Transcription Of Structural Genes Research Articles

BmNPV Bm60 is a key target gene used by a resistant strain of Bombyx mori to inhibit BmNPV proliferation

Bombyx mori nucleopolyhedrovirus (BmNPV) is a pathogen that causes significant losses to the silkworm industry. Numerous antiviral genes and proteins have been identified by studying silkworm resistance to BmNPV. However, the molecular mechanism of silkworm resistance to BmNPV is unclear. We analyzed the differences between the susceptible strain 871 and a near-isogenic resistant strain 871C. The survival of strain 871C was significantly greater than that of 871 after oral and subcutaneous exposure to BmNPV. Strain 871C exhibited a nearly 10,000-fold higher LD50 for BmNPV compared to 871. BmNPV proliferation was significantly inhibited in all tested tissues of strain 871C using HE strain and fluorescence analysis. Strain 871C exhibited cellular resistance to BmNPV rather than peritrophic membrane or serum resistance. Strain 871C suppressed the expression of the viral early gene Bm60. This led to the inhibition of BmNPV DNA replication and late structural gene transcription based on the cascade regulation of baculovirus gene expression. Bm60 could also interact with the viral DNA binding protein and alkaline nuclease, as well as host proteins Methylcrotonoyl-CoA carboxylase subunit alpha, mucin-2-like protein, and 30 K-8. Overexpression of 30 K-8 significantly inhibited BmNPV proliferation. These results increase understanding of the molecular mechanism behind silkworm resistance to BmNPV and suggest targets for the breeding of resistant silkworm strains and the controlling pest of Lepidoptera.

Light irradiation changes the regulation pattern of BtCrgA on carotenogenesis in Blakeslea trispora.

CrgA has been shown to be a negative regulator of carotenogenesis in some filamentous fungi, while light irradiation is an inducible environmental factor for carotenoid biosynthesis. To clarify the relationship between CrgA and light-inducible carotenogenesis in Blakeslea trispora, the cis-acting elements of the btcrgA promoter region were investigated, followed by the analyses of correlation between the expression of btcrgA and carotenoid structural genes under different irradiation conditions. A variety of cis-acting elements associated with light response was observed in the promoter region of btcrgA, and transcription of btcrgA and carotenoid structural genes under different irradiation conditions was induced by white light with a clear correlation. Then, RNA interference and overexpression of btcrgA were performed to investigate their effects on carotenogenesis at different levels under irradiation and darkness. The analyses of transcription and enzyme activities of carotenoid structural gene, and accumulation of carotenoids among btcrgA-interfered, btcrgA-overexpressed, and wild-type strains under irradiation and darkness indicate that btcrgA negatively regulates the synthesis of carotenoid in darkness, while promotes the carotenogenesis under irradiation regardless of reduced or overexpression of btcrgA .

MiR156b-targeted VvSBP8/13 functions downstream of the abscisic acid signal to regulate anthocyanins biosynthesis in grapevine fruit under drought.

Anthocyanins are the primary color components of grapevine berries and wines. In cultivation practices, a moderate water deficit can promote anthocyanin accumulation in red grape skins. Our previous study showed that abscisic acid (ABA) plays a key role in this process. Herein, we identified a microRNA, vv-miR156b, that is generated in grapevine berries in response to drought stress, along with increasing anthocyanin content and biosynthetic structural gene transcripts. In contrast, vv-miR156b short tandem target mimic (STTM) function-loss callus exhibits the opposite phenotype. Results from in vivo and in vitro experiments revealed that the ABA-signaling-regulated transcription factor VvAREB2 binds directly to the ABA-responsive element (ABRE) of the MIR156b promoter and activates miR156b expression. Furthermore, two miR156b downstream targets, VvSBP8 and VvSBP13, exhibited reduced grape anthocyanin content in their overexpressors but there was a contrary result in their CRISPR-edited lines, the decrease in anthocyanin content was rescued in miR156b and SBP8/13 double overexpressors. We further demonstrated that both VvSBP8 and VvSBP13, encoding transcriptional repressors, displayed sufficient ability to interact with VvMYC1 and VvMYBA1, thereby interfering with MYB-bHLH-WD (MBW) repeat transcriptional complex formation, resulting in the repression of anthocyanin biosynthesis. Our findings demonstrate a direct functional relationship between ABA signaling and the miR156-SBP-MBW complex regulatory module in driving drought-induced anthocyanin accumulation in grape berries.

