Northern Blot Analysis Research Articles

Real-Time Functional Bioimaging of Neuron-Specific microRNA Dynamics During Neuronal Differentiation Using a Dual Luciferase Reporter

The capability of monitoring the neuronal differentiation process in living cells is crucial to the understanding of neuronal development and the practical application of cell therapy for the treatment of neurodegenerative disorders. Current research methods, including northern blot and real-time PCR analysis, have been extensively employed to quantify miRNA expression during cellular processes. However, these methods require the cell destruction and could not provide dynamic information of miRNA expression and function in living organisms. In the present study, we developed a dual luciferase reporter to monitor the expression pattern of neuron-specific miRNA-9 and miRNA-124a during neuronal differentiation in vitro and in vivo. The miRNA-responsive reporter is designed to encode a firefly luciferase (Fluc) gene containing miRNA target sequences and a renilla luciferase (Rluc) gene for normalization. These two genes are independent modules and transcribed by two different promoters, which enables precise sense miRNA activity without mutual transcription interference. We demonstrated that the functional activation of miRNA-9 and miRNA-124a during neurogenesis is visualized by the reduction of Fluc bioluminescence signal in P19 cells and nude mice without Rluc signal change, suggesting that miRNA-9 and miRNA-124a specifically downregulates their targets in accordance with their expression. Our dual luciferase-based miRNA imaging system provides a useful tool to quantitatively and continuously monitor miRNA activity during various biological processes. Funding Statement: This work was supported by National Natural Science Foundation of China (No. 81571721, 81772010) and The National Key Research and Development Program of China (973 Program) (Grant No. 2017YFA0205202). Declaration of Interests: No potential conflicts of interest were disclosed. Ethics Approval Statement: All animal studies were implemented with the Guild for the Care and Use of Laboratory Animals approved by Xidian University

Identification and subcellular location of an RNA silencing suppressor encoded by mulberry crinkle leaf virus

Mulberry crinkle leaf virus (MCLV) is a novel geminivirus recently identified from the woody plant mulberry (Morus alba L.). Little is known about the functions of the proteins encoded by the MCLV genome. Here, all the MCLV-encoded proteins were examined for the ability to suppress gene silencing by an agroinfiltration assay in combination with northern blot analysis of green fluorescent protein (GFP) mRNA and western blot analysis. Of the six proteins, only one protein, V3, which has been predicted to play a role in viral movement, was found to suppress the gene silencing induced by a sense GFP gene in Nicotiana benthamiana 16c. The minimal amino acid sequence of V3 that maintains suppressor activity was also determined by constructing truncated mutants lacking different lengths of the amino acid sequences at the N- or C-terminus of the V3 protein. The results showed that the 94 N-terminal amino acid residues of V3 are sufficient to maintain V3 suppressor activity. In addition, the subcellular location of the V3 protein was investigated by confocal laser scanning microscopy after the expression of a V3-RFP fused protein in leaf epidermal cells of N. benthamiana. The results indicated that the V3 protein localized not only to the cytoplasm but also to the nucleus of N. benthamiana, implying that V3 can shuttle between the nucleus and the cytoplasm. Deletion mutant analysis indicated that a putative nuclear localization signal (NLS) between aa 118–134 might be responsible for the nuclear distribution of the V3 protein. Given the importance of RNA silencing in plant-virus interactions, the identification of a silencing suppressor of MCLV should be valuable in understanding the pathogenicity and molecular biology of this virus.

In silico 'fishing' using known small regulatory RNA (sRNA) candidates as the decoy from Escherichia coli, Salmonella typhi and Salmonella typhimurium manifested 14 novel sRNA candidates in the orthologous region of Proteus mirabilis.

Proteus mirabilis, a gram-negative bacterium of the family Enterobacteriaceae, is a leading cause of urinary tract infection (UTI) with rapid development of multi-drug resistance. Identification of small regulatory RNAs (sRNAs), which belongs to a class of RNAs that do not translate into a protein, could permit the comprehension of the regulatory roles this molecules play in mediating pathogenesis and multi-drug resistance of the organism. In this study, comparative sRNA analysis across three different members of Enterobacteriaceae (Escherichia coli, Salmonella typhi and Salmonella typhimurium) was carried out to identify the sRNA homologs in P. mirabilis. A total of 232 sRNA genes that were reported in E. coli, S. typhi and S. typhimurium were subjected to comparative analysis against P. mirabilis HI4320 genome. We report the detection of 14 sRNA candidates, conserved in the orthologous regions of P. mirabilis, that are not included in Rfam database. Northern-blot analysis was carried out for selected three sRNA candidates from the current investigation and three known sRNA from Rfam of P. mirabilis. The expression pattern of the six sRNA candidates shows that they are growth stage-dependant. To the best of our knowledge, this is the first report on the identification of sRNA candidates in P. mirabilis.

