RNA editing underlies genetic risk of common inflammatory diseases.

A major challenge in human genetics is to identify the molecular mechanisms of trait-associated and disease-associated variants. To achieve this, quantitative trait locus (QTL) mapping of genetic variants with intermediate molecular phenotypes such as gene expression and splicing have been widely adopted1,2. However, despite successes, the molecular basis for a considerable fraction of trait-associated and disease-associated variants remains unclear3,4. Here we show that ADAR-mediated adenosine-to-inosine RNA editing, a post-transcriptional event vital for suppressing cellular double-stranded RNA (dsRNA)-mediated innate immune interferon responses5-11, is an important potential mechanism underlying genetic variants associated with common inflammatory diseases. We identified and characterized 30,319 cis-RNA editing QTLs (edQTLs) across 49 human tissues. These edQTLs were significantly enriched in genome-wide association study signals for autoimmune and immune-mediated diseases. Colocalization analysis of edQTLs with disease risk loci further pinpointed key, putatively immunogenic dsRNAs formed by expected inverted repeat Alu elements as well as unexpected, highly over-represented cis-natural antisense transcripts. Furthermore, inflammatory disease risk variants, in aggregate, were associated with reduced editing of nearby dsRNAs and induced interferon responses in inflammatory diseases. This unique directional effect agrees with the established mechanism that lack of RNA editing by ADAR1 leads to the specific activation of the dsRNA sensor MDA5 and subsequent interferon responses and inflammation7-9. Our findings implicate cellular dsRNA editing and sensing as a previously underappreciated mechanism of common inflammatory diseases.

A phosphate-binding pocket within the platform-PAZ-connector helix cassette of human Dicer.

We have solved two families of crystal structures of the human Dicer "platform-PAZ-connector helix" cassette in complex with small interfering RNAs (siRNAs). The structures possess two adjacently positioned pockets: a 2 nt 3'-overhang-binding pocket within the PAZ domain (3' pocket) and a phosphate-binding pocket within the platform domain (phosphate pocket). One family of complexes contains a knob-like α-helical protrusion, designated "hDicer-specific helix," that separates the two pockets and orients the bound siRNA away from the surface of Dicer, which could be indicative of a product release/transfer state. In the second complex, the helical protrusion is melted/disordered and the bound siRNA is aligned toward the surface of Dicer, suggestive of a cleavage-competent state. These structures allow us to propose that the transition from the cleavage-competent to the postulated product release/transfer state may involve release of the 5'-phosphate from the phosphate pocket while retaining the 3' overhang in the 3' pocket.

Soybean NIMA-Related Kinase1 Promotes Plant Growth and Improves Salt and Cold Tolerance.

NEK (NIMA-related kinase) is known as a family of serine/threonine kinases which mainly participate in microtubule-related mitotic events in fungi, mammals and other eukaryotes. Our previous studies found that Arabidopsis NEK6 plays an important role in plant response to abiotic stress. We further investigated roles of the NEK family in soybean and found that at least eight members can respond to abiotic stresses. Among them, only GmNEK1, a novel NEK member which is distantly related to Arabidopsis NEK6, enhanced plant growth and promoted salt and cold tolerance in transgenic Arabidopsis plants. The growth of soybean plants harboring GmNEK1-overexpressing hairy roots under saline condition was also improved. A series of stress-related genes including RH3, CORI3 and ALDH10A8 were found to be up-regulated in GmNEK1-overexpressing Arabidopsis plants and soybean hairy roots. Moreover, soybean plants with GmRH3-overexpressing hairy roots exhibited increased salt tolerance, while soybean plants with GmRH3-RNAi (RNA interference) roots were more sensitive to salt stress than the wild-type plants. Our study uncovers a novel role for GmNEK1 in promoting plant adaptive growth under adverse conditions at least partially through up-regulation of GmRH3. Manipulation of these genes in soybean or other crops may improve growth and production under stress conditions.

Phytoactinopolyspora endophytica gen. nov., sp. nov., a halotolerant filamentous actinomycete isolated from the roots of Glycyrrhiza uralensis F.

