Total transcriptome response for tyrosol exposure in Aspergillus nidulans.

Although tyrosol is a quorum-sensing molecule of Candida species, it has antifungal activity at supraphysiological concentrations. Here, we studied the effect of tyrosol on the physiology and genome-wide transcription of Aspergillus nidulans to gain insight into the background of the antifungal activity of this compound. Tyrosol efficiently reduced germination of conidia and the growth on various carbon sources at a concentration of 35 mM. The growth inhibition was fungistatic rather than fungicide on glucose and was accompanied with downregulation of 2199 genes related to e.g. mitotic cell cycle, glycolysis, nitrate and sulphate assimilation, chitin biosynthesis, and upregulation of 2250 genes involved in e.g. lipid catabolism, amino acid degradation and lactose utilization. Tyrosol treatment also upregulated genes encoding glutathione-S-transferases (GSTs), increased specific GST activities and the glutathione (GSH) content of the cells, suggesting that A. nidulans can detoxify tyrosol in a GSH-dependent manner even though this process was weak. Tyrosol did not induce oxidative stress in this species, but upregulated "response to nutrient levels", "regulation of nitrogen utilization", "carbon catabolite activation of transcription" and "autophagy" genes. Tyrosol may have disturbed the regulation and orchestration of cellular metabolism, leading to impaired use of nutrients, which resulted in growth reduction.

Argonaute and Dicer are essential for communication between Trichoderma atroviride and fungal hosts during mycoparasitism.

Trichoderma species are known for their mycoparasitic activity against phytopathogenic fungi that cause significant economic losses in agriculture. During mycoparasitism, Trichoderma spp. recognize molecules produced by the host fungus and release secondary metabolites and hydrolytic enzymes to kill and degrade the host's cell wall. Here, we explored the participation of the Trichoderma atroviride RNAi machinery in the interaction with six phytopathogenic fungi of economic importance. We determined that both Argonaute-3 and Dicer-2 play an essential role during mycoparasitism. Using an RNA-Seq approach, we identified that perception, detox, and cell wall degradation depend on the T. atroviride-RNAi when interacting with Alternaria alternata, Rhizoctonia solani AG2, and R. solani AG5. Furthermore, we constructed a gene co-expression network that provides evidence of two gene modules regulated by RNAi, which play crucial roles in essential processes during mycoparasitism. In addition, based on small RNA-seq, we conclude that siRNAs regulate amino acid and carbon metabolism and communication during the Trichoderma-host interaction. Interestingly, our data suggest that siRNAs might regulate allorecognition (het) and transport genes in a cross-species manner. Thus, these results reveal a fine-tuned regulation in T. atroviride dependent on siRNAs that is essential during the biocontrol of phytopathogenic fungi, showing a greater complexity of this process than previously established.IMPORTANCEThere is an increasing need for plant disease control without chemical pesticides to avoid environmental pollution and resistance, and the health risks associated with the application of pesticides are increasing. Employing Trichoderma species in agriculture to control fungal diseases is an alternative plant protection strategy that overcomes these issues without utilizing chemical fungicides. Therefore, understanding the biocontrol mechanisms used by Trichoderma species to antagonize other fungi is critical. Although there has been extensive research about the mechanisms involved in the mycoparasitic capability of Trichoderma species, there are still unsolved questions related to how Trichoderma regulates recognition, attack, and defense mechanisms during interaction with a fungal host. In this work, we report that the Argonaute and Dicer components of the RNAi machinery and the small RNAs they process are essential for gene regulation during mycoparasitism by Trichoderma atroviride.

MoNOT3 Subunit Has Important Roles in Infection-Related Development and Stress Responses in Magnaporthe oryzae.