MiR-1307-3p suppresses the chondrogenic differentiation of human adipose-derived stem cells by targeting BMPR2.

MicroRNAs (miRs) are involved in several physiological processes, including chondrogenic differentiation, however, their expression and roles in the chondrogenic differentiation of human adipose-derived stem cells (hADSCs) remain to be fully elucidated to date. Our previous study showed that miR-1307-3p was significantly downregulated during chondrogenic differentiation by microarray and northern blot analysis. The present study aimed to investigate the effects of miR-1307-3p on chondrogenic differentiation and the underlying mechanisms. First, the decreased expression of miR-1307-3p was confirmed by reverse transcription-quantitative polymerase chain reaction analysis. Subsequently, gain- and loss-of-function of miR-1307-3p experiments showed that the overexpression of miR-1307-3p suppressed the deposition of cartilage matrix proteoglycans and decreased the expression of cartilage-related markers, including sex determining region Y-box 9, collagen type II α1 chain and aggrecan, whereas the knockdown of miR-1307-3p had the opposite effect. In addition, bone morphogenetic protein receptor type 2 (BMPR2) was identified as a target of miR-1307-3p. Further mechanistic investigations showed that miR-1307-3p attenuated the chondrogenic differentiation of hADSCs at least partly by inhibiting BMPR2-mothers against decapentaplegic signaling pathways. In conclusion, the findings revealed that miR-1307-3p inhibited the chondrogenic differentiation of hADSCs by targeting BMPR2 and its down-stream signaling pathway, which may provide novel therapeutic clues for the treatment of cartilage injury.

Characterization of cDNA clones encoding major histocompatibility class II receptors from walleye (Sander vitreus)

The teleost major histocompatibility (MH) class II receptor presents peptides from exogenous sources to CD4+ T cells, leading to the initiation of the adaptive immune response. The genes encoding MH class II have been identified in a number of teleost species, but not in walleye, an important recreational fish and commercial fishery in North America. In this study, we cloned and characterized the sequences encoding walleye MH class II α and β chains. These sequences contained all of the domains typical for functional MH class II α and β chain proteins, and aligned with other teleost sequences of MH class II. The walleye MH class II α amino acid sequence, along with other members of the Supraorder Percomorpharia, contains a high concentration of methionine residues in the beginning of the leader peptide. Southern blotting indicated that there is more than one gene copy for both MH class II α and β, while northern blotting analysis of both genes showed that expression of these genes is greatest in lymphoid tissues and at potential entry points for pathogens. These results help to further the understanding of MH class II receptors in teleosts, and could prove useful in the study of disease issues in walleye such as dermal sarcoma virus.

A Major Facilitator Superfamily Transporter Regulated by the Stress-Responsive Transcription Factor Yap1 Is Required for Resistance to Fungicides, Xenobiotics, and Oxidants and Full Virulence in Alternaria alternata.

Alternaria alternata relies on the ability to produce a host-selective toxin and to detoxify reactive oxygen species to successfully colonize the host. An A. alternata major facilitator superfamily transporter designated AaMFS54 was functionally characterized by analysis of loss- and gain-of-function mutations to better understand the factors required for fungal pathogenesis. AaMFS54 was originally identified from a wild-type expression library after being subtracted with that of a mutant impaired for the oxidative stress-responsive transcription regulator Yap1. AaMFS54 contains 14 transmembrane helixes. Fungal mutant lacking AaMFS54 produced fewer conidia and increased sensitivity to many potent oxidants (potassium superoxide and singlet-oxygen generating compounds), xenobiotics (2,3,5-triiodobenzoic acid and 2-chloro-5-hydroxypyridine), and fungicides (clotrimazole, fludioxonil, vinclozolin, and iprodione). AaMFS54 mutant induced necrotic lesion sizes similar to those induced by wild-type on leaves of susceptible citrus cultivars after point inoculation with spore suspensions. However, the mutant produced smaller colonies and less fluffy hyphae on the affected leaves. Virulence assays on citrus leaves inoculated by spraying with spores revealed that AaMFS54 mutant induced less severe lesions than wild-type, indicating the requirement of AaMFS54 in pathogenesis. All defective phenotypes were restored in a strain re-acquiring a functional copy of AaMFS54. Northern blotting analysis revealed that the expression of AaMFS54 was suppressed by xenobiotics. The current studies indicate that the Yap1-mediated transporter plays a role in resistance to toxic oxidants and fungicides in A. alternata. The relationships of MFS transporters with other regulatory components conferring stress resistance and A. alternata pathogenesis are also discussed.

