Mechanistic insights into the interactions of TAX1BP1 with RB1CC1 and mammalian ATG8 family proteins.

TAX1BP1, a multifunctional autophagy adaptor, plays critical roles in different autophagy processes. As an autophagy receptor, TAX1BP1 can interact with RB1CC1, NAP1, and mammalian ATG8 family proteins to drive selective autophagy for relevant substrates. However, the mechanistic bases underpinning the specific interactions of TAX1BP1 with RB1CC1 and mammalian ATG8 family proteins remain elusive. Here, we find that there are two distinct binding sites between TAX1BP1 and RB1CC1. In addition to the previously reported TAX1BP1 SKICH (skeletal muscle and kidney enriched inositol phosphatase (SKIP) carboxyl homology)/RB1CC1 coiled-coil interaction, the first coiled-coil domain of TAX1BP1 can directly bind to the extreme C-terminal coiled-coil and Claw region of RB1CC1. We determine the crystal structure of the TAX1BP1 SKICH/RB1CC1 coiled-coil complex and unravel the detailed binding mechanism of TAX1BP1 SKICH with RB1CC1. Moreover, we demonstrate that RB1CC1 and NAP1 are competitive in binding to the TAX1BP1 SKICH domain, but the presence of NAP1's FIP200-interacting region (FIR) motif can stabilize the ternary TAX1BP1/NAP1/RB1CC1 complex formation. Finally, we elucidate the molecular mechanism governing the selective interactions of TAX1BP1 with ATG8 family members by solving the structure of GABARAP in complex with the non-canonical LIR (LC3-interacting region) motif of TAX1BP1, which unveils a unique binding mode between LIR and ATG8 family protein. Collectively, our findings provide mechanistic insights into the interactions of TAX1BP1 with RB1CC1 and mammalian ATG8 family proteins and are valuable for further understanding the working mode and function of TAX1BP1 in autophagy.

Mechanistic insights into the subversion of the linear ubiquitin chain assembly complex by the E3 ligase IpaH1.4 of Shigella flexneri.

Shigella flexneri, a gram-negative bacterium, is the major culprit of bacterial shigellosis and causes a large number of human infection cases and deaths worldwide annually. For evading the host immune response during infection, S. flexneri secrets two highly similar E3 ligases, IpaH1.4 and IpaH2.5, to subvert the linear ubiquitin chain assembly complex (LUBAC) of host cells, which is composed of HOIP, HOIL-1L, and SHARPIN. However, the detailed molecular mechanism underpinning the subversion of the LUBAC by IpaH1.4/2.5 remains elusive. Here, we demonstrated that IpaH1.4 can specifically recognize HOIP and HOIL-1L through its leucine-rich repeat (LRR) domain by binding to the HOIP RING1 domain and HOIL-1L ubiquitin-like (UBL) domain, respectively. The determined crystal structures of IpaH1.4 LRR/HOIP RING1, IpaH1.4 LRR/HOIL-1L UBL, and HOIP RING1/UBE2L3 complexes not only elucidate the binding mechanisms of IpaH1.4 with HOIP and HOIL-1L but also unveil that the recognition of HOIP by IpaH1.4 can inhibit the E2 binding of HOIP. Furthermore, we demonstrated that the interaction of IpaH1.4 LRR with HOIP RING1 or HOIL-1L UBL is essential for the ubiquitination of HOIP or HOIL-1L in vitro as well as the suppression of NF-κB activation by IpaH1.4 in cells. In summary, our work elucidated that in addition to inducing the proteasomal degradation of LUBAC, IpaH1.4 can also inhibit the E3 activity of LUBAC by blocking its E2 loading and/or disturbing its stability, thereby providing a paradigm showing how a bacterial E3 ligase adopts multiple tactics to subvert the key LUBAC of host cells.

Promoter variations of ClERF1 gene determines flesh firmness in watermelon.

