Identification of orphan histidine kinases that impact sporulation and enterotoxin production by Clostridium perfringens type F strain SM101 in a pathophysiologically-relevant ex vivo mouse intestinal contents model.

When causing food poisoning or antibiotic-associated diarrhea, Clostridium perfringens type F strains must sporulate to produce C. perfringens enterotoxin (CPE) in the intestines. C. perfringens is thought to use some of its seven annotated orphan histidine kinases to phosphorylate Spo0A and initiate sporulation and CPE production. We previously demonstrated the CPR0195 orphan kinase, but not the putative CPR1055 orphan kinase, is important when type F strain SM101 initiates sporulation and CPE production in modified Duncan-Strong (MDS) sporulation medium. Since there is no small animal model for C. perfringens sporulation, the current study used diluted mouse intestinal contents (MIC) to develop an ex vivo sporulation model and employed this model to test sporulation and CPE production by SM101 CPR0195 and CPR1055 null mutants in a pathophysiologically-relevant context. Surprisingly, both mutants still sporulated and produced CPE at wild-type levels in MIC. Therefore, five single null mutants were constructed that cannot produce one of the previously-unstudied putative orphan kinases of SM101. Those mutants implicated CPR1316, CPR1493, CPR1953 and CPR1954 in sporulation and CPE production by SM101 MDS cultures. Phosphorylation activity was necessary for CPR1316, CPR1493, CPR1953 and CPR1954 to affect sporulation in those MDS cultures, supporting their identity as kinases. Importantly, only the CPR1953 or CPR1954 null mutants exhibited significantly reduced levels of sporulation and CPE production in MIC cultures. These phenotypes were reversible by complementation. Characterization studies suggested that, in MDS or MIC, the CPR1953 and CPR1954 mutants produce less Spo0A than wild-type SM101. In addition, the CPR1954 mutant exhibited little or no Spo0A phosphorylation in MDS cultures. These studies, i) highlight the importance of using pathophysiologically-relevant models to investigate C. perfringens sporulation and CPE production in a disease context and ii) link the CPR1953 and CPR1954 kinases to C. perfringens sporulation and CPE production in disease-relevant conditions.

Genome-wide identification and expression pattern analysis of MIKC-Type MADS-box genes in Chionanthus retusus, an androdioecy plant.

BACKGROUND: The MADS-box gene family is widely distributed in the plant kingdom, and its members typically encoding transcription factors to regulate various aspects of plant growth and development. In particular, the MIKC-type MADS-box genes play a crucial role in the determination of floral organ development and identity recognition. As a type of androdioecy plant, Chionanthus retusus have unique gender differentiation. Manifested as male individuals with only male flowers and female individuals with only bisexual flowers. However, due to the lack of reference genome information, the characteristics of MIKC-type MADS-box genes in C. retusus and its role in gender differentiation of C. retusus remain largely unknown. Therefore, it is necessary to identify and characterize the MADS-box gene family within the genome of the C. retusus. RESULTS: In this study, we performed a genome-wide identification and analysis of MIKC-type MADS-box genes in C. retusus (2n = 2x = 46), utilizing the latest reference genome, and studied its expression pattern in individuals of different genders. As a result, we identified a total of 61 MIKC-type MADS-box genes in C. retusus. 61 MIKC-type MADS-box genes can be divided into 12 subfamilies and distributed on 18 chromosomes. Genome collinearity analysis revealed their conservation in evolution, while gene structure, domains and motif analysis indicated their conservation in structure. Finally, based on their expression patterns in floral organs of different sexes, we have identified that CrMADS45 and CrMADS60 may potentially be involved in the gender differentiation of C. retusus. CONCLUSIONS: Our studies have provided a general understanding of the conservation and characteristics of the MIKC-type MADS-box genes family in C. retusus. And it has been demonstrated that members of the AG subfamily, CrMADS45 and CrMADS60, may play important roles in the gender differentiation of C. retusus. This provides a reference for future breeding efforts to improve flower types in C. retusus and further investigate the role of MIKC-type MADS-box genes in gender differentiation.

Revealing the Synergistic Effect of Cation and Anion Vacancies on Enhanced Fenton-Like Reaction: The Electron Density Modulation of O 2p-Co 3d Bands.