The multifunctional carbon catabolite repression negative on TATA-box-less complex (CCR4-NOT) is a multi-subunit complex present in all eukaryotes, including fungi. This complex plays an essential role in gene expression; however, a functional study of the CCR4-NOT complex in the rice blast fungus Magnaporthe oryzae has not been conducted. Seven genes encoding the putative CCR4-NOT complex were identified in the M. oryzae genome. Among these, a homologous gene, MoNOT3, was overexpressed during appressorium development in a previous study. Deletion of MoNOT3 in M. oryzae resulted in a significant reduction in hyphal growth, conidiation, abnormal septation in conidia, conidial germination, and appressorium formation compared to the wild-type. Transcriptional analyses suggest that the MoNOT3 gene affects conidiation and conidial morphology by regulating COS1 and COM1 in M. oryzae. Furthermore, Δmonot3 exhibited a lack of pathogenicity, both with and without wounding, which is attributable to deficiencies in the development of invasive growth in planta. This result was also observed in onion epidermal cells, which are non-host plants. In addition, the MoNOT3 gene was involved in cell wall stress responses and heat shock. Taken together, these observations suggest that the MoNOT3 gene is required for fungal infection-related cell development and stress responses in M. oryzae.

Effects of ScRgt1-Like DNA-binding transcription factor SpRgt1 (SPCC320.03) on Hexose transporters gene expression in Schizosaccharomyces pombe.

Glucose, which plays an essential role in carbon and energy metabolism in eukaryotes, is vital in directing various energy-consuming cellular processes. In S. cerevisiae, transcription factors involved in regulating hexose transporters and their mechanisms of action under different carbon sources were revealed in detail. However, there is limited information on these processes in S. pombe. In this study, the effect of SPCC320.03 (named SpRgt1), the ortholog of ScRgt1 whose molecular mechanism is known in detail in S. cerevisiae, on the transcriptional regulation of hexose transporters (ght1-8) dependent on different carbon sources was investigated. We measured the transcript levels of ght1-8 using the qPCR technique and performed relative evaluation in S. pombe strains (parental, rgt1 deleted mutant, rgt1 overexpressed, and vectoral rgt1 carrying mutant). We aimed to investigate the transcriptional changes caused by the protein product of the rgt1 (SPCC320.03) gene in terms of ght1-8 genes in strains that are grown in different carbon sources (2% glucose, 2% glycerol + 0.1% glucose, and 2% gluconate). Here, we show that SpRgt1 is involved in the regulation of the ght3, ght4, ght6, and ght7 genes but that the ght1, ght2, ght5, and ght8 gene expression vary depending on carbon sources, independently of SpRgt1.

Single-Molecule Fluorescent In Situ Hybridization (smFISH) for RNA Detection in the Fungal Pathogen Candida albicans.

Candida albicans is the most prevalent human fungal pathogen. Its pathogenicity is linked to the ability of C. albicans to reversibly change morphology and to grow as yeast, pseudohyphae, or hyphal cells in response to environmental stimuli. Understanding the molecular regulation controlling those morphological switches remains a challenge that, if solved, could help eradicate C. albicans infections.While numerous studies investigated gene expression changes occurring during C. albicans morphological switches using bulk approaches (e.g., RNA sequencing), here we describe a single-cell and single-molecule RNA imaging and analysis protocol to measure absolute mRNA counts in morphologically intact cells. To detect endogenous mRNAs in single fixed cells, we optimized a single-molecule fluorescent in situ hybridization (smFISH) protocol for C. albicans, which allows one to quantify the differential expression of mRNAs in yeast, pseudohyphae, or hyphal cells. We quantified the expression of two mRNAs, a cell cycle-controlled mRNA (CLB2) and a transcription factor (EFG1), which show expression changes in the different morphological cell types and nutrient conditions. In this protocol, we described in detail the major steps of this approach: growth and fixation, hybridization, imaging, cell segmentation, and mRNA spot analysis. Raw data is provided with the protocol to favor reproducibility. This approach could benefit the molecular characterization of C. albicans and other filamentous fungi, pathogenic or nonpathogenic.

Coordination of two regulators SscA and VosA in Aspergillus nidulans conidia.

