and diameters were thereafter documented through photography and measurement, respectively. The fungal hyphae were examined under a microscope (Wang et al. 2024 ). For the conidiation assay, various strains of U. virens were independently inoculated into 75 mL of PSB medium and incubated in a shaker at 150 rpm for seven days at 28°C. The culture was filtered into a 50 mL sterile centrifuge tube using sterilized three-layer lens paper. The conidia were harvested and resuspended in PSB medium to a concentration of 10 6 spores/mL. A 100 µL aliquot of the spore suspension was uniformly spread on PSA agar plates and incubated at 28°C. Conidial germination was monitored using a microscope, and germination rates were calculated at various stages of growth (Long et al. 2024 ). 2.4 Quantification of ustilaginoidins by high-performance liquid chromatography (HPLC) U. virens strains were cultured at 28°C for 28 days on PSA plates covered with cellophane or on complete medium (CM) plates supplemented with 1% glucose, sucrose, D-fructose, or trehalose. The mycelia were collected in a 50 mL centrifuge tube containing 30 mL of ethyl acetate, then incubated at 28°C for 24 hours on a shaker at 150 rpm. Subsequently, the mixture was concentrated to dryness, reconstituted in acetonitrile, and filtered through a 0.22 µm filter (Wang et al. 2024 ). Samples were then analyzed using an LC-20A HPLC system (Shimadzu, Japan). 2.5 Stress tolerance assays Different strains of U. virens were cultured on YT medium plates supplemented with 0.015% sodium dodecyl sulfate (SDS), 2.5 mM H 2 O 2 , 70 µg/mL Congo red, or 0.25 M NaCl at 28°C for 14 days as described (Wang et al. 2024 ). Growth inhibition rates were calculated by measuring colony diameters on at least three plates. 2.6 Virulence assay Various strains of U. virens , including wild-type, gene-knockout, complemented, and overexpression, were cultured in 75 mL of PSB medium and incubated in a shaker at 150 rpm and 28°C for 7 days. To homogenize the culture, a blender was used to prepare a hyphal and conidial suspension containing 2×10 6 conidia/mL, which was injected into panicles (1 mL per panicle) of the susceptible rice cultivar JN853, approximately 1 week before the heading stage. The inoculated panicles were observed and photographed approximately 1 month after inoculation (Liu et al. 2023 ; Long et al. 2024 ). 2.7 RNA isolation and RT-qPCR Total RNA was extracted from 14-day-old U. virens cultures using an Ultrapure RNA Kit (CWBIO). Complementary DNA (cDNA) synthesis was performed with a PrimeScript reverse transcription kit (TaKaRa, RR047A, Japan) following the manufacturer's standard protocols. Quantitative reverse transcription PCR (RT-qPCR) was conducted to assess Ugs gene expression levels utilizing the Fast SYBR mix (CWBIO, CW0955M, Jiangsu, China) on a LightCycler® 96 system (Roche, Basel, Switzerland) as previously described (Wang et al. 2025 ). The α-tubulin gene was utilized as the internal control, and the relative gene expression levels were quantified employing the 2 −ΔΔCt method (Rao et al. 2013 ). 2.8 Yeast one-hybrid assay The yeast one-hybrid assay was conducted by using the LexA one-hybrid system following the procedure described earlier (Wanke et al. 2009). Briefly, the UvCreA coding sequence was cloned into the pB42AD vector as a prey, while multiple gene promoters were cloned into the pLacZi vector. These constructs were co-transformed into chemically competent EGY48 cells (Coolaber CC302, Beijing, China) according to the manufacturer’s instructions. Transformants were grown on SD medium lacking Trp and Ura. Positive interactions were confirmed by β-galactosidase activity using X-gal. 2.9 Electrophoretic mobility shift assay (EMSA) The coding sequence of UvCreA was subcloned into the pGEX4T-1 vector for expression of glutathione S-transferase (GST) tagged proteins. The construct was introduced into Escherichia coli BL21 (DE3) cells (Sangon, A330305, Shanghai, China). The recombinant protein was purified as described (Yu et al. 2025 ). EMSA was conducted using a chemiluminescent EMSA kit (Beyotime, GS009, Shanghai, China). Briefly, the forward primers were labeled with biotin at the 5' end to generate double-stranded probes. Unlabeled and biotin-labeled mutant probes were used as controls. The biotin-labeled probe and mutant probe were diluted to 0.2 µM for further use, and the unlabeled probe was diluted to 1 µM (10×) and 2 µM (100 ×) for competition assays. The mixture sample was analyzed by polyacrylamide gel electrophoresis. 2.10 Chromatin immunoprecipitation (ChIP) and qPCR ChIP was performed as previously reported (Tang et al. 2021 ). The 14-day-old cultures of both the wild-type and UvCreA -overexpressing strains were subjected to treatment with 1% formaldehyde for 10 minutes to facilitate cross-linking. Subsequently, 150 mM glycine was introduced to terminate the cross-linking process. The samples were then sequentially washed with distilled water, followed by extraction buffer I, extraction buffer II, and finally, extraction buffer Ⅲ. Subsequently, the samples were pelleted at 4°C and resuspended in nucleus lysis buffer. Genomic DNA was fragmented to a size range of 200–500 bp utilizing a sonication device (Diagenode Bioruptor, Belgium), followed by centrifugation at 16,000 g at 4°C for 10 minutes. The supernatant was subsequently diluted with ChIP dilution buffer and divided into three aliquots. One aliquot was kept as input, while the other two were incubated with anti-FLAG (Abcam, Ab205606, Cambridge, UK) and protein A/G beads (Abcam, ab286842, Cambridge, UK) or with anti-IgG antibodies at 4°C overnight on a rotary shaker. Following incubation, the beads were pelleted by centrifugation at 3,000g for 30 seconds and subjected to sequential washes with distinct washing buffers, including (i) low-salt buffer, (ii) high-salt buffer, and (iii) LiCl buffer. The bead-bound DNA was eluted by TE buffer. Finally, ChIP-qPCR was conducted utilizing immunoprecipitated DNA as the template, employing Taq Pro Universal SYBR qPCR Master Mix (Vazyme) and a LightCycler 96 (Roche, Basel, Switzerland). The enrichment fold was determined by comparing the signal from the immunoprecipitated (IP) sample using an anti-FLAG antibody to that of the negative control (IgG). 2.11 Statistical analyses All data were analyzed with GraphPad Prism 9.5 (La Jolla, CA), while IBM SPSS Statistics 26 (IBM, New York) was employed to present the data as mean ± standard deviation (SD). Statistically significant differences ( P < 0.05) were evaluated through one-way ANOVA, followed by Tukey’s test or Student’s t -test. 3. Results 3.1 Carbon sources pose distinct effects on ustilaginoidin accumulation and mycelial growth in U. virens For the assessment of the impact of various carbon sources on ustilaginoidin biosynthesis, the wild-type strain was cultivated on CM or modified CM medium plates, wherein sucrose was replaced with glucose, D-fructose, or trehalose as the exclusive carbon source. Ustilaginoidins were extracted from cultures grown for 28 days on various medium plates and were then quantified through HPLC. Among the tested carbon sources, U. virens generated the highest amount of ustilaginoidins when culturing on CM medium with D-fructose as the sole carbon source. In contrast, the fungus produced the lowest level of ustilaginoidins when cultured on a glucose-containing CM medium (Fig. 1 A). In addition, we compared the expression levels of UvPKS1 and UgsJ in the wild-type strain when culturing on four types of carbon source media for 14 days. RT-qPCR analysis revealed that across all four carbon sources, the expression of UvPKS1 and UgsJ was highest in D-fructose and trehalose, followed by sucrose, and lowest in glucose-containing medium (Fig. 1 B). These results demonstrated a significant effect of different carbon sources on ustilaginoidin production. 3.2 UvCreA is a putative zinc finger transcription factor in U. virens CCR, in filamentous fungi, is a key mechanism for regulating carbon source utilization, which is often mediated by the transcription factor CreA (Huang et al. 2025 ). First, we identified UvCreA as the transcription factor regulating CCR in U. virens through structural analysis. UvCreA contains two conserved zinc finger (ZF) domains, ZF1 and ZF2, located in the N-terminal region (Figure S1 A). These domains are predicted to be critical for DNA binding and protein-protein interactions. Second, a phylogenetic tree was constructed using the maximum-likelihood method for CreA homologs across different fungal species. Protein sequences were aligned using MEGA X, and the tree topology shows that UvCreA (XP_042997845.1) is evolutionarily closest to the homolog from Metarhizium anisopliae . By contrast, CreA homologs in Aspergillus form a distinct branch (Figure S1 B). Subsequently, the three-dimensional structure of UvCreA was predicted using AlphaFold2, revealing a complex folding pattern with multiple α-helices and β-sheets characteristic of zinc finger-containing proteins (Figure S1 C). Collectively, UvCreA is a putative transcription factor comprising two ZF domains for CCR regulation. 3.3 UvCreA regulates mycelial growth, conidial production, and germination rates in U. virens To identify the roles of UvCreA , the mutants with UvCreA- deletion were created via homologous recombination (Figure S2 A). Subsequently, the plasmid-borne UvCreA gene construct with a strong promoter, UV_08266 , was created and introduced into the protoplasts of both the wild-type and Δ UvcreA mutant strains using PEG-mediated transformation. Two UvCreA -complemented strains (CΔ UvcreA -64-15 and CΔ UvcreA -64-18) and two UvCreA -overexpressing strains (OE-UvCreA-FLAG-16 and OE-UvCreA-FLAG-18) were successfully verified by immunoblot analysis (Figure S2 B). After 14 days of culturing, colony morphology was observed for the wild-type, Δ UvcreA -4/64, CΔ UvcreA -64, and OE-UvCreA-FLAG strains (Fig. 2 A). The colony diameters were also measured, and we found that the Δ UvcreA -4 and − 64 mutants had smaller colony diameters than the wild-type strain (Fig. 2 A), whereas the mutants produced more conidia per conidiophore than the wild type (Fig. 2 B). The CΔ UvcreA -64 strain can restore these phenotypes near the wild-type levels. In addition, the colony diameter of the overexpressing strain was significantly greater than that of the wild-type strain (Fig. 2 C). Conidiation assays showed that the Δ UvcreA -4 and Δ UvcreA -64 mutants produce noticeably more conidiospores than the wild-type strain after 7 days of shaking culture. Interestingly, the UvCreA-FLAG -overexpressing strain generated far fewer conidia compared to the wild-type (Fig. 2 D). Additionally, we observed that the Δ UvcreA -4 and Δ UvcreA -64 mutants had much higher germination rates in PSA medium, while the overexpressing strain’s germination rate was significantly lower than that of the wild-type (Fig. 2 E). These findings suggest that UvCreA supports mycelial growth, but suppresses conidiogenesis and conidial germination in U. virens . 3.4 UvCreA positively regulates ustilaginoidin biosynthesis and pathogenicity in U. virens To investigate the role of UvCreA in the pathogenicity and ustilaginoidin production in U. virens , the amounts of ustilaginoidins produced by wild-type, UvCreA knockout, complemented, and overexpressing strains were measured. After culturing on PSA plates for 28 days, the mycelia were harvested for ustilaginoidin extraction using ethyl acetate. HPLC analysis showed that the Δ UvcreA mutant strains had significantly lower ustilaginoidin production compared with the wild-type strain. The CΔ UvcreA -64 complemented strain partially recovered the ability to produce ustilaginoidins. In contrast, the strain overexpressing UvCreA produced more ustilaginoidins than the wild type (Fig. 3 A). Additionally, the expression levels of genes involved in ustilaginoidin biosynthesis, including UgsR1, UgsR2, UgsO, UvPKS1, UgsZ, UgsT, UgsH , and UgsJ , were measured using RT-qPCR. The results showed that in Δ UvcreA -4 and Δ UvcreA -64 mutants, these Ugs genes were significantly downregulated, whereas in the CΔ UvcreA strain, the transcript levels of the tested genes were similar to or higher than those in the wild type (Fig. 3 B). Inoculation assays demonstrated that rice panicles inoculated with Δ UvcreA mutants exhibited significantly fewer false smut balls in comparison with the wild-type strain (Fig. 3 C-D). Conversely, the OE-UvCreA-FLAG strain produced a greater number of false smut balls than both the wild-type and CΔ UvcreA -64 complemented strains. These findings imply that UvCreA plays a positive role in the biosynthesis of ustilaginoidins and contributes to the virulence of U. virens . 3.5 UvCreA negatively regulates tolerance to various stress factors Growth phenotypes of the wild-type, Δ UvcreA -4, Δ UvcreA -64, and CΔ UvcreA -64 strains were observed under various stress conditions to investigate the role of UvCreA in stress tolerance, including oxidative stress (2.5 mM H₂O₂), cell wall stress (70 µg/mL Congo red), cell membrane stress (0.015% SDS), and osmotic stress (0.25 M NaCl) (Fig. 4 A). The Δ UvcreA mutants showed significantly lower relative growth inhibition rates than the wild-type and CΔ UvcreA -64 strains under all tested stress conditions. Specifically, under H₂O₂ and Congo red stress conditions, Δ UvcreA -4 and Δ UvcreA -64 exhibited negative growth inhibition rates. these findings indicate that the UvCreA -knockout mutants demonstrate greater tolerance to H₂O₂ and Congo red than the wild-type strain on YT medium plates. In comparison, when subjected to SDS and NaCl, the inhibition rates of the Δ UvcreA − 4 and Δ UvcreA − 64 strains were notably lower than those of the wild-type strain (Fig. 4 B). Collectively, these results indicate that deletion of UvCreA reduces the sensitivity of U. virens to oxidative, cell wall, membrane, and osmotic stresses. 