Effect of Mosaic Knockout of Phytoene Desaturase Gene NtPDS on Biosynthesis of Carotenoids in Nicotiana tabacum L.

Carotenoids are essential participants in photosynthesis and photo protection as well as growth, development, and stress response in plants. Phytoene desaturase (PDS; EC 1.3.5.5) is an enzyme that catalyzes the first stage of desaturation of 15-cis-phytoene (a precursor to all carotenoids). In this work, we examined for the first time the effect of PDS knockout in the genome of Nicotiana tabacum L. using the CRISPR-Cas9 system on activity of downstream genes involved in biosynthesis of carotenoids. Nine transgenic lines of tobacco were obtained with a mosaic editing of gene NtPDS; three versions of indels (350^351→ins^g#, 350^351→ins^t#, and t351→del(1nt)#) in the exon II resulting in the synthesis of a shortened non-functional version of the protein. The lines were characterized by spotted, green-white pigmentation of the leaves, altered flowering time and morphology as well as a reduced content of carotenoids and chlorophylls in the leaf tissue. A rise in the level of transcripts of phytoene synthase gene NtPSY2 was shown in late-flowering lines as compared to control material. Edited line L29 with the latest flowering showed a considerable increase in the level of transcripts of downstream structural genes of carotenogenesis. A reduction in the carotenoid content in the leaves with mosaic editing was accompanied by a decrease in the level of expression of MADS-box gene NtSEP1, the product of which presumably participates in regulation of transcription of the carotenoid biosynthesis genes. The obtained results may be used for further studies of regulation of biosynthesis of carotenoids and apocarotenoids in Solanaceae.

Elucidating the Differentiation Synthesis Mechanisms of Differently Colored Resistance Quinoa Seedings Using Metabolite Profiling and Transcriptome Analysis.

Quinoa (Chenopodium quinoa wild.), a dicotyledonous plant native to the Andes, is an increasingly popular pseudograin owing to its high nutritional value, stress resistance capabilities, and gluten-free properties. In this study, we aimed to explore the dynamic changes in different varieties of quinoa at the seedling stage and their regulatory networks. Here, we found that the leaves of quinoa showed obvious coloration after 45 days, and four quinoa seedling types (red, white, yellow, and black) were subjected to ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and transcriptome sequencing to identify their differentially expressed genes and metabolites. A total of 29 differential metabolites and 19 genes (14 structural and 5 regulatory genes) were identified, and consistent differences were observed in the flavonoid, phenolic acid, and alkaloid metabolites in the different quinoa types. These differential metabolites were significantly enriched in flavonoid and flavonol biosynthesis, flavonoid biosynthesis, and phenylpropanoid biosynthesis pathways. In addition, real-time fluorescence quantitative PCR (RT-qPCR) technology was used to detect the expression of four structural genes involved in the flavonoid biosynthesis pathway and four regulatory genes (interaction network). The results revealed that the structural and regulatory gene transcript levels in the flavonoid pathway were higher in the red quinoa cultivars than in the white, yellow, and black. Additionally, the differences in the leaves of these four quinoa cultivars were mainly due to differences in flavonoid, phenolic acid, and alkaloid accumulation. Our findings provide a basis for understanding the accumulation and coloration mechanisms of flavonoids, phenolic acids, and alkaloids in quinoa seedlings of different colors and also provide a theoretical basis for future investigations.

Optimization of Anthocyanin Production in Tobacco Cells.

Plant cell cultures have emerged as a promising tool for producing active molecules due to their numerous advantages over traditional agricultural methods. Flavonols, and anthocyanin pigments in particular, together with other phenolic compounds such as chlorogenic acid, are known for their beneficial health properties, mainly due to their antioxidant, antimicrobial, and anti-inflammatory activities. The synthesis of these molecules is finely regulated in plant cells and controlled at the transcriptional level by specific MYB and bHLH transcription factors that coordinate the transcription of structural biosynthetic genes. The co-expression of peach PpMYB10.1 and PpbHLH3 in tobacco was used to develop tobacco cell lines showing high expression of both the peach transgenes and the native flavonol structural genes. These cell lines were further selected for fast growth. High production levels of chlorogenic acid, anthocyanins (mainly cyanidin 3-rutinoside), and other phenolics were also achieved in pre-industrial scale-up trials. A single-column-based purification protocol was developed to produce a lyophile called ANT-CA, which was stable over time, showed beneficial effects on cell viability, and had antioxidant, anti-inflammatory, antibacterial, and wound-healing activities. This lyophile could be a valuable ingredient for food or cosmetic applications.

