Multiprotein Bridging Factor Research Articles

Genome-wide identification, in-silico characterisation and expression analysis of multiprotein bridging factor 1 gene family members in rice

Genome-wide identification, in-silico characterisation and expression analysis of multiprotein bridging factor 1 gene family members in rice

A lily heat-inducible multi-protein bridging factor, LlMBF1c, plays a crucial role in plant thermotolerance

Lily (Lilium spp.) is popular for its colorful flowers and exquisite scents. Nonetheless, high temperatures often severely reduce its yield production and quality. The implementation of biotechnological approaches to manipulate the expression of key heat-resistant genes is an effective way to improve the thermotolerance of plants. Here, we isolated a gene encoding for a multi-protein bridging factor 1c (MBF1c) from L. longiflorum 'White Heaven' (LlMBF1c), which was highly similar to MBF1c from Elaeis guineensis (EgMBF1c). LlMBF1c harbors conserved MBF1 and helix-turn-helix (HTH) domains. Moreover, the expression of LlMBF1c and its promoter activity were enhanced under high-temperature conditions. Further analysis indicated that LlMBF1c is a transcriptional repressor in both yeast and Nicotiana benthamiana. Its protein was located in the nucleus and cytoplasm of N. benthamiana leaf cells. Overexpression of LlMBF1c in lily and Arabidopsis resulted in enhanced thermotolerance in these plants. By contrast, silencing LlMBF1c reduced the thermotolerance of lily. Our results identified an important candidate gene that can be utilized to develop thermotolerant lily germplasm.

Broad Complex-Z2 directly activates BmMBF2 to inhibit the silk protein synthesis in the silkworm, Bombyx mori

Silk proteins, as natural macromolecules, have extensive applications in biomaterials and biomedicine. In the silkworm, the expression of silk protein genes is negatively associated with ecdysone during the molt stage, while it is positively correlated with juvenile hormone during the intermolt stage. In our previous study, overexpression of an isoform Z2 of Broad Complex (BmBrC-Z2), an ecdysone early response factor, significantly reduced the expression of silk protein genes. However, the underlying regulatory mechanism remains unclear. In this study, we conducted transcriptomic analysis and found that overexpressing BmBrC-Z2 significantly upregulated the expression level of multiprotein bridging factor 2 (BmMBF2), an inhibitor of fibroin heavy chain (FibH). Further investigations revealed that BmBrC-Z2 directly regulated BmMBF2 by binding to cis-regulatory elements, as demonstrated using Dual-Luciferase Reporter Gene Assay, EMSA, and ChIP-PCR assay. Additionally, when using the CRISPR/Cas9 system to knock out BmMBF2, silk protein genes were significantly upregulated during the molt stage of mutant larvae. These findings uncover the negative regulation of silk protein synthesis by the ecdysone signaling cascade, specifically through the manipulation of BmMBF2 expression during the molt stage. This study enhances to our understanding of the temporal regulatory mechanism governing silk protein synthesis and offers a potential strategy for improving silk yield.

Transcription Cofactor CsMBF1c Enhances Heat Tolerance of Cucumber and Interacts with Heat-Related Proteins CsNFYA1 and CsDREB2.

Multiprotein bridging factor 1 (MBF1) is a very important transcription factor (TF) in plants, whose members influence numerous defense responses. Our study found that MBF1c in Cucurbitaceae was highly conserved. CsMBF1c expression was induced by temperature, salt stress, and abscisic acid (ABA) in cucumber. Overexpressed CsMBF1c enhanced the heat resistance of a cucumber, and the Csmbf1c mutant showed decreased resistance to high temperatures (HTs). CsMBF1c played an important role in stabilizing the photosynthetic system of cucumber under HT, and its expression was significantly associated with heat-related TFs and genes related to protein processing in the endoplasmic reticulum (ER). Protein interaction showed that CsMBF1c interacted with dehydration-responsive element binding protein 2 (CsDREB2) and nuclear factor Y A1 (CsNFYA1). Overexpression of CsNFYA1 in Arabidopsis improved the heat resistance. Transcriptional activation of CsNFYA1 was elevated by CsMBF1c. Therefore, CsMBF1c plays an important regulatory role in cucumber's resistance to high temperatures.

