Bacillus cereus NJ01 induces SA- and ABA-mediated immunity against bacterial pathogens through the EDS1-WRKY18 module.

Emerging evidence suggests a beneficial role of rhizobacteria in ameliorating plant disease resistance in an environment-friendly way. In this study, we characterize a rhizobacterium, Bacillus cereus NJ01, that enhances bacterial pathogen resistance in rice and Arabidopsis. Transcriptome analyses show that root inoculation of NJ01 induces the expression of salicylic acid (SA)- and abscisic acid (ABA)-related genes in Arabidopsis leaves. Genetic evidence showed that EDS1, PAD4, and WRKY18 are required for B. cereus NJ01-induced bacterial resistance. An EDS1-PAD4 complex interacts with WRKY18 and enhances its DNA binding activity. WRKY18 directly binds to the W box in the promoter region of the SA biosynthesis gene ICS1 and ABA biosynthesis genes NCED3 and NCED5 and contributes to the NJ01-induced bacterial resistance. Taken together, our findings indicate a role of the EDS1/PAD4-WRKY18 complex in rhizobacteria-induced disease resistance.

Diversity of Culturable Bacteria from the Coral Reef Areas in the South China Sea and Their Agar-Degrading Abilities.

The South China Sea (SCS) is abundant in marine microbial resources with high primary productivity, which is crucial for sustaining the coral reef ecosystem and the carbon cycle. Currently, research on the diversity of culturable bacteria in the SCS is relatively extensive, yet the culturable bacteria in coral reefs has been poorly understood. In this study, we analyzed the bacterial community structure of seawater samples among Daya Bay (Fujian Province), Qionghai (Hainan Province), Xisha Islands, and the southern South China Sea based on culturable methods and detected their abilities for agar degradation. There were 441 bacterial strains, belonging to three phyla, five classes, 43 genera, and 101 species, which were isolated by marine agar 2216E (MA; Becton Dickinson). Strains within Gammaproteobacteria were the dominant group, accounting for 89.6% of the total bacterial isolates. To investigate vibrios, which usually correlated with coral health, 348 isolates were obtained from TCBS agar, and all isolates were identified into three phylum, three classes, 14 orders, 25 families, and 48 genera. Strains belonging to the genus Vibrio had the greatest number (294 strains), indicating the high selectivity of TCBS agar for vibrios. Furthermore, nineteen strains were identified as potentially novel species according to the low 16S rRNA gene similarity (<98.65%), and 28 strains (15 species) had agar-degrading ability. These results indicate a high diversity of culturable bacteria in the SCS and a huge possibility to find novel and agar-degrading species. Our study provides valuable microbial resources to maintain the stability of coral ecosystems and investigate their roles in the marine carbon cycle.

Vibrio methylphosphonaticus sp. nov., a methylphosphonate-decomposing bacterium isolated from surface seawater in the Xisha Islands, PR China.

A Gram-stain-negative, facultative anaerobic, methylphosphonate-decomposing, motile by a polar flagellum and rod-shaped marine bacterium, designated S4B1T, was isolated from the surface seawater collected from the Yongle Atoll (Xisha Islands, PR China). The pairwise alignment showed the highest sequence similarity of 97.5 and 96.6 % to Vibrio aestuarianus subsp. cardii 12_122_3T3T and Vibrio atypicus HHS02T, respectively. Phylogenetic analysis based on 16S rRNA gene and the phylogenomic analysis of single-copy genes showed that strain S4B1T belonged to the genus Vibrio and formed a close branch with Vibrio qingdaonensis ZSDZ65T. Growth of strain S4B1T occurred at 4-30 °C (optimum, 28 °C), at pH 6.0-8.0 (optimum, pH 7.0) and in the presence of 2-7 % (w/v) NaCl (optimum, 3 %). The predominant fatty acids (>10 %) were C16 : 0, iso-C16 : 0 and summed feature 3 (C16 : 1 ω7c or/and C16 : 1 ω6c). The DNA G+C content of the assembled genomic sequence was 44.3 mol%. Average nucleotide identity (ANI) values between S4B1T and its reference species were lower than the threshold for species delineation (95-96 %), in which its highest ANI value with V. qingdaonensis ZSDZ65T was 87.0 %. In silico DNA-DNA hybridization further showed that strain S4B1T had less than 70 % similarity to its relatives. On the basis of the polyphasic evidence, strain S4B1T is proposed to represent a novel species of the genus Vibrio, for which the name Vibrio methylphosphonaticus sp. nov. is proposed. The type strain is S4B1T (=KCTC 92311T=MCCC 1K06168T).

