Molecular insights into the function of the viral RNA silencing suppressor HCPro

Potyviral helper component proteinase (HCPro) is a well‐characterized suppressor of antiviral RNA silencing, but its mechanism of action is not yet fully understood. In this study, we used affinity purification coupled with mass spectrometry to identify binding partners of HCPro in potyvirus‐infected plant cells. This approach led to identification of various HCPro interactors, including two key enzymes of the methionine cycle, S–adenosyl‐l –methionine synthase and S–adenosyl‐l –homocysteine hydrolase. This finding, together with the results of enzymatic activity and gene knockdown experiments, suggests a mechanism in which HCPro complexes containing viral and host proteins act to suppress antiviral RNA silencing through local disruption of the methionine cycle. Another group of HCPro interactors identified in this study comprised ribosomal proteins. Immunoaffinity purification of ribosomes demonstrated that HCPro is associated with ribosomes in virus‐infected cells. Furthermore, we show that HCPro and ARGONAUTE1 (AGO1), the core component of the RNA‐induced silencing complex (RISC), interact with each other and are both associated with ribosomes in planta. These results, together with the fact that AGO1 association with ribosomes is a hallmark of RISC‐mediated translational repression, suggest a second mechanism of HCPro action, whereby ribosome‐associated multiprotein complexes containing HCPro relieve viral RNA translational repression through interaction with AGO1.     

Enhanced S-adenosyl-l-methionine production in Saccharomyces cerevisiae by spaceflight culture, overexpressing methionine adenosyltransferase and optimizing cultivation

Aims: S‐adenosyl‐l ‐methionine (SAM) is an important biochemical molecule with great potential in the pharmacological and chemotherapeutic fields. In this study, our aims were to enhance SAM production in Saccharomyces cerevisiae. Methods and Results: Through spaceflight culture, a SAM‐accumulating strain, S. cerevisiae H5M147, was isolated and found to produce 86·89% more SAM than its ground control strain H5. Amplified fragment length polymorphism (AFLP) analysis demonstrated that there were genetic variations between strain H5M147 and its ground control. Through recombinant DNA technology, the heterologous gene encoding methionine adenosyltransferase was integrated into the genome of strain H5M147. The recombinant strain H5MR83 was selected because its SAM production was increased by 42·98% when compared to strain H5M147. Furthermore, cultivation conditions were optimized using the one‐factor‐at‐a‐time and Taguchi methods. Under optimal conditions, strain H5MR83 yielded 7·76 g l^?1 of SAM in shake flask, an increase of 536·07% when compared to the strain H5. Furthermore, 9·64 g l^?1 of SAM was produced in fermenter cultivation. Conclusions: A new SAM‐accumulating strain, S. cerevisiae H5MR83, was obtained through spaceflight culture and genetic modification. Under optimal conditions, SAM production was increased to a relative high level in our study. Significance and Impact of the Study: Through comprehensive application of multiple methods including spaceflight culture, genetic modification and optimizing cultivation, the yield of SAM could be increased by 6·4 times compared to that in the control strain H5. The obtained S. cerevisiae H5MR83 produced 7·76 g l^?1 of SAM in the flask cultures, a significant improvement on previously reported results. The SAM production period with S. cerevisiae H5MR83 was 84 h, which is shorter than previously reported results. Saccharomyces cerevisiae H5MR83 has considerable potential for use in industrial applications.     

Overexpression of S‐adenosyl‐l‐methionine synthetase increased tomato tolerance to alkali stress through polyamine metabolism

S‐adenosyl‐l ‐methionine (SAM) synthetase is the key enzyme involved in the biosynthesis of SAM, which serves as a common precursor for polyamines (PAs) and ethylene. A SAM synthetase cDNA (SlSAMS1) was introduced into the tomato genome using the Agrobacterium tumefaciens transformation method. Transgenic plants overexpressing SlSAMS1 exhibited a significant increase in tolerance to alkali stress and maintained nutrient balance, higher photosynthetic capacity and lower oxidative stress compared with WT lines. Both in vivo and in vitro experiments indicated that the function of SlSAMS1 mainly depended on the accumulation of Spd and Spm in the transgenic lines. A grafting experiment showed that rootstocks from SlSAMS1‐overexpressing plants provided a stronger root system, increased PAs accumulation, essential elements absorption, and decreased Na⁺ absorption in the scions under alkali stress. As a result, fruit set and yield were significantly enhanced. To our knowledge, this is the first report to provide evidence that SlSAMS1 positively regulates tomato tolerance to alkali stress and plays a major role in modulating polyamine metabolism, resulting in maintainability of nutrient and ROS balance.     