Airborne fungal spores are a major cause of fungal diseases in humans, animals, and plants as well as contamination of foods. Previous studies found a variety of regulators including VosA, VelB, WetA, and SscA for sporogenesis and the long-term viability in Aspergillus nidulans. To gain a mechanistic understanding of the complex regulatory mechanisms in asexual spores, here, we focused on the relationship between VosA and SscA using comparative transcriptomic analysis and phenotypic studies. The ΔsscA ΔvosA double-mutant conidia have lower spore viability and stress tolerance compared to the ΔsscA or ΔvosA single mutant conidia. Deletion of sscA or vosA affects chitin levels and mRNA levels of chitin biosynthetic genes in conidia. In addition, SscA and VosA are required for the dormant state of conidia and conidial germination by modulating the mRNA levels of the cytoskeleton and development-associated genes. Overall, these results suggest that SscA and VosA play interdependent roles in governing spore maturation, dormancy, and germination in A. nidulans.

Hypothetical protein MAA_07646 is required for stress resistance and pathogenicity in Metarhizium robertsii.

Metarhizium robertsii, a vital entomopathogenic fungus for pest management, relies on various virulence-related proteins for infection. Identifying these proteins, especially those with unknown functions, can illuminate the fungus's virulence mechanisms. Through RNA-seq, we discovered that the hypothetical protein MAA_07646 was significantly upregulated during appressorium formation in M. robertsii. In this study, we characterized MAA_07646, finding its presence in both the nucleus and cytoplasm. Surprisingly, it did not affect vegetative growth, conidiation, or chemical tolerance. However, it played a role in heat and UV radiation sensitivity. Notably, ΔMAA_07646 exhibited reduced virulence in Galleria mellonella larvae due to impaired appressorium formation and decreased expression of virulence-related genes. In conclusion, MAA_07646 contributes to thermotolerance, UV resistance, and virulence in M. robertsii. Understanding its function sheds light on the insecticidal potential of M. robertsii's hypothetical proteins.

MoMkk1 and MoAtg1 dichotomously regulating autophagy and pathogenicity through MoAtg9 phosphorylation in Magnaporthe oryzae.

Autophagy is a central biodegradation pathway critical in eliminating intracellular cargo to maintain cellular homeostasis and improve stress resistance. At the same time, the key component of the mitogen-activated protein kinase cascade regulating cell wall integrity signaling MoMkk1 has an essential role in the autophagy of the rice blast fungus Magnaporthe oryzae. Still, the mechanism of how MoMkk1 regulates autophagy is unclear. Interestingly, we found that MoMkk1 regulates the autophagy protein MoAtg9 through phosphorylation. MoAtg9 is a transmembrane protein subjected to phosphorylation by autophagy-related protein kinase MoAtg1. Here, we provide evidence demonstrating that MoMkk1-dependent MoAtg9 phosphorylation is required for phospholipid translocation during isolation membrane stages of autophagosome formation, an autophagic process essential for the development and pathogenicity of the fungus. In contrast, MoAtg1-dependent phosphorylation of MoAtg9 negatively regulates this process, also impacting growth and pathogenicity. Our studies are the first to demonstrate that MoAtg9 is subject to MoMkk1 regulation through protein phosphorylation and that MoMkk1 and MoAtg1 dichotomously regulate autophagy to underlie the growth and pathogenicity of M. oryzae.IMPORTANCEMagnaporthe oryzae utilizes multiple signaling pathways to promote colonization of host plants. MoMkk1, a cell wall integrity signaling kinase, plays an essential role in autophagy governed by a highly conserved autophagy kinase MoAtg1-mediated pathway. How MoMkk1 regulates autophagy in coordination with MoAtg1 remains elusive. Here, we provide evidence that MoMkk1 phosphorylates MoAtg9 to positively regulate phospholipid translocation during the isolation membrane or smaller membrane structures stage of autophagosome formation. This is in contrast to the negative regulation of MoAtg9 by MoAtg1 for the same process. Intriguingly, MoMkk1-mediated MoAtg9 phosphorylation enhances the fungal infection of rice, whereas MoAtg1-dependant MoAtg9 phosphorylation significantly attenuates it. Taken together, we revealed a novel mechanism of autophagy and virulence regulation by demonstrating the dichotomous functions of MoMkk1 and MoAtg1 in the regulation of fungal autophagy and pathogenicity.

The MoLfa1 Protein Regulates Fungal Development and Septin Ring Formation in Magnaporthe oryzae.