Selection of Stable Reference Genes for QRT-PCR in Tree Peony 'Doulv' and Functional Analysis of PsCUC3.

(1) Background: Tree peonies display extensive cultivar diversity due to widespread hybridization, resulting in a complex genetic architecture. This complexity complicates the selection of universal reference genes across different cultivars for qRT-PCR analyses. Paeonia suffruticosa 'Doulv', notable for its unique green blooms in China, exhibits chlorosis post-flowering and features petaloid stamens and pistils. (2) Methods: Based on published literature and RNA-seq data from 'Doulv', nine candidate reference genes-ACT (Actin), TUB (β-Tubulin), UBC (Ubiquitin Conjugating Enzyme), UBQ (Ubiquitin), UPL (Ubiquitin Protein Ligase), PP2A (Protein Phosphatase 2A), PP2C (Protein Phosphatase 2C), MBF1A (Multiprotein Bridging Factor 1A), and GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase)-were selected. Their expression stability was assessed across various tissues and developmental stages of 'Doulv' flowers using qRT-PCR, with evaluations conducted via GeNorm_v3.5, NormFinder_v20, and BestKeeper_v1.0. Gene cloning and expression analyses of PsCUC3, including its subcellular localization, were performed. (3) Results: GAPDH and ACT were identified as the most stable reference genes in petaloid stamens across various developmental stages of 'Doulv', whereas UBC and MBF1A were optimal across different tissues. Notably, specific conserved amino acids in PsCUC3 from 'Doulv' diverged from those in NAM/CUC3 proteins of other species, impacting its protein structure. PsCUC3 expression analysis revealed no correlation with chlorophyll content in petaloid stamens but an association with petaloid organ development. Furthermore, PsCUC3 was predominantly localized in the nucleus. (4) Conclusions: This study comprehensively evaluated suitable reference genes using GeNorm_v3.5, NormFinder_v20, and BestKeeper_v1.0 software, establishing a robust qRT-PCR detection system for 'Doulv' peony. These results provide a solid experimental foundation for further research on 'Doulv' peony. Building on this experimental foundation, the functional analysis of the PsCUC3 gene was conducted. The findings suggest a potential association between the PsCUC3 gene and floral morphology alterations in 'Doulv', identifying PsCUC3 as crucial for understanding the molecular mechanisms influencing floral structure in tree peonies.

SfREPAT38, a pathogen response gene (REPAT), is involved in immune response of Spodoptera frugiperda larvae through mediating Toll signalling pathway.

REPAT (response to pathogen) is an immune-associated gene family that plays important roles in insect immune response to pathogens. Although nine REPAT genes have been identified in Spodoptera frugiperda (Lepidoptera: Noctuidae) currently, their functions and mechanisms in the immune response to pathogens still remain unclear. Therefore, SfREPAT38, a pathogen response gene (REPAT) of S. frugiperda, was characterised and its function was analysed. The results showed that SfREPAT38 contains a signal peptide and a transcription activator MBF2 (multi-protein bridging factor 2) domain. Quantitative real-time polymerase chain reaction analysis showed that SfREPAT38 was highly expressed in the sixth-instar larvae (L6) and was the highest in expression in the midgut of L6. We found that the expression of SfREPAT38 could be activated by challenge with four microbial pathogens (Bacillus thuringiensis, Metarhizium anisopliae, Spodoptera exigua nuclearpolyhedrosis and Escherichia coli), except 12 h after E. coli infection. Furthermore, the SfREPAT38 expression levels significantly decreased at 24, 48 and 72 h after SfREPAT38 dsRNA injection or feeding. Feeding with SfREPAT38 dsRNA significantly decreased the weight gain of S. frugiperda, and continuous feeding led to the death of S. frugiperda larvae from the fourth day. Moreover, SfREPAT38 dsRNA injection resulted in a significant decrease of weight gain on the fifth day. Silencing SfREPAT38 gene down-regulated the expression levels of immune genes belonging to the Toll pathway, including SPZ, Myd88, DIF, Cactus, Pell and Toll18W. After treatment with SfREPAT38 dsRNA, S. frugiperda became extremely sensitive to the B. thuringiensis infection, and the survival rate dramatically increased, with 100% mortality by the eighth day. The weight of S. frugiperda larvae was also significantly lower than that of the control groups from the second day onwards. In addition, the genes involved in the Toll signalling pathway and a few antibacterial peptide related genes were down-regulated after treatment. These results showed that SfREPAT38 is involved in the immune response of S. frugiperda larvae through mediating Toll signalling pathway.