Defect engineering is considered as a flexible and effective mean to improve the performance of Fenton-like reactions. Herein, a simple method is employed to synthesize Co3O4 catalysts with Co-O vacancy pairs (VP) for peroxymonosulfate (PMS) activation. Multi-scaled characterization, experimental, and simulation results jointly revealed that the cation vacancies-VCo contributed to enhanced conductivity and anion vacancies-VO provided a new active center for the 1O2 generation. Co3O4-VP can optimize the O 2p and Co 3d bands with the strong assistance of synergistic double vacancies to reduce the reaction energy barrier of the "PMS → Co(IV) = O → 1O2" pathway, ultimately triggering the stable transition of mechanism. Co3O4-VP catalysts with radical-nonradical collaborative mechanism achieve the synchronous improvement of activity and stability, and have good environmental robustness to favor water decontamination applications. This result highlights the possibility of utilizing anion and cation vacancy engineering strategies to rational design Co3O4-based materials widely used in catalytic reactions.

Formation and prediction of heterocyclic amines and N-nitrosamines in smoked sausages using back propagation artificial neural network.

BACKGROUND: Heterocyclic amines (HAs) and N-nitrosamines (NAs) are formed easily during the thermal processing of food, and epidemiological studies have demonstrated that consuming HAs and NAs increases the risk of cancer. However, there are few studies on the application of back propagation artificial neural network (BP-ANN) models to simultaneously predict the content of HAs and NAs in sausages. This study aimed to investigate the effects of cooking time and temperature, smoking time and temperature, and fat-to-lean ratio on the formation of HAs and NAs in smoked sausages, and to predict their total content based on the BP-ANN model. RESULTS: With an increase in processing time, processing temperature and fat ratio, the content of HAs and NAs in smoked sausages increased significantly, while the content of HA precursors and nitrite residues decreased significantly. The optimal network topology of the BP-ANN model was 5-11-2, the correlation coefficient values for training, validation, testing and all datasets were 0.99228, 0.99785, 0.99520 and 0.99369, respectively, and the mean squared error value of the best validation performance was 0.11326. The bias factor and the accuracy factor were within acceptable limits, and the predicted values approximated the true values, indicating that the model has good predictive performance. CONCLUSION: The contents of HAs and NAs in smoked sausages were significantly influenced by the cooking conditions, smoking conditions and fat ratio. The BP-ANN model has high application value in predicting the contents of HAs and NAs in sausages, which provides a theoretical basis for the suppression of carcinogen formation. © 2024 Society of Chemical Industry.

NanJ Is the Major Sialidase for Clostridium perfringens Type F Food Poisoning Strain 01E809.

Clostridium perfringens type F strains cause food poisoning (FP) when they sporulate and produce C. perfringens enterotoxin (CPE) in the intestines. Most type F FP strains carry a chromosomal cpe gene (c-cpe strains). C. perfringens produces up to three different sialidases, named NanH, NanI, and NanJ, but some c-cpe FP strains carry only nanJ and nanH genes. This study surveyed a collection of such strains and showed that they produce sialidase activity when cultured in Todd-Hewitt broth (TH) (vegetative cultures) or modified Duncan-Strong (MDS) medium (sporulating cultures). Sialidase null mutants were constructed in 01E809, a type F c-cpe FP strain carrying the nanJ and nanH genes. Characterization of those mutants identified NanJ as the major sialidase of 01E809 and showed that, in vegetative and sporulating cultures, nanH expression affects nanJ expression and vice versa; those regulatory effects may involve media-dependent changes in transcription of the codY or ccpA genes but not nanR. Additional characterization of these mutants demonstrated the following: (i) NanJ contributions to growth and vegetative cell survival are media dependent, with this sialidase increasing 01E809 growth in MDS but not TH; (ii) NanJ enhances 24-h vegetative cell viability in both TH and MDS cultures; and (iii) NanJ is important for 01E809 sporulation and, together with NanH, CPE production in MDS cultures. Lastly, NanJ was shown to increase CPE-induced cytotoxicity and CH-1 pore formation in Caco-2 cells. Collectively, these results suggest that NanJ may have a contributory role in FP caused by type F c-cpe strains that carry the nanH and nanJ genes.

Genome-wide identification and analysis of the SUPPRESSOR of MAX2 1-LIKE gene family and its interaction with DWARF14 in poplar.

