Search Results (141)

The innate immune response is an important defense against invading pathogens. Stimulator of interferon gene (STING) plays an important role in the cyclic GMP-AMP synthase (cGAS)-mediated activation of type I IFN responses. However, some viruses have evolved the ability to inhibit the function of STING and evade the host antiviral defenses. Understanding both the mechanism of action and the viruses targets of STING effector is important because of their importance to evade the host antiviral defenses. In this study, the STING (IpSTING) of Ictalurus punctatus was first identified and characterized. Subsequently, the yeast two-hybrid system (Y2HS) was used to screen for proteins from channel catfish virus (CCV, Ictalurid herpesvirus 1) that interact with IpSTING. The ORFs of the CCV were cloned into the pGBKT7 vector and expressed in the AH109 yeast strain. The bait protein expression was validated by autoactivation, and toxicity investigation compared with control (AH109 yeast strain transformed with empty pGBKT7 and pGADT7 vector). Two positive candidate proteins, ORF41 and ORF65, were identified through Y2HS screening as interacting with IpSTING. Their interactions were further validated using co-immunoprecipitation (Co-IP). This represented the first identification of interactions between IpSTING and the CCV proteins ORF41 and ORF65. The data advanced our understanding of the functions of ORF41 and ORF65 and suggested that they might contribute to the evasion of host antiviral defenses. However, the interaction mechanism between IpSTING, and CCV proteins ORF41 and ORF65 still needs to be further explored. Full article

Chaperonin 60 proteins plays an important role in plant growth and development as well as the response to abiotic stress. As part of the protein homeostasis system, molecular chaperones have attracted increasing attention in recent years due to their involvement in the folding and assembly of key proteins in photosynthesis. However, little is known about the function of maize chaperonin 60 protein. In the study, a gene encoding the chaperonin 60 proteins was cloned from the maize inbred line B73, and named ZmCpn60-3. The gene was 1, 818 bp in length and encoded a protein consisting of 605 amino acids. Phylogenetic analysis showed that ZmCpn60-3 had high similarity with OsCPN60-1, belonging to the β subunits of the chloroplast chaperonin 60 protein family, and it was predicted to be localized in chloroplasts. The ZmCpn60-3 was highly expressed in the stems and tassels of maize, and could be induced by exogenous plant hormones, mycotoxins, and pathogens; Overexpression of ZmCpn60-3 in Arabidopsis improved the resistance to Pst DC3000 by inducing the hypersensitive response and the expression of SA signaling-related genes, and the H₂O₂ and the SA contents of ZmCpn60-3-overexpressing Arabidopsis infected with Pst DC3000 accumulated significantly when compared to the wild-type controls. Experimental data demonstrate that flg22 treatment significantly upregulated transcriptional levels of the PR1 defense gene in ZmCpn60-3-transfected maize protoplasts. Notably, the enhanced resistance phenotype against Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) in ZmCpn60-3-overexpressing transgenic lines was specifically abolished by pretreatment with ABT, a salicylic acid (SA) biosynthetic inhibitor. Our integrated findings reveal that this chaperonin protein orchestrates plant immune responses through a dual mechanism: triggering a reactive oxygen species (ROS) burst while simultaneously activating SA-mediated signaling cascades, thereby synergistically enhancing host disease resistance. Additionally, yeast two-hybrid assay preliminary data indicated that ZmCpn60-3 might bind to ZmbHLH118 and ZmBURP7, indicating ZmCpn60-3 might be involved in plant abiotic responses. The results provided a reference for comprehensively understanding the resistance mechanism of ZmCpn60-3 in plant responses to abiotic or biotic stress. Full article

