Electrophoretic Mobility Shift Assay Research Articles

Hydroxycinnamic Acids Activate RpfB-Dependent Quorum Sensing Signal Turnover in Phytopathogen Xanthomonas campestris.

Phytopathogen Xanthomonas campestris pv campestris (Xcc) uses various mechanisms, including the quorum sensing (QS) system, to regulate infection and colonize cruciferous host plants. Hydroxycinnamic acids (HCAs) are widely distributed in the plant kingdom, but their effect on the diffusible signaling factor family QS signal of Xcc remains unknown. In this study, we report that HCAs activate RpfB-dependent QS signal turnover via the sensor protein HepR, which in turn activates the HepABCD resistance-nodulation-division efflux pump to remove HCAs in Xcc. Exogenous addition of three HCAs induced DSF signal turnover during the late growth phase of Xcc in XYS medium. Furthermore, HCAs-induced DSF turnover was dependent on the DSF-degrading enzyme RpfB and the sensor HepR. Additionally, Xcc pumps HCAs through the HepABCD efflux pump. An electrophoretic mobility shift assay demonstrated that HCAs interfere with the binding of HepR to the Phep promoter. This work provides insight into the molecular interactions between Xcc and cruciferous plants.

Structural insights into dynamics of the BMV TLS aminoacylation

Brome Mosaic Virus (BMV) utilizes a tRNA-like structure (TLS) within its 3’ untranslated region to mimic host tRNA functions, aiding aminoacylation and viral replication. This study explores the structural dynamics of BMV TLS interacting with tyrosyl-tRNA synthetase (TyrRS) during aminoacylation. Using cryo-EM, we capture multiple states of the TLS-TyrRS complex, including unbound TLS, pre-1a, post-1a, and catalysis states, with resolutions of 4.6 Å, 3.5 Å, 3.7 Å, and 3.85 Å, respectively. These structural comparisons indicate dynamic changes in both TLS and TyrRS. Upon binding, TLS undergoes dynamic rearrangements, particularly with helices B3 and E pivoting, mediated by the unpaired A36 residue, ensuring effective recognition by TyrRS. The dynamic changes also include a more compact arrangement in the catalytic center of TyrRS and the insertion of 3’ CCA end into the enzyme’s active site, facilitating two-steps aminoacylation. Enzymatic assays further demonstrated the functional importance of TLS-TyrRS interactions, with mutations in key residues significantly impacting aminoacylation efficiency. Furthermore, Electrophoretic Mobility Shift Assay (EMSA) demonstrated that BMV TLS binds elongation factors EF1α and EF2, suggesting a multifaceted strategy to exploit host translational machinery. These findings not only enhance our knowledge of virus-host interactions but also offer potential targets for antiviral drug development.

Identification of a functional vitamin D response element in the promoter of goose anti-Müllerian hormone gene.

Anti-Müllerian hormone (AMH) plays an important role in avian ovarian follicle development. The high mRNA expression of AMH in avian ovarian prehierarchical follicles helps prevent premature granulosa cell differentiation. Vitamin D3 was reported to downregulate AMH mRNA expression in granulosa cells of prehierarchical follicles in hens; however, the underlying molecular mechanism remains unknown. In this study, AMH mRNA expression was investigated in granulosa cells of prehierarchical follicles in geese under vitamin D3 induction. A potential vitamin D response element (VDRE) present in the goose AMH promoter was identified using luciferase activity, electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP) assays. The results showed that AMH mRNA expressions were downregulated by vitamin D3 in granulosa cells of prehierarchical follicles in geese. A classical VDRE-like sequence was identified in the goose AMH promoter by in silico analyses. Luciferase activity assays revealed that the putative VDRE is a negative regulatory element. Vitamin D receptor (VDR) and retinoic X receptor (RXR) are necessary to decrease the AMH promoter activity induced by vitamin D3. The EMSA and ChIP assays demonstrated that the VDR/RXR complex directly binds to the putative VDRE. These results suggest that vitamin D3 downregulates AMH mRNA expression via a functional VDRE that binds the VDR/RXR heterodimer in goose ovarian prehierarchical follicles. These results provide new insights into the regulatory mechanisms of vitamin D3 in avian ovarian follicular development.