Sedimentary Vibrio Blooms in the Xisha Islands May Associate with the 2020 Coral Bleaching Event.

Coral reefs are among the most biodiverse ecosystems, providing habitats for various organisms. Studies on coral bleaching have been increasing recently, but little is known about the distribution and community assembly of coral pathogenic bacteria (e.g., several Vibrio species). We elucidated the distribution pattern and interaction relationships of total bacteria and Vibrio spp. in sediments from the Xisha Islands, which are characterized by their high coverage and diversity of coral resources. Vibrio spp. showed significantly higher relative abundance values in the Xisha Islands (1.00 × 108 copies/g) than in other areas (approximately 1 × 104 to 9.04 × 105 copies/g), indicating that the coral bleaching event of 2020 may have promoted the bloom of vibrios. A spatial shift in community composition was observed between the northern (Photobacterium rosenbergii and Vibrio ponticus) and southern (Vibrio ishigakensis and Vibrio natriegens) sites, accompanied by a clear distance-decay pattern. The spatial distance and coral species (e.g., Acroporidae and Fungiidae) had much greater correlations with the Vibrio community than did environmental factors. However, complex mechanisms may exist in the community assembly of Vibrio spp. due to the large proportion of unexplained variation. Stochastic processes may play an important role, as shown by the neutral model. Vibrio harveyi had the highest relative abundance (77.56%) and niche breadth, compared to other species, and it was negatively correlated with Acroporidae, likely reflecting its strong competitive ability and adverse effects on specific corals. Our study provides insights into the bloom and underlying assembly mechanisms of sedimentary vibrios in the Xisha Islands, thereby contributing to identify the potential indicator of coral bleaching and provide inspiration for the environmental management of coral reef areas. IMPORTANCE Coral reefs exert important roles in maintaining the sustainability of marine ecosystems but decline worldwide due to various drivers, especially pathogenic microorganisms. Here, we investigated the distribution pattern and interactions of total bacteria and Vibrio spp. in the sediments from Xisha Islands during the coral bleaching event of 2020. Our results showed that the abundances of Vibrio (1.00 × 108 copies/g) were high across the whole sites, indicating the bloom of sedimentary Vibrio spp. Coral pathogenic Vibrio species were abundant in the sediments, likely reflecting adverse effects on several kinds of corals. The compositions of the Vibrio spp. were separated by geographical location, which was mainly attributable to the spatial distance and coral species. Overall, this work contributes by providing evidence for the outbreak of coral pathogenic vibrios. The pathogenic mechanism of the dominant species (especially V. harveyi) should be comprehensively considered by laboratory infection experiments in the future.

Suppression of ETI by PTI priming to balance plant growth and defense through an MPK3/MPK6-WRKYs-PP2Cs module.

Pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) are required for host defense against pathogens. Although PTI and ETI are intimately connected, the underlying molecular mechanisms remain elusive. In this study, we demonstrate that flg22 priming attenuates Pseudomonas syringae pv. tomato DC3000 (Pst) AvrRpt2-induced hypersensitive cell death, resistance, and biomass reduction in Arabidopsis. Mitogen-activated protein kinases (MAPKs) are key signaling regulators of PTI and ETI. The absence of MPK3 and MPK6 significantly reduces pre-PTI-mediated ETI suppression (PES). We found that MPK3/MPK6 interact with and phosphorylate the downstream transcription factor WRKY18, which regulates the expression of AP2C1 and PP2C5, two genes encoding protein phosphatases. Furthermore, we observed that the PTI-suppressed ETI-triggered cell death, MAPK activation, and growth retardation are significantly attenuated in wrky18/40/60 and ap2c1 pp2c5 mutants. Taken together, our results suggest that the MPK3/MPK6-WRKYs-PP2Cs module underlies PES and is essential for the maintenance of plant fitness during ETI.