E2F transcription factors regulate cell cycle progression by influencing the expression of proteins required for the G₁-S phase transition and DNA synthesis with its heterodimeric partners (DP1 or DP2). The dimerization domain is the E2Fs and DP1 protein interaction interface and is believed to function in protein dimerization. In this study, eight E2F transcription factors (PcE2F1–8) of large yellow croaker Pseudosciaena crocea and one dimerization partner (PcDP1) are identified in the genome of large yellow croakers. The prediction of E2Fs conserved domains revealed that PcE2F1–6 has one DNA-binding domain (DBD) and one dimerization-binding domain (DD), while PcE2F7–8 only possess two duplicate DBDs but not DD, indicating that E2F7–8 cannot form the E2F/DP1 heterodimer. To explore whether PcDP1 is a partner of PcE2F1–6, the ORF of PcE2F1–6 was cloned. Subsequently, its sequence characteristics, the expression pattern in healthy fish, and subcellular co-localization were analyzed, and an interaction between PcDP1 and PcE2F1–6 were detected directly by yeast two-hybrid and BiFC. The PcE2F1, PcE2F2, PcE2F3, PcE2F4, PcE2F5, and PcE2F6 genes encode a protein of 454, 448, 444, 392, 362, and 396 amino acids, respectively, with accession numbers QFZ93593.1, QFZ93594.1, QFZ93595.1, QFZ93596.1, QFZ93597.1, and QFZ93598.1, respectively. Sequence characteristics analysis found that PcE2F1–5 but not PcE2F6 proteins share the pocket protein-binding domain sequestering in dimerization domains and transactivation domains. The PcE2F1,2,4 proteins possess one nuclear localization signal (NLS), and PcE2F3 protein possess two NLSs, but there is no NLS in PcE2F5 and 6 protein. Moreover, PcE2F4 also contains one NES. However, PcE2F1–6 proteins were all located in nucleus by using Euk-mPloc 2.0 programs and were confirmed by performing the Cherry and EGFP reporter assay. Regarding co-expression of DP1, only E2F4 can transfer DP1’s subcellular location from cytoplasm to the nucleus. RT-qPCR analysis indicated that PcE2F1–6 are constitutively and tissue specifically expressed in all of the tissues tested of a healthy large yellow croaker. The PcE2F1–6, except for PcE2F3, mRNA levels were all detected higher in the liver. PcE2F1–4 were also highly specifically expressed in the kidney, PcE2F4,6 in the brain, and PcE2F5 in the spleen of a healthy large yellow croaker, respectively. Using a yeast two-hybrid system, PcE2F4 interacting with PcDP1 was identified. The interaction between PcE2F4 and PcDP1 was further confirmed by a bimolecular fluorescence complementation (BiFC) assay. Collectively, these results indicate that an interaction between PcE2F4 and PcDP1 was detected, which may form heterodimer E2F4/DP1 to regulate cell cycles and immune-related pathways in large yellow croakers. Full article

The areca palm (Areca catechu L.), a medicinal tropical crop, hosts three novel viruses, areca palm necrotic ringspot virus (ANRSV), areca palm necrotic spindle-spot virus (ANSSV), and ANRSV2, which form a new genus Arepavirus in the family Potyviridae. Both viruses feature a unique tandem leader protease arrangement (HCPro1-HCPro2). To elucidate HCPro2’s role, this study identified its interaction partners in infected cells using affinity purification coupled with liquid chromatography-tandem mass spectrometry, a yeast two-hybrid system, and co-immunoprecipitation. Thirteen host proteins and five viral factors (HCPro1, 6K2, VPg, NIa-Pro, NIb) were validated as HCPro2 interactors. Among the host proteins interacting with HCPro2, the expression of five genes (NbeIF4A, NbSAMS1α, NbTEF1α, NbUEP1, and NbRan2) was upregulated under the condition of viral infection, while the expression of another five genes (NbpsbS1, NbPGK, NbchIP, NbClpC1A, and NbCysPrx) was downregulated. Functional assays showed that silencing NbeIF4A or NbPGK significantly reduced viral accumulation in Nicotiana benthamiana. These findings reveal HCPro2’s network of virus-host interaction, highlighting its critical role in viral pathogenesis. Further exploration of these interactions may clarify the evolutionary significance of tandem leader proteases and inform novel plant antiviral strategies. Full article