Septins play a key regulatory role in cell division, cytokinesis, and cell polar growth of the rice blast fungus (Magnaporthe oryzae). We found that the organization of the septin ring, which is essential for appressorium-mediated infection in M. oryzae, requires long-chain fatty acids (LCFAs), which act as mediators of septin organization at membrane interfaces. However, it is unclear how septin ring formation and LCFAs regulate the pathogenicity of the rice blast fungus. In this study, a novel protein was named MoLfa1 because of its role in LCFAs utilization. MoLfa1 affects the utilization of LCFAs, lipid metabolism, and the formation of the septin ring by binding with phosphatidylinositol phosphates (PIPs), thereby participating in the construction of penetration pegs of M. oryzae. In addition, MoLfa1 is localized in the endoplasmic reticulum (ER) and interacts with the ER-related protein MoMip11 to affect the phosphorylation level of Mps1. (Mps1 is the core protein in the MPS1-MAPK pathway.) In conclusion, MoLfa1 affects conidia morphology, appressorium formation, lipid metabolism, LCFAs utilization, septin ring formation, and the Mps1-MAPK pathway of M. oryzae, influencing pathogenicity.

Control of meiotic entry by dual inhibition of a key mitotic transcription factor.

The mitosis to meiosis transition requires dynamic changes in gene expression, but whether and how the mitotic transcriptional machinery is regulated during this transition is unknown. In budding yeast, SBF and MBF transcription factors initiate the mitotic gene expression program. Here, we report two mechanisms that work together to restrict SBF activity during meiotic entry: repression of the SBF-specific Swi4 subunit through LUTI-based regulation and inhibition of SBF by Whi5, a functional homolog of the Rb tumor suppressor. We find that untimely SBF activation causes downregulation of early meiotic genes and delays meiotic entry. These defects are largely driven by the SBF-target G1 cyclins, which block the interaction between the central meiotic regulator Ime1 and its cofactor Ume6. Our study provides insight into the role of SWI4LUTI in establishing the meiotic transcriptional program and demonstrates how the LUTI-based regulation is integrated into a larger regulatory network to ensure timely SBF activity.

A feedback regulation of FgHtf1-FgCon7 loop in conidiogenesis and development of Fusarium graminearum.

The transcription factor FgHtf1 is important for conidiogenesis in Fusarium graminearum and it positively regulates the expression of the sporulation-related gene FgCON7. However, the regulatory mechanism underlying its functions is still unclear. The present study intends to uncover the functional mechanism of FgHtf1 in relation to FgCon7 in F. graminearum. We demonstrated that FgCON7 serves as a target gene for FgHtf1. Interestingly, FgCon7 also binds the promoter region of FgHTF1 to negatively regulate its expression, thus forming a negative-feedback loop. We demonstrated that FgHtf1 and FgCon7 have functional redundancy in fungal development. FgCon7 localizes in the nucleus and has transcriptional activation activity. Deletion of FgCON7 significantly reduces conidia production. 4444 genes were regulated by FgCon7 in ChIP-Seq, and RNA-Seq revealed 4430 differentially expressed genes in FgCON7 deletion mutant, with CCAAT serving as a consensus binding motif of FgCon7 to the target genes. FgCon7 directly binds the promoter regions of FgMSN2, FgABAA, FgVEA and FgSMT3 genes and regulates their expression. These genes were found to be important for conidiogenesis. To our knowledge, this is the first study that unveiled the mutual regulatory functions of FgCON7 and FgHTF1 to form a negative-feedback loop, and how the loop mediates sporulation in F. graminearum.

Factors governing the transcriptome changes and chronological lifespan of fission yeast during phosphate starvation.