A novel endophytic actinomycete, designated strain EGI 60009T, was isolated from the roots of Glycyrrhiza uralensis F. collected from Xinjiang Province, north-west China. The isolate was able to grow in the presence of 0-9% (w/v) NaCl. Strain EGI 60009T had particular morphological properties: the substrate mycelia fragmented into rod-like elements and aerial mycelia differentiated into short spore chains. ll-2, 6-Diaminopimelic acid was the cell-wall diamino acid and rhamnose, galactose and glucose were the cell-wall sugars. MK-9(H4) was the predominant menaquinone. The major fatty acids of strain EGI 60009T were iso-C15 : 0, anteiso-C15 : 0, anteiso-C17 : 0, iso-C17 : 0, iso-C17 : 1 and I/anteiso-C17 : 0 B. Mycolic acids were absent. The DNA G+C content of strain EGI 60009T was 70.4 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain EGI 60009T belongs to the family Jiangellaceae and formed a distinct clade in the phylogenetic tree. 16S rRNA gene sequence similarities between strain EGI 60009T and other members of the genera Jiangella and Haloactinopolyspora were 96.1-96.4 and 95.7-96.0%, respectively. Based on these results and supported by morphological, physiological and chemotaxonomic data and numerous phenotypic differences, a novel species of a new genus, Phytoactinopolyspora endophytica gen. nov., sp. nov., is proposed. The type strain of Phytoactinopolyspora endophytica is EGI 60009T ( = KCTC 29657T = CPCC204078T).

Structural insights into mechanisms of the small RNA methyltransferase HEN1.

RNA silencing is a conserved regulatory mechanism in fungi, plants and animals that regulates gene expression and defence against viruses and transgenes. Small silencing RNAs of approximately 20-30 nucleotides and their associated effector proteins, the Argonaute family proteins, are the central components in RNA silencing. A subset of small RNAs, such as microRNAs and small interfering RNAs (siRNAs) in plants, Piwi-interacting RNAs in animals and siRNAs in Drosophila, requires an additional crucial step for their maturation; that is, 2'-O-methylation on the 3' terminal nucleotide. A conserved S-adenosyl-l-methionine-dependent RNA methyltransferase, HUA ENHANCER 1 (HEN1), and its homologues are responsible for this specific modification. Here we report the 3.1 A crystal structure of full-length HEN1 from Arabidopsis in complex with a 22-nucleotide small RNA duplex and cofactor product S-adenosyl-l-homocysteine. Highly cooperative recognition of the small RNA substrate by multiple RNA binding domains and the methyltransferase domain in HEN1 measures the length of the RNA duplex and determines the substrate specificity. Metal ion coordination by both 2' and 3' hydroxyls on the 3'-terminal nucleotide and four invariant residues in the active site of the methyltransferase domain suggests a novel Mg(2+)-dependent 2'-O-methylation mechanism.

Structural basis for piRNA 2'-O-methylated 3'-end recognition by Piwi PAZ (Piwi/Argonaute/Zwille) domains.

Argonaute and Piwi proteins are key players in the RNA silencing pathway, with the former interacting with micro-RNAs (miRNAs) and siRNAs, whereas the latter targets piwi-interacting RNAs (piRNAs) that are 2'-O-methylated (2(')-OCH(3)) at their 3' ends. Germline-specific piRNAs and Piwi proteins play a critical role in genome defense against transposable elements, thereby protecting the genome against transposon-induced defects in gametogenesis and fertility. Humans contain four Piwi family proteins designated Hiwi1, Hiwi2, Hiwi3, and Hili. We report on the structures of Hili-PAZ (Piwi/Argonaute/Zwille) domain in the free state and Hiwi1 PAZ domain bound to self-complementary 14-mer RNAs (12-bp + 2-nt overhang) containing 2(')-OCH(3) and 2'-OH at their 3' ends. These structures explain the molecular basis underlying accommodation of the 2(')-OCH(3) group within a preformed Hiwi1 PAZ domain binding pocket, whose hydrophobic characteristics account for the preferential binding of 2(')-OCH(3) over 2'-OH 3' ends. These results contrast with the more restricted binding pocket for the human Ago1 PAZ domain, which exhibits a reverse order, with preferential binding of 2'-OH over 2(')-OCH(3) 3' ends.

Synergistic Plant-Microbe Interactions between Endophytic Actinobacteria and Their Role in Plant Growth Promotion and Biological Control of Cotton under Salt Stress.