Potential of granular complexes of lime and montmorillonite for stabilizing soil cadmium and the underlying mechanisms.

Cadmium (Cd) contaminated soils were widely remediated by alkaline materials in powder, while the effects of granular materials are still unknown. This study was conducted to prepare granular materials based on hydrated lime and montmorillonite with ratios of 1:1, 1:2, and 1:3 (LM1, LM2, and LM3); their effects and mechanisms on stabilizing Cd in hydroponic, pot, and field conditions were further explored. The results showed that powdery materials caused intense pH elevations within 30-60 min and dissolved-Cd reductions within 8-100 min. However, granular materials significantly delayed these effects; the highest solution pH and lowest dissolved-Cd occurred after 250 min. The LM1 granules induced a much higher reduction of dissolved-Cd (99.8%) than that in the LM2 (53.6%) and LM3 granules (14.3%) due to the generation of more cadmium carbonate precipitates. Additionally, the soil pH gradually decreased after an intense elevation induced by powdery materials, but the LM1 granules maintained the soil pH at approximately 7.0, resulting in a lower level of CaCl2-extractable Cd (0.03 mg kg-1) than the LM1 powder (0.22 mg kg-1) after 30 d of cultivation. Similar to lime powder, a small spatial variation (Std. of 3.45) of DGT (diffusive gradient in thin films) extractable Cd in soil profile was observed in the LM1 granules, revealing a homogeneous stabilization effect induced by the LM1 granules. Accordingly, the LM1 granules induced a higher reduction in brown rice Cd (50.9%) than that in the LM1 powders (35.1%). Thus, the granular material of hydrated lime and montmorillonite (1:1) h the potential to replace lime powder in the remediation of Cd-contaminated field.

Experimental Realization of a Quantum Refrigerator Driven by Indefinite Causal Orders.

Indefinite causal order (ICO) is playing a key role in recent quantum technologies. Here, we experimentally study quantum thermodynamics driven by ICO on nuclear spins using the nuclear magnetic resonance system. We realize the ICO of two thermalizing channels to exhibit how the mechanism works, and show that the working substance can be cooled or heated albeit it undergoes thermal contacts with reservoirs of the same temperature. Moreover, we construct a single cycle of the ICO refrigerator based on the Maxwell's demon mechanism, and evaluate its performance by measuring the work consumption and the heat energy extracted from the low-temperature reservoir. Unlike classical refrigerators in which the coefficient of performance (COP) is perversely higher the closer the temperature of the high-temperature and low-temperature reservoirs are to each other, the ICO refrigerator's COP is always bounded to small values due to the nonunit success probability in projecting the ancillary qubit to the preferable subspace. To enhance the COP, we propose and experimentally demonstrate a general framework based on the density matrix exponentiation (DME) approach, as an extension to the ICO refrigeration. The COP is observed to be enhanced by more than 3 times with the DME approach. Our Letter demonstrates a new way for nonclassical heat exchange, and paves the way towards construction of quantum refrigerators on a quantum system.

Functional Analysis of 3-Dehydroquinate Dehydratase/Shikimate Dehydrogenases Involved in Shikimate Pathway in Camellia sinensis.

Polyphenols play an important role in the astringent taste of tea [Camellia sinensis (L.)] infusions; catechins in phenolic compounds are beneficial to health. The biosynthesis of gallic acid (GA), a precursor for polyphenol synthesis, in tea plants remains unknown. It is well known that 3-dehydroquinate dehydratase/shikimate dehydrogenase (DQD/SDH) is a key enzyme for catalyzing the conversion of 3-dehydroshikimate (3-DHS) to shikimate (SA); it also potentially participates in GA synthesis in a branch of the SA pathway. In this study, four CsDQD/SDH proteins were produced in Escherichia coli. Three CsDQD/SDHs had 3-DHS reduction and SA oxidation functions. Notably, three CsDQD/SDHs showed individual differences between the catalytic efficiency of 3-DHS reduction and SA oxidation; CsDQD/SDHa had higher catalytic efficiency for 3-DHS reduction than for SA oxidation, CsDQD/SDHd showed the opposite tendency, and CsDQD/SDHc had almost equal catalytic efficiency for 3-DHS reduction and SA oxidation. In vitro, GA was mainly generated from 3-DHS through nonenzymatic conversion. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis showed that CsDQD/SDHc and CsDQD/SDHd expression was correlated with GA and 1-O-galloyl-ß-D-glucose accumulation in C. sinensis. These results revealed the CsDQD/SDHc and CsDQD/SDHd genes are involved in GA synthesis. Finally, site-directed mutagenesis exhibited the mutation of residues Ser-338 and NRT to Gly and DI/LD in the SDH unit is the reason for the low activity of CsDQD/SDHb for 3-DHS reduction and SA oxidation.