Metabolomic and transcriptomice analyses of flavonoid biosynthesis in apricot fruits.

Flavonoids, as secondary metabolites in plants, play important roles in many biological processes and responses to environmental factors. Apricot fruits are rich in flavonoid compounds, and in this study, we performed a combined metabolomic and transcriptomic analysis of orange flesh (JN) and white flesh (ZS) apricot fruits. A total of 222 differentially accumulated flavonoids (DAFs) and 15855 differentially expressed genes (DEGs) involved in flavonoid biosynthesis were identified. The biosynthesis of flavonoids in apricot fruit may be regulated by 17 enzyme-encoding genes, namely PAL (2), 4CL (9), C4H (1), HCT (15), C3'H (4), CHS (2), CHI (3), F3H (1), F3'H (CYP75B1) (2), F3'5'H (4), DFR (4), LAR (1), FLS (3), ANS (9), ANR (2), UGT79B1 (6) and CYP81E (2). A structural gene-transcription factor (TF) correlation analysis yielded 3 TFs (2 bHLH, 1 MYB) highly correlated with 2 structural genes. In addition, we obtained 26 candidate genes involved in the biosynthesis of 8 differentially accumulated flavonoids metabolites in ZS by weighted gene coexpression network analysis. The candidate genes and transcription factors identified in this study will provide a highly valuable molecular basis for the in-depth study of flavonoid biosynthesis in apricot fruits.

Yellow Petal locus GaYP promotes flavonol biosynthesis and yellow coloration in petals of Asiatic cotton (Gossypium arboreum)

Yellow Petal locus GaYP is located on chromosome 11 and encodes a Sg6 R2R3-MYB transcription factor, which promotes flavonol biosynthesis and yellow coloration in Asiatic cotton petals. Petal color is pivotal to ornamental value and reproduction of plants. Yellow coloration in plant petals is mainly attributed to colorants including carotenoids, aurones and some flavonols. To date, the genetic regulatory mechanism of flavonol biosynthesis in petals is still to be elucidated. Here, we employed Asiatic cottons with or without deep yellow coloration in petals to address this question. Multi-omic and biochemical analysis revealed significantly up-regulated transcription of flavonol structural genes and increased levels of flavonols, especially gossypetin and 6-hydroxykaempferol, in yellow petals of Asiatic cotton. Furthermore, the Yellow Petal gene (GaYP) was mapped on chromosome 11 by using a recombinant inbred line population. It was found that GaYP encoded a transcriptional factor belonging to Sg6 R2R3-MYB proteins. GaYP could bind to the promoter of flavonol synthase gene (GaFLS) and activate the transcription of downstream genes. Knocking out of GaYP or GaFLS homologs in upland cotton largely eliminated flavonol accumulation and pale yellow coloration in petals. Our results indicated that flavonol synthesis, up-regulated by the R2R3-MYB transcription activator GaYP, was the causative factor for yellow coloration of Asiatic cotton petals. In addition, knocking out of GaYP homologs also led to decrease in anthocyanin accumulation and petal size in upland cotton, suggesting that GaYP and its homologs might modulate developmental or physiological processes beyond flavonol biosynthesis.

Moritella viscosa early infection and transcriptional responses of intraperitoneal vaccinated and unvaccinated Atlantic salmon

Skin is the first line of defence against waterborne pathogens, such as Moritella viscosa, which is the causative agent of winter ulcer disease in Atlantic salmon (Salmo salar L.). The present study revealed that vaccination protects against both M. viscosa-induced mortality and skin ulceration, but protection varied according to the strain used in the vaccine formulation. Examination of skin tissue 4 days post M. viscosa challenge indicated that M. viscosa initiated infection by colonising the scale surface. Sequencing the transcripts of the variable region of the IgM heavy chain did not detect effect of vaccination or M. viscosa challenge on antibody repertoire and B cell trafficking from the lymphatic organs to skin. Skin layer-specific responses were examined with 44 K oligonucleotide microarray. Skin responded to vaccination and M. viscosa challenge by increasing transcription of structural genes, stimulating metabolism, and regulation of developmental processes. Immune responses to dead (vaccine) and live M. viscosa were very similar and no pathogen-specific changes were found. Combined use of skin transcriptional profiles and image analysis revealed differences in the infected skin layers between the study groups. M. viscosa was likely retained in the epidermis of vaccinated salmon, whereas the dermis was colonised in unvaccinated fish. Furthermore, decreased expression of lymphocyte-specific and antiviral genes in the dermis indicated possible evasion of immune-related cells from skin in salmon challenged with M. viscosa. Our data also indicated that intraperitoneal vaccination may prime the humoral innate response, which enables a rapid response in the dermis layer of skin during M. viscosa invasion.