Indolic glucosinolates are a group of plant secondary metabolites found in Brassica vegetables, and their breakdown products could act as important anti-cancer and defense compounds against biotic stresses. Transcriptional regulation plays a key role in modulating the biosynthesis of indolic glucosinolates in the model plant Arabidopsis, but little is known about the transcriptional regulatory landscape of these glucosinolates in Brassica vegetables. In this study, we selected and functionally validated the important biosynthetic gene BolCYP83B1 from the indolic glucosinolate pathway in cabbage. Through a yeast one-hybrid assay, we systemically screened and identified upstream regulators of BolCYP83B1 in cabbage with BolANTs as the top candidates for further functional validation. Two homologs of BolANTs, BolANT1 and BolANT3, were confirmed to bind the promoter of BolCYP83B1 via both a yeast one-hybrid assay and an LUC assay. The overexpression of BolANT3 in cabbage significantly increased the accumulation of indolic glucosinolates, while the virus-induced gene silencing (VIGS) of BolANT3 significantly reduced the accumulation of indolic glucosinolates in cabbage. Our work provides valuable insights into the transcriptional regulatory mechanisms of indolic glucosinolates in Brassica vegetables. Full article

Background: Rice bacterial leaf blight, caused by the Gram-negative bacterium Xanthomonas oryzae pv. Oryzae (Xoo), significantly impacts rice production. To address this disease, research efforts have focused on discovering and utilizing novel disease-resistant genes and examining their functional mechanisms. Methods and Results: In this study, a variety of bacterial strains were utilized. CX28-3, AX-11, JC12-2, and X10 were isolated from the high-altitude japonica rice-growing region on the Yunnan Plateau. Additionally, PXO61, PXO86, PXO99, and PXO339, sourced from the International Rice Research Institute (IRRI), were included in the analysis. To evaluate the resistance characteristics of Haonuoyang, artificial leaf cutting and inoculation methods were applied. Results indicated that Haonuoyang exhibited broad-spectrum resistance. Additionally, to explore the genetic mechanisms of resistance, the TFAIII-type transcription factor OsZFPH was cloned from Haonuoyang using PCR amplification. The subcellular localization method identified the precise location of the OsZFPH gene within the cell. The expression of OsZFPH was induced by Xoo stress. The overexpression of OsZFPH resulted in increased activities of enzymes, including SOD, CAT, and POD, while silencing the gene led to reduced enzyme activities. Furthermore, the hormones SA (salicylic acid), JA (jasmonic acid), and GA (gibberellin) were shown to positively regulate the gene expression. Protein interactions with OsZFPH were verified through a yeast two-hybrid system and BiFC technology. Hap5, which aligned with the sequence of Haonuoyang, was found to belong to a haplotype consisting of Jingang 30, 40 resequenced rice varieties, 18 Oryza rufipogon, and 29 Oryza granulata. Conclusions: The findings of this study emphasize the vital role of OsZFPH in rice resistance to bacterial leaf blight. The identification of broad-spectrum resistance in Haonuoyang and the understanding of OsZFPH gene functions provide valuable insights for the future development of rice varieties with improved resistance to this destructive disease. Full article

Microalgae possess the remarkable ability to autotrophically grow, utilizing atmospheric carbon dioxide (CO₂) for photosynthesis, thereby converting solar energy into chemical energy and releasing oxygen. This capacity makes them an effective tool for mitigating industrial CO₂ emissions. Mathematical models are crucial for predicting microalgal growth kinetics and thus assessing their potential as industrial CO₂ sequestration agents under controlled conditions. This study innovatively evaluated the effect of continuously supplying CO₂ from winemaking processes on microalgal cultivation and biomass production, demonstrating a novel approach to both carbon capture and the valorization of a valuable by-product. To analyze microalgal growth kinetics, three mathematical models were employed: Logistic, First Order Plus Dead Time, and Second Order Plus Dead Time. Optimal parameter values for each model were identified using a hybrid search algorithm developed by our research group. First, an integrated microvinification system was established, utilizing two microalgae species, Chlorella spp. (FAUBA-17) and Desmodesmus spinosus (FAUBA-4), in conjunction with yeast fermenters. This system facilitated a comparison of the biomass kinetics of these two microalgae species, selecting Chlorella spp. (FAUBA-17) as the most suitable candidate for subsequent cultivation. A pilot-scale vertical column photobioreactor was then constructed and installed at the Casimiro Wines boutique winery in Angaco, San Juan, Argentina. After 15 days of operation within the photobioreactor, a biomass growth of 1.04 ± 0.05 g/L and 1.07 ± 0.1 g/L was obtained in Photobioreactors 1 and 2, respectively. This novel integrated approach to CO₂ capture in the winemaking process is unprecedented. These findings highlight the potential for producing high-value microalgal biomass, promoting the establishment of a local biorefinery and fostering a circular economy and sustainable social development. Full article