CsMYB5a and CsMYB5e from Camellia sinensis differentially regulate anthocyanin and proanthocyanidin biosynthesis.

Tea is one of the most widely consumed nonalcoholic beverages worldwide. Polyphenols are nutritional compounds present in the leaves of tea plants. Although numerous genes are functionally characterized to encode enzymes that catalyze the formation of diverse polyphenolic metabolites, transcriptional regulation of those different pathways such as late steps of the proanthcoyanidin (PA) pathway remains unclear. In this study, using different tea transcriptome databases, we screened at least 140 R2R3-MYB transcription factors (TFs) and grouped them according to the basic function domains of the R2R3 MYB TF superfamily. Among 140 R2R3 TFs, CsMYB5a and CsMYB5e were chosen for analysis because they may be involved in PA biosynthesis regulation. CsMYB5a-overexpressing tobacco plants exhibited downregulated anthocyanin accumulation but a high polymeric PA content in the flowers. Overexpression of CsMYB5e in tobacco plants did not change the anthocyanin content but increased the dimethylaminocinnamaldehyde-stained PA content. RNA-seq and qRT-PCR analyses revealed that genes related to PA and anthocyanin biosynthesis pathways were markedly upregulated in both CsMYB5a- and CsMYB5e-overexpressing flowers. Three UGTs and four GSTs were identified as involved in PA and anthocyanin glycosylation and transportation in transgenic plants. These results provide new insights into the regulation of PA and anthocyanin biosynthesis in Camellia sinensis.

Grass carp (Ctenopharyngodon idella) STAT3 regulates the eIF2α phosphorylation through interaction with PKR.

In mammals, STAT3 (Signal transducer and activator of transcription 3) plays an important role in growth, multiplication, differentiation and participates in inflammation, tumorigenesis, metabolic disorders and immune response. STAT3 is a protein that shuttles between the nucleus and cytoplasm. Compared to the STAT3 in cell nucleus, we did not know the function of STAT3 in cytoplasm for a long time. Some recent studies have shown that cytoplasmic STAT3 regulates autophagy through the interaction with the double-stranded RNA-activated protein kinase (PKR), which plays an important role in cellular antiviral response. Fish is a good target for developmental and comparative immunology. In the present study, we found that the expression of grass carp (Ctenopharyngodon idella) STAT3 (CiSTAT3) was ubiquitous and significantly up-regulated under the stimulation of poly I:C. To explore the potential function of fish cytoplasmic STAT3 in the antiviral signaling pathways, in this paper we analyzed the relationship between cytoplasmic CiSTAT3 and CiPKR. We demonstrated that the CiSTAT3 can combine with CiPKR in vivo and in vitro. The SH2 domain of CiSTAT3 and the C-terminus of CiPKR play an important role in this process. Moreover, the dimer of CiSTAT3 and CiPKR was formed under normal circumstances, however, it was dissociated under the induction of poly I:C. So, we guessed the binding of CiSTAT3 and CiPKR may regulate cell viability. It has also been shown that overexpression of CiSTAT3 in CIK cells can significantly reduce the level of p-eIF2α. On the contrary, the siRNA-mediated knockdown of CiSTAT3 and Stattic induction in CIK cells can up-regulate the p-eIF2α level. To further understand the relationship between CiSTAT3 and p-eIF2α level, we carried out the CiPKR-knockdown experiment. The result indicated that CiSTAT3 regulated the level of p-eIF2α through binding to CiPKR. In addition, overexpression of CiSTAT3 in CIK cells was able to improve the cell viability. These results above unraveled the molecular mechanism of fish cytoplasmic STAT3 regulating the eIF2α phosphorylation and cell viability. Therefore, the function of fish cytoplasmic STAT3 is similar to those of mammals.