BACKGROUND: Strigolactones (SLs) are important phytohormones that can regulate branch development in plants. Although SUPPRESSOR of MAX2 1-LIKE proteins (SMXLs) play a crucial role in SL signaling transduction, the SMXL gene family has not been well characterized in poplar. RESULTS: In this study, 12 members of the poplar SMXL gene family were identified and phylogenetically classified into four clades. Motif and 3D structural analyses revealed that PtSMXL proteins are structurally very conserved; however, the P-loop NTPase domain at the C-terminal was found to vary substantially among clades. A genomic collinearity analysis indicated that PtSMXL gene family members have expanded during recent genome doubling events in poplar, with all gene pairs subsequently undergoing purifying selection. According to a Cis-element analysis, PtSMXL promoters contain many light-responsive elements. In an expression pattern analysis, all 12 PtSMXL genes displayed tissue-specific expression, especially PtSMXL8a. PtSMXL7b expression was significantly downregulated after axillary bud growth begins. In addition, the expressions of PtSMXL7b and PtSMXL8a were highly induced by 2 µM GR24, a synthetic SL analog, thus suggesting that these genes are involved in SL-regulated axillary bud growth. In a yeast two-hybrid assay, only PtSMXL7b in clade II was able to interact with the SL receptor PtD14a in an SL dependent manner, which indicates that PtSMXL7b may be the functional homolog of D53/SMXL6/7/8 in poplar. Finally, we established its ability to affect axillary bud growth by constructing poplar overexpressing the PtSMXL7b gene. CONCLUSIONS: Our findings may inform future research on the functions of SMXLs in poplar, especially with respect to branch development.

Variations and patterns of C and N stoichiometry in the first five root branch orders across 218 woody plant species.

Despite the vital role in carbon (C) sequestration and nutrient retention, variations and patterns in root C and nitrogen (N) stoichiometry of the first five root orders across woody plant species remains unclear. We compiled a dataset to explore variations and patterns of root C and N stoichiometry in the first five orders of 218 woody plant species. Across the five orders, root N concentrations were greater in deciduous, broadleaf, and arbuscular mycorrhizal species than in evergreen, coniferous species, and ectomycorrhizal association species, respectively. Contrasting trends were found for root C : N ratios. Most root branch orders showed clear latitudinal and altitudinal trends in root C and N stoichiometry. There were opposite patterns in N concentrations between latitude and altitude. Such variations were mainly driven by plant species, and climatic factors together. Our results indicate divergent C and N use strategies among plant types and convergence and divergence in the patterns of C and N stoichiometry between latitude and altitude across the first five root orders. These findings provide important data on the root economics spectrum and biogeochemical models to improve understanding and prediction of climate change effects on C and nutrient dynamics in terrestrial ecosystems.

Species richness disparity in tropical terrestrial herbaceous floras: Evolutionary insight from Collabieae (Orchidaceae).

Species richness is spatially heterogeneous even in the hyperdiverse tropical floras. The main cause of uneven species richness among the four tropical regions are hot debated. To date, higher net diversification rates and/or longer colonization time have been usually proposed to contribute to this pattern. However, there are few studies to clarify the species richness patterns in tropical terrestrial floras. The terrestrial tribe Collabieae (Orchidaceae) unevenly distributes in the tropical regions with a diverse and endemic center in Asia. Twenty-one genera 127 species of Collabieae and 26 DNA regions were used to reconstruct the phylogeny and infer the biogeographical processes. We compared the topologies, diversification rates and niche evolutionary rates of Collabieae and regional lineages on empirical samplings and different simulated samplings fractions respectively. Our results suggested that the Collabieae originated in Asia at the earliest Oligocene, and then independently spread to Africa, Central America, and Oceania since the Miocene via long-distance dispersal. These results based on empirical data and simulated data were similar. BAMM, GeoSSE and niche analyses inferred that the Asian lineages had higher net diversification and niche evolutionary rates than those of Oceanian and African lineages on the empirical and simulated analyses. Precipitation is the most important factor for Collabieae, and the Asian lineage has experienced more stable and humid climate, which may promote the higher net diversification rate. Besides, the longer colonization time may also be associated with the Asian lineages' diversity. These findings provided a better understanding of the regional diversity heterogeneity in tropical terrestrial herbaceous floras.