PgWRKY44, a pearl millet WRKY transcription factor-Calmodulin module, enhances salt and drought stress resilience in transgenic plants

WRKY transcription factors (TFs) regulate signal transduction pathways during stress response and can also modulate the activity of downstream genes through binding to their cognate W-box elements [(T) TGAC(C/T)]. Previous studies have identified and in-silico characterized WRKY family members in millets. However, their functional elucidation and molecular mechanism in millets remain vastly unexplored. In this study, a pearl millet WRKY TF (PgWRKY44) belonging to Group IId was characterized, and its ectopic expression in Arabidopsis was found to be positively regulating abiotic stress tolerance in transgenic plants through ABA-mediated signalling. Also, reduced accumulation of reactive oxygen species (ROS) and up-regulation of stress-related genes confirmed improved defense systems of transgenic plants upon abiotic stress treatments. Functional network analysis and expression data indicated towards co-regulation of multiprotein bridging factor (MBF1C), HSFs, and calmodulin (CAM) members with PgWRKY44 in response to osmotic stress. Yeast one-hybrid also confirmed W-box-dependent binding of PgWRKY44. These findings enriched our understanding of the PgWRKY44 functions in pearl millet and exhibited its potential application in developing climate-resilient crop plants.

HD-Zip I protein LlHOX6 antagonizes homeobox protein LlHB16 to attenuate basal thermotolerance in lily.

Homeodomain-leucine zipper (HD-Zip) I transcription factors are crucial for plant responses to drought, salt, and cold stresses. However, how they are associated with thermotolerance remains mostly unknown. We previously demonstrated that lily (Lilium longiflorum) LlHB16 (HOMEOBOX PROTEIN 16) promotes thermotolerance, whereas the roles of other HD-Zip I members are still unclear. Here, we conducted a transcriptomic analysis and identified a heat-responsive HD-Zip I gene, LlHOX6 (HOMEOBOX 6). We showed that LlHOX6 represses the establishment of basal thermotolerance in lily. LlHOX6 expression was rapidly activated by high temperature, and its protein localized to the nucleus. Heterologous expression of LlHOX6 in Arabidopsis (Arabidopsis thaliana) and overexpression in lily reduced their basal thermotolerance. In contrast, silencing LlHOX6 in lily elevated basal thermotolerance. Cooverexpressing or cosilencing LlHOX6 and LlHB16 in vivo compromised their functions in modulating basal thermotolerance. LlHOX6 interacted with itself and with LlHB16, although heterologous interactions were stronger than homologous ones. Notably, LlHOX6 directly bounds DNA elements to repress the expression of the LlHB16 target genes LlHSFA2 (HEAT STRESS TRANSCRIPTION FACTOR A2) and LlMBF1c (MULTIPROTEIN BRIDGING FACTOR 1C). Moreover, LlHB16 activated itself to form a positive feedback loop, while LlHOX6 repressed LlHB16 expression. The LlHOX6-LlHB16 heterooligomers exhibited stronger DNA binding to compete for LlHB16 homooligomers, thus weakening the transactivation ability of LlHB16 for LlHSFA2 and LlMBF1c and reducing its autoactivation. Altogether, our findings demonstrate that LlHOX6 interacts with LlHB16 to limit its transactivation, thereby impairing heat stress responses in lily.

Genome-Wide Analysis of MBF1 Family Genes in Five Solanaceous Plants and Functional Analysis of SlER24 in Salt Stress.