Starvation of Schizosaccharomyces pombe for inorganic phosphate elicits adaptive transcriptome changes in which mRNAs driving ribosome biogenesis, tRNA biogenesis, and translation are globally downregulated, while those for autophagy and phosphate mobilization are upregulated. Here, we interrogated three components of the starvation response: upregulated autophagy; the role of transcription factor Pho7 (an activator of the PHO regulon); and upregulated expression of ecl3, one of three paralogous genes (ecl1, ecl2, and ecl3) collectively implicated in cell survival during other nutrient stresses. Ablation of autophagy factor Atg1 resulted in early demise of phosphate-starved fission yeast, as did ablation of Pho7. Transcriptome profiling of phosphate-starved pho7Δ cells highlighted Pho7 as an activator of genes involved in phosphate acquisition and mobilization, not limited to the original three-gene PHO regulon, and additional starvation-induced genes (including ecl3) not connected to phosphate dynamics. Pho7-dependent gene induction during phosphate starvation tracked with the presence of Pho7 DNA-binding elements in the gene promoter regions. Fewer ribosome protein genes were downregulated in phosphate-starved pho7Δ cells versus WT, which might contribute to their shortened lifespan. An ecl3Δ mutant elicited no gene expression changes in phosphate-replete cells and had no impact on survival during phosphate starvation. By contrast, pan-ecl deletion (ecl123Δ) curtailed lifespan during chronic phosphate starvation. Phosphate-starved ecl123Δ cells experienced a more widespread downregulation of mRNAs encoding aminoacyl tRNA synthetases vis-à-vis WT or pho7Δ cells. Collectively, these results enhance our understanding of fission yeast phosphate homeostasis and survival during nutrient deprivation.

Regulatory functions of homeobox domain transcription factors in fungi.

Homeobox domain (HD) proteins present a crucial involvement in morphological differentiation and other functions in eukaryotes. Most HD genes encode transcription factors (TFs) that orchestrate a regulatory role in cellular and developmental decisions. In fungi, multiple studies have increased our understanding of these important HD regulators in recent years. These reports have revealed their role in fungal development, both sexual and asexual, as well as their importance in governing other biological processes in these organisms, including secondary metabolism, pathogenicity, and sensitivity to environmental stresses. Here, we provide a comprehensive review of the current knowledge on the regulatory roles of HD-TFs in fungi, with a special focus on Aspergillus species.

The regulatory pathway of transcription factor MYB36 from Trichoderma asperellum Tas653 resistant to poplar leaf blight pathogen Alternaria alternata Aal004.

In fungi, MYB transcription factors (TFs) mainly regulate growth, development, and resistance to stress. However, as major disease-resistance TFs, they have rarely been studied in biocontrol fungi. In this study, MYB36 of Trichoderma asperellum Tas653 (Ta) was shown to respond strongly to the stress caused by Alternaria alternata Aa1004. Compared with wild-type Ta (Ta-Wt), the inhibition rate of the MYB36 knockout strain (Ta-Kn) on Aa1004 decreased by 11.06%; the superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities decreased by 82.15 U/g, 0.19 OD470/min/g, and 1631.2 µmol/min/g, respectively. The MYB36 overexpression strain (Ta-Oe) not only enhanced hyperparasitism on Aa1004, caused its hyphae to swell, deform, or even rupture, but also reduced the incidence rate of poplar leaf blight. MYB36 regulates downstream (TFs, detoxification genes, defense genes, and other antifungal-related genes by binding to the cis-acting elements "ACAT" and "ATCG". Zinc finger TFs, as the main antifungal TFs, account for 90% of the total TFs, and Zn37.5 (23.24-) and Zn83.7 (23.18-fold) showed the greatest expression difference when regulated directly by MYB36. The detoxification genes mainly comprised 11 major major facilitator superfamily (MFS) genes, among which MYB36 directly increased the expression levels of three genes by more than 2-3.44-fold. The defense genes mainly encoded cytochrome P450 (P450) and hydrolases. e.g., P45061.3 (2-10.95-), P45060.2 (2-7.07-), and Hyd44.6 (2-2.30-fold). This study revealed the molecular mechanism of MYB36 regulation of the resistance of T. asperellum to A. alternata and provides theoretical guidance for the biocontrol of poplar leaf blight and the anti-disease mechanism of biocontrol fungi.

The histone deacetylase Hda1 affects oxidative and osmotic stress response as well as mycoparasitic activity and secondary metabolite biosynthesis in Trichoderma atroviride.