Transcription factor MdGTL1a accelerates starch degradation by promoting the MdBam8 expression in postharvest apple fruit.

Starch degradation plays a central role in fruit ripening. The β-amylase (Bam) catalysts starch degradation of apple fruit. However, the molecular mechanism regulating Bam gene expression in apples remains unclear. Using yeast one-hybrid library screening, we identified a transcription factor MdGTL1a that directly binds to the MdBam8 promoter. This bind was verified by using an electrophoretic mobility shift assay and confirmed to enhance the promotor activity of MdBam8 by promoter-β-glucuronidase transactivation assay. Transient over-expression of MdGTL1a activated MdBam8 expression and further enhanced starch degradation in apple flesh. Subcellular localization analyses in tobacco protoplasts demonstrated that the nucleus was the exclusive location of the MdGTL1a-GFP fusion protein. Exogenous salicylic acid treatment decreased MdGTL1a expression and resulted in higher starch content in apples, which was consistent with the fruit ripening. It is concluded that salicylic acid treatment could enhance apple storage quality by inhibiting starch degradation through a crucial transcription factor, MdGTL1a.

YafN-YafO toxin-antitoxin system contributes to stress resistance and virulence of avian pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC) is a major threat to the poultry industry, causing bloodstream and extraintestinal infections. Type II toxin-antitoxin (TA) systems are known to aid bacterial pathogens in adapting to stress, promoting persister cell formation, and enhancing virulence. While type II TA systems have been extensively studied in many pathogens, APEC-derived TAs have received limited attention. Our study focused on the YafN-YafO type II TA system in APEC O2 strain E058. Using bacterial two-hybrid and pull-down assays, we confirmed the interaction between YafN and YafO. Electrophoretic mobility shift assays (EMSA) and lacZ fusion reporter assays demonstrated that YafN negatively autoregulates its own operon. The deletion of yafNO resulted in a significant reduction in persister cell formation under antibiotic and environmental stress (P < 0.01). Moreover, the yafNO mutant showed a ∼3-fold reduction in survival within chicken macrophages and attenuated virulence in chicken infection models, with a 44-fold increase in LD50 and ∼80-fold reduction in bacterial loads in blood and tissues (P < 0.01). These results demonstrate that the YafN-YafO is an active type II TA system in APEC E058, contributing to both stress resistance and virulence. Targeting this system could offer a novel strategy for controlling APEC infections.

WRKY45 positively regulates salinity and osmotic stress responses in Arabidopsis.

Salt damage is a major issue that causes a decline in crop yield. WRKY transcription factors (TFs) extensively regulate plant biotic and abiotic stress responses, growth, and development. WRKY45 is crucial in regulating leaf senescence, low phosphorus responses, and cadmium stress response in Arabidopsis. However, the involvement of WRKY45 in salinity and osmotic stress responses is unclear. Here, we report that WRKY45 plays a vital role in responding to salinity and osmotic stress. NaCl and sorbitol treatments upregulate WRKY45 expression. Furthermore, the overexpression of WRKY45 (WRKY45-OXs) may enhance the tolerance of Arabidopsis to salinity and osmotic stress. Moreover, the root length, fresh weight, chlorophyll, and proline content were significantly higher in WRKY45-OXs than in the wide type (WT) Col-0 plants after salt or PEG treatment, whereas malondialdehyde and reactive oxygen species (ROS) levels were significantly lower than in the WT plants. Correspondingly, the overexpression of WRKY45 modulated the expression of stress-responsive genes. Dual luciferase assay and electrophoretic mobility shift assay further confirmed that WRKY45 can activate the promoter of RD29A by directly binding to specific W-box cis-acting elements. Overall, our experimental evidence suggesting that WRKY45 mainly acts as a key regulator coordinating the response to high salinity and osmotic stress through mechanisms dependent on ABA signaling along with enhanced antioxidant capacity.