Cross-cleavage activity of Cas6b in crRNA processing of two different CRISPR-Cas systems in Methanosarcina mazei Gö1

ABSTRACTThe clustered regularly interspaced short palindromic repeat (CRISPR) system is a prokaryotic adaptive defense system against foreign nucleic acids. In the methanoarchaeon Methanosarcina mazei Gö1, two types of CRISPR-Cas systems are present (type I-B and type III-C). Both loci encode a Cas6 endonuclease, Cas6b-IB and Cas6b-IIIC, typically responsible for maturation of functional short CRISPR RNAs (crRNAs). To evaluate potential cross cleavage activity, we biochemically characterized both Cas6b proteins regarding their crRNA binding behavior and their ability to process pre-crRNA from the respective CRISPR array in vivo. Maturation of crRNA was studied in the respective single deletion mutants by northern blot and RNA-Seq analysis demonstrating that in vivo primarily Cas6b-IB is responsible for crRNA processing of both CRISPR arrays. Tentative protein level evidence for the translation of both Cas6b proteins under standard growth conditions was detected, arguing for different activities or a potential non-redundant role of Cas6b-IIIC within the cell. Conservation of both Cas6 endonucleases was observed in several other M. mazei isolates, though a wide variety was displayed. In general, repeat and leader sequence conservation revealed a close correlation in the M. mazei strains. The repeat sequences from both CRISPR arrays from M. mazei Gö1 contain the same sequence motif with differences only in two nucleotides. These data stand in contrast to all other analyzed M. mazei isolates, which have at least one additional CRISPR array with repeats belonging to another sequence motif. This conforms to the finding that Cas6b-IB is the crucial and functional endonuclease in M. mazei Gö1.Abbreviations:sRNA: small RNA; crRNA: CRISPR RNA; pre-crRNAs: Precursor CRISPR RNA; CRISPR: clustered regularly interspaced short palindromic repeats; Cas: CRISPR associated; nt: nucleotide; RNP: ribonucleoprotein; RBS: ribosome binding site

Near-infrared fluorescent northern blot.

Northern blot analysis detects RNA molecules immobilized on nylon membranes through hybridization with radioactive 32P-labeled DNA or RNA oligonucleotide probes. Alternatively, nonradioactive northern blot relies on chemiluminescent reactions triggered by horseradish peroxidase (HRP) conjugated probes. The use of regulated radioactive material and the complexity of chemiluminescent reactions and detection have hampered the adoption of northern blot techniques by the wider biomedical research community. Here, we describe a sensitive and straightforward nonradioactive northern blot method, which utilizes near-infrared (IR) fluorescent dye-labeled probes (irNorthern). We found that irNorthern has a detection limit of ∼0.05 femtomoles (fmol), which is slightly less sensitive than 32P-Northern. However, we found that the IR dye-labeled probe maintains the sensitivity after multiple usages as well as long-term storage. We also present alternative irNorthern methods using a biotinylated DNA probe, a DNA probe labeled by terminal transferase, or an RNA probe labeled during in vitro transcription. Furthermore, utilization of different IR dyes allows multiplex detection of different RNA species. Therefore, irNorthern represents a more convenient and versatile tool for RNA detection compared to traditional northern blot analysis.