The mycoparasitic fungus Trichoderma atroviride is applied in agriculture as a biostimulant and biologic control agent against fungal pathogens that infest crop plants. Secondary metabolites are among the main agents determining the strength and progress of the mycoparasitic attack. However, expression of most secondary metabolism-associated genes requires specific cues, as they are silent under routine laboratory conditions due to their maintenance in an inactive heterochromatin state. Therefore, histone modifications are crucial for the regulation of secondary metabolism. Here, we functionally investigated the role of the class II histone deacetylase encoding gene hda1 of T. atroviride by targeted gene deletion, phenotypic characterization, and multi-omics approaches. Deletion of hda1 did not result in obvious phenotypic alterations but led to an enhanced inhibitory activity of secreted metabolites and reduced mycoparasitic abilities of T. atroviride against the plant-pathogenic fungi Botrytis cinerea and Rhizoctonia solani. The ∆hda1 mutants emitted altered amounts of four volatile organic compounds along their development, produced different metabolite profiles upon growth in liquid culture, and showed a higher susceptibility to oxidative and osmotic stress. Moreover, hda1 deletion affected the expression of several notable gene categories such as polyketide synthases, transcription factors, and genes involved in the HOG MAPK pathway.IMPORTANCEHistone deacetylases play crucial roles in regulating chromatin structure and gene transcription. To date, classical-Zn2+ dependent-fungal histone deacetylases are divided into two classes, of which each comprises orthologues of the two sub-groups Rpd3 and Hos2 and Hda1 and Hos3 of yeast, respectively. However, the role of these chromatin remodelers in mycoparasitic fungi is poorly understood. In this study, we provide evidence that Hda1, the class II histone deacetylases of the mycoparasitic fungus Trichoderma atroviride, regulates its mycoparasitic activity, secondary metabolite biosynthesis, and osmotic and oxidative stress tolerance. The function of Hda1 in regulating bioactive metabolite production and mycoparasitism reveals the importance of chromatin-dependent regulation in the ability of T. atroviride to successfully control fungal plant pathogens.

Multiple roles for the cytoplasmic C-terminal domains of the yeast cell surface receptors Rgt2 and Snf3 in glucose sensing and signaling.

The plasma membrane proteins Rgt2 and Snf3 are glucose sensing receptors (GSRs) that generate an intracellular signal for the induction of gene expression in response to high and low extracellular glucose concentrations, respectively. The GSRs consist of a 12-transmembrane glucose recognition domain and a cytoplasmic C-terminal signaling tail. The GSR tails are dissimilar in length and sequence, but their distinct roles in glucose signal transduction are poorly understood. Here, we show that swapping the tails between Rgt2 and Snf3 does not alter the signaling activity of the GSRs, so long as their tails are phosphorylated in a Yck-dependent manner. Attachment of the GSR tails to Hxt1 converts the transporter into a glucose receptor; however, the tails attached to Hxt1 are not phosphorylated by the Ycks, resulting in only partial signaling. Moreover, in response to non-fermentable carbon substrates, Rgt2 and Hxt1-RT (RT, Rgt2-tail) are efficiently endocytosed, whereas Snf3 and Hxt1-ST (ST, Snf3-tail) are endocytosis-impaired. Thus, the tails are important regulatory domains required for the endocytosis of the Rgt2 and Snf3 glucose sensing receptors triggered by different cellular stimuli. Taken together, these results suggest multiple roles for the tail domains in GSR-mediated glucose sensing and signaling.

Identification of plb1 mutation that extends longevity via activating Sty1 MAPK in Schizosaccharomyces pombe.

To understand the lifespan of higher organisms, including humans, it is important to understand lifespan at the cellular level as a prerequisite. So, fission yeast is a good model organism for the study of lifespan. To identify the novel factors involved in longevity, we are conducting a large-scale screening of long-lived mutant strains that extend chronological lifespan (cell survival in the stationary phase) using fission yeast. One of the newly acquired long-lived mutant strains (No.98 mutant) was selected for analysis and found that the long-lived phenotype was due to a missense mutation (92Phe → Ile) in the plb1+ gene. plb1+ gene in fission yeast is a nonessential gene encoding a homolog of phospholipase B, but its functions under normal growth conditions, as well as phospholipase B activity, remain unresolved. Our analysis of the No.98 mutant revealed that the plb1 mutation reduces the integrity of the cellular membrane and cell wall and activates Sty1 via phosphorylation.