Identification and characterization of the auxin-response factor family in moso bamboo reveals that PeARF41 negatively regulates second cell wall formation.

Auxin response factors (ARFs) are key transcriptional factors mediating the transcriptional of auxin-related genes that play crucial roles in a range of plant metabolic activities. The characteristics of 47 PeARFs, identified in moso bamboo and divided into three classes, were evaluated. Structural feature analysis showed that intron numbers ranged from 3 to 14, while Motif 1, 2, 7 and 10 were highly conserved, altogether forming DNA-binding and ARF domains. Analysis of RNA-seq from different tissues revealed that PeARFs showed tissue-specificity. Additionally, abundant hormone-response and stress-related elements were enriched in promoters of PeARFs, supporting the hypothesis that the expression of PeARFs was significantly activated or inhibited by ABA and cold treatments. Further, PeARF41 overexpression inhibited SCW formation by reducing hemicellulose, cellulose and lignin contents. Moreover, a co-expression network, containing 28 genes with PeARF41 at its core was predicted, and the results of yeast one hybridization (Y1H), electrophoretic mobility shift assay (EMSA) and dual-luciferase (Dul-LUC) assays showed that PeARF41 bound the PeSME1 promoter to inhibit its expression. We conclude that a 'PeARF41-PeSME1' regulatory cascade mediates SCW formation. Our findings provided a solid theoretical foundation for further research on the role of PeARFs.

Polysulfide and persulfide-mediated activation of the PERK-eIF2α-ATF4 pathway increases Sestrin2 expression and reduces methylglyoxal toxicity.

Unfolded protein response (UPR) is activated in cells under endoplasmic reticulum (ER) stress. One sensor protein involved in this response is PERK, which is activated through its redox-dependent oligomerization. Prolonged UPR activation is associated with the development and progression of various diseases, making it essential to understanding the redox regulation of PERK. Sulfane sulfur, such as polysulfides and persulfides, can modify the cysteine residues and regulate the function of various proteins. However, the regulatory mechanism and physiological effects of sulfane sulfur on the PERK-eIF2α-ATF4 pathway remain poorly understood. This study focuses on the persulfidation of PERK to elucidate the effects of polysulfides on the PERK-eIF2α-ATF4 pathway and investigate its cytoprotective mechanism. Here, we demonstrated that polysulfide treatment promoted the oligomerization of PERK and PTP1B in neuronal cells using western blotting under nonreducing conditions. We also observed that l-cysteine, a biological source of sulfane sulfur, promoted the oligomerization of PERK and the knockdown of CBS and 3-MST, two sulfane sulfur-producing enzymes, and reduced PERK oligomerization induced by l-cysteine treatment. Furthermore, the band shift assay and LC-MS/MS studies revealed that polysulfides and persulfides induce PTP1B and PERK persulfidation. Additionally, polysulfides promoted eIF2α phosphorylation and ATF4 accumulation in the nucleus, suggesting that polysulfides activate the PERK-eIF2α-ATF4 pathway in neuronal cells. Moreover, polysulfides protected neuronal cells from methylglyoxal-induced toxicity, and this protective effect was reduced when the expression of Sestrin2, regulated by ATF4 activity, was suppressed. This study identified a novel mechanism for the activation of the PERK-eIF2α-ATF4 pathway through persulfidation by polysulfides and persulfides. Interestingly, activation of this pathway overcame the toxicity of methylglyoxal in dependence on Sestrin2 expression. These findings deepen our understanding of neuronal diseases involving ER stress and UPR disturbance and may inspire new therapeutic strategies.

Maize ZmWRKY71 gene positively regulates drought tolerance through reactive oxygen species homeostasis.

