Oligomerization of Cry9Aa in solution without receptor binding, is not related with insecticidal activity

Background: Bacillus thuringiensis Cry toxins bind with different insect midgut proteins leading to toxin oligomerization, membrane insertion and pore formation. However, different Cry toxins had been shown to readily form high molecular weight oligomers or aggregates in solution in the absence of receptor interaction. The role of Cry oligomers formed in solution remains uncertain. The Cry9A proteins show high toxicity against different Lepidoptera, and no-cross resistance with Cry1A. Results: Cry9Aa655 protein formed oligomers easily in solution mediated by disulfide bonds, according to SDS-PAGE analysis under non-reducing and reducing conditions. However, oligomerization is not observed if Cry9Aa655 is activated with trypsin, suggesting that cysteine residues, C14 and C16, located in the N-terminal end that is processed during activation participate in this oligomerization. To determine the role of these residues on oligomerization and in toxicity single and double alanine substitution were constructed. In contrast to single C14A and C16A mutants, the double C14A\u2013C16A mutant did not form oligomers in solution. Toxicity assays against Plutella xylostella showed that the C14A\u2013C16A mutant had a similar insecticidal activity as the Cry9Aa655 protein indicating the oligomers of Cry9Aa formed in solution in the absence of receptor binding are not related with toxicity. Conclusions: The aggregation of Cry9Aa655 polypeptides was mediated by disulfide bonds. Cry9Aa655 C14 and C16C are involved in oligomerization in solution. These aggregate forms are not related to the mode of action of Cry9Aa leading to toxicity

On non-surjective coarse isometries between Banach spaces

Assume that X; Y are real Banach spaces, Y has uniform convexity of type p (≥ 1), and f : X → Y is a standard coarse isometry. In this paper, we show that if∫ ∞      εf(S) 1/P                     __________ ds < ∞,    1      S1 + 1/pthen there is a linear isometry U : X → Y so that‖ f (x) - U x ‖ = o (‖ x ‖), as ‖ x ‖ → ∞,where εf : ℝ+ → ℝ+ is defined byεf(t) = sup {l ‖ f (x) - f (y) ‖ - ‖ l : x,y ∈ X ‖ x - y ‖ ≤ t }.Representation properties of coarse isometries in free ultrafilter limits on ℕ are also discussed.Mathematics Subject Classification (2010): Primary 46B04, 46B20, 47A58; Secondary 46A20.Keywords: Coarse isometry, stability, uniform convexity, Banach spac

Bidirectional lncRNA Transfer between Cuscuta Parasites and Their Host Plant

Dodder species (Cuscuta spp.) are holoparasites that have extensive material exchange with their host plants through vascular connections. Recent studies on cross-species transfer have provided breakthrough insights, but little is known about the interaction mechanisms of the inter-plant mobile substances in parasitic systems. We sequenced the transcriptomes of dodder growing on soybean hosts to characterize the long non-coding RNA (lncRNA) transfer between the two species, and found that lncRNAs can move in high numbers (365 dodder lncRNAs and 14 soybean lncRNAs) in a bidirectional manner. Reverse transcription-polymerase chain reaction further confirmed that individual lncRNAs were trafficked in the dodder–soybean parasitic system. To reveal the potential functions of mobile transcripts, the Gene Ontology terms of mobile lncRNA target genes were predicted, and mobile dodder target genes were found to be mainly enriched in “metabolic process”, “catalytic activity”, “signaling”, and “response to stimulus” categories, whereas mobile soybean target genes were enriched in organelle-related categories, indicating that specific mobile lncRNAs may be important in regulating dodder parasitism. Our findings reveal that lncRNAs are transferred between dodder and its host soybean plants, which may act as critical regulators to coordinate the host–dodder interaction at the whole parasitic level

Integrative Analyses of Transcriptomes and Metabolomes Reveal Associated Genes and Metabolites with Flowering Regulation in Common Vetch (<i>Vicia sativa</i> L.)

As an important source of protein for livestock and human consumption, Vicia sativa is cultivated worldwide, but its seed production is hampered at high altitudes because of the short frost-free period. Flowering represents the transition from a vegetative to a reproductive period, and early flowering benefits plant seed production at high altitudes. However, the molecular mechanisms of flowering regulation in V. sativa remain elusive. In the present study, two V. sativa accessions with different flowering characteristics were used: Lan3 (early-flowering) was cultivated by our laboratory, and 503 (late-flowering) was selected from 222 V. sativa accessions after three years of field experiments. The shoot samples (shoot tip length = 10 cm) of these two accessions were collected 63, 70, and 77 days after sowing, and the molecular regulatory mechanism of the flowering process was identified by integrative analyses of the transcriptomes and metabolomes. Kyoto Encyclopedia of Genes and Genomes enrichment showed that the synthesis and signal transduction of plant hormone pathways were the most enriched pathways in 4274 differentially expressed genes (DEGs) and in 259 differential metabolites between Lan3 and 503. Moreover, the contents of three metabolites related to salicylic acid biosynthesis and the transcription levels of two DEGs related to salicylic acid signal transduction in Lan3 were higher than those in 503. Further verification in various accessions indicated that salicylic acid metabolism may be involved in the flowering regulation process of V. sativa. These findings provide valuable information for understanding the flowering mechanism and for promoting breeding research in V. sativa

DROUGHT-INDUCED UNKNOWN PROTEIN 1 positively modulates drought tolerance in cultivated alfalfa (Medicago sativa L.)