Functional Analysis of Two Flavanone-3-Hydroxylase Genes from Camellia sinensis: A Critical Role in Flavonoid Accumulation.

Flavonoids are major secondary metabolites in Camellia sinensis. Flavanone-3-hydroxylase (F3H) is a key enzyme in flavonoid biosynthesis in plants. However, its role in the flavonoid metabolism in C. sinensis has not been well studied. In this study, we cloned two F3Hs from C. sinensis, named CsF3Ha and CsF3Hb, where CsF3Ha containing 1107 bases encoded 368 amino acids, and CsF3Hb containing 1071 bases encoded 357 amino acids. Enzymatic activity analysis showed both recombinant CsF3H enzymes in Escherichia coli could convert naringenin and eriodictyol into dihydrokaempferol (DHK) and dihydroquercetin (DHQ), respectively. The expression profiles showed that CsF3Ha and CsF3Hb were highly expressed in the tender leaves of tea plants. Under different abiotic stresses, the two CsF3Hs were induced remarkably by ultraviolet (UV) radiation, sucrose, and abscisic acid (ABA). In the seeds of CsF3Hs transgenic Arabidopsis thaliana, the concentration of most flavonol glycosides and oligomeric proanthocyanidins increased significantly, while the content of monocatechin derivatives decreased. The present study revealed that CsF3Hs played critical roles in flavonoid biosynthesis in tea plants.

Endoplasmic Reticulum Transmembrane Proteins ZDHHC1 and STING Both Act as Direct Adaptors for IRF3 Activation in Teleost.

IFN regulatory factor (IRF)3 is a central regulator for IFN-ß expression in different types of pathogenic infections. Mammals have various pathogenic sensors that are involved in monitoring pathogen intrusions. These sensors can trigger IRF3-mediated antiviral responses through different pathways. Endoplasmic reticulum-associated proteins stimulator of IFN gene (STING) and zinc finger DHHC-type containing 1 (ZDHHC1) are critical mediators of IRF3 activation in response to viral DNA infections. In this study, grass carp STING and ZDHHC1 were found to have some similar molecular features and subcellular localization, and both were upregulated upon stimulation with polyinosinic:polycytidylic acid, B-DNA, or Z-DNA. Based on these results, we suggest that grass carp STING and ZDHHC1 might possess some properties similar to their mammalian counterparts. Overexpression of ZDHHC1 and STING in Ctenopharyngodon idella kidney cells upregulated IFN expression, whereas knockdown of IRF3 inhibited IFN activation. In addition, coimmunoprecipitation and GST pull-down assays demonstrated that STING and ZDHHC1 can interact separately with IRF3 and promote the dimerization and nuclear translocation of IRF3. Furthermore, we also found that small interfering RNA-mediated knockdown of STING could inhibit the expression of IFN and ZDHHC1 in fish cells. Similarly, knockdown of STING resulted in inhibition of the IFN promoter. In contrast, ZDHHC1 knockdown also inhibited IFN expression but had no apparent effect on STING, which indicates that STING is necessary for IFN activation through ZDHHC1. In conclusion, STING and ZDHHC1 in fish can respond to viral DNA or RNA molecules in cytoplasm, as well as activate IRF3 and, eventually, trigger IFN expression.

Characterization and Expression Profiling of Camellia sinensis Cinnamate 4-hydroxylase Genes in Phenylpropanoid Pathways.

Cinnamate 4-hydroxylase (C4H), a cytochrome P450-dependent monooxygenase, participates in the synthesis of numerous polyphenoid compounds, such as flavonoids and lignins. However, the C4H gene number and function in tea plants are not clear. We screened all available transcriptome and genome databases of tea plants and three C4H genes were identified and named CsC4Ha, CsC4Hb, and CsC4Hc, respectively. Both CsC4Ha and CsC4Hb have 1518-bp open reading frames that encode 505-amino acid proteins. CsC4Hc has a 1635-bp open reading frame that encodes a 544-amino acid protein. Enzymatic analysis of recombinant proteins expressed in yeast showed that the three enzymes catalyzed the formation of p-coumaric acid (4-hydroxy trans-cinnamic acid) from trans-cinnamic acid. Quantitative real-time PCR (qRT-PCR) analysis showed that CsC4Ha was highly expressed in the 4th leaf, CsC4Hb was highly expressed in tender leaves, while CsC4Hc was highly expressed in the young stems. The three CsC4Hs were induced with varying degrees by abiotic stress treatments. These results suggest they may have different subcellular localization and different physiological functions.