Drought stress severely affects plant growth and yield. The plant-specific WRKY transcription factors play an important role in regulating the plant response to abiotic stresses. In this study, we identified a group I WRKY gene from maize, designated ZmWRKY71. Real-time quantitative reverse transcription-PCR analysis revealed that ZmWRKY71 was predominantly expressed in the roots and was induced by drought. ZmWRKY71 was localized in the nucleus and showed transcriptional activity in yeast. Heterologous overexpression of ZmWRKY71 improved drought tolerance in yeast and Arabidopsis. Compared with the wild type, the overexpression lines showed a higher survival rate under drought stress with reduced malondialdehyde content and elevated antioxidant enzyme activities. In contrast, mutation of ZmWRKY71 in maize leads to increased sensitivity to drought stress, reduced survival, elevated concentrations of reactive oxygen species, and increased malondialdehyde content. RNA-sequencing analysis revealed that the expression patterns of genes associated with translation, membrane, and oxidoreductase activity pathways were altered under drought stress. Yeast one-hybrid, dual-luciferase, and electrophoretic mobility shift assays confirmed that ZmWRKY71 was capable of directly binding to the W-box element in the promoter region of ZmPOD42 (Zm00001eb330550). Taken together, the results show that ZmWRKY71 positively regulates maize drought tolerance. This research enriches the drought tolerance gene pool for maize and provides a theoretical basis for maize drought tolerance breeding.

TaERFL1a enhances drought resilience through DHAR-mediated ASA-GSH biosynthesis in wheat.

Wheat is one of the important cereal crops around the world, but it often suffers from abiotic stresses, which threaten food security. Thus, it is critical to identify the genes that determine drought tolerance in wheat. AP2/ERFs are known to regulate drought stress in various crops. In this study, TaERFL1a-overexpressing wheat transgenic lines (TaERFL1a-OEs) were used to determine drought resilience mechanism. After 12d without watering, the growth phenotype of TaERFL1a-OEs was better than that of the wild type (WT), whose activities of superoxide dismutase and catalase, and contents of ascorbate acid (ASA) and glutathione (GSH) were significantly increased, while malondialdehyde content was significantly decreased. Transcriptome analysis revealed that 28,520 genes were differentially expressed between TaERFL1a-OEs and WT under drought condition. Further analysis found that these DEGs were involved in multiple stress-response processes, especially in the ASA-GSH pathway. qPCR revealed that the expression levels of GPX, DHAR, and MDHAR, which are suggested to be participated in ASA-GSH biosynthesis, were significantly up-regulated in TaERFL1a-OEs under drought stress, especially the DHAR gene. Moreover, dual-luciferase and luciferase complementation imaging revealed that TaERFL1a was more promoted DHAR transcription to a greater extent than other genes. Furthermore, yeast one-hybrid, electrophoretic mobility shift assay, and chromatin immunoprecipitation combined with qPCR revealed that TaERFL1a regulates DHAR expression by binding to the cis-element ERF in DHAR promoter and promotes the transcription of later in vivo and in vitro. Overall, our results provided molecular regulatory evidence for TaERFL1a in wheat drought stress and suggested candidate genes for improving drought-tolerant wheat breeding.

A transcription factor SlWRKY71 activated the H2S generating enzyme SlDCD1 enhancing the response to Pseudomonas syringae pv DC3000 in tomato leaves.

H2S is a well-known gaseous signaling molecule that plays important roles in plant response to biotic stresses. Pseudomonas syringae pv tomato (Pst) could cause enormous loss, while whether H2S could modulate plant defense against Pst is still unclear. By CRISPR/Cas9, the Sldcd1 gene editing mutant showed reduced endogenous H2S content and attenuated resistance, whereas treatment with exogenous H2S could enhance the resistance. A transcription factor, SlWRKY71, was screened and identified to promote the transcription of SlDCD1 via yeast one-hybrid, dual-luciferase reporter system, electrophoretic mobility shift assays, and transient overexpression. Here, it was found that exogenous H2S relieved the symptoms of bacterial speck disease in tomato leaves, conferring tolerance to Pst. DC3000, and the expression of the H2S-producing enzyme SlDCD1 was significantly induced. The Slwrky71 mutant also showed reduced defense in tomato leaves against Pst. DC3000, whereas SlWRKY71-OE tomato leaves showed increased tolerance. Transient overexpression of SlDCD1 in the context of Slwrky71 with exogenous H2S treatment has stronger resistance, and the overexpression of SlWRKY71 in the context of Sldcd1 showed relatively weak disease resistance, and with the addition of H2S enhanced the effect. Therefore, we concluded that SlWRKY71 could activate SlDCD1 expression and promote endogenous H2S production, thereby improving tomato leaves resistance to Pst. DC3000.