Crystal structure of Streptococcus pneumoniae Sp1610, a putative tRNA methyltransferase,in complex with S‐adenosyl‐L‐methionine

Streptococcus pneumoniae Sp1610, a Class‐I fold S‐adenosylmethionine (AdoMet)‐dependent methyltransferase, is a member of the COG2384 family in the Clusters of Orthologous Groups database, which catalyzes the methylation of N¹‐adenosine at position 22 of bacterial tRNA. We determined the crystal structure of Sp1610 in the ligand‐free and the AdoMet‐bound forms at resolutions of 2.0 and 3.0 Å, respectively. The protein is organized into two structural domains: the N‐terminal catalytic domain with a Class I AdoMet‐dependent methyltransferase fold, and the C‐terminal substrate recognition domain with a novel fold of four α‐helices. Observations of the electrostatic potential surface revealed that the concave surface located near the AdoMet binding pocket was predominantly positively charged, and thus this was predicted to be an RNA binding area. Based on the results of sequence alignment and structural analysis, the putative catalytic residues responsible for substrate recognition are also proposed.     

Crystal structure of the Legionella pneumophila Lpg2936 in complex with the cofactor S‐adenosyl‐L‐methionine reveals novel insights into the mechanism of RsmE family methyltransferases

The methylation of U1498 located in the 16S ribosomal RNA of Escherichia coli is an important modification affecting ribosomal activity. RsmE methyltransferases methylate specifically this position in a mechanism that requires an S‐adenosyl‐L‐methionine (AdoMet) molecule as cofactor. Here we report the structure of Apo and AdoMet‐bound Lpg2936 from Legionella pneumophila at 1.5 and 2.3 Å, respectively. The protein comprises an N‐terminal PUA domain and a C‐terminal SPOUT domain. The latter is responsible for protein dimerization and cofactor binding. Comparison with similar structures suggests that Lpg2936 is an RsmE‐like enzyme that can target the equivalent of U1498 in the L. pneumophila ribosomal RNA, thereby potentially enhancing ribosomal activity during infection‐mediated effector production. The multiple copies of the enzyme found in both structures reveal a flexible conformation of the bound AdoMet ligand. Isothermal titration calorimetry measurements suggest an asymmetric two site binding mode. Our results therefore also provide unprecedented insights into AdoMet/RsmE interaction, furthering our understanding of the RsmE catalytic mechanism.     

Structures of the Neisseria meningitides methionine‐binding protein MetQ in substrate‐free form and bound to l‐ and d‐methionine isomers

The bacterial periplasmic methionine‐binding protein MetQ is involved in the import of methionine by the cognate MetNI methionine ATP binding cassette (ABC) transporter. The MetNIQ system is one of the few members of the ABC importer family that has been structurally characterized in multiple conformational states. Critical missing elements in the structural analysis of MetNIQ are the structure of the substrate‐free form of MetQ, and detailing how MetQ binds multiple methionine derivatives, including both l ‐ and d ‐methionine isomers. In this study, we report the structures of the Neisseria meningitides MetQ in substrate‐free form and in complexes with l ‐methionine and with d ‐methionine, along with the associated binding constants determined by isothermal titration calorimetry. Structures of the substrate‐free (N238A) and substrate‐bound N. meningitides MetQ are related by a “Venus‐fly trap” hinge‐type movement of the two domains accompanying methionine binding and dissociation. l ‐ and d ‐methionine bind to the same site on MetQ, and this study emphasizes the important role of asparagine 238 in ligand binding and affinity. A thermodynamic analysis demonstrates that ligand‐free MetQ associates with the ATP‐bound form of MetNI ～40 times more tightly than does liganded MetQ, consistent with the necessity of dissociating methionine from MetQ for transport to occur.     

Induction of systemic disease resistance in Nicotiana benthamiana by the cyclodipeptides cyclo (l‐Pro‐l‐Pro) and cyclo (d‐Pro‐d‐Pro)

Cyclodipeptides, formed from two amino acids by cyclodehydration, are produced naturally by many organisms, and are known to possess a large number of biological activities. In this study, we found that cyclo (l ‐Pro‐l ‐Pro) and cyclo (d ‐Pro‐d ‐Pro) (where Pro is proline) could induce defence responses and systemic resistance in Nicotiana benthamiana. Treatment with the two cyclodipeptides led to a reduction in disease severity by Phytophthora nicotianae and Tobacco mosaic virus (TMV) infections compared with controls. Both cyclopeptides triggered stomatal closure, induced reactive oxygen species production and stimulated cytosolic calcium ion and nitric oxide production in guard cells. In addition, the application of cyclodipeptides significantly up‐regulated the expression of the plant defence gene PR‐1a and the PR‐1a protein, and increased cellular salicylic acid (SA) levels. These results suggest that the SA‐dependent defence pathway is involved in cyclodipeptide‐mediated pathogen resistance in N. benthamiana. We report the systemic resistance induced by cyclodipeptides, which sheds light on the potential of cyclodipeptides for the control of plant diseases.     