Bacterial endophytes are well-acknowledged inoculants to promote plant growth and enhance their resistance toward various pathogens and environmental stresses. In the present study, 71 endophytic strains associated with the medicinal plant Thymus roseus were screened for their plant growth promotion (PGP), and the applicability of potent strains as bioinoculant has been evaluated. Regarding PGP traits, the percentage of strains were positive for the siderophore production (84%), auxin synthesis (69%), diazotrophs (76%), phosphate solubilization (79%), and production of lytic enzymes (i.e., cellulase (64%), lipase (62%), protease (61%), chitinase (34%), and displayed antagonistic activity against Verticillium dahliae (74%) in vitro. The inoculation of strain XIEG05 and XIEG12 enhanced plant tolerance to salt stress significantly (p < 0.05) through the promotion of shoot, root development, and reduced the activities of antioxidant enzymes (SOD, POD, and CAT), compared with uninoculated controls in vivo. Furthermore, inoculation of strain XIEG57 was capable of reducing cotton disease incidence (DI) symptoms caused by V. dahliae at all tested salt concentrations. The GC-MS analysis showed that many compounds are known to have antimicrobial and antifungal activity. Our findings provide valuable information for applying strains XIEG05 and XIEG12 as bioinoculant fertilizers and biological control agent of cotton under saline soil conditions.

The Metabolic Potential of Endophytic Actinobacteria Associated with Medicinal Plant Thymus roseus as a Plant-Growth Stimulator.

Bio-fertilizer practice considers not only economical but also environmentally friendly, sustainable agriculture. Endophytes can play important beneficiary roles in plant development, directly, indirectly, or synergistically. In this study, the majority of our endophytic actinobacteria were able to possess direct plant growth-promoting (PGP) traits, including auxin (88%), ammonia (96%), siderophore production (94%), and phosphate solubilization (24%), along with cell-wall degrading enzymes such as protease (75%), cellulase (81%), lipase (81%), and chitinase (18%). About 45% of tested strains have an inhibitory effect on the phytopathogen Fusarium oxysporum, followed by 26% for Verticillium dahlia. Overall, our results showed that strains XIEG63 and XIEG55 were the potent strains with various PGP traits that caused a higher significant increase (p ≤ 0.05) in length and biomass in the aerial part and roots of tomato and cotton, compared to the uninoculated plants. Our data showed that the greatest inhibition percentages of two phytopathogens were achieved due to treatment with strains XIEG05, XIEG07, XIEG45, and XIEG51. The GC-MS analysis showed that most of the compounds were mainly alkanes, fatty acid esters, phenols, alkenes, and aromatic chemicals and have been reported to have antifungal activity. Our investigation emphasizes that endophytic actinobacteria associated with medicinal plants might help reduce the use of chemical fertilization and potentially lead to increased agricultural productivity and sustainability.

Structural basis for 5'-end-specific recognition of guide RNA by the A. fulgidus Piwi protein.

RNA interference (RNAi) is a conserved sequence-specific gene regulatory mechanism mediated by the RNA-induced silencing complex (RISC), which is composed of a single-stranded guide RNA and an Argonaute protein. The PIWI domain, a highly conserved motif within Argonaute, has been shown to adopt an RNase H fold critical for the endonuclease cleavage activity of RISC. Here we report the crystal structure of Archaeoglobus fulgidus Piwi protein bound to double-stranded RNA, thereby identifying the binding pocket for guide-strand 5'-end recognition and providing insight into guide-strand-mediated messenger RNA target recognition. The phosphorylated 5' end of the guide RNA is anchored within a highly conserved basic pocket, supplemented by the carboxy-terminal carboxylate and a bound divalent cation. The first nucleotide from the 5' end of the guide RNA is unpaired and stacks over a conserved tyrosine residue, whereas successive nucleotides form a four-base-pair RNA duplex. Mutation of the corresponding amino acids that contact the 5' phosphate in human Ago2 resulted in attenuated mRNA cleavage activity. Our structure of the Piwi-RNA complex, and that determined elsewhere, provide direct support for the 5' region of the guide RNA serving as a nucleation site for pairing with target mRNA and for a fixed distance separating the RISC-mediated mRNA cleavage site from the anchored 5' end of the guide RNA.