Ctenopharyngodon idella p53 mediates between NF-κB and PKR at the transcriptional level.

p53, NF-κB and PKR are well-known to be involved in antiviral response. Although p53 has been reported in fish, its role in the regulation of NF-κB and PKR is not well understood. Here, we cloned and characterized the full length of cDNA sequence of grass carp (Ctenopharyngodon idella) p53 (Cip53) and its promoter sequence. The full length cDNA of Cip53 was 1879 bp with an ORF of 1116 bp encoding a polypeptide of 371 amino acids. Phylogenetic tree analysis revealed that Cip53 shares high homology with Dario rerio p53 (Drp53). Similar to those of Cip65 and CiPKR, the expression of Cip53 in CIK cells was significantly up-regulated after stimulation with poly I:C. To further understand the roles of fish p53 in the transcriptional control of NF-κB and PKR, Cip53 and Cip65 were expressed in E. coli BL21 and purified by affinity chromatography with the Ni-NTA His-Bind resin. In vitro, gel mobility shift assays demonstrated that the high affinity interaction between Cip65 and Cip53 promoter. Similarly, Cip53 bound to CiPKR promoter with high affinity. Dual-luciferase reporter assays showed that Cip65 activated Cip53 promoter and Cip53 activated CiPKR promoter, respectively. In addition, the role of p53 in p65-p53-PKR transcription pathway was explored. When Cip53 was knockdown in CIK cells, the mRNA levels of Cip65 and CiPKR were decreased. Taken together, p53 may play pivotal roles in transcription pathway of NF-κB and PKR in fish.

Ctenopharyngodon idella IRF2 and ATF4 down-regulate the transcriptional level of PRKRA.

PRKRA (interferon-inducible double-stranded RNA-dependent protein kinase activator A) is a protective protein which regulates the adaptation of cells to ER stress and virus-stimulated signaling pathways by activating PKR. In the present study, a grass carp (Ctenopharyngodon idella) PRKRA full-length cDNA (named CiPRKRA, KT891991) was cloned and identified. The full-length cDNA is comprised of a 5' UTR (36 bp), a 3' UTR (350 bp) and the longest ORF (882 bp) encoding a polypeptide of 293 amino acids. The deduced amino acid sequence of CiPRKRA contains three typical dsRNA binding motifs (dsRBM). Phylogenetic tree analysis revealed a closer evolutionary relationship of CiPRKRA with other fish PRKRA, especially with Danio rerio PRKRA. qRT-PCR showed that CiPRKRA was significantly up-regulated after stimulation with tunicamycin (Tm) and Poly I:C in C. idella kidney (CIK) cells. To further study its transcriptional regulation, the partial promoter sequence of CiPRKRA (1463 bp) containing one ISRE and one CARE was cloned by Tail-PCR. Subsequently, grass carp IRF2 (CiIRF2) and ATF4 (CiATF4) were expressed in Escherichia coli BL21 and purified by affinity chromatography with the Ni-NTA His-Bind Resin. In vitro, both CiIRF2 and CiATF4 bound to CiPRKRA promoter with high affinity by gel mobility shift assays, revealing that IRF2 and ATF4 might be potential transcriptional regulatory factors for CiPRKRA. Dual-luciferase reporter assays were applied to further investigate the transcriptional regulation of CiPRKRA in vivo. Recombinant plasmid of pGL3-PRKRAPro was constructed and transiently co-transfected into CIK cells with pcDNA3.1-CiIRF2 and pcDNA3.1-CiATF4, respectively. The results showed that both CiIRF2 and CiATF4 significantly decreased the luciferase activity of pGL3-PRKRAPro, suggesting that they play a negative role in CiPRKRA transcription.