The recent coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has exerted considerable impact on global health. To prepare for rapidly mutating viruses and for the forthcoming pandemic, effective therapies targeting the critical stages of the viral life cycle need to be developed. Viruses are dependent on the interaction between the receptor-binding domain (RBD) of the viral Spike (S) protein (S-RBD) and the angiotensin-converting enzyme 2 (ACE2) receptor to efficiently establish infection and the following replicate. Targeting this interaction provides a promising strategy to inhibit the entry process of the virus, which in turn has both preventive and therapeutic effects. In this study, we developed a robust and straightforward assay based on the Yeast-Two Hybrid system (Y2H) for identifying inhibitors targeting the S-RBD-ACE2 interaction of SARS-CoV-2. Through high-throughput screening, two compounds were identified as potential entry inhibitors. Among them, IMB-1C was superior in terms of pseudovirus entry inhibition and toxicity. It could bind to both ACE2 and S-RBD and induce conformational change in the S-RBD+ACE2 complex. This is the first study to verify the feasibility of utilizing the Y2H system to discover potent SARS-CoV-2 inhibitors targeting the receptor recognition stage. This approach may also be applied in the discovery of other virus receptor recognition inhibitors. Full article

The ubiquitin-proteasome system (UPS) is a key protein degradation pathway in eukaryotes, in which E3 ubiquitin ligases mediate protein ubiquitination, directly or indirectly targeting substrate proteins to regulate various biological processes, including plant growth, hormone signaling, immune responses, and adaptation to abiotic stress. In this study, we identified plant U-box protein 51 in Solanum tuberosum (StPUB51) as an E3 ubiquitin ligase through transcriptomic analysis, and used it as a candidate gene for gene-function analysis. Quantitative real-time PCR (qRT-PCR) was used to examine StPUB51 expression across different tissues, and its expression patterns under simulated drought stress induced by polyethylene glycol (PEG 6000) were assessed. Transgenic plants overexpressing StPUB51 and plants with down-regulated StPUB51 expression were generated to evaluate drought tolerance. The activities of key antioxidant enzymes-superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) as well as malondialdehyde (MDA) content in transgenic plants’ leaves were measured under drought conditions. Protein–protein interactions involving StPUB51 were explored via yeast two-hybrid (Y2H) screening, with interaction verification by bimolecular fluorescence complementation (BiFC). StPUB51 was predominantly expressed in stems, with lower expression observed in tubers, and its expression was significantly upregulated in response to 20% PEG-6000 simulated drought. Subcellular localization assays revealed nuclear localization of the StPUB51 protein. Under drought stress, StPUB51-overexpressing plants exhibited enhanced SOD, POD, and CAT activities and reduced MDA levels, in contrast to plants with suppressed StPUB51 expression. Y2H and BiFC analyses identified two interacting proteins, StSKP2A and StGATA1, which may be functionally linked to StPUB51. Collectively, these findings suggest that StPUB51 plays a positive regulatory role in drought tolerance, enhancing resilience in potato growth and stress adaptation. Full article