Comparative and Functional Analyses Reveal Conserved and Variable Regulatory Systems That Control Lasalocid Biosynthesis in Different Streptomyces Species.

Lasalocid, a representative polyether ionophore, has been successfully applied in veterinary medicine and animal husbandry and also displays promising potential for cancer therapy. Nevertheless, the regulatory system governing lasalocid biosynthesis remains obscure. Here, we identified two conserved (lodR2 and lodR3) and one variable (lodR1, found only in Streptomyces sp. strain FXJ1.172) putative regulatory genes through a comparison of the lasalocid biosynthetic gene cluster (lod) from Streptomyces sp. FXJ1.172 with those (las and lsd) from Streptomyces lasalocidi. Gene disruption experiments demonstrated that both lodR1 and lodR3 positively regulate lasalocid biosynthesis in Streptomyces sp. FXJ1.172, while lodR2 plays a negative regulatory role. To unravel the regulatory mechanism, transcriptional analysis and electrophoretic mobility shift assays (EMSAs) along with footprinting experiments were performed. The results revealed that LodR1 and LodR2 could bind to the intergenic regions of lodR1-lodAB and lodR2-lodED, respectively, thereby repressing the transcription of the lodAB and lodED operons, respectively. The repression of lodAB-lodC by LodR1 likely boosts lasalocid biosynthesis. Furthermore, LodR2 and LodE constitute a repressor-activator system that senses changes in intracellular lasalocid concentrations and coordinates its biosynthesis. LodR3 could directly activate the transcription of key structural genes. Comparative and parallel functional analyses of the homologous genes in S. lasalocidi ATCC 31180T confirmed the conserved roles of lodR2, lodE, and lodR3 in controlling lasalocid biosynthesis. Intriguingly, the variable gene locus lodR1-lodC from Streptomyces sp. FXJ1.172 seems functionally conserved when introduced into S. lasalocidi ATCC 31180T. Overall, our findings demonstrate that lasalocid biosynthesis is tightly controlled by both conserved and variable regulators, providing valuable guidance for further improving lasalocid production. IMPORTANCE Compared to its elaborated biosynthetic pathway, the regulation of lasalocid biosynthesis remains obscure. Here, we characterize the roles of regulatory genes in lasalocid biosynthetic gene clusters of two distinct Streptomyces species and identify a conserved repressor-activator system, LodR2-LodE, which could sense changes in the concentration of lasalocid and coordinate its biosynthesis with self-resistance. Furthermore, in parallel, we verify that the regulatory system identified in a new Streptomyces isolate is valid in the industrial lasalocid producer and thus applicable for the construction of high-yield strains. These findings deepen our understanding of regulatory mechanisms involved in the production of polyether ionophores and provide novel clues for the rational design of industrial strains for scaled-up production.

UVA Enhanced Promotive Effects of Blue Light on the Antioxidant Capacity and Anthocyanin Biosynthesis of Pak Choi

Anthocyanins are widely common natural antioxidants and represent an important economic feature in vegetables, but the potential response of UVA–blue co-irradiation on the anthocyanin biosynthesis of pak choi is not clear. Here, we investigated the effects of the supplement of four doses of UVA to blue light on growth, metabolites and the anthocyanin biosynthesis of two cultivars of pak choi. The results revealed that supplementing UVA light to blue light positively affected the growth of the pak choi and elevated the soluble protein content and antioxidant capacity. Especially, when compared with a monochromatic blue light, the anthocyanin synthesis was enhanced with an increase in UVA light strength, which reached a peak value at the strength of 10 μmol·m−2·s−1. Further study revealed that the UVA–blue co-irradiation enhanced the transcription of partial light-induced and anthocyanin structural genes. The intraspecific difference in the expression patterns of MYB1 and PAP1 were observed in these two tested cultivars. MYB1 was significantly up-regulated in red-leaf pak choi, but down-regulated in purple-leaf pak choi. On the contrary, PAP1 was significantly up-regulated in purple-leaf pak choi, but down-regulated in red-leaf pak choi. To sum up, this study established an efficient pre-harvest lighting strategy to elevate the economic value of pak choi.