Alfalfa is the most widely cultivated perennial legume forage crop worldwide. Drought is one of the major environmental factors influencing alfalfa productivity. However, the molecular mechanisms underlying alfalfa responses to drought stress are still largely unknown. This study identified a drought-inducible gene of unknown function, designated as Medicago sativa DROUGHT-INDUCED UNKNOWN PROTEIN 1 (MsDIUP1). MsDIUP1 was localized to the nucleus, chloroplast, and plasma membranes. Overexpression of MsDIUP1 in Arabidopsis resulted in increased tolerance to drought, with higher seed germination, root length, fresh weight, and survival rate than in wild-type (WT) plants. Consistently, analysis of MsDIUP1 over-expression (OE) alfalfa plants revealed that MsDIUP1 also increased tolerance to drought stress, accompanied by physiological changes including reduced malondialdehyde (MDA) content and increased osmoprotectants accumulation (free proline and soluble sugar), relative to the WT. In contrast, disruption of MsDIUP1 expression by RNA interference (RNAi) in alfalfa resulted in a drought-hypersensitive phenotype, with a lower chlorophyll content, higher MDA content, and less osmoprotectants accumulation than that of the WT. Transcript profiling of alfalfa WT, OE, and RNAi plants during drought stress showed differential responses for genes involved in stress signaling, antioxidant defense, and osmotic adjustment. Taken together, these results reveal a positive role for MsDIUP1 in regulating drought tolerance

Oligomerization of Cry9Aa in solution without receptor binding, is not related with insecticidal activity

Background: Bacillus thuringiensis Cry toxins bind with different insect midgut proteins leading to toxin oligomerization, membrane insertion and pore formation. However, different Cry toxins had been shown to readily form high molecular weight oligomers or aggregates in solution in the absence of receptor interaction. The role of Cry oligomers formed in solution remains uncertain. The Cry9A proteins show high toxicity against different Lepidoptera, and no-cross resistance with Cry1A. Results: Cry9Aa655 protein formed oligomers easily in solution mediated by disulfide bonds, according to SDS-PAGE analysis under non-reducing and reducing conditions. However, oligomerization is not observed if Cry9Aa655 is activated with trypsin, suggesting that cysteine residues, C14 and C16, located in the N-terminal end that is processed during activation participate in this oligomerization. To determine the role of these residues on oligomerization and in toxicity single and double alanine substitution were constructed. In contrast to single C14A and C16A mutants, the double C14A–C16A mutant did not form oligomers in solution. Toxicity assays against Plutella xylostella showed that the C14A–C16A mutant had a similar insecticidal activity as the Cry9Aa655 protein indicating the oligomers of Cry9Aa formed in solution in the absence of receptor binding are not related with toxicity. Conclusions: The aggregation of Cry9Aa655 polypeptides was mediated by disulfide bonds. Cry9Aa655 C14 and C16C are involved in oligomerization in solution. These aggregate forms are not related to the mode of action of Cry9Aa leading to toxicity

Transcriptome and Coexpression Network Analyses Provide In-Sights into the Molecular Mechanisms of Hydrogen Cyanide Synthesis during Seed Development in Common Vetch (Vicia sativa L.)

The common vetch (Vicia sativa L.) seed is an ideal plant-based protein food for humans, but its edible value is mainly limited by the presence of cyanogenic glycosides that hydrolyze to produce toxic hydrogen cyanide (HCN), and the genes that regulate HCN synthesis in common vetch are unknown. In this study, seeds from common vetch at 5, 10, 15, 20, 25, 30, and 35 days after anthesis were sampled, and the seven stages were further divided into five developmental stages, S1, S2, S3, S4, and S5, based on morphological and transcriptome analyses. A total of 16,403 differentially expressed genes were identified in the five developmental stages. The HCN contents of seeds in these five stages were determined by alkaline titration, and weighted gene coexpression network analysis was used to explain the molecular regulatory mechanism of HCN synthesis in common vetch seeds. Eighteen key regulatory genes for HCN synthesis were identified, including the VsGT2, VsGT17 and CYP71A genes, as well as the VsGT1 gene family. VsGT1, VsGT2, VsGT17 and CYP71A jointly promoted HCN synthesis, from 5 to 25 days after anthesis, with VsGT1-1, VsGT1-4, VsGT1-11 and VsGT1-14 playing major roles. The HCN synthesis was mainly regulated by VsGT1, from 25 to 35 days after anthesis. As the expression level of VsGT1 decreased, the HCN content no longer increased. In-depth elucidation of seed HCN synthesis lays the foundations for breeding common vetch with low HCN content

Cryo-EM structures reveal the molecular basis of receptor-initiated coxsackievirus uncoating

Enterovirus uncoating receptors bind at the surface depression ("canyon") that encircles each capsid vertex causing the release of a host-derived lipid called "pocket factor" that is buried in a hydrophobic pocket formed by the major viral capsid protein, VP1. Coxsackievirus and adenovirus receptor (CAR) is a universal uncoating receptor of group B coxsackieviruses (CVB). Here, we present five high-resolution cryoEM structures of CVB representing different stages of virus infection. Structural comparisons show that the CAR penetrates deeper into the canyon than other uncoating receptors, leading to a cascade of events: collapse of the VP1 hydrophobic pocket, high-efficiency release of the pocket factor and viral uncoating and genome release under neutral pH, as compared with low pH. Furthermore, we identified a potent therapeutic antibody that can neutralize viral infection by interfering with virion-CAR interactions, destabilizing the capsid and inducing virion disruption. Together, these results define the structural basis of CVB cell entry and antibody neutralization