MiR-378a-3p sensitizes ovarian cancer cells to cisplatin through targeting MAPK1/GRB2

Ovarian cancer has gradually become one of the commonest gynecological tumor in the world. Although various therapies have been developed by researchers, the chemoresistance of ovarian cancer is still a huge challenge. MircroRNAs (miRNAs) have been widely studied due to their anti-oncogenic functions. MiR-378a-3p has been reported to sensitize breast cancer cells to chemotherapy. Here, we hypothesized that miR-378a-3p is a potential chemosensitizer in ovarian cancer. Firstly, miR-378a-3p was uncovered to down-regulated in ovarian cancer tissues and cell lines through using qRT-PCR analysis and northern blot analysis. According to the result of Kaplan Meier analysis, low expression of miR-378a-3p is closely associated with unfavorable prognosis of ovarian cancer patients. Subsequently, gain-of function assays indicated that miR-378a-3p suppressed cell proliferation and promoted cell apoptosis. Moreover, miR-378a-3p was found to enhance cisplatin sensitivity of ovarian cancer cells. Mechanism investigations suggested that MAPK1 and GRB2 are two targets of miR-378a-3p. Finally, rescue assays revealed that MAPK1 and GRB2 can reverse the effects of miR-378a-3p on chemosensitivity of ovarian cancer cells. In conclusion, miR-378a-3p enhanced the sensitivity of ovarian cancer cells to cisplatin through targeting MAPK1 and GRB2.

The Long Noncoding RNA Lncenc1 Maintains Naive States of Mouse ESCs by Promoting the Glycolysis Pathway.

SummaryThe naive embryonic stem cells (nESCs) display unique characteristics compared with the primed counterparts, but the underlying molecular mechanisms remain elusive. Here we investigate the functional roles of Lncenc1, a highly abundant long noncoding RNA in nESCs. Knockdown or knockout of Lncenc1 in mouse nESCs leads to a significantly decreased expression of core pluripotency genes and a significant reduction of colony formation capability. Furthermore, upon the depletion of Lncenc1, the expression of glycolysis-associated genes is significantly reduced, and the glycolytic activity is substantially impaired, as indicated by a more than 50% reduction in levels of glucose consumption, lactate production, and extracellular acidification rate. Mechanistically, Lncenc1 interacts with PTBP1 and HNRNPK, which regulate the transcription of glycolytic genes, thereby maintaining the self-renewal of nESCs. Our results demonstrate the functions of Lncenc1 in linking energy metabolism and naive state of ESCs, which may enhance our understanding of the molecular basis underlying naive pluripotency.

Regeneration and Genetic Transformation Systems of Durum Wheat, Triticum durum

Wheat is a major cereal crop for humans but quite recalcitrant in transformation. Establishment of regeneration system in wheat using immature embryos is not easy and time/cost-consuming. Herein, we developed a regeneration and transformation system using mature seeds in four pasta wheat cultivars. The MS medium with 2.0 mg/l 2,4-D and 2 mg/l BA was the optimum medium for developing shoots from calli. Wheat cultivars showed different regeneration frequencies response due to their genetic makeup. The cultivar Sohag-3 produced the highest regeneration frequency (93.2%) among the tested cultivars. Developed cultivars Sohag-3 and ACSAD1105 mature embryos were co-cultivated with Agrobacterium tumefaciens strain GV3101 with the binary vector pISV2678 harboring the bar gene and β-glucuronidase (gus) gene. The transformation efficiencies were 12.3 and 9.1% for cultivars Sohag-3 and ACSAD1105, respectively. The polymerase chain reaction (with specific primers for the transgenes) and the dot blot hybridization were used to confirm the integration of the transgene in transformed plants. The transformation percentages were reduced according to their expression and reached 5.6 and 4.6% for cultivars Sohag-3 and ACSAD1105, respectively. RT-PCR and northern blot analysis confirmed the expression of the gus gene only in the transgenic plants. The procedures developed in this study demonstrate the ability to produce transgenic wheat plants expressing the gus gene; hence, this protocol could be used to regenerate transgenic wheat plants expressing desirable and selective genes.

Cloning and expression of the lectin gene from the mushroom Agrocybe aegerita and the activities of recombinant lectin in the resistance of shrimp white spot syndrome virus infection

Lectin is a protein with multiple functions. In this study, the full-length cDNA of the Agrocybe aegerita lectin (AAL) gene was cloned, recombinant AAL (AAL-His) was expressed, and the activities of AAL-His were analyzed. Northern blot analysis showed that the major AAL transcript is approximately 900 bp. Sequence analysis showed that the coding region of AAL is 489 bp with a transcription start site located 39 nucleotides upstream of the translation initiation codon. In an agglutination test, AAL-His agglutinated rabbit erythrocytes at 12.5 μg/ml. AAL-His also showed antiviral activity in protecting shrimp from white spot syndrome virus (WSSV) infection. This anti-WSSV effect might be due to the binding of AAL-His on WSSV virions via the direct interactions with four WSSV structural proteins, VP39B, VP41B, VP53A and VP216. AAL demonstrates the potential for development as an anti-WSSV agent for shrimp culture. It also implies that these four AAL interaction WSSV proteins may play important roles in virus infection.