Autophagy inhibitor 3-methyladenine mitigates the severity of colitis in aged mice by inhibiting autophagy.

The elderly ulcerative colitis (UC) patients pose unique challenges due to their comorbidities, diminished functional capacity, and heightened risk of treatment-related complications. Thus, finding a safe and effective treatment for this age group is crucial. This study investigates the role of autophagy in the pathogenesis of UC in young and elderly patients, and explores the therapeutic potential and mechanisms of autophagy modulators in aged mice with dextran sulfate sodium (DSS)-induced colitis. Colonic biopsies were collected from young and old UC patients as well as comparable healthy subjects. Young (6-8 weeks) and aged (56 weeks) C57BL/6 mice were treated with DSS to induce acute colitis model. The autophagy inhibitor 3-methyladenine was administered intraperitoneally to aged DSS-induced mice. The autophagy activity was detected by the protein expressions of LC3B-II, p62 and ATG5 by western blot and immunohistochemistry. The levels of TNF -α, IL-6, CCL4, CXCL12 and CD86 were measured by qRT-PCR. The transcriptional activity of NF-κB was measured by electrophoretic mobility shift assay (EMSA). Increased autophagy activity was observed in aged DSS-induced mice. Treatment with 3-methyladenine suppressed autophagy in intestinal epithelial cells (IECs) and alleviated colitis severity. Additionally, 3-methyladenine reduced macrophage recruitment, decreased IL-6 levels, and inhibited NF-κB signaling, thereby mitigating inflammation. Significant differences in autophagy activity were identified between young and aged DSS-induced mice. These findings underscore the potential therapeutic benefits of autophagy inhibition in elderly UC patients.

A concept of natural genome reconstruction. Part 2. Effect of extracellular double-stranded DNA fragments on hematopoietic stem cells

In this part of the study, the first component of the concept of “natural genome reconstruction” is being proven. It was shown with mouse and human model organisms that CD34+ hematopoietic bone marrow progenitors take up fragments of extracellular double-stranded DNA through a natural mechanism. It is known that the process of internalization of extracellular DNA fragments involves glycocalyx structures, which include glycoproteins/protein glycans, glycosylphosphatidylinositol-anchored proteins and scavenger receptors. The bioinformatic analysis conducted indicates that the main surface marker proteins of hematopoietic stem cells belong to the indicated groups of factors and contain specific DNA binding sites, including a heparin-binding domain and clusters of positively charged amino acid residues. A direct interaction of CD34 and CD84 (SLAMF5) glycoproteins, markers of hematopoietic stem cells, with double-stranded DNA fragments was demonstrated using an electrophoretic mobility shift assay system. In cells negative for CD34, which also internalize fragments, concatemerization of the fragments delivered into the cell occurs. In this case, up to five oligonucleotide monomers containing 9 telomeric TTAGGG repeats are stitched together into one structure. Extracellular fragments delivered to hematopoietic stem cells initiate division of the original hematopoietic stem cell in such a way that one of the daughter cells becomes committed to terminal differentiation, and the second retains its low-differentiated status. After treatment of bone marrow cells with hDNAgr, the number of CD34+ cells in the colonies increases to 3 % (humans as the model organism). At the same time, treatment with hDNAgr induces proliferation of blood stem cells and their immediate descendants and stimulates colony formation (mouse, rat and humans as the model organisms). Most often, the granulocyte-macrophage lineage of hematopoiesis is activated as a result of processing extracellular double-stranded DNA. The commitment process is manifested by the appearance and repair of pangenomic single-strand breaks. The transition time in the direction of differentiation (the time it takes for pangenomic single-strand breaks to appear and to be repaired) is about 7 days. It is assumed that at the moment of initiation of pangenomic single-strand breaks, a “recombinogenic situation” ensues in the cell and molecular repair and recombination mechanisms are activated. In all experiments with individual molecules, recombinant human angiogenin was used as a comparison factor. In all other experiments, one of the experimental groups consisted of hematopoietic stem cells treated with angiogenin.