The IV subfamily of receptor-like cytoplasmic kinase (RLCK-IV), known as calcium-binding receptor-like cytoplasmic kinases (CRCKs), plays a vital role in plant signal transduction, particularly in coordinating growth and responses to abiotic stresses. However, our comprehension of CRCK genes in Populus deltoides, a species characterized as fast-growing and pest-resistant but with drought intolerance, is limited. Here, we identify 6 members of the CRCK subfamily on a genome-wide scale in P. deltoides, denoted as PdeCRCK1–PdeCRCK6. An evolutionary and structural analysis revealed highly conserved kinase catalytic domains across all PdeCRCKs, characterized by calmodulin (CaM)-binding sites and serine (Ser)/threonine (Thr) phosphorylation sites. The cis-acting elements of promoters indicated the presence of responsive elements for plant hormones, abiotic stresses, and transcription factor binding sites, which is supported by the distinct transcriptional expression patterns of PdeCRCKs under abscisic acid (ABA), polyethylene glycol (PEG), and mannitol treatments. A transient overexpression of PdeCRCK3/5/6 in tobacco (Nicotiana benthamiana) leaves indicated their involvement in reactive oxygen species (ROS) scavenging, polyamine gene synthesis, and ABA signaling pathway modulation. Immunoprecipitation–Mass Spectrometry (IP–MS) and a yeast two-hybrid (Y2H) assay showed that PdeCRCK6 interacted with AAA-type ATPase proteins and ubiquitin, suggesting its potential function in being involved in chloroplast homeostasis and the 26S ubiquitin protease system. Taken together, these findings offer a comprehensive analysis of the RLCK-IV subfamily members in P. deltoides, especially laying a foundation for revealing the potential mechanism of PdeCRCK6 in response to osmotic stresses and accelerating the molecular design breeding of drought tolerance in poplar. Full article

The extremely rapid development of understanding and technology that led to the containment of the COVID-19 pandemic resulted from collaborative efforts in the fields of Betacoronavirus pandemicum (SARS-CoV-2) biology, pharmacology, vaccinology, and medicine. Perhaps surprisingly, much of the research was conducted using simple and efficient yeast models. In this manuscript, we describe how yeast, eukaryotic microorganisms, have been used to research this global challenge, focusing on the therapeutic potential of the studies discussed herein. Thus, we outline the role of yeast in studying viral protein interactions with the host cell proteome, including the binding of the SARS-CoV-2 virus spike protein to the human ACE2 receptor and its modulation. The production and exploration of viral antigens in yeast systems, which led to the development of two approved COVID-19 vaccines, are also detailed. Moreover, yeast platforms facilitating the discovery and production of single-domain antibodies (nanobodies) against SARS-CoV-2 are described. Methods guiding modern and efficient drug discovery are explained at length. In particular, we focus on studies designed to search for inhibitors of the main protease (Mpro), a unique target for anti-coronaviral therapies. We highlight the adaptability of the techniques used, providing opportunities for rapid modification and implementation alongside the evolution of the SARS-CoV-2 virus. Approaches introduced in yeast systems that may have universal potential application in studies of emerging viral diseases are also described. Full article

Cytoplasmic male sterile (CMS) systems have great potential in hybrid seed production in many plants. However, the incompatibility between the cytoplasmic and nuclear genes and the availability of fewer CMS lines limit the system’s application in cotton heterosis. Therefore, the present study sequenced the mitochondrial (mt) genomes of a novel cotton (Gossypium hirsutum L.) CMS line J4A and its cytoplasmic nuclear homologous maintainer line J4B to investigate the mechanism underlying CMS and improve its application. A novel CMS gene, orf116b, was identified in the CMS line. Ectopic expression of orf116b in Escherichia coli suppressed growth, while its overexpression in Arabidopsis, rice, tobacco, and cotton led to complete or partial male sterility. Further analysis of anthers revealed mt disruption in J4A with higher levels of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA), but lower levels of ATP and ribosomal protein (RP) than in J4B. Finally, a membrane-based yeast two-hybrid (MYTH) assay and bimolecular fluorescence complementation (BiFC) assays demonstrated that orf116b probably interacts with an anther-specific protein, LAT52. These observations collectively proved that orf116b is associated with early and stable pollen abortion in cotton, providing a foundation for further research on cotton fertility restoration and heterosis breeding. Full article