Genetic engineering of sunflower (Helianthus annuus L.) for resistance to necrosis disease through deployment of the TSV coat protein gene

Necrosis disease incited by tobacco streak virus (TSV) causes significant yield losses in sunflower in the tropics and sub-tropics. Genetic engineering of sunflower through deployment of the coat protein gene of TSV (TSV-CP) was attempted along with neomycin phosphotransferase gene (nptII) for selection of putative transformants on kanamycin. Split cotyledons from decoated mature seeds were used as target tissues for Agrobacterium tumefaciens-mediated transformation and putative transformed shoots were obtained with an average frequency of 3.3%. Presence of the introduced transgene was identified by PCR; expression was determined through RT-PCR, northern blot analysis and quantitative Real-time PCR (qRT-PCR); stable integration was ascertained through Southern blot analysis of plants in T0 to T4 generations. Out of 102 positive T0 events, 20 events were carried to T1 generation from which five events (CP-S-237, CP-S-247, CP-S-481, CP-S-648 and CP-S-753) were advanced till T4 generation. Challenge inoculations of plants of event No 481 showed resistance to necrosis disease and plants grew to maturity in TSV-CP transgenics while control plants (untransformed) showed mortality within 1–2 weeks following inoculation. Expression analysis of the TSV-CP and nptII genes in different tissues at flowering and seed setting stages revealed constitutive expression of the transgene till seed maturation. One event (No 481) was selected for transfer of the TSV-CP gene into agronomically superior genotypes.

Cloning and characterization of SucNHX1, a novel vacular Na+/H+ antiporter from the halophyte Suaeda corniculata that enhances the saline-alkali tolerance in Arabidopsis by its overexpression

Vacuolar Na+/H+ antiporters play central roles in plant saline (NaCl) stress response. However, the functional characterizations of antiporters involved in plant saline-alkali (Na2CO3/NaHCO3) tolerance are limited. In this study, SucNHX1, a Na+/H+ antiporter gene was isolated from Suaeda corniculata, a halophyte native to the saline-alkali soil, and further studied in transgenic Arabidopsis thaliana. The SucNHX1 gene contains 2123 nucleotides with an open reading frame of 1665 nucleotides (GenBank accession number: DQ512716), encoding a polypeptide of 554 amino acids with a predicted molecular mass of 61.2 kDa. A northern blot analysis indicated that the enhanced mRNA accumulation of SucNHX1 was induced by sodium carbonate (Na2CO3) rather than by NaCl, polyethylene glycol or abscisic acid in S. corniculata seedlings. SucNHX1 was localized in membrane, nucleus and cytoplasm when it was transiently expressed in tobacco mesophyll cells. The expression of SucNHX1 in salt sensitive yeast mutant ΔenaΔnhx1 which lost of yeast vacuolar Na+/H+ antiporter ScNHX1 showed functional complementation. The transgenic Arabidopsis overexpressing SucNHX1 had higher Na2CO3 and NaCl tolerance levels than the wild-type plants. The expression patern of SucNHX1 under Na2CO3 and high NaCl stress in transgenic plants showed that SucNHX1 higher expressed under stress condition than normal control. The analysis of ion contents indicated that under Na2CO3 and NaCl conditions, there were more Na+ in the roots and higher K+/Na+ ratio in the leaves in the transgenic plants than in the wild-type plants. Thus, SucNHX1 may play a significant role in plant saline-alkali tolerance.

MicroRNA‑130a inhibits the proliferation, migration and invasive ability of hepatocellular carcinoma cells by downregulating Rho‑kinase 2.