3.6 UvCreA positively regulates the expression of ustilaginoidin biosynthetic genes through binding gene promoters As a transcription factor, UvCreA regulates the expression of numerous genes. Therefore, we predicted the UvCreA-binding cis- elements through the JASPAR database ( https://jaspar.elixir.no/ ) and found that S(G/C)Y(T/C)GGR(G/A)G is a putative cis- element (Fig. 5 A). Furthermore, we predicted the number of UvCreA-binding cis -elements in the promoter regions of the ustilaginoidin synthesis-related genes (Table S2 ). To investigate the effect of UvCreA on expression of ustilaginoidin biosynthetic genes, we chose the UgsJ and UvPKS1 genes for validation. The coding sequence of UvCreA and the promoter regions (~ 1.5 kb) of UgsJ and UvPKS1 genes were amplified to construct the vectors pB42AD- UvCreA , pLacZi- UgsJ Pro , and pLacZi- UvPKS1 Pro , respectively. The subsequent yeast one-hybrid assay revealed that the yeast colonies co-transformed with pB42AD- UvCreA /pLacZi- UgsJ Pro and with pB42AD- UvCreA /pLacZi- UvPKS1 Pro turned blue in the presence of X-Gal (Fig. 5 B), indicating that UvCreA binds the promoter regions of UvPKS1 and UgsJ . To further determine whether UvCreA binds to the cis -elements in the promoters of UvPKS1 and UgsJ , we performed electrophoretic mobility shift assays. Primers containing the cis -element GTGGGG or CCCCAC, with 7-bp flanking regions on either side, were designed. These primers were biotin-labelled as EMSA probes. When in vitro purified GST-UvCreA was incubated with a biotin-labeled probe derived from the UvPKS1 or UgsJ promoter in EMSA, distinct band shifts were observed. In addition, the shifted bands gradually faded when increasing amounts of unlabeled probes were added. While GST-UvCreA was incubated with biotin-labeled mutant probes as negative controls, the shift bands disappeared (Fig. 5 C-D). The results collectively indicate that UvCreA directly binds the cis -elements in the promoters of UvPKS1 and UgsJ . To confirm that UvCreA specifically binds to the UvPKS1 and UgsJ promoters, we conducted ChIP assays using the OE-UvCreA-FLAG strain. Genomic DNA was fragmented and then incubated with anti-FLAG antibody or IgG antibody in the presence of protein A/G beads. ChIP-qPCR was performed using bead-bound DNA as a template to investigate whether UvCreA enriches the promoter regions of UvPKS1 and UgsJ . We designed individually two pairs of primers to validate CreA enrichment in the promoter regions of UvPKS1 and UgsJ . The UvPKS1 Pro -1 and UgsJ Pro -1 primers were used to amplify regions containing the cis -element motifs, whereas the UvPKS1 Pro -2 and UgsJ Pro -2 primers amplify control regions without the cis -element (Fig. 5 E). The results showed that the UvPKS1 and UgsJ promoter regions immunoprecipitated by anti-FLAG antibody were significantly enriched compared with the anti-IgG control (Fig. 5 F). These results indicate that UvCreA directly and specifically binds to the UvPKS1 and UgsJ promoters. 4. Discussion During infection, fungi have to utilize limited nutrients efficiently to complete the infection process. Carbon sources are one of the most important nutrients. Numerous empirical studies have shown that carbon source utilization regulates various physiological phenotypes, including growth rate, conidial yield, and secondary metabolite synthesis (Achimón et al. 2019 ; Achimón et al. 2021 ; Ruiz et al. 2010 ). CreA plays a pivotal role as a key regulatory factor in this process (Brown et al. 2014 ). Starch and sugar metabolism provide important carbon sources for plant pathogens during infection, which is closely related to pathogenicity. The transcriptome and metabolome correlation analysis reveals that four metabolites in rice, including glucose-6-phosphate, trehalose, sucrose, and D-fructose, are significantly different before and after U. virens infection (Ozcengiz et al. 2013). Ustilaginoidins are major mycotoxins produced by U. virens , and pose a severe impact on human and animal health and rice quality (Sun et al. 2017 ). Studies have shown that the type of carbon source directly affects the production of mycotoxins (Szilágyi et al. 2013 ). In Penicillium chrysogenum , glucose, sucrose, and to a certain extent, fructose all have a negative impact on the biosynthesis of penicillin (Ruiz-Villafán et al. 2022 ). Penicillin biosynthesis in P. chrysogenum is subject to CCR. Specifically, glucose has been shown to strongly inhibit penicillin production by down-regulating the expression of the biosynthetic gene cluster ( pcbAB , pcbC , penDE ) (Ozcengiz et al. 2013; Ruiz-Villafán et al. 2022 ). In this study, we demonstrated that the wild-type strain produced higher levels of ustilaginoidins on D-fructose and trehalose-containing media than on glucose or sucrose-containing media (Fig. 1 A). This might be attributed to glucose and sucrose being preferred or superior carbon sources in filamentous fungi (Fernandez et al. 2012 ), whereas D-fructose and trehalose are alternative carbon sources (Nihashi et al. 1984). Next, we investigated the role of UvCreA in the pathogenicity of U. virens . Consistent with a previous study (Xie et al. 2024 ), we found that deletion of UvCreA reduced growth rate and pathogenicity in U. virens . More interestingly, conidial yield and germination rate of Δ UvcreA mutants were significantly increased (Fig. 2 ). By contrast, in the rice blast fungus Magnaporthe oryzae , the deletion of MoCreA results in reduced growth, conidial yield, and delayed growth (Wanke et al. 2009). The Cre1- deletion mutant in Trichoderma reesei also exhibits morphological changes, including reduced ascospore formation (Nakari-Setälä et al. 2009 ). These findings indicate that CreA has diverse regulatory mechanisms for conidiation across fungal species, providing a new perspective on its role in carbon source utilization. Moreover, CreA is widely involved in regulating stress tolerance in filamentous fungi, but its specific functions may vary across species. The Δ creA mutant in P. chrysogenum is more sensitive to calcofluor white (CFW) than the wild type. In Valsa mali , CFW-induced cell wall stress significantly inhibits the growth of Δ creA , indicating that CreA may participate in maintaining cell wall integrity (Fasoyin et al. 2018 ). In our study, the Δ UvcreA mutant exhibited significantly enhanced tolerance to osmotic stress (NaCl), oxidative stress (H₂O₂), cell wall stress (Congo red), and membrane stress (SDS) (Fig. 4 ). This result is consistent with the phenotypic trait of Δ creA in A. flavus , which shows enhanced tolerance to salt and oxidative stresses. These differences indicate that CreA's regulatory role in stress responses shows significant species specificity in filamentous fungi. Secondary metabolites are synthesized during pathogen infection by enzymes encoded by biosynthetic gene clusters and are harmful to the host's physiological activities and growth (Grau et al. 2018 ). As a global transcription factor, CreA is presumably involved in secondary metabolism by regulating the expression of biosynthetic gene clusters. The mycotoxin deoxynivalenol (DON) produced by Fusarium graminearum is a triterpene compound synthesized by 15 TRI genes (Fernandez et al. 2012 ). The promoters of 10 TRI genes, including TRI6 , in the TRI gene cluster contain typical CreA-binding cis -elements 5' -SYGGRG − 3' (Hou Rui et al. 2018), suggesting that CreA regulates DON biosynthesis by regulating the expression of TRI genes. The biosynthesis of ustilaginoidin is regulated by the Ugs gene cluster in U. virens (Wang et al. 2024 ). Consequently, we investigated whether CreA regulates the expression of the Ugs gene cluster. Through yeast one-hybrid assay, EMSA, chromatin immunoprecipitation, and quantitative PCR, we confirmed that UvCreA regulates the expression of UvPKS1 and UgsJ by directly binding to the cis -elements 5' -SYGGRG − 3' in the promoters of these Ugs genes (Fig. 5 B). Similarly, CreA positively regulates aflatoxin biosynthesis in A. flavus (Wanke et al. 2009). Altogether, these results indicate that CreA is necessary for the synthesis of secondary metabolites and the utilization of carbon sources. Numerous genes in fungal genomes contain the SYGGRG motif, which is specifically recognized by CreA (Antoniêto et al. 2014 ; Cubero et al. 1994). Besides acting as a transcriptional repressor, CreA acts as a transcription factor that activates the expression of certain genes (Antoniêto et al. 2014 ). Therefore, the functions of CreA in regulating various fungal processes and activities remain to be further investigated. In this study, UvCreA, a key transcription factor in carbon source utilization, positively regulates ustilaginoidins biosynthesis, which may depend on the type of carbon sources (Fig. 1 ). This finding not only elucidates the molecular pathway through which carbon source types regulate ustilaginoidin synthesis via UvCreA but also provides critical insights into how the composition of carbon sources in rice panicles during the infection of rice by U. virens influences ustilaginoidin production by U. virens in field conditions. Specifically, the later stages of infection may involve the accumulation of greater amounts of trehalose or D-fructose in rice panicles, creating favorable conditions for ustilaginoidin biosynthesis by U. virens . Future studies could investigate the correlation between dynamic changes in carbon source composition in rice panicles and ustilaginoidin accumulation, offering a theoretical basis for developing targeted strategies to mitigate mycotoxin contamination. In conclusion, our results show that UvCreA positively regulates the growth, development, and pathogenicity of U. virens , but negatively regulates conidial production and germination, and tolerance to various stresses. As a global transcription factor, UvCreA positively regulates the biosynthesis of ustilaginoidins in U. virens by directly binding to the promoters in the Ugs gene cluster. Declarations A cknowledgements We thank the platform support from the College of Plant Protection at Jilin Agricultural University and China Agricultural University, and thank Wenxian Sun and Ling Liu for their funding of the project. Author contributions W.S. and L.L. designed the experiments. X.B., Z.P., and J.W. performed the experiments and collected data. Y.D., J.W., M.O., M.Z., R.C., X.T., Y.Z., and Y.L. performed data analyses. X.B. wrote the manuscript, and W.S., L.L., D.L., and M.Z.N.D. revised the manuscript. This submitted version of the manuscript was reviewed and completed by all authors. Funding The work is supported by the National Natural Science Foundation of China grant (31630064), the China Agricultural Research System (CARS01), the Science and Technology Development Project of Jilin Province (20240304122SF), the Jilin Provincial Department of Education-Science and Technology Project (JJKH20230407KJ), and the 111 project (D17014). Availability of data and materials The datasets used and/or analyzed in this study are available from the corresponding authors on request. Ethical approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References Achimón F, Dambolena J S, Zygadlo J A, Pizzolitto R P. Carbon sources as factors affecting the secondary metabolism of the maize pathogen Fusarium verticillioides . Lwt. 2019;115:108470. https://doi.org/10.1016/j.lwt.2019.108470. Achimón F, Krapacher C R, Jacquat A G, Pizzolitto R P, Zygadlo J A. Carbon sources to enhance the biosynthesis of useful secondary metabolites in Fusarium verticillioides submerged cultures . World Journal of Microbiology and Biotechnology. 2021;37:78. https://doi.org/10.1007/s11274-021-03044-z. Antoniêto A C C, Dos Santos Castro L, Silva-Rocha R, Persinoti G F, Silva R N. Defining the genome-wide role of CRE1 during carbon catabolite repression in T richoderma reesei using RNA-seq analysis . Fungal Genet. Biol. 2014;73:93-103. https://doi.org/10.1016/j.fgb.2014.10.009. Benocci T, Aguilar-Pontes M V, Zhou M, Seiboth B, de Vries R P. Regulators of plant biomass degradation in ascomycetous fungi . Biotechnol. Biofuels. 2017;10:1-25. https://doi.org/10.1186/s13068-017-0841-x. Brown N A, Ries L N A, Goldman G H. How nutritional status signalling coordinates metabolism and lignocellulolytic enzyme secretion . Fungal Genet. Biol. 2014;72:48-63. https://doi.org/10.1016/j.fgb.2014.06.012. Chen X, Hai D, Tang J, Liu H, Huang J, Luo C, et al. UvCom1 is an important regulator required for development and infection in the rice false smut fungus Ustilaginoidea virens . Phytopathology. 2020;110:483-93. https://doi.org/10.1094/PHYTO-05-19-0179-R. Cubero B, Scazzocchio C. Two different, adjacent and divergent zinc finger binding sites are necessary for CREA‐mediated carbon catabolite repression in the proline gene cluster of A spergillus nidulans . The Embo Journal. 1994;13:407-15. https://doi.org/10.1002/j.1460-2075.1994.tb06275.x. Fasoyin O E, Wang B, Qiu M, Han X, Chung K, Wang S. Carbon catabolite repression gene CreA regulates morphology, aflatoxin biosynthesis and virulence in A spergillus flavus . Fungal Genet. Biol. 2018;115:41-51. https://doi.org/10.1016/j.fgb.2018.04.008. Fernandez J, Wright J D, Hartline D, Quispe C F, Madayiputhiya N, Wilson R A. Principles of carbon catabolite repression in the rice blast fungus: Tps1, Nmr1-3, and a MATE–family pump regulate glucose metabolism during infection . Plos Genet. 2012;8:e1002673. https://doi.org/10.1371/journal.pgen.1002673. Fu X, Wang W, Li Y, Wang X, Wang B. Development of a monoclonal antibody with equal reactivity to ustiloxins A and B for quantification of main cyclopeptide mycotoxins in rice samples . Food Control. 2018;92:201-7. https://doi.org/10.1016/j.foodcont.2018.04.048. Grau M F, Entwistle R, Chiang Y, Ahuja M, Oakley C E, Akashi T, et al. Hybrid transcription factor engineering activates the silent secondary metabolite gene cluster for (+)-Asperlin in Aspergillus nidulans . Acs Chem. Biol. 2018;13:3193-205. https://doi.org/10.1021/acschembio.8b00679. Hou R, Wang C. The function of the carbon metabolism regulator FgCreA in F usarium graminearum . Scientia Agricultura Sinica . 2018;51: 257-67. https://doi.org/10.3864/j.issn.0578-1752.2018.02.006. Huang Z, Wang Q, Li Y, Huang P, Liao J, Wang J, et al. A multilayered regulatory network mediated by protein phosphatase 4 controls carbon catabolite repression and de-repression in Magnaporthe oryzae . Commun. Biol. 2025;8:130. https://doi.org/10.1038/s42003-025-07581-3. Jiang C, Li X, Fang A, Fu Y, Li C, Dong H, et al. An Ustilaginoidea virens nuclear effector SCRE7 inhibits rice immunity via suppressing OsLBD11/12‐promoted transcription of OsCPS2 . Plant Biotechnol. J. 2026;24:1305-21. https://doi.org/10.1111/pbi.70413. Li W L, Li L Y, Feng A Q, Zhu X Y, Li J X. Rice false smut fungus, Ustilaginoidea virens , inhibits pollen germination and degrades the integuments of rice ovule . American Journal of Plant Sciences. 2013;4:2295-304. https://doi.org/10.4236/ajps.2013.412284. Li Y, Wang M, Liu Z, Zhang K, Cui F, Sun W. Towards understanding the biosynthetic pathway for ustilaginoidin mycotoxins in Ustilaginoidea virens . Environ. Microbiol. 2019;21:2629-43. https://doi.org/10.1111/1462-2920.14572. Lin X, Bian Y, Mou R, Cao Z, Cao Z, Zhu Z, et al. Isolation, identification, and characterization of Ustilaginoidea virens from rice false smut balls with high ustilotoxin production potential . J. Basic Microbiol. 