BACKGROUND: Flesh firmness is a critical factor that influences fruit storability, shelf-life and consumer's preference as well. However, less is known about the key genetic factors that are associated with flesh firmness in fresh fruits like watermelon. RESULTS: In this study, through bulk segregant analysis (BSA-seq), we identified a quantitative trait locus (QTL) that influenced variations in flesh firmness among recombinant inbred lines (RIL) developed from cross between the Citrullus mucosospermus accession ZJU152 with hard-flesh and Citrullus lanatus accession ZJU163 with soft-flesh. Fine mapping and sequence variations analyses revealed that ethylene-responsive factor 1 (ClERF1) was the most likely candidate gene for watermelon flesh firmness. Furthermore, several variations existed in the promoter region between ClERF1 of two parents, and significantly higher expressions of ClERF1 were found in hard-flesh ZJU152 compared with soft-flesh ZJU163 at key developmental stages. DUAL-LUC and GUS assays suggested much stronger promoter activity in ZJU152 over ZJU163. In addition, the kompetitive allele-specific PCR (KASP) genotyping datasets of RIL populations and germplasm accessions further supported ClERF1 as a possible candidate gene for fruit flesh firmness variability and the hard-flesh genotype might only exist in wild species C. mucosospermus. Through yeast one-hybrid (Y1H) and dual luciferase assay, we found that ClERF1 could directly bind to the promoters of auxin-responsive protein (ClAux/IAA) and exostosin family protein (ClEXT) and positively regulated their expressions influencing fruit ripening and cell wall biosynthesis. CONCLUSIONS: Our results indicate that ClERF1 encoding an ethylene-responsive factor 1 is associated with flesh firmness in watermelon and provide mechanistic insight into the regulation of flesh firmness, and the ClERF1 gene is potentially applicable to the molecular improvement of fruit-flesh firmness by design breeding.

Editing of ORF138 restores fertility of Ogura cytoplasmic male sterile broccoli via mitoTALENs.

Cytoplasmic male sterility (CMS), encoded by the mitochondrial open reading frames (ORFs), has long been used to economically produce crop hybrids. However, the utilization of CMS also hinders the exploitation of sterility and fertility variation in the absence of a restorer line, which in turn narrows the genetic background and reduces biodiversity. Here, we used a mitochondrial targeted transcription activator-like effector nuclease (mitoTALENs) to knock out ORF138 from the Ogura CMS broccoli hybrid. The knockout was confirmed by the amplification and re-sequencing read mapping to the mitochondrial genome. As a result, knockout of ORF138 restored the fertility of the CMS hybrid, and simultaneously manifested a cold-sensitive male sterility. ORF138 depletion is stably inherited to the next generation, allowing for direct use in the breeding process. In addition, we proposed a highly reliable and cost-effective toolkit to accelerate the life cycle of fertile lines from CMS-derived broccoli hybrids. By applying the k-mean clustering and interaction network analysis, we identified the central gene networks involved in the fertility restoration and cold-sensitive male sterility. Our study enables mitochondrial genome editing via mitoTALENs in Brassicaceae vegetable crops and provides evidence that the sex production machinery and its temperature-responsive ability are regulated by the mitochondria.

Prevalence and impact of viral myocarditis in patients with severe fever with thrombocytopenia syndrome.

To explore the association and impact between viral myocarditis and mortality in patients with severe fever with thrombocytopenia syndrome. A dynamic analysis was conducted between fatal group and nonfatal group regarding the daily epidemiology data, clinical symptoms, and electrocardiogram (ECG), echocardiogram, and laboratory findings. Outcomes of patients with and without viral myocarditis were compared. The association between viral myocarditis and mortality was analyzed. Among 183 severe fever with thrombocytopenia syndrome patients, 32 were in the fatal group and 151 in the nonfatal group; there were 26 (81.25%) with viral myocarditis in the fatal group, 66 (43.70%) with viral myocarditis in the nonfatal group (p < 0.001), 79.35% of patients had abnormal ECG results. The abnormal rate of ECG in the fatal group was 100%, and in the nonfatal group was 74.83%. Univariate analysis found that the number of risk factors gradually increased on Day 7 of the disease course and reached the peak on Day 10. Combined with the dynamic analysis of the disease course, alanine aminotransferase, aspartate aminotransferase, creatine kinase, creatine kinase fraction, lactate dehydrogenase, hydroxybutyrate dehydrogenase, neutrophil count, serum creatinine, Na, Ca, carbon dioxide combining power, amylase, lipase, activated partial thromboplastin time and thrombin time had statistically significant impact on prognosis. The incidence of fever with thrombocytopenia syndrome combined with viral myocarditis is high, especially in the fatal group of patients. Viral myocarditis is closely related to prognosis and is an early risk factor. The time point for changes in myocarditis is Day 7 of the course of the disease.