MicroRNA‑130a (miR‑130a) has been reported to be downregulated in hepatocellular carcinoma (HCC). However, the roles and underlying tumor‑suppressive mechanisms of miR‑130a in the pathogenesis of HCC remain unclear. In the current study, reduced expression of miR‑130a was observed in tumor tissues of patients with HCC in addition to in four HCC cell lines, BEL‑7402, MHCC97H, HepG2 and Huh7. Results of methyl thiazolyl tetrazolium (MTT) assays identified decreased growth rates of MHCC97H and HepG2 cells transfected with miR‑130a mimics. The invitro colony formation assays demonstrated that the number of colonies formed by cells transfected with miR‑130a mimics and cells transfected with miR‑130a inhibitors was lower and higher, respectively, than that formed by the cells transfected with miR‑negative control. In addition, it was identified that overexpression of miR‑130a reduced the migration and invasiveness of MHCC97H and HepG2 cells. Luciferase reporter assays demonstrated that miR‑130a directly targeted the 3'‑untranslated region of Rho‑kinase 2 (ROCK2) mRNA. Northern and western blot analyses indicated that miR‑130a could modulate the mRNA and protein expression of ROCK2. Additionally, small‑interfering RNA‑mediated knockdown of ROCK2 decreased the proliferation, migration and invasiveness of MHCC97H and HepG2 cells. Overall, these observation suggest that miR‑130a is a regulator of ROCK2 and can inhibit proliferation, migration and invasive ability of HCC cells, at least in part, by suppressing the expression of ROCK2. The current study provides further insight into the molecular mechanisms of HCC pathogenesis and suggests a new potential biotarget for HCC treatment.

DROSHA Knockout Leads to Enhancement of Viral Titers for Vectors Encoding miRNA-Adapted shRNAs

RNAi-based gene therapy using miRNA-adapted short hairpin RNAs (shRNAmiR) is a powerful approach to modulate gene expression. However, we have observed low viral titers with shRNAmiR-containing recombinant vectors and hypothesized that this could be due to cleavage of viral genomic RNA by the endogenous microprocessor complex during virus assembly. To test this hypothesis, we targeted DROSHA, the core component of the microprocessor complex, and successfully generated monoallelic and biallelic DROSHA knockout (KO) HEK293T cells for vector production. DROSHA KO was verified by polymerase chain reaction (PCR) and western blot analysis. We produced lentiviral vectors containing Venus with or without shRNA hairpins and generated virus supernatants using DROSHA KO packaging cells. We observed an increase in the fluorescence intensity of hairpin-containing Venus transcripts in DROSHA KO producer cells consistent with reduced microprocessor cleavage of encoded mRNA transcripts, and recovery in the viral titer of hairpin-containing vectors compared with non-hairpin-containing constructs. We confirmed the absence of significant shRNAmiR processing by northern blot analysis and showed that this correlated with an increase in the amount of full-length vector genomic RNA. These findings may have important implications in future production of viral shRNAmiR-containing vectors for RNAi-based therapy.

Identification of heat shock-inducible transcripts and Hop (Hsp-organizing protein)-interacting proteins in orchardgrass (Dactylis glomerata L.)

Plants are continuously exposed to temperature changes and biotic/abiotic stresses. Current global warming dynamics cause extreme temperature fluctuations that severely affect crop productivity. Orchardgrass (Dactylis glomerata L.) is one of the most important perennial grasses adapted for cool temperatures. Orchardgrass displays rapid establishment, vigorous growth, and rapid recovery after cutting or grazing, but it is sensitive to heat stress. In this study, we isolated 23 heat shock-inducible partial gene fragments from orchardgrass using a differential-display RT-PCR and a reverse northern blotting. Those full-length cDNAs encode major molecular chaperones (Hsp90s, Hsp70, and small Hsp) and Hsp90 co-chaperones (Hop, p23, and Aha) were identified from a heat-treated orchardgrass cDNA library and analyzed those expressions by northern blot analysis. In addition, we further characterized Hop, which plays as an important adapter that organizes the Hsp90 and Hsp70 complex in the Hsp90-associated heteromultimeric chaperone complex. Orchardgrass Hop (DgHop) transcript levels were enhanced by heat shock stress and dramatically reduced by cold, salt, or dehydration stress. DgHop contains three conserved tetratricopeptide repeat domains, which are involved in interactions with major Hsps. Yeast two-hybrid and pull-down analysis indicated that the major DgHop client proteins were Hsp90 and Hsp70, and DgHop directly interacted with DgHsp90 and DgHsp70. Our data provide new genetic information on heat shock responses in orchardgrass. We suggest that this information contribute to generating heat shock-tolerant forage crops.