2018;58:670-8. https://doi.org/10.1002/jobm.201800167. Liu L, Wang B, Duan G, Wang J, Pan Z, Ou M, et al. Histone deacetylase UvHST2 is a global regulator of secondary metabolism in Ustilaginoidea virens . J. Agric. Food. Chem. 2023;71:13124-36. https://doi.org/10.1021/acs.jafc.3c01782. Long Z, Wang P, Yu Q, Wang B, Li D, Yang C, et al. The histone deacetylase UvHOS2 regulates vegetative growth, conidiation, ustilaginoidin synthesis, and pathogenicity in Ustilaginoidea virens . Phytopathol. Res. 2024;6:16. https://doi.org/10.1186/s42483-024-00230-3. Meng J, Sun W, Mao Z, Xu D, Wang X, Lu S, et al. Main ustilaginoidins and their distribution in rice false smut balls . Toxins. 2015;7:4023-34. https://doi.org/10.3390/toxins7104023. Meshram M K, Wasule D L. Method for extracting genomic DNA from filaments fungi. Annals of Plant Physiology. 2010;24:108-9. Mogensen J, Nielsen H B, Hofmann G, Nielsen J. Transcription analysis using high-density micro-arrays of A spergillus nidulans wild-type and C re A mutant during growth on glucose or ethanol . Fungal Genet. Biol. 2006;43:593-603. https://doi.org/ 10.1016/j.fgb.2006.03.003. Momany M, Westfall P J, Abramowsky G. Aspergillus nidulans swo mutants show defects in polarity establishment, polarity maintenance and hyphal morphogenesis . Genetics. 1999;151:557-67. https://doi.org/10.1093/genetics/151.2.557. Nakari-Setälä T, Paloheimo M, Kallio J, Vehmaanperä J, Penttilä M, Saloheimo M. Genetic modification of carbon catabolite repression in Trichoderma reesei for improved protein production . Appl. Environ. Microbiol. 2009;75:4853-60. https://doi.org/10.1128/AEM.00282-09. New A M, Cerulus B, Govers S K, Perez-Samper G, Zhu B, Boogmans S, et al. Different levels of catabolite repression optimize growth in stable and variable environments . Plos Biol. 2014;12:e1001764. https://doi.org/10.1371/journal.pbio.1001764. Nihashi J, Fujita Y. Catabolite repression of inositol dehydrogenase and gluconate kinase syntheses in Bacillus subtilis . Biochimica Et Biophysica Acta (BBA) - General Subjects. 1984;798:88-95. https://doi.org/10.1016/0304-4165(84)90014-X. Ozcengiz G, Demain A L. Recent advances in the biosynthesis of penicillins, cephalosporins and clavams and its regulation . Biotechnol. Adv. 2013;31:287-311. https://doi.org/10.1016/j.biotechadv.2012.12.001. Rao X, Huang X, Zhou Z, Lin X. An improvement of the 2ˆ(-delta delta CT) method for quantitative real-time polymerase chain reaction data analysis . Biostat Bioinforma Biomath. 2013;3:71-85. https://doi.org/10.1016/S0920-5489(99)92176-1 Ruiz B, Chávez A, Forero A, García-Huante Y, Romero A, Sánchez M, et al. Production of microbial secondary metabolites: regulation by the carbon source . Crit. Rev. Microbiol. 2010;36:146-67. https://doi.org/10.3109/10408410903489576. Ruiz-Villafán B, Cruz-Bautista R, Manzo-Ruiz M, Passari A K, Villarreal-Gómez K, Rodríguez-Sanoja R, et al. Carbon catabolite regulation of secondary metabolite formation, an old but not well-established regulatory system . Microb. Biotechnol. 2022;15:1058-72. https://doi.org/10.1111/1751-7915.13791. Xie S, Shi H, Wen H, Liu Zhi, Qiu J, Jiang N, et al. Carbon catabolite repressor UvCreA is required for development and pathogenicity in Ustilaginoidea virens . Rice Sci. 2024;31:203-14. https://doi.org/10.1016/j.rsci.2023.11.008. Sun W, Wang A, Xu D, Wang W, Meng J, Dai J, et al. New ustilaginoidins from rice false smut balls caused by Villosiclava virens and their phytotoxic and cytotoxic activities . J. Agric. Food. Chem. 2017;65:5151-60. https://doi.org/10.1021/acs.jafc.7b01791. Szilágyi M, Miskei M, Karányi Z, Lenkey B, Pócsi I, Emri T. Transcriptome changes initiated by carbon starvation in Aspergillus nidulans . Microbiology. 2013;159:176-90. https://doi.org/10.1099/mic.0.062935-0. Tang G, Yuan J, Wang J, Zhang Y, Xie S, Wang H, et al. Fusarium BP1 is a reader of H3K27 methylation . Nucleic. Acids. Res. 2021;49:10448-64. https://doi.org/10.1093/nar/gkab844. Tang Y, Jin J, Hu D, Yong M, Xu Y, He L. Elucidation of the infection process of Ustilaginoidea virens (teleomorph: Villosiclava virens ) in rice spikelets . Plant Pathol. 2013;62:1-8. https://doi.org/10.1111/j.1365-3059.2012.02629.x. Wang B, Bai X, Zhang M, Liu X, Dara M, Liu L, et al. Ustilaginoidin d induces acute toxicity and hepatotoxicity in mice . Toxins. 2025;17. https://doi.org/10.3390/toxins17050250. Wang B, Duan G, Liu L, Long Z, Bai X, Ou M, et al. UvHOS3‐mediated histone deacetylation is essential for virulence and negatively regulates ustilaginoidin biosynthesis in Ustilaginoidea virens . Mol. Plant Pathol. 2024;25:e13429. https://doi.org/10.1111/mpp.13429. Wang B, Liu L, Li Y, Zou J, Li D, Zhao D, et al. Ustilaginoidin D induces hepatotoxicity and behaviour aberrations in zebrafish larvae . Toxicology. 2021;456:152786. https://doi.org/10.1016/j.tox.2021.152786. Wang G, Wang Y, Yang B, Zhang C, Zhang H, Xing F, et al. Carbon catabolite repression gene AoCreA regulates morphological development and ochratoxin a biosynthesis responding to carbon sources in A spergillus ochraceus . Toxins. 2020;12:697. https://doi.org/10.3390/toxins12110697. Wang W, Fan J, Jeyakumar J M J. Rice false smut: an increasing threat to grain yield and quality . Protecting Rice Grains in the Post-Genomic Era. 2019;89-108. https://doi.org/10.5772/ intechopen.84862. Wanke D, Harter K. Analysis of plant regulatory DNA sequences by the yeast-one-hybrid assay. Methods in Molecular Biology. 2009;479:291. https://doi.org/10.1007/978-1-59745-289-2_19. Wen H, Shi H, Jiang N, Qiu J, Lin F, Kou Y. Antifungal mechanisms of silver nanoparticles on mycotoxin producing rice false smut fungus . Iscience. 2023;26:105763. https://doi.org/10.1016/j.isci.2022.105763. Xu Y, Guan X, Han Z, Zhou L, Zhang Y, Asad M A U, et al. Combined effect of nitrogen fertilizer application and high temperature on grain quality properties of cooked rice . Front. Plant Sci. 2022;13:874033. https://doi.org/10.3389/fpls.2022.874033. Yoav S, Salame T M, Feldman D, Levinson D, Ioelovich M, Morag E, et al. Effects of c re1 modification in the white-rot fungus P leurotus ostreatus PC 9 : altering substrate preference during biological pretreatment . Biotechnol. Biofuels. 2018;11:1-16. https://doi.org/10.1186/s13068-018-1209-6. Yu S, Zhang S, Zheng X, Li X, Wang J, Duan G, et al. Ustilaginoidea virens suppresses floral immunity through promoting GA biosynthesis by the effector SCRE9 . New Phytol. 2025;248:3115-31. https://doi.org/10.1111/nph.70414. Yu W, Pei R, Zhou J, Zeng B, Tu Y, He B. Molecular regulation of fungal secondary metabolism . World Journal of Microbiology and Biotechnology. 2023;39:204. https://doi.org/10.1007/s11274-023-03649-6. Zhou L, Lu S, Shan T, Wang P, Wang S. .Chemistry and biology of mycotoxins from rice false smut pathogen. Mycotoxins: Properties, Applications and Hazards. 2012. https://doi.org/10.1021/acs.jafc.5c05629. Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-9251727\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":616735892,\"identity\":\"b99ce2f2-2e52-4242-978c-c687beed686a\",\"order_by\":0,\"name\":\"xiaolong Bai\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jilin Agricultural University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"xiaolong\",\"middleName\":\"\",\"lastName\":\"Bai\",\"suffix\":\"\"},{\"id\":616735893,\"identity\":\"c20e2c52-a06f-453c-b763-ea4c738491b6\",\"order_by\":1,\"name\":\"Zequn Pan\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jilin Agricultural University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Zequn\",\"middleName\":\"\",\"lastName\":\"Pan\",\"suffix\":\"\"},{\"id\":616735894,\"identity\":\"9173bb6c-2dc9-4862-834e-84c420c0d2f0\",\"order_by\":2,\"name\":\"Ying Du\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jilin Agricultural University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Ying\",\"middleName\":\"\",\"lastName\":\"Du\",\"suffix\":\"\"},{\"id\":616735895,\"identity\":\"8a34303d-ee44-4e4c-b871-ac73a0e4acb1\",\"order_by\":3,\"name\":\"Muhammad Zulqar Nain Dara\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jilin Agricultural University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Muhammad\",\"middleName\":\"Zulqar Nain\",\"lastName\":\"Dara\",\"suffix\":\"\"},{\"id\":616735896,\"identity\":\"3b0119aa-3ca1-45b4-82b1-377dedc6bd5f\",\"order_by\":4,\"name\":\"Jing Wang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jilin Agricultural University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Jing\",\"middleName\":\"\",\"lastName\":\"Wang\",\"suffix\":\"\"},{\"id\":616735897,\"identity\":\"c18d80ef-35d6-4d4e-840c-fba8bfc6de96\",\"order_by\":5,\"name\":\"Mingming Ou\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jilin Agricultural University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Mingming\",\"middleName\":\"\",\"lastName\":\"Ou\",\"suffix\":\"\"},{\"id\":616735898,\"identity\":\"831540ce-294c-4647-8588-91445fdf1441\",\"order_by\":6,\"name\":\"Min Zhang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jilin Agricultural University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Min\",\"middleName\":\"\",\"lastName\":\"Zhang\",\"suffix\":\"\"},{\"id\":616735899,\"identity\":\"a07e5cd7-1055-4de8-8b7c-986ec16a2aea\",\"order_by\":7,\"name\":\"Rui Cheng\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jilin Agricultural University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Rui\",\"middleName\":\"\",\"lastName\":\"Cheng\",\"suffix\":\"\"},{\"id\":616735900,\"identity\":\"a37be612-ddd3-4754-9224-6dc1aed63e4c\",\"order_by\":8,\"name\":\"Xuxuan Tian\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jilin Agricultural University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Xuxuan\",\"middleName\":\"\",\"lastName\":\"Tian\",\"suffix\":\"\"},{\"id\":616735901,\"identity\":\"334fff91-38bc-44f4-b757-336afc4003f6\",\"order_by\":9,\"name\":\"Yingchi Zhang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jilin Agricultural University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Yingchi\",\"middleName\":\"\",\"lastName\":\"Zhang\",\"suffix\":\"\"},{\"id\":616735902,\"identity\":\"2ee188ec-8258-406a-887e-8d4b3f78ccc7\",\"order_by\":10,\"name\":\"Yuhan Liu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jilin Agricultural University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Yuhan\",\"middleName\":\"\",\"lastName\":\"Liu\",\"suffix\":\"\"},{\"id\":616735903,\"identity\":\"1341d735-ff1d-4493-8300-59fc28c96ebe\",\"order_by\":11,\"name\":\"Dayong Li\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jilin Agricultural University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Dayong\",\"middleName\":\"\",\"lastName\":\"Li\",\"suffix\":\"\"},{\"id\":616735904,\"identity\":\"297820c5-02f4-4d9a-afd8-0576e1e6edd4\",\"order_by\":12,\"name\":\"Ling Liu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jilin Agricultural University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Ling\",\"middleName\":\"\",\"lastName\":\"Liu\",\"suffix\":\"\"},{\"id\":616735905,\"identity\":\"8171b84e-b836-4d3e-a6b0-354ba62a9c44\",\"order_by\":13,\"name\":\"Wenxian Sun\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAo0lEQVRIiWNgGAWjYHACxgcQOoF4LcwGJGthkyBNi8GN9GcVP2oOM/Cz5xgw/NxBjJYzZ8xu9hw7zCDZ88aAsfcMEVrMjvew3WZgOwy0LseAmbGNGC2H2Z8VM/w7zGBPvJbjDWZAlUBbJIjVYn/mjLFkb186j8SZZwUHe4nRIjkj/eGHH9+s5fjbkzc++EmMFhjgAREHSNAwCkbBKBgFowAfAABXxDOrx9RhjgAAAABJRU5ErkJggg==\",\"orcid\":\"\",\"institution\":\"Jilin Agricultural University\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Wenxian\",\"middleName\":\"\",\"lastName\":\"Sun\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2026-03-28 10:08:20\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-9251727/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-9251727/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":106403020,\"identity\":\"7aeb5796-ef4a-4da7-98dc-0de133ea43df\",\"added_by\":\"auto\",\"created_at\":\"2026-04-08 09:13:24\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":117682,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eDifferent carbon sources pose distinct effects on ustilaginoidin biosynthesis in \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003eU. virens\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003e.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(A) HPLC analysis reveals the ustilaginoidin accumulation levels in the wild-type strain when cultured on media containing various carbon sources. (B) The expression levels of the key polyketide synthase gene \\u003cem\\u003eUvPKS1\\u003c/em\\u003e for ustilaginoidins biosynthesis, and the methyltransferase gene \\u003cem\\u003eUgsJ\\u003c/em\\u003eunder diverse carbon sources, including glucose, sucrose, D-fructose, and trehalose, were detected in the WT strain. Different letters (a-c) indicate statistically significant differences in gene expression levels (\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.05), as determined by one-way ANOVA followed by Tukey’s test.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9251727/v1/9508b7182db8bc4cc64f6256.png\"},{\"id\":106993938,\"identity\":\"5d1b28c5-1f37-472d-9a8c-9f28fdd34275\",\"added_by\":\"auto\",\"created_at\":\"2026-04-15 15:00:32\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":553557,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eUvCreA regulates hyphal growth, conidiation and conidial germination in \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003eU. virens\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003e.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(A) Colony morphology of the wild-type, Δ\\u003cem\\u003eUvcreA\\u003c/em\\u003e, \\u003cem\\u003eUvCreA\\u003c/em\\u003e-complemented, and overexpressing strains after 14 days of culturing at 28 °C on PSA plates. (B) Hyphal and conidiophore morphology of different strains. Scale bar, 100 mm. (C-E) Colony diameter (C), conidial production (D), and conidial germination rate (E) of the wild-type, Δ\\u003cem\\u003eUvcreA\\u003c/em\\u003e-4/64, CΔ\\u003cem\\u003eUvcreA\\u003c/em\\u003e-64, and OE-UvCreA-FLAG strains were assessed. Statistically significant differences among groups are indicated by different letters (a-c) based on one-way ANOVAfollowed by Tukey’s test(\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.05).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9251727/v1/86f95b769c8021f74a1c6b92.png\"},{\"id\":106231810,\"identity\":\"f622eb29-c32c-40a2-88ce-582165c68b08\",\"added_by\":\"auto\",\"created_at\":\"2026-04-06 12:47:33\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":181355,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eUvCreA positively regulates ustilaginoidin biosynthesis and virulence in \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003eU. virens\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003e.