Orchestration of hydrogen peroxide and nitric oxide in brassinosteroid‐mediated systemic virus resistance in Nicotiana benthamiana

Brassinosteroids (BRs) play essential roles in modulating plant growth, development and stress responses. Here, involvement of BRs in plant systemic resistance to virus was studied. Treatment of local leaves in Nicotiana benthamiana with BRs induced virus resistance in upper untreated leaves, accompanied by accumulations of H₂O₂ and NO. Scavenging of H₂O₂ or NO in upper leaves blocked BR‐induced systemic virus resistance. BR‐induced systemic H₂O₂ accumulation was blocked by local pharmacological inhibition of NADPH oxidase or silencing of respiratory burst oxidase homolog gene NbRBOHB, but not by systemic NADPH oxidase inhibition or NbRBOHA silencing. Silencing of the nitrite‐dependent nitrate reductase gene NbNR or systemic pharmacological inhibition of NR compromised BR‐triggered systemic NO accumulation, while local inhibition of NR, silencing of NbNOA1 and inhibition of NOS had little effect. Moreover, we provide evidence that BR‐activated H₂O₂ is required for NO synthesis. Pharmacological scavenging or genetic inhibiting of H₂O₂ generation blocked BR‐induced systemic NO production, but BR‐induced H₂O₂ production was not sensitive to NO scavengers or silencing of NbNR. Systemically applied sodium nitroprusside rescued BR‐induced systemic virus defense in NbRBOHB‐silenced plants, but H₂O₂ did not reverse the effect of NbNR silencing on BR‐induced systemic virus resistance. Finally, we demonstrate that the receptor kinase BRI1(BR insensitive 1) is an upstream component in BR‐mediated systemic defense signaling, as silencing of NbBRI1 compromised the BR‐induced H₂O₂ and NO production associated with systemic virus resistance. Together, our pharmacological and genetic data suggest the existence of a signaling pathway leading to BR‐mediated systemic virus resistance that involves local Respiratory Burst Oxidase Homolog B (RBOHB)‐dependent H₂O₂ production and subsequent systemic NR‐dependent NO generation.     

Analysis of ethylene‐induced gene regulation during carposporogenesis in the red seaweed Grateloupia imbricata (Rhodophyta)

Ethylene favors carposporogenesis in the red seaweed Grateloupia imbricata. Analyses of cystocarp development in vitro in thalli treated with ethylene suggest an interconnection between polyamine and ethylene biosynthesis pathways. Yet, little is known about molecular mechanisms underlying carposporogenesis. Here, we used droplet digital PCR to analyze genes encoding enzymes related to polyamine (Spermidine [Spd] synthase) and ethylene (ACC synthase) synthesis; a pivotal compound of both pathways (S‐adenosyl methionine synthase, SAMS); the gene that encodes amine oxidase, which is involved in polyamine degradation, and a candidate gene involved in seaweed reproduction (ornithine decarboxylase, ODC). In addition, we analyzed genes encoding proteins related to stress and reactive oxygen species, ascorbate peroxidase (APX), cytochrome P450 and WD 40. We characterized gene expression in fertilized and fertile thalli from G. imbricata that were exposed to ethylene for 15 min at two time points after treatment (1 and 7 d). The differential gene expression of SAMS, Spd synthase, ACC synthase, and cytochrome P450 was related to disclosure and development of cystocarps in fertilized thalli that transitioned from having no visible cystocarps at 1 d to developing cystocarps at 7 d. Likewise, cytochrome P450 was associated with cystocarp disclosure and maturation. In addition, amine oxidase and APX were involved in fine‐tuning polyamine and reactive oxygen species during carposporogenesis, respectively, whereas WD 40 did so in relation to ethylene signaling. Expression of the candidate gene ODC was increased when cystocarps were not visible (fertilized thalli, 1d), as previously described. This analysis suggests developmental stage‐specific roles for these genes during carposporogenesis.     