LACCASE35 Enhances Lignification and Resistance Against Pseudomonas syringae pv. actinidiae Infection in Kiwifruit.

Kiwifruit bacterial canker, a highly destructive disease caused by Pseudomonas syringae pv. actinidiae (Psa), seriously affects kiwifruit (Actinidia spp.) production. Lignin deposition in infected cells serves as a defense mechanism, effectively suppressing pathogen growth. However, the underlying process remains unclear. In this study, we determined that Psa infection leads to a significant increase in S-lignin accumulation in kiwifruit. The S/G ratio in lignin was higher in a Psa-resistant cultivar than in a Psa-sensitive cultivar. Furthermore, kiwifruit laccase 35 (AcLac35), encoding an enzyme in the lignin biosynthesis pathway with characteristic laccase activity, showed tissue-specific expression in plants and was upregulated following infection by Psa. Overexpressing AcLac35 in kiwifruit leaves resulted in greater lignin content than in wild-type leaves, leading to the formation of thicker cell walls, and also activated plant-pathogen interactions and MAPK pathways, thereby enhancing resistance against Psa infection. Yeast one-hybrid assays, dual-LUC reporter assays, electrophoretic mobility shift assays, and transient injection experiments indicated that SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 9 (AcSPL9) can bind to the AcLac35 promoter, thereby positively regulating its expression. Moreover, overexpression of AcSPL9 increased lignin accumulation in kiwifruit leaves, enhancing resistance to Psa, while virus-induced gene silencing of AcSPL9 expression reduced this resistance. Our findings reveal the function of AsSPL9-AcLac35 in kiwifruit, providing insight into enhancing resistance against Psa in kiwifruit.

MarR family regulator LcbR2 activates lincomycin biosynthesis in multiple ways.

Lincomycin, produced by the actinomycete Streptomyces lincolnensis, is highly effective against Gram-positive bacteria and protozoans, making it widely used in clinical settings. This study identified LcbR2, a MarR family transcriptional regulator, as an activator of lincomycin biosynthesis. Knocking out the lcbR2 gene reduced lincomycin production by 63.0 % without affecting growth or morphology. Quantitative real-time PCR, electrophoretic mobility shift assays, and XylE reporter assays demonstrated that LcbR2 binds to a 13-bp imperfect palindromic sequence -TTGCCnnnnnCAA-, repressing the expression of lcbR2 Further analysis revealed that LcbR2 directly activates the expression of lincomycin biosynthesis genes (lmbD, lmbJ, lmbK, lmbV, and lmbW), enhancing lincomycin production. It also regulates lincomycin resistance genes (lmrA and lmrB), increasing the self-tolerance of S. lincolnensis to lincomycin. Additionally, LcbR2 modulates other regulatory genes (lmbU, adpA, aflQ1, bldD, and lcbR1), affecting lincomycin production in a cascade manner. LcbR2 also influences the expression of genes related to carbon, nitrogen, phosphorus, and sulfur metabolism, indirectly impacting lincomycin production. Moreover, the binding of LcbR2 to DNA can be attenuated by apramycin. This study thus characterized LcbR2 as a novel transcriptional regulator with a broad regulatory scope.