The coiled-coil protein gene WPRb confers recessive resistance to Cucumber green mottle mosaic virus.

Cucumber green mottle mosaic virus (CGMMV) is one of the major global quarantine viruses and causes severe symptoms in Cucurbit crops, particularly with regard to fruit decay. However, the genetic mechanisms that control plant resistance to CGMMV have yet to be elucidated. Here, we found that WPRb, a weak chloroplast movement under blue light 1 and plastid movement impaired 2-related protein family gene, is recessively associated with CGMMV resistance in watermelon (Citrullus lanatus). We developed a reproducible marker based on a single non-synonymous substitution (G1282A) in WPRb, which can be used for marker-assisted selection for CGMMV resistance in watermelon. Editing of WPRb conferred greater tolerance to CGMMV. We found WPRb targets to the plasmodesmata (PD) and biochemically interacts with the CGMMV movement protein, facilitating viral intercellular movement by affecting the permeability of PD. Our findings enable us to genetically control CGMMV resistance in planta by using precise genome editing techniques targeted to WPRb.

Pan-genome analysis sheds light on structural variation-based dissection of agronomic traits in melon crops.

Sweetness and appearance of fresh fruits are key palatable and preference attributes for consumers and are often controlled by multiple genes. However, fine-mapping the key loci or genes of interest by single genome-based genetic analysis is challenging. Herein, we present the chromosome-level genome assembly of 1 landrace melon accession (Cucumis melo ssp. agrestis) with wild morphologic features and thus construct a melon pan-genome atlas via integrating sequenced melon genome datasets. Our comparative genomic analysis reveals a total of 3.4 million genetic variations, of which the presence/absence variations (PAVs) are mainly involved in regulating the function of genes for sucrose metabolism during melon domestication and improvement. We further resolved several loci that are accountable for sucrose contents, flesh color, rind stripe, and suture using a structural variation (SV)-based genome-wide association study. Furthermore, via bulked segregation analysis (BSA)-seq and map-based cloning, we uncovered that a single gene, (CmPIRL6), determines the edible or inedible characteristics of melon fruit exocarp. These findings provide important melon pan-genome information and provide a powerful toolkit for future pan-genome-informed cultivar breeding of melon.

A large presence/absence variation in the promotor of the ClLOG gene determines trichome elongation in watermelon.

KEY MESSAGE: The ClLOG gene encoding a cytokinin riboside 5'-monophosphate phosphoribohydrolase determines trichome length in watermelon, which is associated with its promoter variations. Trichomes, which are differentiated from epidermal cells, are special accessory structures that cover the above-ground organs of plants and possibly contribute to biotic and abiotic stress resistance. Here, a bulked segregant analysis (BSA) of an F2 population with significant variations in trichome length was undertaken. A 1.84-Mb candidate region on chromosome 10 was associated with trichome length. Resequencing and fine-mapping analyses indicated that a 12-kb structural variation in the promoter of Cla97C10G203450 (ClLOG) led to a significant expression difference in this gene in watermelon lines with different trichome lengths. In addition, a virus-induced gene silencing analysis confirmed that ClLOG positively regulated trichome elongation. These findings provide new information and identify a potential target gene for controlling multicellular trichome elongation in watermelon.

Development of an HSV-1 production process involving serum-reduced culturing and bead-to-bead transfer.