Interaction of IRF9 and STAT2 synergistically up-regulates IFN and PKR transcription in Ctenopharyngodon idella.

IRF9 is a key factor in the JAK-STAT pathway. Under the stimulation of type I IFN, IRF9 interacts with STAT1 and STAT2 to form the IFN-I-stimulated gene factor 3 (ISGF3) which activates the transcription of ISG. However, many studies also showed that the dimmer IRF9/STAT2 rather than the tripolymer IRF9/STAT1/STAT2 acts as the ISGF3 in cells in response to IFN signals. In the present study, the full-length cDNA sequence of IRF9 (termed CiIRF9, KT601055) and STAT2 (term CiSTAT2, KT781914) from grass carp were cloned and identified. A low level of constitutive expression of CiIRF9 was detected by RT-PCR in grass carp tissues, but it was significantly up-regulated by LPS and poly I:C stimulation. In vitro, a high-affinity interaction between CiIRF9 and the promoter of CiIFN or CiPKR was demonstrated by gel mobility shift assay. In vivo, the promoter activities of CiIFN and CiPKR were not only increased by transient transfection of CiIRF9, but also prominently increased by co-transfection of CiIRF9 and CiSTAT2. Moreover, the interaction of CiIRF9 and CiSTAT2 was further investigated by in vivo and in vitro protein interaction assays. Recombinant CiIRF9 and CiSTAT2, both tagged with FLAG (or HA), were expressed in HEK 293T cells by transient transfection experiment. Co-immunoprecipitation assays showed that CiIRF9 can interact with CiSTAT2 in vivo. Soluble GST-ST2-936 (containing the N-terminal and coiled-coil domain of CiSTAT2) was expressed and purified from E. coli. A GST pull-down assay suggested that GST-tagged ST2-936 efficiently bound to FLAG-tagged IRF9. The data indicated that interaction of IRF9 and STAT2 synergistically up-regulated the transcriptional level of IFN and ISG genes.

Identification and characterization of a constitutively expressed Ctenopharyngodon idella ADAR1 splicing isoform (CiADAR1a).

As one member of ADAR family, ADAR1 (adenosine deaminase acting on RNA 1) can convert adenosine to inosine within dsRNA. There are many ADAR1 splicing isoforms in mammals, including an interferon (IFN) inducible ∼150 kD protein (ADAR1-p150) and a constitutively expressed ∼110 kD protein (ADAR1-p110). The structural diversity of ADAR1 splicing isoforms may reflect their multiple functions. ADAR1 splicing isoforms were also found in fish. In our previous study, we have cloned and identified two different grass carp ADAR1 splicing isoforms, i.e. CiADAR1 and CiADAR1-like, both of them are IFN-inducible proteins. In this paper, we identified a novel CiADAR1 splicing isoform gene (named CiADAR1a). CiADAR1a gene contains 15 exons and 14 introns. Its full-length cDNA is comprised of a 5' UTR (359 bp), a 3' UTR (229 bp) and a 2952 bp ORF encoding a polypeptide of 983 amino acids with one Z-DNA binding domain, three dsRNA binding motifs and a highly conserved hydrolytic deamination domain. CiADAR1a was constitutively expressed in Ctenopharyngodon idella kidney (CIK) cells regardless of Poly I:C stimulation by Western blot assay. In normal condition, CiADAR1a was found to be present mainly in the nucleus. After treatment with Poly I:C, it gradually shifted to cytoplasm. To further investigate the mechanism of transcriptional regulation of CiADAR1a, we cloned and identified its promoter sequence. The transcriptional start site of CiADAR1a is mapped within the truncated exon 2. CiADAR1a promoter is 1303 bp in length containing 4 IRF-Es. In the present study, we constructed pcDNA3.1 eukaryotic expression vectors with IRF1 and IRF3 and co-transfected them with pGL3-CiADAR1a promoter into CIK cells. The results showed that neither the over-expression of IRF1 or IRF3 nor Poly I:C stimulation significantly impacted CiADAR1a promoter activity in CIK cells. Together, according to the molecular and expression characteristics, subcellular localization and transcriptional regulatory mechanism, we deduced that CiADAR1a shared a high degree of homology with mammalian ADAR1-p110.