Multiprotein bridging factor 1 (MBF1) is an ancient family of transcription coactivators that play a crucial role in the response of plants to abiotic stress. In this study, we analyzed the genomic data of five Solanaceae plants and identified a total of 21 MBF1 genes. The expansion of MBF1a and MBF1b subfamilies was attributed to whole-genome duplication (WGD), and the expansion of the MBF1c subfamily occurred through transposed duplication (TRD). Collinearity analysis within Solanaceae species revealed collinearity between members of the MBF1a and MBF1b subfamilies, whereas the MBF1c subfamily showed relative independence. The gene expression of SlER24 was induced by sodium chloride (NaCl), polyethylene glycol (PEG), ABA (abscisic acid), and ethrel treatments, with the highest expression observed under NaCl treatment. The overexpression of SlER24 significantly enhanced the salt tolerance of tomato, and the functional deficiency of SlER24 decreased the tolerance of tomato to salt stress. SlER24 enhanced antioxidant enzyme activity to reduce the accumulation of reactive oxygen species (ROS) and alleviated plasma membrane damage under salt stress. SlER24 upregulated the expression levels of salt stress-related genes to enhance salt tolerance in tomato. In conclusion, this study provides basic information for the study of the MBF1 family of Solanaceae under abiotic stress, as well as a reference for the study of other plants.

A long noncoding RNA HILinc1 enhances pear thermotolerance by stabilizing PbHILT1 transcripts through complementary base pairing

As global warming intensifies, heat stress has become a major environmental constraint threatening crop production and quality worldwide. Here, we characterize Heat-induced long intergenic noncoding RNA 1 (HILinc1), a cytoplasm-enriched lincRNA that plays a key role in thermotolerance regulation of pear (Pyrus spp.). HILinc1 Target 1 (PbHILT1) which is the target transcript of HILinc1, was stabilized via complementary base pairing to upregulate its expression. PbHILT1 could bind to Heat shock transcription factor A1b (PbHSFA1b) to enhance its transcriptional activity, leading to the upregulation of a major downstream transcriptional regulator, Multiprotein bridging factor 1c (PbMBF1c), during heat response. Transient overexpressing of either HILinc1 or PbHILT1 increases thermotolerance in pear, while transient silencing of HILinc1 or PbHILT1 makes pear plants more heat sensitive. These findings provide evidences for a new regulatory mechanism by which HILinc1 facilitates PbHSFA1b activity and enhances pear thermotolerance through stabilizing PbHILT1 transcripts.

Membrane-Fluidization-Dependent and -Independent Pathways Are Involved in Heat-Stress-Inducible Gene Expression in the Marine Red Alga Neopyropia yezoensis

Heat stress responses are complex regulatory processes, including sensing, signal transduction, and gene expression. However, the exact mechanisms of these processes in seaweeds are not well known. We explored the relationship between membrane physical states and gene expression in the red alga Neopyropia yezoensis. To analyze heat-stress-induced gene expression, we identified two homologs of the heat-inducible high temperature response 2 (HTR2) gene in Neopyropia seriata, named NyHTR2 and NyHTR2L. We found conservation of HTR2 homologs only within the order Bangiales; their products contained a novel conserved cysteine repeat which we designated the Bangiales cysteine-rich motif. A quantitative mRNA analysis showed that expression of NyHTR2 and NyHTR2L was induced by heat stress. However, the membrane fluidizer benzyl alcohol (BA) did not induce expression of these genes, indicating that the effect of heat was not due to membrane fluidization. In contrast, expression of genes encoding multiprotein-bridging factor 1 (NyMBF1) and HSP70s (NyHSP70-1 and NyHSP70-2) was induced by heat stress and by BA, indicating that it involved a membrane-fluidization-dependent pathway. In addition, dark treatment under heat stress promoted expression of NyHTR2, NyHTR2L, NyMBF1, and NyHSP70-2, but not NyHSP70-1; expression of NyHTR2 and NyHTR2L was membrane-fluidization-independent, and that of other genes was membrane-fluidization-dependent. These findings indicate that the heat stress response in N. yezoensis involves membrane-fluidization-dependent and -independent pathways.

The transcriptional coactivator CmMBF1c is required for waterlogging tolerance in Chrysanthemum morifolium.