Herpes simplex virus type 1 (HSV-1) plays an important role in the field of gene therapy and viral vaccines, especially as an oncolytic virus. However, the mass production of HSV-1 viral vectors remains a challenge in the industry. In this study, a microcarrier-mediated serum-reduced medium culture was used to improve the bioprocess of HSV-1 production and increase HSV-1 yields. The composition of the culture media, which included a basal medium, serum concentration, and glutamine additive, was optimized. The process was successfully conducted in a 1 L bioreactor, and virus production was threefold greater than that of conventional processes with a 10% serum medium. The bead-to-bead transfer process was also developed to further increase scalability. In spinner flasks, the detachment rate increased from 49.4 to 80.6% when combined agitation was performed during digestion; the overall recovery proportion increased from 37.9 to 71.1% after the operational steps were optimized. Specifically, microcarrier loss was reduced during aspiration and transfer, and microcarriers and detached cells were separated with filters. Comparable cell growth was achieved with the baseline process using 2D culture as the inoculum by exchanging the subculture medium. To increase virus production after bead-to-bead transfer, critical parameters, including shear stress during digestion, TrypLE and EDTA concentrations in the subculture, and the CCI, were identified from 47 parameters via correlation analysis and principal component analysis. The optimized bead-to-bead transfer process achieved an average of 90.4% overall recovery and comparable virus production compared to that of the baseline process. This study is the first to report the optimization of HSV-1 production in Vero cells cultured on microcarriers in serum-reduced medium after bead-to-bead transfer. KEY POINTS: • An HSV-1 production process was developed that involves culturing in serum-reduced medium, and this process achieved threefold greater virus production than that of traditional processes. • An indirect bead-to-bead transfer process was developed with over 90% recovery yield in bioreactors. • HSV-1 production after bead-to-bead transfer was optimized and was comparable to that achieved with 2D culture as inoculum.

Control of Compensation Temperature in CoGd Films through Hydrogen and Oxygen Migration under Gate Voltage.

Electrical control of magnetic properties is crucial for low-energy memory and logic spintronic devices. We find that the magnetic properties of ferrimagnetic CoGd can be altered through ionic liquid gating. Gate voltages manipulate the opposite magnetic moments in Co and Gd sublattices and induce a giant magnetic compensation temperature change of more than 200 K in Pt/CoGd/Pt heterostructures. The electrically controlled dominant magnetic sublattice allows voltage-induced magnetization switching. Both experiments and theoretical calculations demonstrate that the significant modulations of compensation temperature are relevant to the reduced Gd moments due to the presence of hydrogen ions at positive voltages as well as the enhanced Co moments and reduced Gd moments due to the injection of oxygen ions at negative voltages. These findings expand the possibilities for all-electric and reversible magnetization control in the field of spintronics.

Structural variation of GL1 gene determines the trichome formation in Brassica juncea.

KEY MESSAGE: A 448 kb region on chromosome B02 was delimited to be associated with trichome trait in Brassica juncea, in which the BjuVB02G54610 gene with a structural variation of 3 kb structure variation (SV) encoding a MYB transcription factor was predicted as the possible candidate gene. Mustards (Brassica juncea) are allopolyploid crops in the worldwide, and trichomes are essential quality attributes that significantly influence its taste and palpability in vegetable-use cultivars. As important accessory tissues from specialized epidermal cells, trichomes also play an important role in mitigating biotic and abiotic stresses. In this study, we constructed a F2 segregating population using YJ27 with intensive trichome leaves and 03B0307 with glabrous leaves as parents. By bulked segregant analysis (BSA-seq), we obtained a 2.1 Mb candidate region on B02 chromosome associated with the trichome or glabrous trait formation. Then, we used 13 Kompetitive Allele Specific PCR (KASP) markers for fine mapping and finally narrowed down the candidate region to about 448 kb in length. Interestingly, among the region, there was a 3 kb sequence deletion that located on the BjuVB02G54610 gene in the F2 individuals with trichome leaves. Genotyping results of F2 populations confirmed this deletion (R2 = 81.44%) as a major QTL. Natural population re-sequencing analysis and genotyping results further validated the key role of the 3 kb structure variation (SV) of insertion/deletion type in trichome development in B. juncea. Our findings provide important information on the formation of trichomes and potential target gene for breeding vegetable mustards.

Allelic variations of ClACO gene improve nitrogen uptake via ethylene-mediated root architecture in watermelon.

KEY MESSAGE: The ClACO gene encoding 1-aminocyclopropane-1-carboxylate oxidase enabled highly efficient 15N uptake in watermelon. Nitrogen is one of the most essential nutrient elements that play a pivotal role in regulating plant growth and development for crop productivity. Elucidating the genetic basis of high nitrogen uptake is the key to improve nitrogen use efficiency for sustainable agricultural productivity. Whereas previous researches on nitrogen absorption process are mainly focused on a few model plants or crops. To date, the causal genes that determine the efficient nitrogen uptake of watermelon have not been mapped and remains largely unknown. Here, we fine-mapped the 1-aminocyclopropane-1-carboxylate oxidase (ClACO) gene associated with nitrogen uptake efficiency in watermelon via bulked segregant analysis (BSA). The variations in the ClACO gene led to the changes of gene expression levels between two watermelon accessions with different nitrogen uptake efficiencies. Intriguingly, in terms of the transcript abundance of ClACO, it was concomitant with significant differences in ethylene evolutions in roots and root architectures between the two accessions and among the different genotypic offsprings of the recombinant BC2F1(ZJU132)-18. These findings suggest that ethylene as a negative regulator altered nitrogen uptake efficiency in watermelon by controlling root development. In conclusion, our current study will provide valuable target gene for precise breeding of 'green' watermelon varieties with high-nitrogen uptake efficiencies.