Characterization of Pathway-Specific Regulator NigR for High Yield Production of Nigericin in Streptomyces malaysiensis F913.

Nigericin is a polyether antibiotic with potent antibacterial, antifungal, antimalarial and anticancer activity. NigR, the only regulator in the nigericin biosynthetic gene cluster in Streptomyces malaysiensis F913, was identified as a SARP family regulator. Disruption of nigR abolished nigericin biosynthesis, while complementation of nigR restored nigericin production, suggesting that NigR is an essential positive regulator for nigericin biosynthesis. Overexpression of nigR in Streptomyces malaysiensis led to significant increase in nigericin production compared to the wild-type strain. Nigericin production in the overexpression strain was found to reach 0.56 g/L, which may be the highest nigericin titer reported to date. Transcriptional analysis suggested that nigR is required for the transcription of structural genes in the nig gene cluster; quantitative RT-PCR analysis revealed that the expression of structural genes was upregulated in the nigR overexpression strain. Our study suggested that NigR acts in a positive manner to modulate nigericin production by activating transcription of structural genes and provides an effective strategy for scaling up nigericin production.

ABA signaling mediates 5-aminolevulinic acid-induced anthocyanin biosynthesis in red pear fruits

Peel color is an important quality trait of pears and directly affects its commercial value. Although several studies have reported that exogenous 5-aminolevulinic acid (ALA) induces anthocyanin biosynthesis, the mechanisms involved remain unclear. The purpose of this study was to confirm the effects of ALA on anthocyanin biosynthesis in the red pear ‘Nanhong’ and elucidate the effect of abscisic acid (ABA) on ALA-induced anthocyanin accumulation. Application of ALA solution (100 or 300 mg L−1) promoted arbutin, flavonol, and anthocyanin accumulation, but decreased chlorogenic acid and flavanol content, especially at 300 mg L−1. The structural gene transcriptional levels and transcription factors (PuMYB10 and PubHLH33) associated with anthocyanin synthesis were also enhanced by ALA treatment. Furthermore, ALA treatment increased the endogenous ABA content and upregulated the expression of ABA biosynthesis-related genes. Additionally, the influence of ALA on anthocyanin biosynthesis was removed by the ABA biosynthesis inhibitor fluridone, while the expression of PuMYB10, PuUFGT, and PuGSTF12, which were induced by exogenous ABA and ALA treatments, was suppressed. These results suggest that ABA signaling plays a crucial role in ALA-induced anthocyanin accumulation in red pear fruits. The results can provide a theoretical basis for improving the color and fruit quality of red pears.

Regulation of maltocin synthesis in Stenotrophomonas maltophilia by positive and negative regulators

Maltocin P28, produced by Stenotrophomonas maltophilia P28, is an R-type phage tail-like bacteriocin (PTLB). Its gene cluster consists of 23 putative genes, including nine nonstructural genes and fourteen structural genes. In this work, three nonstructural genes, mpsA, mpsH and mpsR, were found to encode transcriptional regulators to control maltocin P28 synthesis. MpsA activated the transcription of mpsH and lysis genes. MpsH activated the transcription of structural genes. Under normal growth conditions, MpsR repressed the transcription of mpsA and the structural genes, as well as its own. When S. maltophilia P28 was treated with mitomycin C, an immediate and significant decrease in the amount of MpsR was observed, followed by derepressed expression of mpsA, mpsR and structural genes, a marked rise in the expression of all regulatory and structural genes, and finally a clear increase in the maltocin P28 production. Neither the recA gene nor the lexA gene was found to be involved in the induced synthesis of maltocin P28. Our study indicated that a unique mechanism regulates the expression of maltocin genes in S. maltophilia, representing a novel strategy for balancing the expression of PTLB genes in bacteria.