Structural basis for overhang-specific small interfering RNA recognition by the PAZ domain.

Short RNAs mediate gene silencing, a process associated with virus resistance, developmental control and heterochromatin formation in eukaryotes. RNA silencing is initiated through Dicer-mediated processing of double-stranded RNA into small interfering RNA (siRNA). The siRNA guide strand associates with the Argonaute protein in silencing effector complexes, recognizes complementary sequences and targets them for silencing. The PAZ domain is an RNA-binding module found in Argonaute and some Dicer proteins and its structure has been determined in the free state. Here, we report the 2.6 A crystal structure of the PAZ domain from human Argonaute eIF2c1 bound to both ends of a 9-mer siRNA-like duplex. In a sequence-independent manner, PAZ anchors the 2-nucleotide 3' overhang of the siRNA-like duplex within a highly conserved binding pocket, and secures the duplex by binding the 7-nucleotide phosphodiester backbone of the overhang-containing strand and capping the 5'-terminal residue of the complementary strand. On the basis of the structure and on binding assays, we propose that PAZ might serve as an siRNA-end-binding module for siRNA transfer in the RNA silencing pathway, and as an anchoring site for the 3' end of guide RNA within silencing effector complexes.

Beneficial Endophytic Bacterial Populations Associated With Medicinal Plant Thymus vulgaris Alleviate Salt Stress and Confer Resistance to Fusarium oxysporum.

As a result of climate change, salinity has become a major abiotic stress that reduces plant growth and crop productivity worldwide. A variety of endophytic bacteria alleviate salt stress; however, their ecology and biotechnological potential has not been fully realized. To address this gap, a collection of 117 endophytic bacteria were isolated from wild populations of the herb Thymus vulgaris in Sheikh Zuweid and Rafah of North Sinai Province, Egypt, and identified based on their 16S rRNA gene sequences. The endophytes were highly diverse, including 17 genera and 30 species. The number of bacterial species obtained from root tissues was higher (n = 18) compared to stem (n = 14) and leaf (n = 11) tissue. The endophytic bacteria exhibited several plant growth-promoting activities in vitro, including auxin synthesis, diazotrophy, phosphate solubilization, siderophore production, and production of lytic enzymes (i.e., chitinase, cellulase, protease, and lipase). Three endophytes representing Bacillus species associated with T. vulgaris such as EGY05, EGY21, and EGY25 were selected based on their ex-situ activities for growth chamber assays to test for their ability to promote the growth of tomato (Solanum lycopersicum L.) under various NaCl concentrations (50-200 mM). All three strains significantly (P < 0.05) promoted the growth of tomato plants under salt stress, compared to uninoculated controls. In addition, inoculated tomato plants by all tested strains decreased (P < 0.05) the activity of antioxidant enzymes (superoxide dismutase, catalase, and peroxidase). Six strains, representing Bacillus and Enterobacter species EGY01, EGY05, EGY16, EGY21, EGY25, and EGY31 were selected based on in vitro antagonistic activity to F. oxysporum for pot experiments under salt stress. All tested strains reduced the disease severity index (DSI) of tomato plants at all tested salt concentrations. Gas-chromatography/mass-spectrometry analysis of cell-free extracts of B. subtilis (EGY16) showed at least ten compounds were known to have antimicrobial activity, with the major peaks being benzene, 1,3-dimethyl-, p-xylene, dibutyl phthalate, bis (2-ethylhexyl) phthalate, and tetracosane. This study demonstrates that diverse endophytes grow in wild thyme populations and that some are able to alleviate salinity stress and inhibit F. oxysporum pathogenesis, making them promising candidates for biofertilizers and biocontrol agents.

Author Correction: Genome-wide identification of Major Intrinsic Proteins in Glycine soja and characterization of GmTIP2;1 function under salt and water stress.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

An Alfin-like gene from Atriplex hortensis enhances salt and drought tolerance and abscisic acid response in transgenic Arabidopsis.