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(A) HPLC quantification of ustilaginoidins generated by wild-type, Δ\\u003cem\\u003eUvcreA\\u003c/em\\u003e, \\u003cem\\u003eUvCreA\\u003c/em\\u003e-complemented, and overexpressing strains after incubation for 28 days at 28 °C on PSA plates. (B) Expression of ustilaginoidin biosynthesis-related genes in different \\u003cem\\u003eU. virens\\u003c/em\\u003e strains detected via RT-qPCR.Data are shown as mean ± SD (\\u003cem\\u003en \\u003c/em\\u003e= 3). Statistically significant differences were represented by different letters (a-d) according to one-way ANOVA following Tukey’stest (\\u003cem\\u003eP \\u003c/em\\u003e\\u0026lt; 0.05). (C-D) Virulence assays of the wild-type, Δ\\u003cem\\u003eUvcreA\\u003c/em\\u003e-4/64, CΔ\\u003cem\\u003eUvcreA\\u003c/em\\u003e-64, and OE-UvCreA-FLAG strains in the panicles of rice cultivar JN853. Disease symptoms were documented photographically (C), and the quantity of false smut balls was enumerated (D) approximately four weeks following inoculation with \\u003cem\\u003eU. virens\\u003c/em\\u003e.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9251727/v1/ac92dc22773d83069ca440cf.png\"},{\"id\":106231814,\"identity\":\"81685f1b-d211-4988-b6c3-ffc3a1a1dc97\",\"added_by\":\"auto\",\"created_at\":\"2026-04-06 12:47:33\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":362734,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eUvCreA negatively regulates oxidative, cell wall, membrane, and osmotic stresses in \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003eU. virens\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003e.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(A) Colony morphologies of the wild-type, Δ\\u003cem\\u003eUvcreA\\u003c/em\\u003e, and \\u003cem\\u003eUvCreA\\u003c/em\\u003e-complemented strains after culturing on yeast extract-tryptone (YT) medium plates containing various environmental stress factors (2.5mM H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e, 70 μg/mL Congo red, 0.015% SDS, or 0.25 M NaCl) for 14 days. (B) The growth inhibition rates of the wild-type, Δ\\u003cem\\u003eUvcreA\\u003c/em\\u003e, and \\u003cem\\u003eUvCreA\\u003c/em\\u003e-complemented strains in response to various stress factors were assessed. Colony diameters were measured after cultivating the tested \\u003cem\\u003eU. virens\\u003c/em\\u003e strains on YT plates or YT plates supplemented with 2.5 mM H₂O₂, 70 μg/mL Congo red, 0.015% SDS, or 0.25 M NaCl. Data are presented as mean ± SD (\\u003cem\\u003en\\u003c/em\\u003e = 3). Different lowercase letters indicate statistically significant differences in growth inhibition rates, as determined by one-way ANOVA followed by Tukey’s test (\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.05).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9251727/v1/1510596ef7a219763b819d68.png\"},{\"id\":106231813,\"identity\":\"5f5c2624-33cd-43f6-80f6-102fc7ef4109\",\"added_by\":\"auto\",\"created_at\":\"2026-04-06 12:47:33\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":170803,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eUvCreA regulates the expression of \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003eUvPKS1\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003eand \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003eUgsJ\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003e genes by directly binding their promoter.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(A) The putative \\u003cem\\u003ecis\\u003c/em\\u003e-element of the transcription factor UvCreA predicted by JASPAR. (B) Yeast one-hybrid assay to show the binding of UvCreA to the promoters of \\u003cem\\u003eUvPKS1\\u003c/em\\u003e and \\u003cem\\u003eUgsJ\\u003c/em\\u003e genes. (C-D) EMSA to verify that UvCreA binds the \\u003cem\\u003ecis\\u003c/em\\u003e-element in \\u003cem\\u003eUvPKS1\\u003c/em\\u003e (C) and \\u003cem\\u003eUgsJ\\u003c/em\\u003e(D) genes involved in ustilaginoidin biosynthesis. (E) Diagrams to show the positions of putative UvCreA-binding \\u003cem\\u003ecis\\u003c/em\\u003e-elements S (G/C)Y (T/C)GGR (G/A)G in the promoters of \\u003cem\\u003eUvPKS1\\u003c/em\\u003eand \\u003cem\\u003eUgsJ\\u003c/em\\u003e and the primers used for ChIP-qPCR. (F) ChIP-qPCR to detect the enriched promoter regions of the \\u003cem\\u003eUvPKS1\\u003c/em\\u003e and \\u003cem\\u003eUgsJ\\u003c/em\\u003e genes by UvCreA. Asterisks (*) indicate statistically significant differences in enrichment fold (**, \\u003cem\\u003eP \\u003c/em\\u003e\\u0026lt; 0.01; ****, \\u003cem\\u003eP \\u003c/em\\u003e\\u0026lt; 0.0001; Student’s \\u003cem\\u003et\\u003c/em\\u003e-test).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9251727/v1/ce22926af843f27c39e34ade.png\"},{\"id\":108180828,\"identity\":\"9d976336-caef-4882-b946-eb1f2206aa07\",\"added_by\":\"auto\",\"created_at\":\"2026-04-30 08:54:14\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":1858549,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9251727/v1/1ea221ed-88d6-405e-b82a-d5565ca87481.pdf\"},{\"id\":106404014,\"identity\":\"0be7ae90-5792-4037-a839-fcd5a0749eef\",\"added_by\":\"auto\",\"created_at\":\"2026-04-08 09:15:22\",\"extension\":\"docx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":810457,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementaryMaterial.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9251727/v1/d2b5ed8a93156b81873c372e.docx\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"The transcription factor UvCreA regulates ustilaginoidin synthesis and pathogenicity in Ustilaginoidea virens\",\"fulltext\":[{\"header\":\"1. Introduction\",\"content\":\"\\u003cp\\u003eRice false smut (RFS) has become one of the most damaging fungal diseases of rice worldwide, which is caused by \\u003cem\\u003eUstilaginoidea virens\\u003c/em\\u003e. RFS epidemic is largely attributed to a combination of factors, including climate change, excessive use of chemical fertilizers, high-yield cultivation practices, and widespread adoption of hybrid rice varieties (Tang et al. \\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e2013\\u003c/span\\u003e). \\u003cem\\u003eU. virens\\u003c/em\\u003e infects rice florets by colonizing the stamen filaments (Li et al. \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e2013\\u003c/span\\u003e), and covers the rice grains with powdery chlamydospores, known as false smut balls (Chen et al. \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e). \\u003cem\\u003eU. virens\\u003c/em\\u003e produces ustiloxins and ustilaginoidins, besides other multiple types of mycotoxins (Wen et al. \\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e; Zhou et al. \\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e), seriously affecting grain quality (Lin et al. \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eUstilaginoidins mycotoxins are part of the naphtho-γ-pyrone class, which are usually soluble in organic solvents (Meng et al. \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e; Wang et al. \\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). Specifically, Ustilaginoidin D has been demonstrated to induce a substantial delay in yolk sac absorption and hepatic injury in zebrafish (Wang et al. \\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). The biosynthesis of ustilaginoidins is controlled by the \\u003cem\\u003eUgs\\u003c/em\\u003e (\\u003cspan type=\\\"Underline\\\" class=\\\"Underline\\\" name=\\\"Emphasis\\\"\\u003eu\\u003c/span\\u003estila\\u003cspan type=\\\"Underline\\\" class=\\\"Underline\\\" name=\\\"Emphasis\\\"\\u003eg\\u003c/span\\u003einoidin \\u003cspan type=\\\"Underline\\\" class=\\\"Underline\\\" name=\\\"Emphasis\\\"\\u003es\\u003c/span\\u003eynthesis) gene cluster, which contains at least fourteen genes (Li et al. \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). Among them, \\u003cem\\u003eUvPKS1\\u003c/em\\u003e encodes a polyketide synthase that catalyzes the initial step in ustilaginoidin biosynthesis. Knockout of \\u003cem\\u003eUvPKS1\\u003c/em\\u003e in \\u003cem\\u003eU. virens\\u003c/em\\u003e results in complete cessation of ustilaginoidin production, including the intermediates. The gene \\u003cem\\u003eUgsJ\\u003c/em\\u003e encodes a methyltransferase responsible for methylation at the C3-3' side chain of the pyranone ring. In comparison with the wild type, the \\u003cem\\u003eUgsJ\\u003c/em\\u003e-knockout strain generates a greater quantity of ustilaginoidins F, G, and A, which are derivatives that lack the C3-3' methylation modification of the pyranone ring (Li et al. \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). However, the regulatory mechanisms underlying ustilaginoidin biosynthesis remain largely unknown.\\u003c/p\\u003e \\u003cp\\u003eCarbon catabolite repression (CCR) serves as a regulatory mechanism that controls carbon utilization, influencing growth, development, and secondary metabolism in fungi (Xu et al. \\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). The highly conserved transcription factors, catabolite-responsive elements, CreA/Cre1 have been considered as major regulatory factors in the CCR mechanism (Mogensen et al. \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e2006\\u003c/span\\u003e). When preferred carbon sources such as glucose are present, expression of genes encoding hydrolases for secondary carbon sources is suppressed (Yoav et al. \\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). CreA suppresses the transcription of secondary carbon source hydrolase genes through direct bindind with the conserved \\u003cem\\u003ecis\\u003c/em\\u003e-acting element 5'-SYGGRG-3' in the promoter region of the target gene (Benocci et al. \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e). This conserved mechanism enables efficient utilization of carbon sources and represents a crucial transcriptional control system for carbon nutrient selectivity in microorganisms (New et al. \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e). CreA is also involved in growth and development, secondary metabolite synthesis, and pathogenicity in phytopathogenic fungi. \\u003cem\\u003eAspergillus ochraceus\\u003c/em\\u003e contaminates a wide range of foods and feeds by producing ochratoxin A (OTA). AoCreA plays a crucial role in modulating OTA biosynthesis and influences morphological development in response to different carbon sources (Wang et al. \\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e). In \\u003cem\\u003eA. flavus\\u003c/em\\u003e, CreA regulates the biosynthesis of aflatoxin and sclerotium production (Fasoyin et al. \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). A recent study showed that UvCreA plays an important role in virulence and carbon source utilization through transcriptional regulation in \\u003cem\\u003eU. virens\\u003c/em\\u003e, and is a key regulatory factor in CCR (Xie et al. \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). Additionally, CreA responds to carbon source signals and controls the expression of secondary metabolic genes and the production of secondary metabolites across various species. (Yu et al. \\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). Therefore, CreA is implicated not only in the regulation of CCR but also in the biosynthesis of secondary metabolites and fungal pathogenicity. However, limited knowledge exists regarding the molecular mechanisms through which the transcription factor regulates mycotoxin biosynthesis.\\u003c/p\\u003e \\u003cp\\u003eIn this study, we demonstrate that different carbon sources significantly influence ustilaginoidin accumulation. We identify UvCreA as a global regulator affecting fungal growth, conidial production and germination, secondary metabolism, stress response, and pathogenicity. More importantly, UvCreA directly binds to the promoters of \\u003cem\\u003eUvPKS1\\u003c/em\\u003e and \\u003cem\\u003eUgsJ\\u003c/em\\u003e within the \\u003cem\\u003eUgs\\u003c/em\\u003e gene cluster, thereby regulating ustilaginoidin biosynthesis. These findings not only reveal the role of UvCreA in \\u003cem\\u003eU. virens\\u003c/em\\u003e development and pathogenicity but also offer new insights into the molecular mechanisms regulating ustilaginoidin biosynthesis, suggesting possible molecular drug targets for managing rice false smut.\\u003c/p\\u003e\"},{\"header\":\"2. Materials and methods\",\"content\":\"\\u003cp\\u003eBoth nucleotide and amino-acid sequences were retrieved from the NCBI database (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://www.ncbi.nlm.nih.gov/\\u003c/span\\u003e\\u003cspan address=\\\"https://www.ncbi.nlm.nih.gov/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e). The alignment analyses were conducted using DNAMAN 6 (v 6.0) and MEGA Ⅹ (v 10.0). Primer sequences employed in this work are provided in Supplementary Table \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e. The assays conducted in this study were independently repeated three times, unless noted.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.1 Strains and culture media\\u003c/h2\\u003e \\u003cp\\u003eThe strain was cultivated on PSA plates consisting of 200 g/L potato, 20 g/L sucrose, and 20 g/L agar. The \\u003cem\\u003eU. virens\\u003c/em\\u003e strain P1FZ was used as the wild type (WT). Furthermore, strains were cultured on PSA plates (Jiang et al. \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e2026\\u003c/span\\u003e) or complete medium (CM, containing 1 g of yeast extract, 6 g of NaNO₃, 2 g of peptone, 1 g of acid hydrolyzed casein, 1 g of MgSO₄\\u0026middot;7H₂O, 0.5 g of NaCl, and 1.5 g of KH₂PO₄ per liter) supplemented with different carbon sources (10g glucose, sucrose, D-fructose, or trehalose), subsequently incubated at 28\\u0026deg;C, as described earlier (Momany et al. \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e1999\\u003c/span\\u003e).