A deep learning framework for enhancer prediction using word embedding and sequence generation

Enhancers are non-coding DAN fragments that play key roles in gene regulations and can promote the transcription of structural genes, thereby affecting the expression of structural protein catalytic enzymes and regulatory proteins. Accurate identification of enhancers helps to understand the transcription of structural genes and the development of human tumorigenesis, diagnosis and treatment. The enhancer sequences have high position variations and dispersions, and the identification of enhancers is more challenging than other genetic factors. Based on word embedding and sequence generative adversarial networks, a deep learning framework for enhancer identification is proposed. Firstly, considering the small number of sequences in the benchmark dataset, RankGAN is used to amplify the dataset size while maintaining the data characteristics. Then, in view of the similarity between DNA sequence and natural language, DNA sequence is regarded as a sentence composed of multiple “words”, and the word embedding technology FastText is applied to transform it into a numerical matrix. To extract the dependencies and highly abstract features of nucleotides in DNA sequences, a Long Short-Term Memory Convolutional Neural network (LSTM-CNN) is constructed to perform the identification task. On the independent test set, the accuracy and Matthew's correlation coefficient (MCC) for enhancer prediction are 0.7525 and 0.5051, respectively. For the enhancer type prediction, the accuracy and MCC of this method are 0.6972 and 0.3954, respectively. Compared with existing methods, this method achieves more satisfactory results for the prediction of enhancers and their types. This study will further enrich the application of natural language processing in bioinformatics.

Functional Characterization of a Flavone Synthase That Participates in a Kumquat Flavone Metabolon.

Flavones predominantly accumulate as O- and C-glycosides in kumquat plants. Two catalytic mechanisms of flavone synthase II (FNSII) support the biosynthesis of glycosyl flavones, one involving flavanone 2-hydroxylase (which generates 2-hydroxyflavanones for C-glycosylation) and another involving the direct catalysis of flavanones to flavones for O-glycosylation. However, FNSII has not yet been characterized in kumquats. In this study, we identified two kumquat FNSII genes (FcFNSII-1 and FcFNSII-2), based on transcriptome and bioinformatics analysis. Data from in vivo and in vitro assays showed that FcFNSII-2 directly synthesized apigenin and acacetin from naringenin and isosakuranetin, respectively, whereas FcFNSII-1 showed no detectable catalytic activities with flavanones. In agreement, transient overexpression of FcFNSII-2 in kumquat peels significantly enhanced the transcription of structural genes of the flavonoid-biosynthesis pathway and the accumulation of several O-glycosyl flavones. Moreover, studying the subcellular localizations of FcFNSII-1 and FcFNSII-2 demonstrated that N-terminal membrane-spanning domains were necessary to ensure endoplasmic reticulum localization and anchoring. Protein–protein interaction analyses, using the split-ubiquitin yeast two-hybrid system and bimolecular fluorescence-complementation assays, revealed that FcFNSII-2 interacted with chalcone synthase 1, chalcone synthase 2, and chalcone isomerase-like proteins. The results provide strong evidence that FcFNSII-2 serves as a nucleation site for an O-glycosyl flavone metabolon that channels flavanones for O-glycosyl flavone biosynthesis in kumquat fruits. They have implications for guiding genetic engineering efforts aimed at enhancing the composition of bioactive flavonoids in kumquat fruits.

Two Dof transcription factors promote flavonoid synthesis in kumquat fruit by activating C-glucosyltransferase

Although DNA binding with one finger (Dof) constitutes a crucial plant-specific family of transcription factors (TFs) that plays important roles in a wide range of biological processes, the molecular mechanisms underlying Dof regulation of flavonoid biosynthesis in plants remain largely unknown. Here, we characterized 28 Dof genes (FhDof1-FhDof28) from the ‘Hongkong’ kumquat (Fortunella hindsii) cultivar genome. Promoter analysis and transcriptome profiling revealed that four FhDofs — FhDof4, FhDof9, FhDof15, and FhDof16 — may be involved in flavonoid biosynthesis through binding to the flavonoid C-glycosyltransferase (FhCGT) promoter. We cloned homologous genes of four FhDofs, designated as FcDof4, FcDof9, FcDof15, FcDof16, and a homologous gene of FhCGT, designated as FcCGT, from the widely cultivated ‘HuaPi’ kumquat (F. crassifolia). Quantitative reverse transcription-polymerase chain reaction analysis revealed that FcDof4 and FcDof16 were significantly correlated with FcCGT expression during development stages in the ‘HuaPi’ fruit (Pearson’s correlation coefficient > 0.7) and were localized to the nucleus. Results of yeast one-hybrid, electrophoretic mobility shift, and dual-luciferase assays indicated that the two FcDofs trigger FcCGT expression by specifically binding to its promoters. Moreover, transient overexpression of FcDof4 and FcDof16 enhances the transcription of structural genes in the flavonoid biosynthetic pathway and increases C-glycosylflavonoid content. Our results provide strong evidence that the TFs FcDof4 and FcDof16 promote flavonoid synthesis in kumquat fruit by activating FcCGT expression.