Catabolism of l–methionine in the formation of sulfur and other volatiles in melon (Cucumis melo L.) fruit

Sulfur‐containing aroma volatiles are important contributors to the distinctive aroma of melon and other fruits. Melon cultivars and accessions differ in the content of sulfur‐containing and other volatiles. l –methionine has been postulated to serve as a precursor of these volatiles. Incubation of melon fruit cubes with ¹³C‐ and ²H‐labeled l –methionine revealed two distinct catabolic routes into volatiles. One route apparently involves the action of an l ‐methionine aminotransferase and preserves the main carbon skeleton of l ‐methionine. The second route apparently involves the action of an l ‐methionine‐γ–lyase activity, releasing methanethiol, a backbone for formation of thiol‐derived aroma volatiles. Exogenous l ‐methionine also generated non‐sulfur volatiles by further metabolism of α–ketobutyrate, a product of l ‐methionine‐γ–lyase activity. α–Ketobutyrate was further metabolized into l –isoleucine and other important melon volatiles, including non‐sulfur branched and straight‐chain esters. Cell‐free extracts derived from ripe melon fruit exhibited l ‐methionine‐γ–lyase enzymatic activity. A melon gene (CmMGL) ectopically expressed in Escherichia coli, was shown to encode a protein possessing l ‐methionine‐γ–lyase enzymatic activity. Expression of CmMGL was relatively low in early stages of melon fruit development, but increased in the flesh of ripe fruits, depending on the cultivar tested. Moreover, the levels of expression of CmMGL in recombinant inbred lines co‐segregated with the levels of sulfur‐containing aroma volatiles enriched with +1 m/z unit and postulated to be produced via this route. Our results indicate that l ‐methionine is a precursor of both sulfur and non‐sulfur aroma volatiles in melon fruit.     

CYLD negatively regulates cell‐cycle progression by inactivating HDAC6 and increasing the levels of acetylated tubulin

CYLD is a tumour‐suppressor gene that is mutated in a benign skin tumour syndrome called cylindromatosis. The CYLD gene product is a deubiquitinating enzyme that was shown to regulate cell proliferation, cell survival and inflammatory responses, mainly through inhibiting NF‐κB signalling. Here we show that CYLD controls cell growth and division at the G₁/S‐phase as well as cytokinesis by associating with α‐tubulin and microtubules through its CAP‐Gly domains. Translocation of activated CYLD to the perinuclear region of the cell is achieved by an inhibitory interaction of CYLD with histone deacetylase‐6 (HDAC6) leading to an increase in the levels of acetylated α‐tubulin around the nucleus. This facilitates the interaction of CYLD with Bcl‐3, leading to a significant delay in the G₁‐to‐S‐phase transition. Finally, CYLD also interacts with HDAC6 in the midbody where it regulates the rate of cytokinesis in a deubiquitinase‐independent manner. Altogether these results identify a mechanism by which CYLD regulates cell proliferation at distinct cell‐cycle phases.     

Energy–water and seasonal variations in climate underlie the spatial distribution patterns of gymnosperm species richness in China

Studying the pattern of species richness is crucial in understanding the diversity and distribution of organisms in the earth. Climate and human influences are the major driving factors that directly influence the large‐scale distributions of plant species, including gymnosperms. Understanding how gymnosperms respond to climate, topography, and human‐induced changes is useful in predicting the impacts of global change. Here, we attempt to evaluate how climatic and human‐induced processes could affect the spatial richness patterns of gymnosperms in China. Initially, we divided a map of the country into grid cells of 50 × 50 km² spatial resolution and plotted the geographical coordinate distribution occurrence of 236 native gymnosperm taxa. The gymnosperm taxa were separated into three response variables: (a) all species, (b) endemic species, and (c) nonendemic species, based on their distribution. The species richness patterns of these response variables to four predictor sets were also evaluated: (a) energy–water, (b) climatic seasonality, (c) habitat heterogeneity, and (d) human influences. We performed generalized linear models (GLMs) and variation partitioning analyses to determine the effect of predictors on spatial richness patterns. The results showed that the distribution pattern of species richness was highest in the southwestern mountainous area and Taiwan in China. We found a significant relationship between the predictor variable set and species richness pattern. Further, our findings provide evidence that climatic seasonality is the most important factor in explaining distinct fractions of variations in the species richness patterns of all studied response variables. Moreover, it was found that energy–water was the best predictor set to determine the richness pattern of all species and endemic species, while habitat heterogeneity has a better influence on nonendemic species. Therefore, we conclude that with the current climate fluctuations as a result of climate change and increasing human activities, gymnosperms might face a high risk of extinction.     