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.2 Construction of \\u003cem\\u003eUvCreA\\u003c/em\\u003e deletion mutants, complemented, and overexpression strains\\u003c/h2\\u003e \\u003cp\\u003eDeletion of the \\u003cem\\u003eUvCreA\\u003c/em\\u003e gene was accomplished by the CRISPR/Cas9 system and homologous recombination (Jiang et al. \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e2026\\u003c/span\\u003e). The flanking sequences of the target gene and the hygromycin resistance gene (\\u003cem\\u003eHyg\\u003c/em\\u003e) from plasmid pGKO2-\\u003cem\\u003eHyg\\u003c/em\\u003e were independently amplified. Subsequently, these \\u003cem\\u003eUvCreA\\u003c/em\\u003e sequences were fused to the 5\\u0026rsquo; and 3\\u0026rsquo; termini of the \\u003cem\\u003eHyg\\u003c/em\\u003e gene via fusion PCR. Primer pairs specific for the Cas9 target site and sgRNA were designed using an online tool (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://portals.broadinstitute.org/gppx/crispick/public\\u003c/span\\u003e\\u003cspan address=\\\"https://portals.broadinstitute.org/gppx/crispick/public\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e). Following the annealing process, the sgRNA primers were incorporated into the pCAS9-tRp vector, which had been pre-digested with \\u003cem\\u003eBsmB\\u003c/em\\u003e I (Li et al. \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). The PCR products and construct were transformed into \\u003cem\\u003eU. virens\\u003c/em\\u003e protoplasts using the polyethylene glycol method (Fu et al. \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). The mutants were first screened by PCR and then confirmed by Southern blot analysis as described earlier (Meshram et al. 2010). For complementation and overexpression purposes, the coding sequence of \\u003cem\\u003eUvCreA\\u003c/em\\u003e and the robust \\u003cem\\u003eHSP70\\u003c/em\\u003e promoter (OE8266) were cloned into the pY2P102-FLAG vector (Li et al. \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). The constructed plasmid was introduced into the wild-type and Δ\\u003cem\\u003eUvcreA\\u003c/em\\u003e-64 strains. An anti-FLAG antibody (Sigma-Aldrich, 12352203, St. Louis, MO) was used to confirm protein expression by immunoblot analysis.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.3 Determination of growth rate, conidiogenesis, and conidial germination rate\\u003c/h2\\u003e \\u003cp\\u003eMycelial plugs were inoculated onto PSA plates and subsequently cultured at 28\\u0026deg;C for 14 days. The colony morphology and diameters were thereafter documented through photography and measurement, respectively. The fungal hyphae were examined under a microscope (Wang et al. \\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eFor the conidiation assay, various strains of \\u003cem\\u003eU. virens\\u003c/em\\u003e were independently inoculated into 75 mL of PSB medium and incubated in a shaker at 150 rpm for seven days at 28\\u0026deg;C. The culture was filtered into a 50 mL sterile centrifuge tube using sterilized three-layer lens paper. The conidia were harvested and resuspended in PSB medium to a concentration of 10\\u003csup\\u003e6\\u003c/sup\\u003e spores/mL. A 100 \\u0026micro;L aliquot of the spore suspension was uniformly spread on PSA agar plates and incubated at 28\\u0026deg;C. Conidial germination was monitored using a microscope, and germination rates were calculated at various stages of growth (Long et al. \\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.4 Quantification of ustilaginoidins by high-performance liquid chromatography (HPLC)\\u003c/h2\\u003e \\u003cp\\u003e \\u003cem\\u003eU. virens\\u003c/em\\u003e strains were cultured at 28\\u0026deg;C for 28 days on PSA plates covered with cellophane or on complete medium (CM) plates supplemented with 1% glucose, sucrose, D-fructose, or trehalose. The mycelia were collected in a 50 mL centrifuge tube containing 30 mL of ethyl acetate, then incubated at 28\\u0026deg;C for 24 hours on a shaker at 150 rpm. Subsequently, the mixture was concentrated to dryness, reconstituted in acetonitrile, and filtered through a 0.22 \\u0026micro;m filter (Wang et al. \\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). Samples were then analyzed using an LC-20A HPLC system (Shimadzu, Japan).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec7\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.5 Stress tolerance assays\\u003c/h2\\u003e \\u003cp\\u003eDifferent strains of \\u003cem\\u003eU. virens\\u003c/em\\u003e were cultured on YT medium plates supplemented with 0.015% sodium dodecyl sulfate (SDS), 2.5 mM H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e, 70 \\u0026micro;g/mL Congo red, or 0.25 M NaCl at 28\\u0026deg;C for 14 days as described (Wang et al. \\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). Growth inhibition rates were calculated by measuring colony diameters on at least three plates.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.6 Virulence assay\\u003c/h2\\u003e \\u003cp\\u003eVarious strains of \\u003cem\\u003eU. virens\\u003c/em\\u003e, including wild-type, gene-knockout, complemented, and overexpression, were cultured in 75 mL of PSB medium and incubated in a shaker at 150 rpm and 28\\u0026deg;C for 7 days. To homogenize the culture, a blender was used to prepare a hyphal and conidial suspension containing 2\\u0026times;10\\u003csup\\u003e6\\u003c/sup\\u003e conidia/mL, which was injected into panicles (1 mL per panicle) of the susceptible rice cultivar JN853, approximately 1 week before the heading stage. The inoculated panicles were observed and photographed approximately 1 month after inoculation (Liu et al. \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e; Long et al. \\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec9\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.7 RNA isolation and RT-qPCR\\u003c/h2\\u003e \\u003cp\\u003eTotal RNA was extracted from 14-day-old \\u003cem\\u003eU. virens\\u003c/em\\u003e cultures using an Ultrapure RNA Kit (CWBIO). Complementary DNA (cDNA) synthesis was performed with a PrimeScript reverse transcription kit (TaKaRa, RR047A, Japan) following the manufacturer's standard protocols. Quantitative reverse transcription PCR (RT-qPCR) was conducted to assess \\u003cem\\u003eUgs\\u003c/em\\u003e gene expression levels utilizing the Fast SYBR mix (CWBIO, CW0955M, Jiangsu, China) on a LightCycler\\u0026reg; 96 system (Roche, Basel, Switzerland) as previously described (Wang et al. \\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e2025\\u003c/span\\u003e). The \\u003cem\\u003eα-tubulin\\u003c/em\\u003e gene was utilized as the internal control, and the relative gene expression levels were quantified employing the 2\\u003csup\\u003e\\u0026minus;ΔΔCt\\u003c/sup\\u003e method (Rao et al. \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e2013\\u003c/span\\u003e).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.8 Yeast one-hybrid assay\\u003c/h2\\u003e \\u003cp\\u003eThe yeast one-hybrid assay was conducted by using the LexA one-hybrid system following the procedure described earlier (Wanke et al. 2009). Briefly, the \\u003cem\\u003eUvCreA\\u003c/em\\u003e coding sequence was cloned into the pB42AD vector as a prey, while multiple gene promoters were cloned into the pLacZi vector. These constructs were co-transformed into chemically competent EGY48 cells (Coolaber CC302, Beijing, China) according to the manufacturer\\u0026rsquo;s instructions. Transformants were grown on SD medium lacking Trp and Ura. Positive interactions were confirmed by β-galactosidase activity using X-gal.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.9 Electrophoretic mobility shift assay (EMSA)\\u003c/h2\\u003e \\u003cp\\u003eThe coding sequence of \\u003cem\\u003eUvCreA\\u003c/em\\u003e was subcloned into the pGEX4T-1 vector for expression of glutathione S-transferase (GST) tagged proteins. The construct was introduced into \\u003cem\\u003eEscherichia coli\\u003c/em\\u003e BL21 (DE3) cells (Sangon, A330305, Shanghai, China). The recombinant protein was purified as described (Yu et al. \\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e2025\\u003c/span\\u003e). EMSA was conducted using a chemiluminescent EMSA kit (Beyotime, GS009, Shanghai, China). Briefly, the forward primers were labeled with biotin at the 5' end to generate double-stranded probes. Unlabeled and biotin-labeled mutant probes were used as controls. The biotin-labeled probe and mutant probe were diluted to 0.2 \\u0026micro;M for further use, and the unlabeled probe was diluted to 1 \\u0026micro;M (10\\u0026times;) and 2 \\u0026micro;M (100 \\u0026times;) for competition assays. The mixture sample was analyzed by polyacrylamide gel electrophoresis.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.10 Chromatin immunoprecipitation (ChIP) and qPCR\\u003c/h2\\u003e \\u003cp\\u003eChIP was performed as previously reported (Tang et al. \\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). The 14-day-old cultures of both the wild-type and \\u003cem\\u003eUvCreA\\u003c/em\\u003e-overexpressing strains were subjected to treatment with 1% formaldehyde for 10 minutes to facilitate cross-linking. Subsequently, 150 mM glycine was introduced to terminate the cross-linking process. The samples were then sequentially washed with distilled water, followed by extraction buffer I, extraction buffer II, and finally, extraction buffer Ⅲ. Subsequently, the samples were pelleted at 4\\u0026deg;C and resuspended in nucleus lysis buffer. Genomic DNA was fragmented to a size range of 200\\u0026ndash;500 bp utilizing a sonication device (Diagenode Bioruptor, Belgium), followed by centrifugation at 16,000 g at 4\\u0026deg;C for 10 minutes. The supernatant was subsequently diluted with ChIP dilution buffer and divided into three aliquots. One aliquot was kept as input, while the other two were incubated with anti-FLAG (Abcam, Ab205606, Cambridge, UK) and protein A/G beads (Abcam, ab286842, Cambridge, UK) or with anti-IgG antibodies at 4\\u0026deg;C overnight on a rotary shaker. Following incubation, the beads were pelleted by centrifugation at 3,000g for 30 seconds and subjected to sequential washes with distinct washing buffers, including (i) low-salt buffer, (ii) high-salt buffer, and (iii) LiCl buffer. The bead-bound DNA was eluted by TE buffer. Finally, ChIP-qPCR was conducted utilizing immunoprecipitated DNA as the template, employing Taq Pro Universal SYBR qPCR Master Mix (Vazyme) and a LightCycler 96 (Roche, Basel, Switzerland). The enrichment fold was determined by comparing the signal from the immunoprecipitated (IP) sample using an anti-FLAG antibody to that of the negative control (IgG).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.11 Statistical analyses\\u003c/h2\\u003e \\u003cp\\u003eAll data were analyzed with GraphPad Prism 9.5 (La Jolla, CA), while IBM SPSS Statistics 26 (IBM, New York) was employed to present the data as mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;standard deviation (SD). Statistically significant differences (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) were evaluated through one-way ANOVA, followed by Tukey\\u0026rsquo;s test or Student\\u0026rsquo;s \\u003cem\\u003et\\u003c/em\\u003e-test.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"3. Results\",\"content\":\"\\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003e3.1 Carbon sources pose distinct effects on ustilaginoidin accumulation and mycelial growth in \\u003cem\\u003eU. virens\\u003c/em\\u003e\\u003c/h2\\u003e\\n \\u003cp\\u003eFor the assessment of the impact of various carbon sources on ustilaginoidin biosynthesis, the wild-type strain was cultivated on CM or modified CM medium plates, wherein sucrose was replaced with glucose, D-fructose, or trehalose as the exclusive carbon source. Ustilaginoidins were extracted from cultures grown for 28 days on various medium plates and were then quantified through HPLC. Among the tested carbon sources, \\u003cem\\u003eU. virens\\u003c/em\\u003e generated the highest amount of ustilaginoidins when culturing on CM medium with D-fructose as the sole carbon source. In contrast, the fungus produced the lowest level of ustilaginoidins when cultured on a glucose-containing CM medium (Fig. \\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA). In addition, we compared the expression levels of \\u003cem\\u003eUvPKS1\\u003c/em\\u003e and \\u003cem\\u003eUgsJ\\u003c/em\\u003e in the wild-type strain when culturing on four types of carbon source media for 14 days. RT-qPCR analysis revealed that across all four carbon sources, the expression of \\u003cem\\u003eUvPKS1\\u003c/em\\u003e and \\u003cem\\u003eUgsJ\\u003c/em\\u003e was highest in D-fructose and trehalose, followed by sucrose, and lowest in glucose-containing medium (Fig. \\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB). These results demonstrated a significant effect of different carbon sources on ustilaginoidin production.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003e3.2 UvCreA is a putative zinc finger transcription factor in \\u003cem\\u003eU. virens\\u003c/em\\u003e\\u003c/h2\\u003e\\n \\u003cp\\u003eCCR, in filamentous fungi, is a key mechanism for regulating carbon source utilization, which is often mediated by the transcription factor CreA (Huang et al. \\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e2025\\u003c/span\\u003e). First, we identified UvCreA as the transcription factor regulating CCR in \\u003cem\\u003eU. virens\\u003c/em\\u003e through structural analysis. UvCreA contains two conserved zinc finger (ZF) domains, ZF1 and ZF2, located in the N-terminal region (Figure \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003eA). These domains are predicted to be critical for DNA binding and protein-protein interactions. Second, a phylogenetic tree was constructed using the maximum-likelihood method for CreA homologs across different fungal species. Protein sequences were aligned using MEGA X, and the tree topology shows that UvCreA (XP_042997845.1) is evolutionarily closest to the homolog from \\u003cem\\u003eMetarhizium anisopliae\\u003c/em\\u003e. By contrast, CreA homologs in \\u003cem\\u003eAspergillus\\u003c/em\\u003e form a distinct branch (Figure \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003eB). Subsequently, the three-dimensional structure of UvCreA was predicted using AlphaFold2, revealing a complex folding pattern with multiple \\u0026alpha;-helices and \\u0026beta;-sheets characteristic of zinc finger-containing proteins (Figure \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003eC). Collectively, UvCreA is a putative transcription factor comprising two ZF domains for CCR regulation.