NS3 Protein from Rice stripe virus affects the expression of endogenous genes in Nicotiana benthamiana

BackgroundRice stripe virus (RSV) belongs to the genus Tenuivirus. It is transmitted by small brown planthoppers in a persistent and circulative-propagative manner and causes rice stripe disease (RSD). The NS3 protein of RSV, encoded by the viral strand of RNA3, is a viral suppressor of RNA silencing (VSR). NS3 plays a significant role in viral infection, and NS3-transgenic plants manifest resistance to the virus.MethodsThe stability and availability of NS3 produced by transgenic Nicotiana benthamiana was investigated by northern blot analysis. The accumulation of virus was detected by western blot analysis. Transcriptome sequencing was used to identify differentially expressed genes (DEGs) in NS3-transgenic N. benthamiana.ResultsWhen the host plants were inoculated with RSV, symptoms and viral accumulation in NS3-transgenic N. benthamiana were reduced compared with the wild type. Transcriptome analysis identified 2533 differentially expressed genes (DEGs) in the NS3-transgenic N. benthamiana, including 597 upregulated genes and 1936 downregulated genes. These DEGs were classified into three Gene Ontology (GO) categories and were associated with 43 GO terms. KEGG pathway analysis revealed that these DEGs were involved in pathways associated with ribosomes (ko03010), photosynthesis (ko00195), photosynthesis-antenna proteins (ko00196), and carbon metabolism (ko01200). More than 70 DEGs were in these four pathways. Twelve DEGs were selected for RT-qPCR verification and subsequent analysis. The results showed that NS3 induced host resistance by affecting host gene expression.ConclusionNS3, which plays dual roles in the process of infection, may act as a VSR during RSV infection, and enable viral resistance in transgenic host plants. NS3 from RSV affects the expression of genes associated with ribosomes, photosynthesis, and carbon metabolism in N. benthamiana. This study enhances our understanding of the interactions between VSRs and host plants.

The chimeric mitochondrial gene orf182 causes non-pollen-type abortion in Dongxiang cytoplasmic male-sterile rice.

D1-cytoplasmic male sterility (CMS) rice is a sporophytic cytoplasmic male-sterile rice developed from Dongxiang wild rice that exhibits a no-pollen-grain phenotype. A mitochondrial chimeric gene (orf182) was detected by mitochondrial genome sequencing and a comparative analysis. Orf182 is composed of three recombinant fragments, the largest of which is homologous to Sorghum bicolor mitochondrial sequences. In addition, orf182 was found only in wild rice species collected from China. Northern blot analysis showed that orf182 transcripts were affected by Rf genes in the isocytoplasmic restorer line DR7. Western blot analysis showed that the ORF182 product was localized in the mitochondria of the CMS line. An expression cassette containing orf182 fused to a mitochondrial transit peptide induced the maintainer line of male sterility, which lacked pollen grains in the anthers. Furthermore, the invivo expression of orf182 also inhibited the growth of Escherichia coli, with lower respiration rate, excess accumulation of reactive oxygen species and decreased ATP levels. We conclude that the mitochondrial chimeric gene orf182 possesses a unique structure and origin differing from other identified mitochondrial CMS genes, and this gene is connected to non-pollen type of sporophytic male sterility in D1-CMS rice.

Genome-wide screening of potential RNase Y-processed mRNAs in the M49 serotype Streptococcus pyogenes NZ131.

RNase Y is a major endoribonuclease in Group A streptococcus (GAS) and other Gram‐positive bacteria. Our previous study showed that RNase Y was involved in mRNA degradation and processing in GAS. We hypothesized that mRNA processing regulated the expression of important GAS virulence factors via altering their mRNA stabilities and that RNase Y mediated at least some of the mRNA‐processing events. The aims of this study were to (1) identify mRNAs that were processed by RNase Y and (2) confirm the mRNA‐processing events. The transcriptomes of Streptococcus pyogenes NZ131 wild type and its RNase Y mutant (Δrny) were examined with RNA‐seq. The data were further analyzed to define GAS operons. The mRNA stabilities of the wild type and Δrny at subgene level were determined with tiling array analysis. Operons displaying segmental stability in the wild type but not in the Δrny were predicted to be RNase Y processed. Overall 865 operons were defined and their boundaries predicted. Further analysis narrowed down 15 mRNAs potentially processed by RNase Y. A selection of four candidates including folC1 (folylpolyglutamate synthetase), prtF (fibronectin‐binding protein), speG (streptococcal exotoxin G), ropB (transcriptional regulator of speB), and ypaA (riboflavin transporter) mRNAs was examined with Northern blot analysis. However, only folC1 was confirmed to be processed, but it is unlikely that RNase Y is responsible. We conclude that GAS use RNase Y to selectively process mRNA, but the overall impact is confined to selected virulence factors.