Role of Dipolar Organic Cations on Light-triggered Charge Transfer at TiO2 /CH3 NH3 PbI3 Interfaces.

The TiO2 /MAPbI3 (MA=CH3 NH3 ) interfaces have manifested correlation with current-voltage hysteresis in perovskite solar cells (PSCs) under light illumination conditions, but the relations between the photo-induced charge transfer and the collective polarization response of the dipolar MA cations are largely unexplored. In this work, we adopt density functional theory (DFT) and time-dependent DFT approach to study the light-triggered charge transfer across the TiO2 /MAPbI3 interfaces with MAI- and PbI-exposed terminations. It is found that regardless of the surface exposure of the MAPbI3 , the photo-induced charge transfer varies when going from the ground-state geometries to the excited-state configurations. Besides, thanks to the electrostatic interactions between the ends of MA cations and the photogenerated electrons, the photo-induced charge transfer across the interfaces is enhanced (weakened) by the negatively (positively) charged CH3 (NH3 ) moieties of the MA species. Resultantly, the positively charged iodine vacancies at the TiO2 /MAPbI3 interfaces tend to inhibit the charge transfer induced by light. Combining with the energy level alignment which is significantly modulated by the orientation of the MA species at the interfaces, the dipolar MA cations might be a double-edge sword for the hysteresis in PSCs with the TiO2 /MAPbI3 interfaces.

A Modified Method for Transient Transformation via Pollen Magnetofection in Lilium Germplasm.

Lily (Lilium spp.) is a popular ornamental plant. Traditional genetic transformation methods have low efficiency in lily, thus development of a high-efficiency genetic transformation system is important. In this study, a novel transient transformation method involving pollen magnetofection was established and optimized pollen viability, and exogenous gene expression in magnetofected pollen and that of different germplasm were assessed. The highest germination percentage of Lilium regale pollen was 85.73% in medium containing 100 g/L sucrose, 61.5 mg/L H3BO3, and 91.5 mg/L CaCl2. A 1:4 ratio of nanomagnetic beads to DNA plasmid and transformation time of 0.5 h realized the highest transformation efficiency (88.32%). The GFP activity in transformed pollen averaged 69.66%, while that of the control pollen was 0.00%. In contrast to the control, transgenic seedlings obtained by pollination with magnetofected pollen showed strong positive GUS activity with 56.34% transformation efficiency. Among the lily germplasm tested, 'Sweet Surrender' and L. leucanthum had the highest transformation efficiency (85.80% and 54.47%), whereas L. davidii var. willmottiae was not successfully transformed. Transformation efficiency was positively correlated with pollen equatorial diameter and negatively correlated with polar axis/equatorial diameter ratio. The results suggest that pollen magnetofection-mediated transformation can be applied in Lilium but might have species or cultivar specificity.

Integrated Bulk Segregant Analysis, Fine Mapping, and Transcriptome Revealed QTLs and Candidate Genes Associated with Drought Adaptation in Wild Watermelon.