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003e3.3 UvCreA regulates mycelial growth, conidial production, and germination rates in \\u003cem\\u003eU. virens\\u003c/em\\u003e\\u003c/h2\\u003e\\n \\u003cp\\u003eTo identify the roles of \\u003cem\\u003eUvCreA\\u003c/em\\u003e, the mutants with \\u003cem\\u003eUvCreA-\\u003c/em\\u003edeletion were created via homologous recombination (Figure \\u003cspan refid=\\\"MOESM2\\\" class=\\\"InternalRef\\\"\\u003eS2\\u003c/span\\u003eA). Subsequently, the plasmid-borne \\u003cem\\u003eUvCreA\\u003c/em\\u003e gene construct with a strong promoter, \\u003cem\\u003eUV_08266\\u003c/em\\u003e, was created and introduced into the protoplasts of both the wild-type and \\u0026Delta;\\u003cem\\u003eUvcreA\\u003c/em\\u003e mutant strains using PEG-mediated transformation. Two \\u003cem\\u003eUvCreA\\u003c/em\\u003e-complemented strains (C\\u0026Delta;\\u003cem\\u003eUvcreA\\u003c/em\\u003e-64-15 and C\\u0026Delta;\\u003cem\\u003eUvcreA\\u003c/em\\u003e-64-18) and two \\u003cem\\u003eUvCreA\\u003c/em\\u003e-overexpressing strains (OE-UvCreA-FLAG-16 and OE-UvCreA-FLAG-18) were successfully verified by immunoblot analysis (Figure \\u003cspan refid=\\\"MOESM2\\\" class=\\\"InternalRef\\\"\\u003eS2\\u003c/span\\u003eB). After 14 days of culturing, colony morphology was observed for the wild-type, \\u0026Delta;\\u003cem\\u003eUvcreA\\u003c/em\\u003e-4/64, C\\u0026Delta;\\u003cem\\u003eUvcreA\\u003c/em\\u003e-64, and OE-UvCreA-FLAG strains (Fig. \\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA). The colony diameters were also measured, and we found that the \\u0026Delta;\\u003cem\\u003eUvcreA\\u003c/em\\u003e-4 and \\u0026minus;\\u0026thinsp;64 mutants had smaller colony diameters than the wild-type strain (Fig. \\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA), whereas the mutants produced more conidia per conidiophore than the wild type (Fig. \\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB). The C\\u0026Delta;\\u003cem\\u003eUvcreA\\u003c/em\\u003e-64 strain can restore these phenotypes near the wild-type levels. In addition, the colony diameter of the overexpressing strain was significantly greater than that of the wild-type strain (Fig. \\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC).\\u003c/p\\u003e\\n \\u003cp\\u003eConidiation assays showed that the \\u0026Delta;\\u003cem\\u003eUvcreA\\u003c/em\\u003e-4 and \\u0026Delta;\\u003cem\\u003eUvcreA\\u003c/em\\u003e-64 mutants produce noticeably more conidiospores than the wild-type strain after 7 days of shaking culture. Interestingly, the \\u003cem\\u003eUvCreA-FLAG\\u003c/em\\u003e-overexpressing strain generated far fewer conidia compared to the wild-type (Fig. \\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eD). Additionally, we observed that the \\u0026Delta;\\u003cem\\u003eUvcreA\\u003c/em\\u003e-4 and \\u0026Delta;\\u003cem\\u003eUvcreA\\u003c/em\\u003e-64 mutants had much higher germination rates in PSA medium, while the overexpressing strain\\u0026rsquo;s germination rate was significantly lower than that of the wild-type (Fig. \\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eE). These findings suggest that UvCreA supports mycelial growth, but suppresses conidiogenesis and conidial germination in \\u003cem\\u003eU. virens\\u003c/em\\u003e.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec18\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003e3.4 UvCreA positively regulates ustilaginoidin biosynthesis and pathogenicity in \\u003cem\\u003eU. virens\\u003c/em\\u003e\\u003c/h2\\u003e\\n \\u003cp\\u003eTo investigate the role of UvCreA in the pathogenicity and ustilaginoidin production in \\u003cem\\u003eU. virens\\u003c/em\\u003e, the amounts of ustilaginoidins produced by wild-type, \\u003cem\\u003eUvCreA\\u003c/em\\u003e knockout, complemented, and overexpressing strains were measured. After culturing on PSA plates for 28 days, the mycelia were harvested for ustilaginoidin extraction using ethyl acetate. HPLC analysis showed that the \\u0026Delta;\\u003cem\\u003eUvcreA\\u003c/em\\u003e mutant strains had significantly lower ustilaginoidin production compared with the wild-type strain. The C\\u0026Delta;\\u003cem\\u003eUvcreA\\u003c/em\\u003e-64 complemented strain partially recovered the ability to produce ustilaginoidins. In contrast, the strain overexpressing \\u003cem\\u003eUvCreA\\u003c/em\\u003e produced more ustilaginoidins than the wild type (Fig. \\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA). Additionally, the expression levels of genes involved in ustilaginoidin biosynthesis, including \\u003cem\\u003eUgsR1, UgsR2, UgsO, UvPKS1, UgsZ, UgsT, UgsH\\u003c/em\\u003e, and \\u003cem\\u003eUgsJ\\u003c/em\\u003e, were measured using RT-qPCR. The results showed that in \\u0026Delta;\\u003cem\\u003eUvcreA\\u003c/em\\u003e-4 and \\u0026Delta;\\u003cem\\u003eUvcreA\\u003c/em\\u003e-64 mutants, these \\u003cem\\u003eUgs\\u003c/em\\u003e genes were significantly downregulated, whereas in the C\\u0026Delta;\\u003cem\\u003eUvcreA\\u003c/em\\u003e strain, the transcript levels of the tested genes were similar to or higher than those in the wild type (Fig. \\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB).\\u003c/p\\u003e\\n \\u003cp\\u003eInoculation assays demonstrated that rice panicles inoculated with \\u0026Delta;\\u003cem\\u003eUvcreA\\u003c/em\\u003e mutants exhibited significantly fewer false smut balls in comparison with the wild-type strain (Fig. \\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eC-D). Conversely, the OE-UvCreA-FLAG strain produced a greater number of false smut balls than both the wild-type and C\\u0026Delta;\\u003cem\\u003eUvcreA\\u003c/em\\u003e-64 complemented strains. These findings imply that UvCreA plays a positive role in the biosynthesis of ustilaginoidins and contributes to the virulence of \\u003cem\\u003eU. virens\\u003c/em\\u003e.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec19\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003e3.5 UvCreA negatively regulates tolerance to various stress factors\\u003c/h2\\u003e\\n \\u003cp\\u003eGrowth phenotypes of the wild-type, \\u0026Delta;\\u003cem\\u003eUvcreA\\u003c/em\\u003e-4, \\u0026Delta;\\u003cem\\u003eUvcreA\\u003c/em\\u003e-64, and C\\u0026Delta;\\u003cem\\u003eUvcreA\\u003c/em\\u003e-64 strains were observed under various stress conditions to investigate the role of UvCreA in stress tolerance, including oxidative stress (2.5 mM H₂O₂), cell wall stress (70 \\u0026micro;g/mL Congo red), cell membrane stress (0.015% SDS), and osmotic stress (0.25 M NaCl) (Fig. \\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA). The \\u0026Delta;\\u003cem\\u003eUvcreA\\u003c/em\\u003e mutants showed significantly lower relative growth inhibition rates than the wild-type and C\\u0026Delta;\\u003cem\\u003eUvcreA\\u003c/em\\u003e-64 strains under all tested stress conditions. Specifically, under H₂O₂ and Congo red stress conditions, \\u0026Delta;\\u003cem\\u003eUvcreA\\u003c/em\\u003e-4 and \\u0026Delta;\\u003cem\\u003eUvcreA\\u003c/em\\u003e-64 exhibited negative growth inhibition rates. these findings indicate that the \\u003cem\\u003eUvCreA\\u003c/em\\u003e-knockout mutants demonstrate greater tolerance to H₂O₂ and Congo red than the wild-type strain on YT medium plates. In comparison, when subjected to SDS and NaCl, the inhibition rates of the \\u0026Delta;\\u003cem\\u003eUvcreA\\u003c/em\\u003e\\u0026thinsp;\\u0026minus;\\u0026thinsp;4 and \\u0026Delta;\\u003cem\\u003eUvcreA\\u003c/em\\u003e\\u0026thinsp;\\u0026minus;\\u0026thinsp;64 strains were notably lower than those of the wild-type strain (Fig. \\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eB). Collectively, these results indicate that deletion of \\u003cem\\u003eUvCreA\\u003c/em\\u003e reduces the sensitivity of \\u003cem\\u003eU. virens\\u003c/em\\u003e to oxidative, cell wall, membrane, and osmotic stresses.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec20\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003e3.6 UvCreA positively regulates the expression of ustilaginoidin biosynthetic genes through binding gene promoters\\u003c/h2\\u003e\\n \\u003cp\\u003eAs a transcription factor, UvCreA regulates the expression of numerous genes. Therefore, we predicted the UvCreA-binding \\u003cem\\u003ecis-\\u003c/em\\u003eelements through the JASPAR database (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://jaspar.elixir.no/\\u003c/span\\u003e\\u003c/span\\u003e) and found that S(G/C)Y(T/C)GGR(G/A)G is a putative \\u003cem\\u003ecis-\\u003c/em\\u003eelement (Fig. \\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA). Furthermore, we predicted the number of UvCreA-binding \\u003cem\\u003ecis\\u003c/em\\u003e-elements in the promoter regions of the ustilaginoidin synthesis-related genes (Table \\u003cspan refid=\\\"MOESM2\\\" class=\\\"InternalRef\\\"\\u003eS2\\u003c/span\\u003e). To investigate the effect of UvCreA on expression of ustilaginoidin biosynthetic genes, we chose the \\u003cem\\u003eUgsJ\\u003c/em\\u003e and \\u003cem\\u003eUvPKS1\\u003c/em\\u003e genes for validation. The coding sequence of \\u003cem\\u003eUvCreA\\u003c/em\\u003e and the promoter regions (~\\u0026thinsp;1.5 kb) of \\u003cem\\u003eUgsJ\\u003c/em\\u003e and \\u003cem\\u003eUvPKS1\\u003c/em\\u003e genes were amplified to construct the vectors pB42AD-\\u003cem\\u003eUvCreA\\u003c/em\\u003e, pLacZi-\\u003cem\\u003eUgsJ\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003ePro\\u003c/em\\u003e\\u003c/sub\\u003e, and pLacZi-\\u003cem\\u003eUvPKS1\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003ePro\\u003c/em\\u003e\\u003c/sub\\u003e, respectively. The subsequent yeast one-hybrid assay revealed that the yeast colonies co-transformed with pB42AD-\\u003cem\\u003eUvCreA\\u003c/em\\u003e/pLacZi-\\u003cem\\u003eUgsJ\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003ePro\\u003c/em\\u003e\\u003c/sub\\u003e and with pB42AD-\\u003cem\\u003eUvCreA\\u003c/em\\u003e/pLacZi-\\u003cem\\u003eUvPKS1\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003ePro\\u003c/em\\u003e\\u003c/sub\\u003e turned blue in the presence of X-Gal (Fig. \\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eB), indicating that UvCreA binds the promoter regions of \\u003cem\\u003eUvPKS1\\u003c/em\\u003e and \\u003cem\\u003eUgsJ\\u003c/em\\u003e.\\u003c/p\\u003e\\n \\u003cp\\u003eTo further determine whether UvCreA binds to the \\u003cem\\u003ecis\\u003c/em\\u003e-elements in the promoters of \\u003cem\\u003eUvPKS1\\u003c/em\\u003e and \\u003cem\\u003eUgsJ\\u003c/em\\u003e, we performed electrophoretic mobility shift assays. Primers containing the \\u003cem\\u003ecis\\u003c/em\\u003e-element GTGGGG or CCCCAC, with 7-bp flanking regions on either side, were designed. These primers were biotin-labelled as EMSA probes. When in vitro purified GST-UvCreA was incubated with a biotin-labeled probe derived from the \\u003cem\\u003eUvPKS1\\u003c/em\\u003e or \\u003cem\\u003eUgsJ\\u003c/em\\u003e promoter in EMSA, distinct band shifts were observed. In addition, the shifted bands gradually faded when increasing amounts of unlabeled probes were added. While GST-UvCreA was incubated with biotin-labeled mutant probes as negative controls, the shift bands disappeared (Fig. \\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eC-D). The results collectively indicate that UvCreA directly binds the \\u003cem\\u003ecis\\u003c/em\\u003e-elements in the promoters of \\u003cem\\u003eUvPKS1\\u003c/em\\u003e and \\u003cem\\u003eUgsJ\\u003c/em\\u003e.\\u003c/p\\u003e\\n \\u003cp\\u003eTo confirm that UvCreA specifically binds to the \\u003cem\\u003eUvPKS1\\u003c/em\\u003e and \\u003cem\\u003eUgsJ\\u003c/em\\u003e promoters, we conducted ChIP assays using the OE-UvCreA-FLAG strain. Genomic DNA was fragmented and then incubated with anti-FLAG antibody or IgG antibody in the presence of protein A/G beads. ChIP-qPCR was performed using bead-bound DNA as a template to investigate whether UvCreA enriches the promoter regions of \\u003cem\\u003eUvPKS1\\u003c/em\\u003e and \\u003cem\\u003eUgsJ\\u003c/em\\u003e. We designed individually two pairs of primers to validate CreA enrichment in the promoter regions of \\u003cem\\u003eUvPKS1\\u003c/em\\u003e and \\u003cem\\u003eUgsJ\\u003c/em\\u003e. The \\u003cem\\u003eUvPKS1\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003ePro\\u003c/em\\u003e\\u003c/sub\\u003e-1 and \\u003cem\\u003eUgsJ\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003ePro\\u003c/em\\u003e\\u003c/sub\\u003e-1 primers were used to amplify regions containing the \\u003cem\\u003ecis\\u003c/em\\u003e-element motifs, whereas the \\u003cem\\u003eUvPKS1\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003ePro\\u003c/em\\u003e\\u003c/sub\\u003e-2 and \\u003cem\\u003eUgsJ\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003ePro\\u003c/em\\u003e\\u003c/sub\\u003e-2 primers amplify control regions without the \\u003cem\\u003ecis\\u003c/em\\u003e-element (Fig. \\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eE). The results showed that the \\u003cem\\u003eUvPKS1\\u003c/em\\u003e and \\u003cem\\u003eUgsJ\\u003c/em\\u003e promoter regions immunoprecipitated by anti-FLAG antibody were significantly enriched compared with the anti-IgG control (Fig. \\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eF). These results indicate that UvCreA directly and specifically binds to the \\u003cem\\u003eUvPKS1\\u003c/em\\u003e and \\u003cem\\u003eUgsJ\\u003c/em\\u003e promoters.\\u003c/p\\u003e\\n\\u003c/div\\u003e\"},{\"header\":\"4. Discussion\",\"content\":\"\\u003cp\\u003eDuring infection, fungi have to utilize limited nutrients efficiently to complete the infection process. Carbon sources are one of the most important nutrients. Numerous empirical studies have shown that carbon source utilization regulates various physiological phenotypes, including growth rate, conidial yield, and secondary metabolite synthesis (Achim\\u0026oacute;n et al. \\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e; Achim\\u0026oacute;n et al. \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e; Ruiz et al. \\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e2010\\u003c/span\\u003e). CreA plays a pivotal role as a key regulatory factor in this process (Brown et al. \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e). Starch and sugar metabolism provide important carbon sources for plant pathogens during infection, which is closely related to pathogenicity. The transcriptome and metabolome correlation analysis reveals that four metabolites in rice, including glucose-6-phosphate, trehalose, sucrose, and D-fructose, are significantly different before and after \\u003cem\\u003eU. virens\\u003c/em\\u003e infection (Ozcengiz et al. 2013).