The PbDELLA-PbMYB56-PbCYP78A6 Module Regulates GA4+7-Induced Pseudo-Embryo Development and Parthenocarpy in Pear (Pyrus bretschneideri)

Abstract Parthenocarpy can ensure fruit setting without fertilization and generate seedless fruits. PbCYP78A6 has been shown to play a role in gibberellin (GA)-induced parthenocarpy in pears. However, the transcriptional response mechanism of PbCYP78A6 to GA remains unclear. In this study, using a yeast one-hybrid assay combined with co-expression analysis, PbMYB56 was initially identified as a transcription regulator of PbCYP78A6, which was further confirmed by electrophoretic mobility shift assay (EMSA) and dual-luciferase reporter assays. The biofunction of PbMYB56 was further verified using transient transgene tests, stable transgenic pear callus and tomato. PbMYB56 overexpression resulted in reduced cell death and higher fluorescence intensity after fluoresce diacetate (FDA) staining, as well as delayed fruit-drop by increasing PbCYP78A6 expression in un-pollinated pear fruitlets and callus. In contrast, silencing PbMYB56 caused cell death and early fruit-drop with decreased PbCYP78A6 expression. Moreover, after emasculation, heterologous overexpression of PbMYB56 induced parthenocarpy and enlarged seed size in pollinated tomato fruits. Silencing SlMYB56, a homolog of PbMYB56 in tomatoes, resulted in smaller fruit and seed size, and these traits were restored by co-overexpression with PbCYP78A6. Furthermore, we investigated the protein interaction between PbMYB56 and PbDELLA, which is crucial component of the GA signaling pathway. This interaction inhibited PbMYB56-induced transcriptional activation of PbCYP78A6. Co-overexpression of PbMYB56 and PbDELLA contributed to reduced seed development and loss of parthenocarpy potential in tomatoes. Collectively, our study identifies PbDELLA-PbMYB56-PbCYP78A6 as a regulatory module of GA4+7-induced pseudo-embryo and parthenocarpy development, offering insights into the mechanism underlying parthenocarpy formation in pears.

ZmHB53, a Maize Homeodomain-Leucine Zipper I Transcription Factor Family Gene, Contributes to Abscisic Acid Sensitivity and Confers Seedling Drought Tolerance by Promoting the Activity of ZmPYL4.

Plant-specific homeodomain-leucine zipper I (HD-Zip I) transcription factors (TFs) crucially regulate plant drought tolerance. However, their specific roles in maize (Zea mays L.) regulating drought tolerance remain largely unreported. Here, we screened a maize HD-Zip I TF family gene, ZmHB53, and clarified its role in drought stress. ZmHB53 overexpression maize plants exhibited sensitivity to abscisic acid (ABA), tolerant to polyethylene glycol (PEG 6000)-induced stress during germination, along with improved seedling drought resistance. Compared to the wild-type, ZmHB53 overexpression lines show higher water retention, biomass, and survival rates, and reduced water loss and stomatal size under drought, suggesting ZmHB53's role in drought adaptation. DNA affinity purification sequencing (DAP-Seq), yeast one hybrid, electrophoretic mobility shift assay (EMSA), and dual luciferase showed that ZmHB53 directly bound to and upregulated the expression of ABA receptor ZmPYL4. Meanwhile, transgenic plants overexpressing ZmPYL4 also exhibit ABA sensitivity and drought tolerance. The research results provide novel insights into the regulatory role of ZmHB53 and ZmPYL4 in enhancing maize's drought tolerance, establishing a foundation for future validation and potential application of ZmHB53 in strategies to improve maize resistance to drought.