Tannic Acid Inhibits Salmonella enterica Serovar Typhimurium Infection by Targeting the Type III Secretion System.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a zoonotic pathogen that can cause food poisoning and diarrhea in both humans and animals worldwide. The Salmonella pathogenicity island (SPI) genes encoded type III secretion system (T3SS) is important for S. Typhimurium invasion and replication in host cells. Due to the increasing problem of antibiotic resistance, antibiotic treatment for clinical Salmonella infection has gradually been limited. Anti-virulence inhibitors are a promising alternative to antibiotics because they do not easily induce bacterial antibiotic resistance. Here, we systematically evaluated the therapeutic effect of tannic acid (TA) on Salmonella-infected mice and elucidated its anti-infection mechanism. TA treatment improved the survival rate of S. Typhimurium-infected mice and alleviated cecum pathological lesions. In addition, TA inhibited S. Typhimurium invasion to HeLa cells without affecting their growth. Further studies showed that TA could inhibit the expression of sipA and sipB. This inhibition may be implemented by inhibiting the transcription of key regulatory and structural genes of the T3SS. This study provides an alternative anti-virulence strategy for Salmonella infection treatment.

Tbx15/18/22 shares a binding site with Tbx6-r.b to maintain expression of a muscle structural gene in ascidian late embryos

The ascidian larval tail contains muscle cells for swimming. Most of these muscle cells differentiate autonomously. The genetic program behind this autonomy has been studied extensively and the genetic cascade from maternal factors to initiation of expression of a muscle structural gene, Myl.c, has been uncovered; Myl.c expression is directed initially by transcription factor Tbx6-r.b at the 64-cell stage and then by the combined actions of Tbx6-r.b and Mrf from the gastrula to early tailbud stages. In the present study, we showed that transcription of Myl.c continued in late tailbud embryos and larvae, although a fusion protein of Tbx6-r.b and GFP was hardly detectable in late tailbud embryos. A knockdown experiment, reporter assay, and in vitro binding assay indicated that an essential cis-regulatory element of Myl.c that bound Tbx6-r.b in early embryos bound Tbx15/18/22 in late embryos to maintain expression of Myl.c. We also found that Tbx15/18/22 was controlled by Mrf, which constitutes a regulatory loop with Tbx6-r.b. Therefore, our data indicated that Tbx15/18/22 was activated initially under control of this regulatory loop as in the case of Myl.c, and then Tbx15/18/22 maintained the expression of Myl.c after Tbx6-r.b had disappeared. RNA-sequencing of Tbx15/18/22 morphant embryos revealed that many muscle structural genes were regulated similarly by Tbx15/18/22. Thus, the present study revealed the mechanisms of maintenance of transcription of muscle structural genes in late embryos in which Tbx15/18/22 takes the place of Tbx6-r.b.

Multilevel Regulation of Carotenoid Synthesis by Light and Active Oxygen in Blakeslea trispora.

Although Blakeslea trispora has been used for industrial production of β-carotene, the effects of light and oxidative stress on its synthesis have not been fully clarified. The present study focuses on the effects of light and reactive oxygen species (ROS) on carotenoid synthesis and their multilevel regulation in B. trispora. Blue light significantly influenced the intracellular ROS levels, carotenoid contents, and transcription of carotenoid structural genes, while ROS levels were positively correlated with intracellular carotenoid contents and transcriptional levels of carotenoid structural genes. Blue light and ROS were both significant factors affecting carotenoid synthesis with a significant interaction between them. Irradiation by pulsed blue light and (or) addition of generating agents for active oxygen could partially compensate for the inhibition derived from the transcription inhibitor (dactinomycin) and translation inhibitor (cycloheximide) on the multilevel phenotype. Therefore, blue light and ROS act on the transcription and translation of carotenoid structural genes to promote the accumulation of carotenoid in B. trispora.