\\u003c/p\\u003e \\u003cp\\u003eUstilaginoidins are major mycotoxins produced by \\u003cem\\u003eU. virens\\u003c/em\\u003e, and pose a severe impact on human and animal health and rice quality (Sun et al. \\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e). Studies have shown that the type of carbon source directly affects the production of mycotoxins (Szil\\u0026aacute;gyi et al. \\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e2013\\u003c/span\\u003e). In \\u003cem\\u003ePenicillium chrysogenum\\u003c/em\\u003e, glucose, sucrose, and to a certain extent, fructose all have a negative impact on the biosynthesis of penicillin (Ruiz-Villaf\\u0026aacute;n et al. \\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). Penicillin biosynthesis in \\u003cem\\u003eP. chrysogenum\\u003c/em\\u003e is subject to CCR. Specifically, glucose has been shown to strongly inhibit penicillin production by down-regulating the expression of the biosynthetic gene cluster (\\u003cem\\u003epcbAB\\u003c/em\\u003e, \\u003cem\\u003epcbC\\u003c/em\\u003e, \\u003cem\\u003epenDE\\u003c/em\\u003e) (Ozcengiz et al. 2013; Ruiz-Villaf\\u0026aacute;n et al. \\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). In this study, we demonstrated that the wild-type strain produced higher levels of ustilaginoidins on D-fructose and trehalose-containing media than on glucose or sucrose-containing media (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA). This might be attributed to glucose and sucrose being preferred or superior carbon sources in filamentous fungi (Fernandez et al. \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e), whereas D-fructose and trehalose are alternative carbon sources (Nihashi et al. 1984).\\u003c/p\\u003e \\u003cp\\u003eNext, we investigated the role of UvCreA in the pathogenicity of \\u003cem\\u003eU. virens\\u003c/em\\u003e. Consistent with a previous study (Xie et al. \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e), we found that deletion of \\u003cem\\u003eUvCreA\\u003c/em\\u003e reduced growth rate and pathogenicity in \\u003cem\\u003eU. virens\\u003c/em\\u003e. More interestingly, conidial yield and germination rate of Δ\\u003cem\\u003eUvcreA\\u003c/em\\u003e mutants were significantly increased (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). By contrast, in the rice blast fungus \\u003cem\\u003eMagnaporthe oryzae\\u003c/em\\u003e, the deletion of \\u003cem\\u003eMoCreA\\u003c/em\\u003e results in reduced growth, conidial yield, and delayed growth (Wanke et al. 2009). The \\u003cem\\u003eCre1-\\u003c/em\\u003edeletion mutant in \\u003cem\\u003eTrichoderma reesei\\u003c/em\\u003e also exhibits morphological changes, including reduced ascospore formation (Nakari-Set\\u0026auml;l\\u0026auml; et al. \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e2009\\u003c/span\\u003e). These findings indicate that CreA has diverse regulatory mechanisms for conidiation across fungal species, providing a new perspective on its role in carbon source utilization.\\u003c/p\\u003e \\u003cp\\u003eMoreover, CreA is widely involved in regulating stress tolerance in filamentous fungi, but its specific functions may vary across species. The Δ\\u003cem\\u003ecreA\\u003c/em\\u003e mutant in \\u003cem\\u003eP. chrysogenum\\u003c/em\\u003e is more sensitive to calcofluor white (CFW) than the wild type. In \\u003cem\\u003eValsa mali\\u003c/em\\u003e, CFW-induced cell wall stress significantly inhibits the growth of Δ\\u003cem\\u003ecreA\\u003c/em\\u003e, indicating that CreA may participate in maintaining cell wall integrity (Fasoyin et al. \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). In our study, the Δ\\u003cem\\u003eUvcreA\\u003c/em\\u003e mutant exhibited significantly enhanced tolerance to osmotic stress (NaCl), oxidative stress (H₂O₂), cell wall stress (Congo red), and membrane stress (SDS) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). This result is consistent with the phenotypic trait of Δ\\u003cem\\u003ecreA\\u003c/em\\u003e in \\u003cem\\u003eA. flavus\\u003c/em\\u003e, which shows enhanced tolerance to salt and oxidative stresses. These differences indicate that CreA's regulatory role in stress responses shows significant species specificity in filamentous fungi.\\u003c/p\\u003e \\u003cp\\u003eSecondary metabolites are synthesized during pathogen infection by enzymes encoded by biosynthetic gene clusters and are harmful to the host's physiological activities and growth (Grau et al. \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). As a global transcription factor, CreA is presumably involved in secondary metabolism by regulating the expression of biosynthetic gene clusters. The mycotoxin deoxynivalenol (DON) produced by \\u003cem\\u003eFusarium graminearum\\u003c/em\\u003e is a triterpene compound synthesized by 15 \\u003cem\\u003eTRI\\u003c/em\\u003e genes (Fernandez et al. \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e). The promoters of 10 \\u003cem\\u003eTRI\\u003c/em\\u003e genes, including \\u003cem\\u003eTRI6\\u003c/em\\u003e, in the \\u003cem\\u003eTRI\\u003c/em\\u003e gene cluster contain typical CreA-binding \\u003cem\\u003ecis\\u003c/em\\u003e-elements 5' -SYGGRG\\u0026thinsp;\\u0026minus;\\u0026thinsp;3' (Hou Rui et al. 2018), suggesting that CreA regulates DON biosynthesis by regulating the expression of \\u003cem\\u003eTRI\\u003c/em\\u003e genes. The biosynthesis of ustilaginoidin is regulated by the \\u003cem\\u003eUgs\\u003c/em\\u003e gene cluster in \\u003cem\\u003eU. virens\\u003c/em\\u003e (Wang et al. \\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). Consequently, we investigated whether CreA regulates the expression of the \\u003cem\\u003eUgs\\u003c/em\\u003e gene cluster. Through yeast one-hybrid assay, EMSA, chromatin immunoprecipitation, and quantitative PCR, we confirmed that UvCreA regulates the expression of \\u003cem\\u003eUvPKS1\\u003c/em\\u003e and \\u003cem\\u003eUgsJ\\u003c/em\\u003e by directly binding to the \\u003cem\\u003ecis\\u003c/em\\u003e-elements 5' -SYGGRG\\u0026thinsp;\\u0026minus;\\u0026thinsp;3' in the promoters of these \\u003cem\\u003eUgs\\u003c/em\\u003e genes (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eB). Similarly, CreA positively regulates aflatoxin biosynthesis in \\u003cem\\u003eA. flavus\\u003c/em\\u003e (Wanke et al. 2009). Altogether, these results indicate that CreA is necessary for the synthesis of secondary metabolites and the utilization of carbon sources. Numerous genes in fungal genomes contain the SYGGRG motif, which is specifically recognized by CreA (Antoni\\u0026ecirc;to et al. \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e; Cubero et al. 1994). Besides acting as a transcriptional repressor, CreA acts as a transcription factor that activates the expression of certain genes (Antoni\\u0026ecirc;to et al. \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e). Therefore, the functions of CreA in regulating various fungal processes and activities remain to be further investigated.\\u003c/p\\u003e \\u003cp\\u003eIn this study, UvCreA, a key transcription factor in carbon source utilization, positively regulates ustilaginoidins biosynthesis, which may depend on the type of carbon sources (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). This finding not only elucidates the molecular pathway through which carbon source types regulate ustilaginoidin synthesis via UvCreA but also provides critical insights into how the composition of carbon sources in rice panicles during the infection of rice by \\u003cem\\u003eU. virens\\u003c/em\\u003e influences ustilaginoidin production by \\u003cem\\u003eU. virens\\u003c/em\\u003e in field conditions. Specifically, the later stages of infection may involve the accumulation of greater amounts of trehalose or D-fructose in rice panicles, creating favorable conditions for ustilaginoidin biosynthesis by \\u003cem\\u003eU. virens\\u003c/em\\u003e. Future studies could investigate the correlation between dynamic changes in carbon source composition in rice panicles and ustilaginoidin accumulation, offering a theoretical basis for developing targeted strategies to mitigate mycotoxin contamination.\\u003c/p\\u003e \\u003cp\\u003eIn conclusion, our results show that UvCreA positively regulates the growth, development, and pathogenicity of \\u003cem\\u003eU. virens\\u003c/em\\u003e, but negatively regulates conidial production and germination, and tolerance to various stresses. As a global transcription factor, UvCreA positively regulates the biosynthesis of ustilaginoidins in \\u003cem\\u003eU. virens\\u003c/em\\u003e by directly binding to the promoters in the \\u003cem\\u003eUgs\\u003c/em\\u003e gene cluster.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eA\\u003c/strong\\u003e\\u003cstrong\\u003ecknowledgements\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe thank the platform support from the College of Plant Protection at Jilin Agricultural University and China Agricultural University, and thank Wenxian Sun and Ling Liu for their funding of the project.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor contributions\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eW.S. and L.L. designed the experiments. X.B., Z.P., and J.W. performed the experiments and collected data. Y.D., J.W., M.O., M.Z., R.C., X.T., Y.Z., and Y.L. performed data analyses. X.B. wrote the manuscript, and W.S., L.L., D.L., and M.Z.N.D. revised the manuscript. This submitted version of the manuscript was reviewed and completed by all authors.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe work is supported by the National Natural Science Foundation of China grant (31630064), the China Agricultural Research System (CARS01), the Science and Technology Development Project of Jilin Province (20240304122SF), the Jilin Provincial Department of Education-Science and Technology Project (JJKH20230407KJ), and the 111 project (D17014).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAvailability of data and materials\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe datasets used and/or analyzed in this study are available from the corresponding authors on request.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthical approval and consent to participate\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent for publication\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare that they have no competing interests.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n \\u003cli\\u003eAchim\\u0026oacute;n F, Dambolena J S, Zygadlo J A, Pizzolitto R P. Carbon sources as factors affecting the secondary metabolism of the maize pathogen \\u003cem\\u003eFusarium verticillioides\\u003c/em\\u003e\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eLwt. 2019;115:108470. https://doi.org/10.1016/j.lwt.2019.108470.\\u003c/li\\u003e\\n \\u003cli\\u003eAchim\\u0026oacute;n F, Krapacher C R, Jacquat A G, Pizzolitto R P, Zygadlo J A. Carbon sources to enhance the biosynthesis of useful secondary metabolites in \\u003cem\\u003eFusarium verticillioides\\u003c/em\\u003e submerged cultures\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eWorld Journal of Microbiology and Biotechnology. 2021;37:78. https://doi.org/10.1007/s11274-021-03044-z.\\u003c/li\\u003e\\n \\u003cli\\u003eAntoni\\u0026ecirc;to A C C, Dos Santos Castro L, Silva-Rocha R, Persinoti G F, Silva R N. Defining the genome-wide role of CRE1 during carbon catabolite repression in \\u003cem\\u003eT\\u003c/em\\u003e\\u003cem\\u003erichoderma reesei\\u003c/em\\u003e using RNA-seq analysis\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eFungal Genet. Biol. 2014;73:93-103. https://doi.org/10.1016/j.fgb.2014.10.009.\\u003c/li\\u003e\\n \\u003cli\\u003eBenocci T, Aguilar-Pontes M V, Zhou M, Seiboth B, de Vries R P. Regulators of plant biomass degradation in ascomycetous fungi\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eBiotechnol. Biofuels. 2017;10:1-25. https://doi.org/10.1186/s13068-017-0841-x.\\u003c/li\\u003e\\n \\u003cli\\u003eBrown N A, Ries L N A, Goldman G H. How nutritional status signalling coordinates metabolism and lignocellulolytic enzyme secretion\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eFungal Genet. Biol. 2014;72:48-63. https://doi.org/10.1016/j.fgb.2014.06.012.\\u003c/li\\u003e\\n \\u003cli\\u003eChen X, Hai D, Tang J, Liu H, Huang J, Luo C, et al. UvCom1 is an important regulator required for development and infection in the rice false smut fungus \\u003cem\\u003eUstilaginoidea virens\\u003c/em\\u003e\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003ePhytopathology. 2020;110:483-93. https://doi.org/10.1094/PHYTO-05-19-0179-R.\\u003c/li\\u003e\\n \\u003cli\\u003eCubero B, Scazzocchio C. Two different, adjacent and divergent zinc finger binding sites are necessary for CREA‐mediated carbon catabolite repression in the proline gene cluster of \\u003cem\\u003eA\\u003c/em\\u003e\\u003cem\\u003espergillus nidulans\\u003c/em\\u003e.\\u003cem\\u003e\\u0026nbsp;\\u003c/em\\u003eThe Embo Journal. 1994;13:407-15. https://doi.org/10.1002/j.1460-2075.1994.tb06275.x.\\u003c/li\\u003e\\n \\u003cli\\u003eFasoyin O E, Wang B, Qiu M, Han X, Chung K, Wang S. Carbon catabolite repression gene CreA regulates morphology, aflatoxin biosynthesis and virulence in \\u003cem\\u003eA\\u003c/em\\u003e\\u003cem\\u003espergillus flavus\\u003c/em\\u003e\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eFungal Genet. Biol. 2018;115:41-51. https://doi.org/10.1016/j.fgb.2018.04.008.\\u003c/li\\u003e\\n \\u003cli\\u003eFernandez J, Wright J D, Hartline D, Quispe C F, Madayiputhiya N, Wilson R A. Principles of carbon catabolite repression in the rice blast fungus: Tps1, Nmr1-3, and a MATE\\u0026ndash;family pump regulate glucose metabolism during infection\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003ePlos Genet. 2012;8:e1002673. https://doi.org/10.1371/journal.pgen.1002673.\\u003c/li\\u003e\\n \\u003cli\\u003eFu X, Wang W, Li Y, Wang X, Wang B. Development of a monoclonal antibody with equal reactivity to ustiloxins A and B for quantification of main cyclopeptide mycotoxins in rice samples\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eFood Control. 2018;92:201-7. https://doi.org/10.1016/j.foodcont.2018.04.048.\\u003c/li\\u003e\\n \\u003cli\\u003eGrau M F, Entwistle R, Chiang Y, Ahuja M, Oakley C E, Akashi T, et al. Hybrid transcription factor engineering activates the silent secondary metabolite gene cluster for (+)-Asperlin in \\u003cem\\u003eAspergillus nidulans\\u003c/em\\u003e\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eAcs Chem. Biol. 2018;13:3193-205. https://doi.org/10.1021/acschembio.8b00679.\\u003c/li\\u003e\\n \\u003cli\\u003eHou R, Wang C. The function of the carbon metabolism regulator FgCreA in \\u003cem\\u003eF\\u003c/em\\u003e\\u003cem\\u003eusarium graminearum\\u003c/em\\u003e. Scientia Agricultura Sinica\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003e2018;51: 257-67. https://doi.org/10.3864/j.issn.0578-1752.2018.02.006.\\u003c/li\\u003e\\n \\u003cli\\u003eHuang Z, Wang Q, Li Y, Huang P, Liao J, Wang J, et al. A multilayered regulatory network mediated by protein phosphatase 4 controls carbon catabolite repression and de-repression in \\u003cem\\u003eMagnaporthe oryzae\\u003c/em\\u003e\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eCommun. Biol. 2025;8:130. https://doi.org/10.1038/s42003-025-07581-3.