Functional analysis of the type II toxin-antitoxin system ParDE in Streptococcus suis serotype 2

Streptococcus suis (S. suis) is a major pathogen in swine and poses a potential zoonotic threat, which may cause serious diseases. Many toxin-antitoxin (TA) systems have been discovered in S. suis, but their functions have not yet been fully elucidated. In this study, an auto-regulating type II TA system, ParDE, was identified in S. suis serotype 2 strain ZY05719. We constructed a mutant strain, ΔparDE, to explore its functions in bacterial virulence, various stress responses, and biofilm formation capabilities. The toxicity exerted by the toxin ParE can be neutralized by the antitoxin ParD. The β-galactosidase activity analysis indicated that ParDE has an autoregulatory function. An electrophoretic mobility shift assay (EMSA) confirmed that the antitoxin ParD bound to the promoter of ParDE as dimers. In the mouse infection model, the deletion of ParDE in ZY05719 significantly attenuated virulence. ΔparDE also exhibited a reduced anti-oxidative stress ability, and ΔparDE was more susceptible to phagocytosis and killing by macrophages. Moreover, the biofilm formation ability of the ΔparDE strain was significantly enhanced compared to ZY05719. Taken together, these findings indicate that the type II TA system ParDE plays a significant role in the pathogenesis of S. suis, providing new insights into its pathogenic mechanisms.

NAA enhances armillaria gallica growth by modulating nitrogen metabolism through AgZFP48.

Armillaria gallica (A. gallica) is a fungus with both medicinal and edible properties. Previous transcriptome analysis has identified the C2H2-type zinc finger transcription factor as a candidate gene involved in the NAA-induced growth promotion of A. gallica. However, the molecular mechanism underlying the enhancement of A. gallica growth by C2H2 transcription factor in response to NAA treatment remains unclear. In this study, we identified a C2H2-type zinc finger transcription factor gene in A. gallica and investigated its function, aiming to elucidate the mechanism by which C2H2-type zinc finger transcription factors regulate the growth of A. gallica. We identified and characterized a novel C2H2-type zinc finger transcription factor, AgZFP48, in A. gallica and found that AgZFP48 is located in the nucleus, where it acts as a transcription activator. AgZFP48 positively regulated the growth of A. gallica. The potential targets of AgZFP48 were identified by using DNA affinity purification sequencing (DAP-seq). In addition, four candidate genes were selected for Electrophoretic Mobility Shift Assays (EMSA) and luciferase reporter activity assessment. The results showed that AgZFP48 activated the expression of ammonium transporter (AgAMT), glutamine synthetase (AgGS), acetylornithine aminotransferase (AgAcOAT), and amino acid permease (AgAAP) by binding to their promoters or exons. In summary, our results suggest that AgZFP48 promotes nitrogen metabolism in A. gallica by activating the expression of nitrogen metabolism-related genes, thereby regulating the growth of the fungus.

Susceptibility to pseudoexfoliation linked to intronic variant rs4926246 in CACNA1A: Evidence from an Indian population study.

Pseudoexfoliation (PEX) is an age-related, complex systemic disorder of protein aggregopathy. It is characterized by the extracellular fibril depositions, termed PEX fibrils, initially observed in various organ tissues during pseudoexfoliation syndrome (PEXS) and with significant prominence in the eye during advanced pseudoexfoliation glaucoma (PEXG). The study explores the association between CACNA1A (calcium channel, voltage-dependent, P/Q type, alpha 1A subunit) variants and PEX in an Indian population. The investigation involved genotyping one intronic single nucleotide polymorphism (SNP), rs4926244, and three tag SNPs using the Sanger and TaqMan genotyping approaches in a cohort of 300 controls and 300 PEX patients (including 200 PEXS and 100 PEXG cases). Findings from the present study revealed a significant association at both allelic and genotypic levels for rs4926246, whereas rs4926244 showed association only at the genotypic level with PEX. Functional assays demonstrated increased mRNA expression linked to the risk genotype of both variants and luciferase reporter assays indicated an allele-specific regulatory effect of rs4926246.While in silico analysis predicted potential transcription factor binding sites for c-Myc and Hypoxia-inducing factor-1 (HIF-1) at the rs4926246 locus, electrophoretic mobility shift assay (EMSA) validated that only the "T" variant showed the reduced binding affinity with c-Myc compared to the protective variant "C". Our study identifies rs4926246, an intronic variant strongly associated with both PEXS and PEXG, potentially influencing gene expression and protein binding, warranting further investigation into its role in PEX pathogenesis.