Structure-Based Discovery of a Novel Class of Small-Molecule Pure Antagonists of Integrin αVß3.

Inhibitors of integrin αVß3 have therapeutic promise for a variety of diseases. Most αVß3-targeting small molecules patterned after the RGD motif are partial agonists because they induce a high-affinity, ligand-binding conformation and prime the receptor to bind the ligand without an activating stimulus, in part via a charge-charge interaction between their aspartic acid carboxyl group and the metal ion in the metal-ion-dependent adhesion site (MIDAS). Building upon our previous studies on the related integrin αIIbß3, we searched for pure αVß3 antagonists that lack this typical aspartic acid carboxyl group and instead engage through direct binding to one of the coordinating residues of the MIDAS metal ion, specifically ß3 E220. By in silico screening of two large chemical libraries for compounds interacting with ß3 E220, we indeed discovered a novel molecule that does not contain an acidic carboxyl group and does not induce the high-affinity, ligand-binding state of the receptor. Functional and structural characterization of a chemically optimized version of this compound led to the discovery of a novel small-molecule pure αVß3 antagonist that (i) does not prime the receptor to bind the ligand and does not induce hybrid domain swing-out or receptor extension as judged by antibody binding and negative-stain electron microscopy, (ii) binds at the RGD-binding site as predicted by metadynamics rescoring of induced-fit docking poses and confirmed by a cryo-electron microscopy structure of the compound-bound integrin, and (iii) coordinates the MIDAS metal ion via a quinoline moiety instead of an acidic carboxyl group.

Extraction of non-starch lipid from protease-treated wheat flour.

BACKGROUND: Lipids account for 2.0-2.5% of wheat flour by dry weight and affect properties and quality of cereal foods. A new method was developed to extract non-starch lipids from wheat flour. Wheat flour was first hydrolyzed with a protease and followed by extraction of non-starch lipids by water-saturated butanol (WSB). RESULT: Protein hydrolysis by protease followed by extraction of non-starch lipids with WSB increased yield to 1.9 ± 0.3% from 1.0 ± 0.1% with no protease treatment. The lipid profile showed a significant increase in phospholipid compounds extracted with protease hydrolysis (5.9 ± 0.8 nmol·g-1 ) versus without enzymatic treatment (2.4 ± 1.3 nmol g-1 ). CONCLUSION: Improved lipid extraction yield and phospholipid compounds following protease-assisted extraction method provided additional insight towards the understanding of protein-lipid interaction in wheat flour. The new protease-assisted extraction method may be applied to analyzing non-starch lipids in other types of wheat flours and other cereal flours. © 2021 Society of Chemical Industry.

Identifying the Basis for VirS/VirR Two-Component Regulatory System Control of Clostridium perfringens Beta-Toxin Production.

Clostridium perfringens toxin production is often regulated by the Agr-like quorum sensing (QS) system signaling the VirS/VirR two-component regulatory system (TCRS), which consists of the VirS membrane sensor histidine kinase and the VirR response regulator. VirS/VirR is known to directly control expression of some genes by binding to a DNA binding motif consisting of two VirR boxes located within 500 bp of the target gene start codon. Alternatively, the VirS/VirR system can indirectly regulate production levels of other proteins by increasing expression of a small regulatory RNA, VR-RNA. Previous studies demonstrated that C. perfringens beta-toxin (CPB) production by C. perfringens type B and C strains is positively regulated by both the Agr-like QS and the VirS/VirR TCRS, but the mechanism has been unclear. The current study first inactivated the vrr gene encoding VR-RNA to show that VirS/VirR regulation of cpb expression does not involve VR-RNA. Subsequently, bioinformatic analyses identified a potential VirR binding motif, along with a predicted strong promoter, ∼1.4 kb upstream of the cpb open reading frame (ORF). Two insertion sequences were present between this VirR binding motif/promoter region and the cpb ORF. PCR screening of a collection of strains carrying cpb showed that the presence and sequence of this VirR binding motif/promoter is highly conserved among CPB-producing strains. Reverse transcription-PCR (RT-PCR) and a GusA reporter assay showed this VirR binding motif is important for regulating CPB production. These findings indicate that VirS/VirR directly regulates cpb expression via VirS binding to a VirR binding motif located unusually distant from the cpb start codon. IMPORTANCE Clostridium perfringens beta-toxin (CPB) is only produced by type B and C strains. Production of CPB is essential for the pathogenesis of type C-associated infections, which include hemorrhagic necrotizing enteritis and enterotoxemia in both humans and animals. In addition, CPB can synergize with other toxins during C. perfringens gastrointestinal diseases. CPB toxin production is cooperatively regulated by the Agr-like quorum sensing (QS) system and the VirS/VirR two-component regulatory system. This study now reports that the VirS/VirR regulatory cascade directly controls expression of the cpb gene via a process involving a VirR box binding motif located unusually far (∼1.4 kb) upstream of the cpb ORF. This study provides a better understanding of the regulatory mechanisms for CPB production by the VirS/VirR regulatory cascade.