Alfin-like (AL) is a small plant-specific gene family with prominent roles in root growth and abiotic stress response. Here, we aimed to identify novel stress tolerance AL genes from the stress-tolerant species Atriplex hortensis. Totally, we isolated four AhAL genes, all encoding nuclear-localized proteins with cis-element-binding and transrepression activities. Constitutive expression of AhAL1 in Arabidopsis facilitated plants to survive under saline condition, while expressing anyone of the other three AhAL genes led to salt-hypersensitive response, indicating functional divergence of AhAL family. AhAL1 also conferred enhanced drought tolerance, as judged from enhanced survival, improved growth, decreased malonaldehyde (MDA) content and reduced water loss in AhAL1-expressing plants compared to WT. In addition, abscisic acid (ABA)-mediated stomatal closure and inhibition of seed germination and primary root elongation were enhanced in AhAL1-transgenic plants. Further analysis demonstrated that AhAL1 could bind to promoter regions of GRF7, DREB1C and several group-A PP2C genes and repress their expression. Correspondingly, the expression levels of positive stress regulator genes DREB1A, DREB2A and three ABFs were all increased in AhAL1-expressing plants. Based on these results, AhAL1 was identified as a novel candidate gene for improving abiotic stress tolerance of crop plants.

Evaluation of the Antimicrobial Activity of Endophytic Bacterial Populations From Chinese Traditional Medicinal Plant Licorice and Characterization of the Bioactive Secondary Metabolites Produced by Bacillus atrophaeus Against Verticillium dahliae.

Endophytic bacteria associated with medicinal plants possess unique strategies that enhance growth and suvival of host plants, many of which are mediated by distinctive secondary metabolites. These bacteria and their secondary metabolites are important subjects for both basic and applied research aimed at sustainable agriculture. In the present study, 114 endophytic strains isolated from the wild ethnomedicinal plant Glycyrrhiza uralensis (licorice) were screened for their in vitro antimicrobial activities against common fungal pathogens of tomato (Fusarium oxysporum f. sp., Fulvia fulva, Alternaria solani), cotton (Fusarium oxysporum f. sp. Vesinfectum, Verticillium dahliae), pomegranite (Ceratocystis fimbriata), Cymbidinium (Colletotrichum gloeosporioides), and Tsao-ko (Pestalotiopsis microspora and Fusarium graminearum) and the common bacteria Staphylococcus aureus, Bacillus cereus, Salmonella enteritidis, and Escherichia coli. Several Bacillus strains, particularly Bacillus atrophaeus and Bacillus mojavensis, had a broad spectrum of antifungal and antibacterial activity. A total of 16 strains, selected based on broad antimicrobial activity, were shown to contain at least one putative secondary metabolite-encoding gene (i.e., polyketide synthase or non-ribosomal peptide synthetase) and/or one lytic enzyme (i.e., protease, cellulase, lipase, chitinase), which may be important mediators of antagonistic activity against pathogens. Five strains, representing Bacillus atrophaeus and Bacillus mojavensis, were selected for plant growth chamber experiments based on strong in vitro antifungal activities. All five strains significantly reduced disease severity in Arabidopsis thaliana plants challenged with V. dahlia infection. Gas-chromatography/mass-spectrometry analysis of cell-free extracts of Bacillus atrophaeus strain XEGI50 showed that at least 13 compounds were produced only during co-cultivation with V. dahlia, including putative compounds known to have antimicrobial activity, such as 1,2-benzenedicarboxylic acid, bis (2-methylpropyl) ester; 9,12-octadecadienoic acid (Z,Z)-, methyl ester; 9-octadecenoic acid, methyl ester, (E)-; and decanedioic acid, bis(2-ethylhexyl) ester. To our knowledge, this study is the first to report that bacteria isolated from G. uralensis have biocontrol abilities. Our findings provide new insights into the antimicrobial activities of natural endophytes, particularly B. atrophaeus, and suggest this species may a promising candidate as a biocontrol agent to confer resistance to Verticillium wilt disease and other phytopathogens in cotton and other crops.

Production, characterization and structural modification of exopolysaccharide-based bioflocculant by Rhizobium radiobacter SZ4S7S14 and media optimization.