Regulation of TRI5 expression and deoxynivalenol biosynthesis by a long non-coding RNA in Fusarium graminearum.

Deoxynivalenol (DON) is the most frequently detected mycotoxin in cereal grains and processed food or feed. Two transcription factors, Tri6 and Tri10, are essential for DON biosynthesis in Fusarium graminearum. In this study we conduct stranded RNA-seq analysis with tri6 and tri10 mutants and show that Tri10 acts as a master regulator controlling the expression of sense and antisense transcripts of TRI6 and over 450 genes with diverse functions. TRI6 is more specific for regulating TRI genes although it negatively regulates TRI10. Two other TRI genes, including TRI5 that encodes a key enzyme for DON biosynthesis, also have antisense transcripts. Both Tri6 and Tri10 are essential for TRI5 expression and for suppression of antisense-TRI5. Furthermore, we identify a long non-coding RNA (named RNA5P) that is transcribed from the TRI5 promoter region and is also regulated by Tri6 and Tri10. Deletion of RNA5P by replacing the promoter region of TRI5 with that of TRI12 increases TRI5 expression and DON biosynthesis, indicating that RNA5P suppresses TRI5 expression. However, ectopic constitutive overexpression of RNA5P has no effect on DON biosynthesis and TRI5 expression. Nevertheless, elevated expression of RNA5P in situ reduces TRI5 expression and DON production. Our results indicate that TRI10 and TRI6 regulate each other's expression, and both are important for suppressing the expression of RNA5P, a long non-coding RNA with cis-acting inhibitory effects on TRI5 expression and DON biosynthesis in F. graminearum.

Global rewiring of lipid metabolism to produce carotenoid by deleting the transcription factor genes ino2/ino4 in Saccharomyces cerevisiae.

The transcription factor complex INO2 and INO4 in Saccharomyces cerevisiae plays a vital role in lipid biosynthesis by activating multiple genes in the biosynthetic pathways of phospholipid, fatty acid, and sterol. Previous studies have reported conflicting results regarding the effects of ino2 and ino4 gene expression levels on target chemicals. Therefore, this study aimed to examine the influence of different ino2 and ino4 expression levels on carotenoid production (e.g., lycopene), which shares a common precursor, acetyl-CoA, with lipid metabolism. Surprisingly, 2.6- and 1.8-fold increase in lycopene yield in the ino2 and ino4 deletion strains were found, respectively. In contrast, ino2 overexpression did not promote lycopene accumulation. Additionally, there was a decrease in intracellular free fatty acids in the ino2 deletion strain. Comparative transcriptome analysis revealed a significant downregulation of genes related to lipid biosynthesis in the ino2 deletion strain. To our knowledge, this is the first report showing that deletion of transcription factor genes ino2 and ino4 can facilitate lycopene accumulation. These findings hold significant implications for the development of metabolically engineered S. cerevisiae with enhanced carotenoid production.

GTP-dependent regulation of heterochromatin fluctuations at subtelomeric regions in Saccharomyces cerevisiae.

In eukaryotes, single cells in a population display different transcriptional profiles. One of the factors regulating this heterogeneity is the chromatin state in each cell. However, the mechanisms of epigenetic chromatin regulation of specific chromosomal regions remain unclear. Therefore, we used single-cell tracking system to analyze IMD2. IMD2 is located at the subtelomeric region of budding yeast, and its expression is epigenetically regulated by heterochromatin fluctuations. Treatment with mycophenolic acid, an inhibitor of de novo GTP biosynthesis, triggered a decrease in GTP, which caused heterochromatin fluctuations at the IMD2 locus. Interestingly, within individually tracked cells, IMD2 expression state underwent repeated switches even though IMD2 is positioned within the heterochromatin region. We also found that 30% of the cells in a population always expressed IMD2. Furthermore, the addition of nicotinamide, a histone deacetylase inhibitor, or guanine, the GTP biosynthesis factor in salvage pathway of GTP biosynthesis, regulated heterogeneity, resulting in IMD2 expression being uniformly induced or suppressed in the population. These results suggest that gene expression heterogeneity in the IMD2 region is regulated by changes in chromatin structure triggered by slight decreases in GTP.