NanI Sialidase Contributes to the Growth and Adherence of Clostridium perfringens Type F Strain F4969 in the Presence of Adherent Mucus.

Clostridium perfringens type F strains causing nonfoodborne human gastrointestinal diseases (NFD) typically produce NanI sialidase as their major secreted sialidase. Type F NFDs can persist for several weeks, indicating their pathogenesis involves intestinal colonization, including vegetative cell growth and adherence, with subsequent sporulation that fosters enterotoxin production and release. We previously reported that NanI contributes to type F NFD strain adherence and growth using Caco-2 cells. However, Caco-2 cells make minimal amounts of mucus, which is significant because the intestines are coated with adherent mucus. Therefore, it was important to assess if NanI contributes to the growth and adherence of type F NFD strains in the presence of adherent mucus. Consequently, the current study first demonstrated greater growth of nanI-carrying versus non-nanI-carrying type F strains in the presence of HT29-MTX-E12 cells, which produce an adherent mucus layer, versus their parental HT29 cells, which make minimal mucus. Demonstrating the specific importance of NanI for this effect, type F NFD strain F4969 or a complementing strain grew and adhered better than an isogenic nanI null mutant in the presence of HT29-MTX-E12 cells versus HT29 cells. Those effects involved mucus production by HT29-MTX-E12 cells since mucus reduction using N-acetyl cysteine reduced F4969 growth and adherence. Consistent with those in vitro results, NanI contributed to growth of F4969 in the mouse small intestine. By demonstrating a growth and adherence role for NanI in the presence of adherent mucus, these results further support NanI as a potential virulence factor during type F NFDs.

The transcription factor PagLBD3 contributes to the regulation of secondary growth in Populus.

LATERAL ORGAN BOUNDARIES DOMAIN (LBD) genes encode plant-specific transcription factors that participate in regulating various developmental processes. In this study, we genetically characterized PagLBD3 encoding an important regulator of secondary growth in poplar (Populus alba × Populus glandulosa). Overexpression of PagLBD3 increased stem secondary growth in Populus with a significantly higher rate of cambial cell differentiation into phloem, while dominant repression of PagLBD3 significantly decreased the rate of cambial cell differentiation into phloem. Furthermore, we identified 1756 PagLBD3 genome-wide putative direct target genes (DTGs) through RNA sequencing (RNA-seq)-coupled DNA affinity purification followed by sequencing (DAP-seq) assays. Gene Ontology analysis revealed that genes regulated by PagLBD3 were enriched in biological pathways regulating meristem development, xylem development, and auxin transport. Several central regulator genes for vascular development, including PHLOEM INTERCALATED WITH XYLEM (PXY), WUSCHEL RELATED HOMEOBOX4 (WOX4), Secondary Wall-Associated NAC Domain 1s (SND1-B2), and Vascular-Related NAC-Domain 6s (VND6-B1), were identified as PagLBD3 DTGs. Together, our results indicate that PagLBD3 and its DTGs form a complex transcriptional network to modulate cambium activity and phloem/xylem differentiation.

Cryo-Electron Microscopy Structure of the αIIbß3-Abciximab Complex.