Drought stress has detrimental effects on crop productivity worldwide. A strong root system is crucial for maintaining water and nutrients uptake under drought stress. Wild watermelons possess resilient roots with excellent drought adaptability. However, the genetic factors controlling this trait remain uninvestigated. In this study, we conducted a bulk segregant analysis (BSA) on an F2 population consisting of two watermelon genotypes, wild and domesticated, which differ in their lateral root development under drought conditions. We identified two quantitative trait loci (qNLR_Dr. Chr01 and qNLR_Dr. Chr02) associated with the lateral root response to drought. Furthermore, we determined that a small region (0.93 Mb in qNLR_Dr. Chr01) is closely linked to drought adaptation through quantitative trait loci (QTL) validation and fine mapping. Transcriptome analysis of the parent roots under drought stress revealed unique effects on numerous genes in the sensitive genotype but not in the tolerant genotype. By integrating BSA, fine mapping, and the transcriptome, we identified six genes, namely L-Ascorbate Oxidase (AO), Cellulose Synthase-Interactive Protein 1 (CSI1), Late Embryogenesis Abundant Protein (LEA), Zinc-Finger Homeodomain Protein 2 (ZHD2), Pericycle Factor Type-A 5 (PFA5), and bZIP transcription factor 53-like (bZIP53-like), that might be involved in the drought adaptation. Our findings provide valuable QTLs and genes for marker-assisted selection in improving water-use efficiency and drought tolerance in watermelon. They also lay the groundwork for the genetic manipulation of drought-adapting genes in watermelon and other Cucurbitacea species.

LINC00662: A new oncogenic lncRNA with great potential.

LINC00662 is located on chromosome 19q11 and is 2085 bp long. It is a long noncoding RNA (lncRNA) newly discovered. LINC00662 expression is upregulated in at least 14 tumors. In addition, the upregulation of LINC00662 expression is also closely related to the poor prognosis of cancer patients and resistance to radiotherapy and chemotherapy. LINC00662 can act as a ceRNA of at least 8 miRNAs. By regulating these miRNAs and their downstream genes, LINC00662 participates in the regulation of four signaling pathways, including the extracellular signal-regulated kinase (ERK) signaling pathway, the Wnt/ß-catenin signaling pathway, the Hippo signaling pathway, and the SMD signaling pathway. In addition, the abnormal upregulation of LINC00662 can promote the stem-like features of lung cancer cells. LINC00662 can reduce the promoter methylation level of s-adenosylmethionine (SAM)-dependent hepatocellular carcinoma (HCC)-promoting genes by regulating the MAT1A/SAM and AHCY/SAH axes, thereby promoting the activation of oncogenes. This article summarizes the molecular regulation mechanism of LINC00662 in cancer and the diagnostic and prognostic value of LINC00662 in cancer.

An allelic variant in the ACS7 gene promotes primary root growth in watermelon.

KEY MESSAGE: Gene mining in a C. lanatus × C. amarus population revealed one gene, ACS7, linked to primary root elongation in watermelon. Watermelon is a xerophytic crop characterized by a long primary root and robust lateral roots. Therefore, watermelon serves as an excellent model for studying root elongation and development. However, the genetic mechanism underlying the primary root elongation in watermelon remains unknown. Herein, through bulk segregant analysis we identified a genetic locus, qPRL.Chr03, controlling primary root length (PRL) using two different watermelon species (Citrullus lanatus and Citrullus amarus) that differ in their root architecture. Fine mapping revealed that xaa-Pro dipeptidase and 1-aminocyclopropane-1-carboxylate synthase 7 (ACS7) are candidate regulators of the primary root growth. Allelic variation in the delimited region among 193 watermelon accessions indicated that the long-root alleles might only exist in C. amarus. Interestingly, the discrepancy in PRL among the C. amarus accessions was clearly associated with a nonsynonymous single nucleotide polymorphism variant within the ACS7 gene. The ACS7 expression and ethylene levels in the primary root tips suggested that ethylene is a negative regulator of root elongation in watermelon, as supported by the application of 1-aminocyclopropane-1-carboxylate (ACC, the ethylene precursor) or 2-aminoethoxyvinyl glycine (AVG, an ACS inhibitor). To the best of our knowledge, these findings provide the first description of the genetic basis of root elongation in watermelon. The detected markers of the ACS7 gene will facilitate marker-assisted selection for the PRL trait to improve water and nutrient use efficacy in watermelon and beyond.

Porcine reproductive and respiratory syndrome virus non-structural protein 4 cleaves guanylate-binding protein 1 via its cysteine proteinase activity to antagonize GBP1 antiviral effect.