Synthesis of the exopolysaccharide-based bioflocculant Rhizobium radiobacter SZ4S7S14 was researched and medium optimized for enhanced production of bioflocculant. D-Mannose and yeast extract were found to be best carbon and nitrogen sources for maximal yield of bioflocculant. The bioflocculant samples obtained in different media with different carbon and nitrogen sources were further analyzed by SEM-EDX and FT-IR. FT-IR spectroscopy of the bioflocculant samples, obtained in different carbon/nitrogen sources showed slight modification of the structures of biopolymers. SEM micrographs revealed that exopolysaccharide-based bioflocculant of R. radiobacter SZ4S7S14 looks like bricks, and chemical structure of it can be varied due to utilized carbon and nitrogen source.

Genome-wide identification of Major Intrinsic Proteins in Glycine soja and characterization of GmTIP2;1 function under salt and water stress.

In different plant species, aquaporins (AQPs) facilitate water movement by regulating root hydraulic conductivity under diverse stress conditions such as salt and water stresses. To improve survival and yield of crop plants, a detailed understanding of stress responses is imperative and required. We used Glycine soja genome as a tool to study AQPs, considering it shows abundant genetic diversity and higher salt environment tolerance features and identified 62 Gs AQP genes. Additionally, this study identifies major aquaporins responsive to salt and drought stresses in soybean and elucidates their mode of action through yeast two-hybrid assay and BiFC. Under stress condition, the expression analysis of AQPs in roots and leaves of two contrasting ecotypes of soybean revealed diverse expression patterns suggesting complex regulation at transcriptional level. Based on expression analysis, we identify GmTIP2;1 as a potential candidate involved in salinity and drought responses. The overexpression of GmTIP2;1 in Saccharomyces cerevisiae as well as in-planta enhanced salt and drought tolerance. We identified that GmTIP2;1 forms homodimers as well as interacts with GmTIP1;7 and GmTIP1;8. This study augments our knowledge of stress responsive pathways and also establishes GmTIP2;1 as a new stress responsive gene in imparting salt stress tolerance in soybean.

An interlocked dimeric parallel-stranded DNA quadruplex: a potent inhibitor of HIV-1 integrase.

We report on the NMR-based solution structure of the 93del d(GGGGTGGGAGGAGGGT) aptamer, a potent nanomolar inhibitor of HIV-1 integrase. This guanine-rich DNA sequence adopts an unusually stable dimeric quadruplex architecture in K+ solution. Within each 16-nt monomer subunit, which contains one A.(G.G.G.G) pentad sandwiched between two G.G.G.G tetrads, all G-stretches are parallel, and all guanines are anti with the exception of G1, which is syn. Dimer formation is achieved through mutual pairing of G1 of one monomer, with G2, G6, and G13 of the other monomer, to complete G.G.G.G tetrad formation. There are three single-nucleotide double-chain-reversal loops within each monomer fold, such that the first (T5) and third (A12) loops bridge three G-tetrad layers, whereas the second (A9) loop bridges two G-tetrad layers and participates in A.(G.G.G.G) pentad formation. Results of NMR and of integrase inhibition assays on loop-modified sequences allowed us to propose a strategy toward the potential design of improved HIV-1 integrase inhibitors. Finally, we propose a model, based on molecular docking approaches, for positioning the 93del dimeric DNA quadruplex within a basic channel/canyon formed between subunits of a dimer of dimers of HIV-1 integrase.

Crystal structure of A. aeolicus argonaute, a site-specific DNA-guided endoribonuclease, provides insights into RISC-mediated mRNA cleavage.

Argonaute (Ago) proteins constitute a key component of the RNA-induced silencing complex (RISC). We report the crystal structure of Aquifex aeolicus Ago (Aa-Ago) together with binding and cleavage studies, which establish this eubacterial Ago as a bona fide guide DNA strand-mediated site-specific RNA endonuclease. We have generated a stereochemically robust model of the complex, where the guide DNA-mRNA duplex is positioned within a basic channel spanning the bilobal interface, such that the 5' phosphate of the guide strand can be anchored in a basic pocket, and the mRNA can be positioned for site-specific cleavage by RNase H-type divalent cation-coordinated catalytic Asp residues of the PIWI domain. Domain swap experiments involving chimeras of human Ago (hAgo1) and cleavage-competent hAgo2 reinforce the role of the PIWI domain in "slicer" activity. We propose a four-step Ago-mediated catalytic cleavage cycle model, which provides distinct perspectives into the mechanism of guide strand-mediated mRNA cleavage within the RISC.