OBJECTIVE: The αIIbß3 antagonist antiplatelet drug abciximab is the chimeric antigen-binding fragment comprising the variable regions of murine monoclonal antibody 7E3 and the constant domains of human IgG1 and light chain κ. Previous mutagenesis studies suggested that abciximab binds to the ß3 C177-C184 specificity-determining loop (SDL) and Trp129 on the adjacent ß1-α1 helix. These studies could not, however, assess whether 7E3 or abciximab prevents fibrinogen binding by steric interference, disruption of either the αIIbß3-binding pocket for fibrinogen or the ß3 SDL (which is not part of the binding pocket but affects fibrinogen binding), or some combination of these effects. To address this gap, we used cryo-electron microscopy to determine the structure of the αIIbß3-abciximab complex at 2.8 Å resolution. Approach and Results: The interacting surface of abciximab is comprised of residues from all 3 complementarity-determining regions of both the light and heavy chains, with high representation of aromatic residues. Binding is primarily to the ß3 SDL and neighboring residues, the ß1-α1 helix, and ß3 residues Ser211, Val212 and Met335. Unexpectedly, the structure also indicated several interactions with αIIb. As judged by the cryo-electron microscopy model, molecular-dynamics simulations, and mutagenesis, the binding of abciximab does not appear to rely on the interaction with the αIIb residues and does not result in disruption of the fibrinogen-binding pocket; it does, however, compress and reduce the flexibility of the SDL. CONCLUSIONS: We deduce that abciximab prevents ligand binding by steric interference, with a potential contribution via displacement of the SDL and limitation of the flexibility of the SDL residues.

Change of soluble acid invertase gene (SAI-1) haplotype in hybrid sorghum breeding program in China.

Sugar metabolism is the most important and core one which drives plant growth and development. Invertases are key enzymes that regulate sugar metabolism. A still-growing number of studies have revealed that invertases play a crucial role in various aspects of plant growth and development. Crop yield is the product of sugar metabolism; it could be deduced that invertase also regulated the yield formation. So we have done a series of research on soluble acid invertase in sweet sorghum from enzyme activity to gene cloning and functional marker development. In this paper, we sequenced full length of SAI-1 gene in 69 grain sorghum parent lines, trying to see how it differs in their gene sequences and their distribution in related hybrid varieties released in the past. To our surprise, the result showed that B-lines and restore lines (R-line) have almost different SAI-1 haplotype distribution. The change of haplotype of SAI-1 gene is associated with yield gain as with grain sorghum breeding progress, which proved that SAI-1 may take a very important role in yield formation. And we also found the SAI-1 gene tends to become shorter as with the breeding advance, which means short sequence in introns, while exon remains unchanged leading to higher gene efficiency. The best SAI-1 haplotype combination of sorghum hybrid was also found for different planting regions. These findings are of great significance for improving breeding efficiency, understanding heterosis, and germplasm enhancement. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01231-2.

PagGRF12a interacts with PagGIF1b to regulate secondary xylem development through modulating PagXND1a expression in Populus alba × P. glandulosa.

Growth-regulating factors (GRFs) are important regulators of plant development and growth, but their possible roles in xylem development in woody plants remain unclear. Here, we report that Populus alba × Papulus glandulosa PagGRF12a negatively regulates xylem development in poplar. PagGRF12a is expressed in vascular tissues. Compared to non-transgenic control plants, transgenic poplar plants overexpressing PagGRF12a exhibited reduced xylem width and plants with repressed expression of PagGRF12a exhibited increased xylem width. Xylem NAC domain 1 (XND1) encodes a NAC domain transcription factor that regulates xylem development and transcriptional analyses revealed that PagXND1a is highly upregulated in PagGRF12a-overexpressing plants and downregulated in PagGRF12a-suppressed plants, indicating that PagGRF12a may regulate xylem development through PagXND1a. Transient transcriptional assays and chromatin immunoprecipitation-polymerase chain reaction assays confirmed that PagGRF12a directly upregulates PagXND1a. In addition, PagGRF12a interacts with the GRF-Interacting Factor (GIF) PagGIF1b, and this interaction enhances the effects of PagGRF12a on PagXND1a. Our results indicate that PagGRF12a inhibits xylem development by upregulating the expression of PagXND1a.

Transcriptome sequencing of active buds from Populus deltoides CL. and Populus × zhaiguanheibaiyang reveals phytohormones involved in branching.