Detoxification of insecticides,allechemicals and heavy metals by glutathione S‐transferase SlGSTE1 in the gut of Spodoptera litura

Insect glutathione S‐transferases (GSTs) play important roles in detoxifying toxic compounds and eliminating oxidative stress caused by these compounds. In this study, detoxification activity of the epsilon GST SlGSTE1 in Spodoptera litura was analyzed for several insecticides and heavy metals. SlGSTE1 was significantly up‐regulated by chlorpyrifos and xanthotoxin in the midgut of S. litura. The recombinant SlGSTE1 had V_max (reaction rate of the enzyme saturated with the substrate) and K_m (michaelis constant and equals to the substrate concentration at half of the maximum reaction rate of the enzyme) values of 27.95 ± 0.88 μmol/min/mg and 0.87 ± 0.028 mmol/L for glutathione, respectively, and V_max and K_m values of 22.96 ± 0.78 μmol/min/mg and 0.83 ± 0.106 mmol/L for 1‐chloro‐2,4‐dinitrobenzene, respectively. In vitro enzyme indirect activity assay showed that the recombinant SlGSTE1 possessed high binding activities to the insecticides chlorpyrifos, deltamethrin, malathion, phoxim and dichloro‐diphenyl‐trichloroethane (DDT). SlGSTE1 showed higher binding activity to toxic heavy metals cadmium, chromium and lead than copper and zinc that are required for insect normal growth. Western blot analysis showed that SlGSTE1 was induced in the gut of larvae fed with chlorpyrifos or cadmium. SlGSTE1 also showed high peroxidase activity. All the results together indicate that SlGSTE1 may play an important role in the gut of S. litura to protect the insect from the toxic effects of these compounds and heavy metals.     

Beneficial effects of dithiothreitol on relative levels of glutathione S-transferase activity and thiols in oocytes, and cell number, DNA fragmentation and allocation at the blastocyst stage in the mouse

We analyzed the effect of in vitro aging of mouse oocytes in the presence of dithiothreitol (DTT) on relative levels of glutathione S-transferase (GST) activity and thiols in oocytes, and cell number, DNA fragmentation and cellular allocation to the inner cell mass (ICM) and trophectoderm (TE) lineage at the blastocyst stage. Ovulated oocytes from gonadotropin primed hybrid female mice of 6-8 weeks of age were aged in vitro in the presence of 0, 5, 50, or 500 microM DTT for 6 hr prior to insemination. Relative levels of GST activity and thiols in oocytes were determined by confocal laser scanning microscopy, DNA fragmentation using a single-step TUNEL method, and cell allocation to the ICM and TE lineage by blastocyst staining with propidium iodide and Hoechst 33258. Non-aged oocytes exhibited higher relative levels of GST activity and thiols when compared to oocytes aged in the presence of 0, 5, and 50 microM DTT. Day 5 blastocysts from the 5, 50, and 500 microM DTT groups exhibited higher total number of cells, number of ICM cells, and ICM/TE ratio, but lower percentage of number of nuclei with DNA fragmentation/number of ICM cells than blastocyst from the 0 microM DTT group. These data show that DTT counteracts the negative effects of a post-ovulatory aging of mouse oocytes in vitro on relative levels of GST activity and thiols in oocytes, and percentage of number of nuclei with DNA fragmentation/number of ICM cells, total number of cells, number of ICM cells and ICM/TE ratio in Day 5 blastocysts.     

CRISPR/Cas9‐mediated knockout of six glycosyltransferase genes in Nicotiana benthamiana for the production of recombinant proteins lacking β‐1,2‐xylose and core α‐1,3‐fucose

Plants offer fast, flexible and easily scalable alternative platforms for the production of pharmaceutical proteins, but differences between plant and mammalian N‐linked glycans, including the presence of β‐1,2‐xylose and core α‐1,3‐fucose residues in plants, can affect the activity, potency and immunogenicity of plant‐derived proteins. Nicotiana benthamiana is widely used for the transient expression of recombinant proteins so it is desirable to modify the endogenous N‐glycosylation machinery to allow the synthesis of complex N‐glycans lacking β‐1,2‐xylose and core α‐1,3‐fucose. Here, we used multiplex CRISPR/Cas9 genome editing to generate N. benthamiana production lines deficient in plant‐specific α‐1,3‐fucosyltransferase and β‐1,2‐xylosyltransferase activity, reflecting the mutation of six different genes. We confirmed the functional gene knockouts by Sanger sequencing and mass spectrometry‐based N‐glycan analysis of endogenous proteins and the recombinant monoclonal antibody 2G12. Furthermore, we compared the CD64‐binding affinity of 2G12 glycovariants produced in wild‐type N. benthamiana, the newly generated FX‐KO line, and Chinese hamster ovary (CHO) cells, confirming that the glyco‐engineered antibody performed as well as its CHO‐produced counterpart.