Waterlogging is one of the most serious abiotic stressors affecting Chrysanthemum morifolium during its lifespan. However, the molecular mechanisms underlying the waterlogging tolerance of chrysanthemum remain unclear. In this study, we discovered that the transcriptional coactivator MULTIPROTEIN BRIDGING FACTOR 1c (CmMBF1c) was significantly induced by waterlogging stress in chrysanthemums. Promoter sequence analysis and transient dual-luciferase assay using chrysanthemum protoplasts showed that the waterlogging-tolerant cultivar 'Nannongxuefeng' carried more response elements involved in waterlogging and hypoxia stress compared with the waterlogging-sensitive cultivar 'Qinglu', conferring on 'Nannongxuefeng' a stronger hypoxia responsive activity and higher CmMBF1c expression under waterlogging conditions. Subcellular localization and transcriptional activity assays showed that CmMBF1c protein was localized to the nucleus and had no transcriptional activation activity. Overexpression of CmMBF1c in 'Qinglu' enhanced its waterlogging tolerance by promoting its reactive oxygen species (ROS) scavenging ability and maintaining low ROS levels. However, RNAi-mediated knockdown of CmMBF1c in cultivar 'Nannongxuefeng' resulted in the opposite tendency. Yeast two-hybrid screening and tobacco bimolecular fluorescence complementation assays revealed that CmHRE2, a pivotal regulator of hypoxia response, could interact with CmMBF1c. In summary, this study demonstrates that CmMBF1c improves chrysanthemum waterlogging tolerance by regulating its ROS signaling pathway and interacting with CmHRE2. These findings together offer, to our knowledge, new mechanistic insights into chrysanthemum waterlogging tolerance and provide a rational foundation for future research on the genetic improvement of horticultural crops for waterlogging stress tolerance.

Comparative Analysis and Function of Multiprotein Bridging Factor1 (MBF1) Genes in Cotton

Comparative Analysis and Function of Multiprotein Bridging Factor1 (MBF1) Genes in Cotton

Overexpression of TaMBF1c improves thermo-tolerance of perennial ryegrass

High temperature is one of the major threats to global crop production, especially the C3 plant species, such as a widely used forage/turfgrass, perennial ryegrass. MBF1c gene belongs to group II of the highly conserved multiprotein bridging factor1 (MBF1) family, and it has been reported to be involved in various plant abiotic stresses, in particular, heat stress. In the study, a wheat MBF1c (TaMBF1c) was overexpressed in perennial ryegrass, and physiological parameters including antioxidant enzyme activities were investigated under both control (16/22 °C, n/d) and high temperature (42 °C) treatments for 24 and 48 h. The results found that the ectopic expression of TaMBF1c improved thermotolerance of perennial ryegrass, demonstrated by lower leaf electrolyte leakage and lipid peroxidation, better water status, higher chlorophyll content (SPAD index), and net photosynthetic rate under high temperature, while there was no difference between wild type and transgenic perennial ryegrass plants under control condition. The enhanced stress tolerance of transgenic perennial ryegrass was, at least partially associated with its elevated antioxidative capacity under stress. The findings confirmed that MBF1c could confer thermotolerance in C3 perennial grass species, which offers an additional strategy to develop cool-season turfgrass/forage plants to cope with the challenges of higher temperature and more frequent heat waves due to global warming.

StMBF1c positively regulates disease resistance to Ralstonia solanacearum via it’s primary and secondary upregulation combining expression of StTPS5 and resistance marker genes in potato

Multiprotein bridging factor 1 (MBF1) is a transcription coactivator that has a general defense response to pathogens. However, the regulatory mechanisms of MBF1 resistance bacterial wilt remain largely unknown. Here, the role of StMBF1c in potato resistance to Ralstonia solanacearum infection was characterized. qRT-PCR assays indicated that StMBF1c could was elicited by SA, MJ and ABA and the time-course expression pattern of the StMBF1c gene induced by R. solanacearum was found to be twice significant upregulated expression during the early and middle stages of bacterial wilt. Combined with the co-expression analysis of disease-resistant marker genes, gain-of-function and loss-of-function assays demonstrated that StMBF1c was associated with defence priming. Overexpression or silencing the MBF1c could enhance plants resistance or sensitivity to R. solanacearum through inducing or reducing NPR and PR genes related to SA signal pathway. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) experiment results confirmed the interaction of StMBF1c with StTPS5 which played a key role in ABA signal pathway in potato. It is speculated that by combining StTPS5 and resistance marker genes, StMBF1c is activated twice to participate in potato bacterial wilt resistance, in which EPI, PTI involved.