Branching in woody plants affects their ecological benefits and impacts wood formation. To obtain genome-wide insights into the transcriptome changes and regulatory mechanisms associated with branching, we performed high-throughput RNA sequencing to characterize cDNA libraries generated from active buds of Populus deltoides CL. 'zhonglin2025' (BC) and Populus × zhaiguanheibaiyang (NC). NC has more branches than BC and rapid growth. We obtained a total of 198.2 million high-quality clean reads from the NC and BC libraries. We detected 3543 differentially expressed genes (DEGs) between the NC and BC libraries; 1418 were down-regulated and 2125 were up-regulated. Gene ontology functional classification of the DEGs indicated that they included 89 genes that encoded proteins related to hormone biosynthesis, 364 genes related to hormone signaling transduction, and 104 related to the auxin efflux transmembrane transporter. We validated the expression profiles of 16° by real-time quantitative PCR and found that their expression patterns were similar to those obtained from the high-throughput RNA sequencing data. We also measured the hormone content in young buds of BC and NC by high-pressure liquid chromatography. In this study, we identified global hormone regulatory patterns and differences in gene expression between NC and BC, and constructed a hormone regulatory network to explain branching in Populus buds. In addition, candidate genes that may be useful for molecular breeding of particular plant types were identified. Our results will provide a starting point for future investigations into the molecular mechanisms of branching in Populus.

Pollen-mediated gene flow ensures connectivity among spatially discrete sub-populations of Phalaenopsis pulcherrima, a tropical food-deceptive orchid.

BACKGROUND: Gene flow in plants via pollen and seeds is asymmetrical at different geographic scales. Orchid seeds are adapted to long-distance wind dispersal but pollinium transfer is often influenced by pollinator behavior. We combined field studies with an analysis of genetic diversity among 155 physically mapped adults and 1105 F1 seedlings to evaluate the relative contribution of pollen and seed dispersal to overall gene flow among three sub-populations of the food-deceptive orchid Phalaenopsis pulcherrima on Hainan Island, China. RESULTS: Phalaenopsis pulcherrima is self-sterile and predominantly outcrossing, resulting in high population-level genetic diversity, but plants are clumped and exhibit fine-scale genetic structuring. Even so, we detected low differentiation among sub-populations, with polynomial regression analysis suggesting gene flow via seed to be more restricted than that via pollen. Paternity analysis confirmed capsules of P. pulcherrima to each be sired by a single pollen donor, probably in part facilitated by post-pollination stigma obfuscation, with a mean pollen flow distance of 272.7 m. Despite limited sampling, we detected no loss of genetic diversity from one generation to the next. CONCLUSIONS: Outcrossing mediated by deceptive pollination and self-sterility promote high genetic diversity in P. pulcherrima. Long-range pollinia transfer ensures connectivity among sub-populations, offsetting the risk of genetic erosion at local scales.

Aeromonas hydrophila suppresses complement pathways via degradation of complement C3 in bony fish by metalloprotease.

Aeromonas hydrophila is a pathogen that causes high mortality in the grass carp. The complement system, as a frontline defence of innate immunity, plays an important role in the immune response against pathogens. However, the immunity evasion mechanism of A. hydrophila against the complement system of grass carp remains unclear. In this study, we described an additional mechanism used by A. hydrophila GD18 to evade the complement system and survive in grass carp serum. First, A. hydrophila evaded the bactericidal activity of grass carp serum. Second, the haemolytic activity assays showed that A. hydrophila obviously suppressed the alternative pathway, which depended on preventing the formation or disabling the function of the membrane-attack complex (MAC). Further research indicated that A. hydrophila targeted complement C3, the central component of the three complement pathways, and degraded it in the grass carp serum, leading to the inhibition of the complement pathways, which resulted in the serum-resistance of A. hydrophila. Furthermore, cleavage analyses showed that extracellular proteases (ECPases) of A. hydrophila efficiently cleaved purified C3 as well as C3 in grass carp serum. Finally, protease inhibitor studies and mass spectrum analysis identified the secreted metalloprotease elastase (AhE), which was present in large amounts in crude ECPases, as the central molecule responsible for C3 cleavage. Compared to wild strain GD18, the AhE knockout, Δahe was dramatically reduced in the ability of serum resistance. Our findings suggested that A. hydrophila escaped serum-killing by suppressing the complement pathways via the degradation of complement C3 in bony fish, which was related to secreted metalloproteases.