Somatic embryogenesis is valuable for clonal propagation and genetic improvement, and it also serves as an ideal system for studying plant development mechanisms. In Larix kaempferi, microRNA171 and its target gene L. kaempferi SCARECROW-LIKE6 (LaSCL6), which has two alternative splicing variants, can regulate somatic embryogenesis; however, the underlying molecular mechanism is still unknown. In this study, we overexpressed these two LaSCL6 variants in Oryza sativa and Arabidopsis thaliana and then used the RNA-Seq method to screen genes from O. sativa and A. thaliana, whose expression patterns are related to those of LaSCL6 variants. The screened genes were then used to search L. kaempferi proteins to identify the candidate target genes of LaSCL6. After yeast one-hybrid and dual- luciferase transcriptional activity assays, cytochrome P450, family 89, subfamily A, polypeptide 5 (CYP89A5), and wall-associated receptor kinase-like 20 (WAKL20) were confirmed to be the target genes of LaSCL6-var1; in addition, WAKL20 and UDP-glycosyltransferase 85A3 (UGT85A3) were confirmed to be the target genes of LaSCL6-var2. Moreover, APETALA2-like protein 2, a transcription factor from the AP2/ERF family, was shown to interact with LaSCL6-var1 and LaSCL6-var2. Taken together, our results suggest a regulatory network of miR171-LaSCL6. The findings presented here not only provide novel insights into the regulation of the miR171-LaSCL6 module but also explain the mechanism underlying larch somatic embryogenesis and other biological processes. Full article

The transcription factor E2F1 is the principal target of the tumor suppressor pRB. E2F1 promotes cell proliferation by activating growth-promoting genes upon growth stimulation. In contrast, E2F1 contributes to tumor suppression by activating tumor suppressor genes, such as ARF, upon loss of pRB function, a major oncogenic change. The transactivation domain of E2F1 has previously been mapped to the C-terminal region. We show here that the N-terminal region of E2F1 is critical for the activation of tumor suppressor genes. Deletion of the N-terminal region dramatically compromised E2F1 activation of tumor suppressor genes. The N-terminal region showed transactivation ability when fused to the DNA-binding domain of GAL4. A search for novel interacting factors with the N-terminal region, using a yeast two-hybrid system, identified the general transcription factor GTF2H2. Overexpression of GTF2H2 enhanced E2F1 activation of tumor suppressor genes and induction of cell death. Conversely, the knockdown of GTF2H2 compromised both. E2F1 binding enhanced the binding of GTF2H2 to target promoters depending on the integrity of the N-terminal region. Taken together, these results suggest that the N-terminal region of E2F1 contains a novel transactivation domain that mediates the activation of tumor suppressor genes, at least in part, by recruiting GTF2H2. Full article

Pepper (Capsicum annuum L.) suffers severe quality and yield loss from oomycete diseases caused by Phytophthora capsici. CaSGT1 was previously determined to positively regulate the immune response of pepper plants against P. capsici, but by which mechanism remains elusive. In the present study, the potential interacting proteins of CaSGT1 were isolated from pepper using a yeast two-hybrid system, among which CaARP1 was determined to interact with CaSGT1 via bimolecular fluorescence complementation (BiFC) and microscale thermophoresis (MST) assays. CaARP1 belongs to the auxin-repressed protein family, which is well-known to function in modulating plant growth. The transcriptional and protein levels of CaARP1 were both significantly induced by infection with P. capsici. Silencing of CaARP1 promotes the vegetative growth of pepper plants and attenuates its disease resistance to P. capsici, as well as compromising the hypersensitive response-like cell death in pepper leaves induced by PcINF1, a well-characterized typical PAMP from P. capsici. Chitin-induced transient expression of CaARP1 in pepper leaves enhanced its disease resistance to P. capsici, which is amplified by CaSGT1 co-expression as a positive regulator. Taken together, our result revealed that CaARP1 plays a dual role in the pepper, negatively regulating the vegetative growth and positively regulating plant immunity against P. capsici in a manner associated with CaSGT1. Full article