Porcine reproductive and respiratory syndrome (PRRS) is a highly infectious disease caused by PRRS virus (PRRSV) that causes great economic losses to the swine industry worldwide. PRRSV has been recognized to modulate the host antiviral interferon (IFN) response and downstream interferon-stimulated gene expression to intercept the antiviral effect of host cells. Guanylate-binding proteins (GBPs) are IFN-inducible GTPases that exert broad antiviral activity against several DNA and RNA viruses, of which GBP1 is considered to play a pivotal role. However, the role of GBP1 in PRRSV replication remains unknown. The present study showed that overexpression of GBP1 notably inhibited PRRSV infection, while the knockdown of endogenous GBP1 promoted PRRSV infection. The K51 and R48 residues of GBP1 were essential for the suppression of PRRSV replication. Furthermore, GBP1 abrogated PRRSV replication by disrupting normal fibrous actin structures, which was indispensable for effective PRRSV replication. By using a co-immunoprecipitation assay, we found that GBP1 interacted with the non-structural protein 4 (nsp4) protein of PRRSV, and this interaction was mapped to the N-terminal globular GTPase domain of GBP1 and amino acids 1-69 of nsp4. PRRSV infection significantly downregulated GBP1 protein expression in Marc-145 cells, and nsp4, a 3C-like serine proteinase, was responsible for GBP1 cleavage, and the cleaved site was located at glutamic acid 338 of GBP1. Additionally, the anti-PRRSV activity of GBP1 was antagonized by nsp4. Taken together, these findings expand our understanding of the sophisticated interaction between PRRSV and host cells, PRRSV pathogenesis and its mechanisms of evading the host immune response.

K205R specific nanobody-horseradish peroxidase fusions as reagents of competitive ELISA to detect African swine fever virus serum antibodies.

BACKGROUND: African swine fever virus (ASFV) is a highly contagious hemorrhagic disease and often lethal, which has significant economic consequences for the swine industry. Due to lacking of commercial vaccine, the prevention and control of ASF largely depend on early large-scale detection and screening. So far, the commercial ELISA kits have a long operation time and are expensive, making it difficult to achieve large-scale clinical applications. Nanobodies are single-domain antibodies produced by camelid animals, and have unique advantages such as smaller molecular weight, easy genetic engineering modification and low-costing of mass production, thus exhibiting good application prospects. RESULTS: The present study developed a new method for detection of ASFV specific antibodies using nanobody-horseradish peroxidase (Nb-HRP) fusion proteins as probe. By using camel immunization, phage library construction and phage display technology, five nanobodies against K205R protein were screened. Then, Nb-HRP fusion proteins were produced using genetic modification technology. Based on the Nb-HRP fusion protein as specific antibodies against K205R protein, a new type of cELISA was established to detect ASFV antibodies in pig serum. The cut-off value of the cELISA was 34.8%, and its sensitivity, specificity, and reproducibility were good. Furthermore, the developed cELISA exhibited 99.3% agreement rate with the commercial available ELISA kit (kappa value = 0.98). CONCLUSIONS: The developed cELISA method has the advantages of simple operation, rapid and low-costing, and can be used for monitoring of ASFV infection in pigs, thus providing a new method for the prevention and control of ASF.

The casein kinase 2 ß subunit CK2B1 is required for swollen stem formation via cell cycle control in vegetable Brassica juncea.

The swollen stem is a determinant of yield for the stem-type vegetable Brassica juncea that is representative of vegetative organ formation. However, the genetic mechanism underlying swollen stem formation and its regulation remains unknown. In this study, we identified a casein kinase 2 ß subunit 1 (CK2B1) and revealed its role in swollen stem formation. Genotyping analysis revealed that a homozygous variation in the CK2B1 promoter is responsible for swollen stem formation, and the promoter activity of CK2B1 was significantly associated with the variations between swollen stem and non-swollen stem types. CK2B1 was exclusively located in the nucleus and expressed in the stem nodes of the plant. Swollen stem formation was blocked when CK2B1 expression was silenced, and induced in a backcross population carrying a swollen stem genotype, which indicates that CK2B1 is required for swollen stem formation. Cell numbers were increased during swollen stem formation and decreased in CK2B1-silenced expression plant, indicating that CK2B1 regulates swollen stem formation via cell division. CK2B1 directly interacted with E2Fa, a regulator of G1/S transition in the cell cycle, in which CK2 phosphorylates E2Fa. Our results revealed that CK2B1 affects swollen stem formation via the control of the cell cycle. These findings help to elucidate the signals that control swollen stem formation and provide a promising molecular target to enhance the yield of vegetative organ formation.