LlWRKY39 is involved in thermotolerance by activating LlMBF1c and interacting with LlCaM3 in lily (Lilium longiflorum)

WRKY transcription factors (TFs) are of great importance in plant responses to different abiotic stresses. However, research on their roles in the regulation of thermotolerance remains limited. Here, we investigated the function of LlWRKY39 in the thermotolerance of lily (Lilium longiflorum ‘white heaven’). According to multiple alignment analyses, LlWRKY39 is in the WRKY IId subclass and contains a potential calmodulin (CaM)-binding domain. Further analysis has shown that LlCaM3 interacts with LlWRKY39 by binding to its CaM-binding domain, and this interaction depends on Ca2+. LlWRKY39 was induced by heat stress (HS), and the LlWRKY39-GFP fusion protein was detected in the nucleus. The thermotolerance of lily and Arabidopsis was increased with the ectopic overexpression of LlWRKY39. The expression of heat-related genes AtHSFA1, AtHSFA2, AtMBF1c, AtGolS1, AtDREB2A, AtWRKY39, and AtHSP101 was significantly elevated in transgenic Arabidopsis lines, which might have promoted an increase in thermotolerance. Then, the promoter of LlMBF1c was isolated from lily, and LlWRKY39 was found to bind to the conserved W-box element in its promoter to activate its activity, suggesting that LlWRKY39 is an upstream regulator of LlMBF1c. In addition, a dual-luciferase reporter assay showed that via protein interaction, LlCaM3 negatively affected LlWRKY39 in the transcriptional activation of LlMBF1c, which might be an important feedback regulation pathway to balance the LlWRKY39-mediated heat stress response (HSR). Collectively, these results imply that LlWRKY39 might participate in the HSR as an important regulator through Ca2+-CaM and multiprotein bridging factor pathways.

A Novel Multiprotein Bridging Factor 1-Like Protein Induces Cyst Wall Protein Gene Expression and Cyst Differentiation in Giardia lamblia.

The capacity to synthesize a protective cyst wall is critical for infectivity of Giardia lamblia. It is of interest to know the mechanism of coordinated synthesis of three cyst wall proteins (CWPs) during encystation, a differentiation process. Multiprotein bridging factor 1 (MBF1) gene family is a group of transcription coactivators that bridge various transcription factors. They are involved in cell growth and differentiation in yeast and animals, or in stress response in fungi and plants. We asked whether Giardia has MBF1-like genes and whether their products influence gene expression. BLAST searches of the Giardia genome database identified one gene encoding a putative MBF1 protein with a helix-turn-helix domain. We found that it can specifically bind to the AT-rich initiator promoters of the encystation-induced cwp1-3 and myb2 genes. MBF1 localized to cell nuclei and cytoplasm with higher expression during encystation. In addition, overexpression of MBF1 induced cwp1-3 and myb2 gene expression and cyst generation. Mutation of the helixes in the helix-turn-helix domain reduced cwp1-3 and myb2 gene expression and cyst generation. Chromatin immunoprecipitation assays confirmed the binding of MBF1 to the promoters with its binding sites in vivo. We also found that MBF1 can interact with E2F1, Pax2, WRKY, and Myb2 transcription factors that coordinately up-regulate the cwp genes during encystation. Using a CRISPR/Cas9 system for targeted disruption of mbf1 gene, we found a downregulation of cwp1-3 and myb2 genes and decrease of cyst generation. Our results suggest that MBF1 is functionally conserved and positively regulates Giardia cyst differentiation.

Newly identified genes contribute to vanillin tolerance in Saccharomyces cerevisiae.