\\u003c/li\\u003e\\n \\u003cli\\u003eJiang C, Li X, Fang A, Fu Y, Li C, Dong H, et al. An \\u003cem\\u003eUstilaginoidea virens\\u003c/em\\u003e nuclear effector SCRE7 inhibits rice immunity via suppressing OsLBD11/12‐promoted transcription of \\u003cem\\u003eOsCPS2\\u003c/em\\u003e\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003ePlant Biotechnol. J. 2026;24:1305-21. https://doi.org/10.1111/pbi.70413.\\u003c/li\\u003e\\n \\u003cli\\u003eLi W L, Li L Y, Feng A Q, Zhu X Y, Li J X. Rice false smut fungus, \\u003cem\\u003eUstilaginoidea virens\\u003c/em\\u003e, inhibits pollen germination and degrades the integuments of rice ovule\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eAmerican Journal of Plant Sciences. 2013;4:2295-304. https://doi.org/10.4236/ajps.2013.412284.\\u003c/li\\u003e\\n \\u003cli\\u003eLi Y, Wang M, Liu Z, Zhang K, Cui F, Sun W. Towards understanding the biosynthetic pathway for ustilaginoidin mycotoxins in \\u003cem\\u003eUstilaginoidea virens\\u003c/em\\u003e\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eEnviron. Microbiol. 2019;21:2629-43. https://doi.org/10.1111/1462-2920.14572.\\u003c/li\\u003e\\n \\u003cli\\u003eLin X, Bian Y, Mou R, Cao Z, Cao Z, Zhu Z, et al. Isolation, identification, and characterization of \\u003cem\\u003eUstilaginoidea virens\\u003c/em\\u003e from rice false smut balls with high ustilotoxin production potential\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eJ. Basic Microbiol. 2018;58:670-8. https://doi.org/10.1002/jobm.201800167.\\u003c/li\\u003e\\n \\u003cli\\u003eLiu L, Wang B, Duan G, Wang J, Pan Z, Ou M, et al. Histone deacetylase UvHST2 is a global regulator of secondary metabolism in \\u003cem\\u003eUstilaginoidea virens\\u003c/em\\u003e\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eJ. Agric. Food. Chem. 2023;71:13124-36. https://doi.org/10.1021/acs.jafc.3c01782.\\u003c/li\\u003e\\n \\u003cli\\u003eLong Z, Wang P, Yu Q, Wang B, Li D, Yang C, et al. The histone deacetylase UvHOS2 regulates vegetative growth, conidiation, ustilaginoidin synthesis, and pathogenicity in \\u003cem\\u003eUstilaginoidea virens\\u003c/em\\u003e\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003ePhytopathol. Res. 2024;6:16. https://doi.org/10.1186/s42483-024-00230-3.\\u003c/li\\u003e\\n \\u003cli\\u003eMeng J, Sun W, Mao Z, Xu D, Wang X, Lu S, et al. Main ustilaginoidins and their distribution in rice false smut balls\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eToxins. 2015;7:4023-34. https://doi.org/10.3390/toxins7104023.\\u003c/li\\u003e\\n \\u003cli\\u003eMeshram M K, Wasule D L. Method for extracting genomic DNA from filaments fungi.\\u003cem\\u003e\\u0026nbsp;\\u003c/em\\u003eAnnals of Plant Physiology. 2010;24:108-9.\\u003c/li\\u003e\\n \\u003cli\\u003eMogensen J, Nielsen H B, Hofmann G, Nielsen J. Transcription analysis using high-density micro-arrays of \\u003cem\\u003eA\\u003c/em\\u003e\\u003cem\\u003espergillus nidulans\\u003c/em\\u003e wild-type and \\u003cem\\u003eC\\u003c/em\\u003e\\u003cem\\u003ere\\u003c/em\\u003e\\u003cem\\u003eA\\u003c/em\\u003e mutant during growth on glucose or ethanol\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eFungal Genet. Biol. 2006;43:593-603. https://doi.org/ 10.1016/j.fgb.2006.03.003.\\u003c/li\\u003e\\n \\u003cli\\u003eMomany M, Westfall P J, Abramowsky G. \\u003cem\\u003eAspergillus nidulans\\u003c/em\\u003e swo mutants show defects in polarity establishment, polarity maintenance and hyphal morphogenesis\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eGenetics. 1999;151:557-67. https://doi.org/10.1093/genetics/151.2.557.\\u003c/li\\u003e\\n \\u003cli\\u003eNakari-Setälä T, Paloheimo M, Kallio J, Vehmaanperä J, Penttilä M, Saloheimo M. Genetic modification of carbon catabolite repression in\\u003cem\\u003e\\u0026nbsp;Trichoderma reesei\\u0026nbsp;\\u003c/em\\u003efor improved protein production\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eAppl. Environ. Microbiol. 2009;75:4853-60. https://doi.org/10.1128/AEM.00282-09.\\u003c/li\\u003e\\n \\u003cli\\u003eNew A M, Cerulus B, Govers S K, Perez-Samper G, Zhu B, Boogmans S, et al. Different levels of catabolite repression optimize growth in stable and variable environments\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003ePlos Biol. 2014;12:e1001764. https://doi.org/10.1371/journal.pbio.1001764.\\u003c/li\\u003e\\n \\u003cli\\u003eNihashi J, Fujita Y. Catabolite repression of inositol dehydrogenase and gluconate kinase syntheses in \\u003cem\\u003eBacillus subtilis\\u003c/em\\u003e\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eBiochimica Et Biophysica Acta (BBA) - General Subjects. 1984;798:88-95. https://doi.org/10.1016/0304-4165(84)90014-X.\\u003c/li\\u003e\\n \\u003cli\\u003eOzcengiz G, Demain A L. Recent advances in the biosynthesis of penicillins, cephalosporins and clavams and its regulation\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eBiotechnol. Adv. 2013;31:287-311. https://doi.org/10.1016/j.biotechadv.2012.12.001.\\u003c/li\\u003e\\n \\u003cli\\u003eRao X, Huang X, Zhou Z, Lin X. An improvement of the 2\\u0026circ;(-delta delta CT) method for quantitative real-time polymerase chain reaction data analysis\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eBiostat Bioinforma Biomath. 2013;3:71-85. https://doi.org/10.1016/S0920-5489(99)92176-1\\u003c/li\\u003e\\n \\u003cli\\u003eRuiz B, Ch\\u0026aacute;vez A, Forero A, Garc\\u0026iacute;a-Huante Y, Romero A, S\\u0026aacute;nchez M, et al. Production of microbial secondary metabolites: regulation by the carbon source\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eCrit. Rev. Microbiol. 2010;36:146-67. https://doi.org/10.3109/10408410903489576.\\u003c/li\\u003e\\n \\u003cli\\u003eRuiz-Villaf\\u0026aacute;n B, Cruz-Bautista R, Manzo-Ruiz M, Passari A K, Villarreal-G\\u0026oacute;mez K, Rodr\\u0026iacute;guez-Sanoja R, et al. Carbon catabolite regulation of secondary metabolite formation, an old but not well-established regulatory system\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eMicrob. Biotechnol. 2022;15:1058-72. https://doi.org/10.1111/1751-7915.13791.\\u003c/li\\u003e\\n \\u003cli\\u003eXie S, Shi H, Wen H, Liu Zhi, Qiu J, Jiang N, et al. Carbon catabolite repressor UvCreA is required for development and pathogenicity in \\u003cem\\u003eUstilaginoidea virens\\u003c/em\\u003e\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eRice Sci. 2024;31:203-14. https://doi.org/10.1016/j.rsci.2023.11.008.\\u003c/li\\u003e\\n \\u003cli\\u003eSun W, Wang A, Xu D, Wang W, Meng J, Dai J, et al. New ustilaginoidins from rice false smut balls caused by \\u003cem\\u003eVillosiclava virens\\u003c/em\\u003e and their phytotoxic and cytotoxic activities\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eJ. Agric. Food. Chem. 2017;65:5151-60. https://doi.org/10.1021/acs.jafc.7b01791.\\u003c/li\\u003e\\n \\u003cli\\u003eSzil\\u0026aacute;gyi M, Miskei M, Kar\\u0026aacute;nyi Z, Lenkey B, P\\u0026oacute;csi I, Emri T. Transcriptome changes initiated by carbon starvation in \\u003cem\\u003eAspergillus nidulans\\u003c/em\\u003e\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eMicrobiology. 2013;159:176-90. https://doi.org/10.1099/mic.0.062935-0.\\u003c/li\\u003e\\n \\u003cli\\u003eTang G, Yuan J, Wang J, Zhang Y, Xie S, Wang H, et al. Fusarium BP1 is a reader of H3K27 methylation\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eNucleic. Acids. Res. 2021;49:10448-64. https://doi.org/10.1093/nar/gkab844.\\u003c/li\\u003e\\n \\u003cli\\u003eTang Y, Jin J, Hu D, Yong M, Xu Y, He L. Elucidation of the infection process of \\u003cem\\u003eUstilaginoidea virens\\u003c/em\\u003e (teleomorph: \\u003cem\\u003eVillosiclava virens\\u003c/em\\u003e) in rice spikelets\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003ePlant Pathol. 2013;62:1-8. https://doi.org/10.1111/j.1365-3059.2012.02629.x.\\u003c/li\\u003e\\n \\u003cli\\u003eWang B, Bai X, Zhang M, Liu X, Dara M, Liu L, et al. Ustilaginoidin d induces acute toxicity and hepatotoxicity in mice\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eToxins. 2025;17. https://doi.org/10.3390/toxins17050250.\\u003c/li\\u003e\\n \\u003cli\\u003eWang B, Duan G, Liu L, Long Z, Bai X, Ou M, et al. UvHOS3‐mediated histone deacetylation is essential for virulence and negatively regulates ustilaginoidin biosynthesis in \\u003cem\\u003eUstilaginoidea virens\\u003c/em\\u003e\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eMol. Plant Pathol. 2024;25:e13429. https://doi.org/10.1111/mpp.13429.\\u003c/li\\u003e\\n \\u003cli\\u003eWang B, Liu L, Li Y, Zou J, Li D, Zhao D, et al. Ustilaginoidin D induces hepatotoxicity and behaviour aberrations in zebrafish larvae\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eToxicology. 2021;456:152786. https://doi.org/10.1016/j.tox.2021.152786.\\u003c/li\\u003e\\n \\u003cli\\u003eWang G, Wang Y, Yang B, Zhang C, Zhang H, Xing F, et al. Carbon catabolite repression gene AoCreA regulates morphological development and ochratoxin a biosynthesis responding to carbon sources in \\u003cem\\u003eA\\u003c/em\\u003e\\u003cem\\u003espergillus ochraceus\\u003c/em\\u003e\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eToxins. 2020;12:697. https://doi.org/10.3390/toxins12110697.\\u003c/li\\u003e\\n \\u003cli\\u003eWang W, Fan J, Jeyakumar J M J. Rice false smut: an increasing threat to grain yield and quality\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eProtecting Rice Grains in the Post-Genomic Era. 2019;89-108. https://doi.org/10.5772/ intechopen.84862.\\u003c/li\\u003e\\n \\u003cli\\u003eWanke D, Harter K. Analysis of plant regulatory DNA sequences by the yeast-one-hybrid assay.\\u003cem\\u003e\\u0026nbsp;\\u003c/em\\u003eMethods in Molecular Biology. 2009;479:291. https://doi.org/10.1007/978-1-59745-289-2_19.\\u003c/li\\u003e\\n \\u003cli\\u003eWen H, Shi H, Jiang N, Qiu J, Lin F, Kou Y. Antifungal mechanisms of silver nanoparticles on mycotoxin producing rice false smut fungus\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eIscience. 2023;26:105763. https://doi.org/10.1016/j.isci.2022.105763.\\u003c/li\\u003e\\n \\u003cli\\u003eXu Y, Guan X, Han Z, Zhou L, Zhang Y, Asad M A U, et al. Combined effect of nitrogen fertilizer application and high temperature on grain quality properties of cooked rice\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eFront. Plant Sci. 2022;13:874033. https://doi.org/10.3389/fpls.2022.874033.\\u003c/li\\u003e\\n \\u003cli\\u003eYoav S, Salame T M, Feldman D, Levinson D, Ioelovich M, Morag E, et al. Effects of \\u003cem\\u003ec\\u003c/em\\u003e\\u003cem\\u003ere1\\u003c/em\\u003e modification in the white-rot fungus \\u003cem\\u003eP\\u003c/em\\u003e\\u003cem\\u003eleurotus ostreatus\\u003c/em\\u003e\\u003cem\\u003e\\u0026nbsp;PC\\u003c/em\\u003e\\u003cem\\u003e9\\u003c/em\\u003e: altering substrate preference during biological pretreatment\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eBiotechnol. Biofuels. 2018;11:1-16. https://doi.org/10.1186/s13068-018-1209-6.\\u003c/li\\u003e\\n \\u003cli\\u003eYu S, Zhang S, Zheng X, Li X, Wang J, Duan G, et al. \\u003cem\\u003eUstilaginoidea virens\\u003c/em\\u003e suppresses floral immunity through promoting GA biosynthesis by the effector SCRE9\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eNew Phytol. 2025;248:3115-31. https://doi.org/10.1111/nph.70414.\\u003c/li\\u003e\\n \\u003cli\\u003eYu W, Pei R, Zhou J, Zeng B, Tu Y, He B. Molecular regulation of fungal secondary metabolism\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003eWorld Journal of Microbiology and Biotechnology. 2023;39:204. https://doi.org/10.1007/s11274-023-03649-6.\\u003c/li\\u003e\\n \\u003cli\\u003eZhou L, Lu S, Shan T, Wang P, Wang S. .Chemistry and biology of mycotoxins from rice false smut pathogen. Mycotoxins: Properties, Applications and Hazards. 2012. https://doi.org/10.1021/acs.jafc.5c05629.\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Ustilaginoidea virens, Rice false smut, Transcription factor, UvCreA, Pathogenicity, Ustilaginoidins\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-9251727/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-9251727/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003e\\u003cem\\u003eUstilaginoidea virens\\u003c/em\\u003e generates several types of mycotoxins, including ustilaginoidins during infection. The transcription factor CreA plays a central role in carbon catabolite repression, coordinating carbon source utilization during fungal growth, development, and host infection. However, knowledge is limited regarding the specific functions and molecular mechanisms by which CreA regulates mycotoxin biosynthesis and pathogenicity. In this study, we found that the wild-type \\u003cem\\u003eU. virens\\u003c/em\\u003e strain exhibited significantly different ustilaginoidin-producing capacities under distinct carbon sources. The \\u003cem\\u003eUvCreA\\u003c/em\\u003e gene knockout mutants exhibited slowed mycelial growth, decreased pathogenicity and ustilaginoidin production, but produced significantly more conidiospores than the wild type. Consistently, the expression of ustilaginoidin biosynthetic genes was significantly down-regulated in the\\u003cem\\u003e UvCreA\\u003c/em\\u003e mutant. Moreover, yeast one-hybrid and electrophoretic mobility shift assays confirmed that UvCreA binds to the promoter regions of polyketide synthase gene \\u003cem\\u003eUvPKS1 \\u003c/em\\u003eand methyltransferase gene \\u003cem\\u003eUgsJ\\u003c/em\\u003e. ChIP-qPCR showed significant enrichment in promoter regions of \\u003cem\\u003eUvPKS1\\u003c/em\\u003e and \\u003cem\\u003eUgsJ\\u003c/em\\u003e. These findings indicate that UvCreA not only controls ustilaginoidin biosynthesis through directly binding the \\u003cem\\u003eUgs\\u003c/em\\u003e gene promoters, but also positively regulates mycelium growth and pathogenicity in \\u003cem\\u003eU. virens\\u003c/em\\u003e. Therefore, this study provides theoretical insights into the mechanisms of carbon metabolism and mycotoxin biosynthesis in phytopathogenic fungi.\\u003c/p\\u003e\",\"manuscriptTitle\":\"The transcription factor UvCreA regulates ustilaginoidin synthesis and pathogenicity in Ustilaginoidea virens\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2026-04-06 12:47:29\",\"doi\":\"10.21203/rs.3.rs-9251727/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"8d7b0412-481b-484f-9b1f-ae8803dd789d\",\"owner\":[],\"postedDate\":\"April 6th, 2026\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2026-04-15T07:10:27+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2026-04-06 12:47:29\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-9251727\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-9251727\",\"identity\":\"rs-9251727\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}