Exploring the mechanisms of tolerance in microorganisms to vanillin, which is derived from lignin, will benefit the design of robust cell factories that produce biofuels and chemicals using lignocellulosic materials. Our objective was to identify the genes related to vanillin tolerance in Saccharomyces cerevisiae. We investigated the effects on vanillin tolerance of several genes that have site mutations in the highly vanillin-tolerant strain EMV-8 compared to its parental line NAN-27. The results showed that overexpression of GCY1, a gene that encodes an aldo-keto reductase that also has mRNA-binding activity, YPR1, a paralog of GCY1 that encodes an aldo-keto reductase, PEX5, a gene that encodes a peroxisomal membrane signal receptor and MBF1, a gene that encodes a multiprotein bridging factor increase the specific growth rates (μ) by 49%, 41%, 44% and 48 %, respectively, in medium containing 6mmoll-1 vanillin. Among these gene products, Gcy1p and Ypr1p showed NADPH-dependent and NAD(P)H-dependent vanillin reductase activity, respectively. The reductase-inactive mutant Gcy1pY56F also increased vanillin tolerance in S. cerevisiae, suggesting that other mechanisms exist. Although TRS85 and PEX5, genes for which the mRNAs are binding targets of Gcy1p, were shown to be related to vanillin tolerance, both the mRNA and protein levels of these genes were not changed by overexpression of GCY1. The relationship between the mRNA-binding activity of Gcy1p and its positive effect on vanillin tolerance is still not clear. Finally, we found that the point mutation D112A in Mbf1p, which disrupts the binding of Mbf1p and the TATA element-binding protein (TBP), did not decrease the positive effect of Mbf1p on vanillin tolerance. This indicates that the binding of Mbf1p and TBP is not necessary for the positive effect on vanillin tolerance mediated by Mbf1p. We have successfully identified new genes related to vanillin tolerance and provided novel targets that can be used to improve the vanillin tolerance of S. cerevisiae. Moreover, we have extended our understanding of the proteins encoded by these genes.

Study on Interaction Between TATA-Box Binding Protein (TBP), TATA-Box and Multiprotein Bridging Factor 1(MBF1) in Beauveria bassiana by Graphene-Based Electrochemical Biosensors.

The regulation of transcription level is an important step in gene expression process. Beauveria bassiana is a broad-spectrum insecticidal fungi widely used in the biologic control of arthropod. The regulation of its transcription level is a multilevel complex process. Multiprotein bridging factor 1(MBF1) is a transcriptional co-activator that bridges sequence-specific activators and the TATA-box binding protein(TBP), Little is known about the interaction between MBF1, TBP, and TBP binding to DNA(TATA-sequences)in filamentous fungi of Beauveria bassiana, The binding of TBP to TATA-box and TBP to MBF1 was investigated via electrochemical biosensor. Graphene oxide has an electronic mobility that is unattainable for any metal, so it will be highly sensitive as a test electrode. Hence, we developed a simple, sensitive and specific sensor based on an TBP probe and graphene oxide that successfully detected the interaction of TBP and TATA-box or MBF1. From the electrochemical impedance spectroscopy (EIS), we find that the radius will increase when adding TATA-box or MBF1 buffer to the modified TBP protein electrode. When adding no TATA-box or no MBF1, the radius is relatively unchanged. The interaction between TBP and TATA-box or MBF1 was proved based on the results. These data confirmed the specificity of the interactions, (1) our developed graphene-based electrochemical biosensor can be used for monitoring the interaction between TBP and TATA-box or MBF1, (2) TBP can bind to TATA-box, (3) TBP can bind to MBF1, and (4) TBP mediates the interactions of MBF1 to DNA. Therefore, this work provided a label-free, low-cost and simple detection method for the complex process of eukaryotic gene transcription regulation.

The plant MBF1 protein family: a bridge between stress and transcription.

The Multiprotein Bridging Factor 1 (MBF1) proteins are transcription co-factors whose molecular function is to form a bridge between transcription factors and the basal machinery of transcription. MBF1s are present in most archaea and all eukaryotes, and numerous reports show that they are involved in developmental processes and in stress responses. In this review we summarize almost three decades of research on the plant MBF1 family, which has mainly focused on their role in abiotic stress responses, in particular the heat stress response. However, despite the amount of information available, there are still many questions that remain about how plant MBF1 genes, transcripts, and proteins respond to stress, and how they in turn modulate stress response transcriptional pathways.