拟南芥RALF多肽家族的功能多样性初步分析

绿色植物中的快速碱化因子(Rapid alkalinization factor,RALF)为一类进化保守的多肽信号分子,以基因家族形式存在。模式植物拟南芥中至少存在35个RALF基因成员,前期研究显示拟南芥RALF家族的部分成员,比如RALF1/23,RALF4/19可分别作为Cr RLK1L类蛋白受体激酶家族成员FERONIA及BUPS1/2的配体,调控细胞伸长、植物免疫应答及双受精等过程,但是RALF家族其他成员是否具有生物学活性,以及不同成员之间是否具有功能性差异均尚不清楚。因此,本研究异源表达了19个代表性的RALF,并对其生物学活性和功能性差异进行了分析。实验结果表明,19个RALF均对根的生长起到不同程度的抑制作用,进一步挑选了部分代表性RALF成员进行了活性氧(Reactive oxygen species,ROS)迸发及丝裂原活化蛋白激酶(Mitogen-activated protein kinase,MAPK)磷酸化实验分析,实验结果表明,我们确证了11个RALF蛋白参与了MAPK信号的响应,同时,证实16个RALF蛋白抑制了由flg22引起的ROS的释放。此外,不同RALF成员在下胚轴细胞伸长上的作用也存在明显差异,比如RALF10促进下胚轴的伸长。以上研究结果表明不同RALF之间既存在功能冗余性,又存在功能性差异。本研究丰富了对RALF功能复杂性和多样性的认识。     

New Insights into the Diversity of Marine Picoeukaryotes

Over the last decade, culture-independent surveys of marine picoeukaryotic diversity based on 18S ribosomal DNA clone libraries have unveiled numerous sequences of novel high-rank taxa. This newfound diversity has significantly altered our understanding of marine microbial food webs and the evolution of eukaryotes. However, the current picture of marine eukaryotic biodiversity may be significantly skewed by PCR amplification biases, occurrence of rDNA genes in multiple copies within a single cell, and the capacity of DNA to persist as extracellular material. In this study we performed an analysis of the metagenomic dataset from the Global Ocean Survey (GOS) expedition, seeking eukaryotic ribosomal signatures. This PCR-free approach revealed similar phylogenetic patterns to clone library surveys, suggesting that PCR steps do not impose major biases in the exploration of environmental DNA. The different cell size fractions within the GOS dataset, however, displayed a distinct picture. High protistan diversity in the <0.8 µm size fraction, in particular sequences from radiolarians and ciliates (and their absence in the 0.8–3 µm fraction), suggest that most of the DNA in this fraction comes from extracellular material from larger cells. In addition, we compared the phylogenetic patterns from rDNA and reverse transcribed rRNA 18S clone libraries from the same sample harvested in the Mediterranean Sea. The libraries revealed major differences, with taxa such as pelagophytes or picobiliphytes only detected in the 18S rRNA library. MAST (Marine Stramenopiles) appeared as potentially prominent grazers and we observed a significant decrease in the contribution of alveolate and radiolarian sequences, which overwhelmingly dominated rDNA libraries. The rRNA approach appears to be less affected by taxon-specific rDNA copy number and likely better depicts the biogeochemical significance of marine protists.     

Analysis of Uromodulin Polymerization Provides New Insights into the Mechanisms Regulating ZP Domain-mediated Protein Assembly

Uromodulin is the most abundant protein secreted in urine, in which it is found as a high-molecular-weight polymer. Polymerization occurs via its zona pellucida (ZP) domain, a conserved module shared by many extracellular eukaryotic proteins that are able to assemble into matrices. In this work, we identified two motifs in uromodulin, mapping in the linker region of the ZP domain and in between protein cleavage and glycosylphosphatidylinositol (GPI)-anchoring sites, which regulate its polymerization. Indeed, mutations in either module led to premature intracellular polymerization of a soluble uromodulin isoform, demonstrating the inhibitory role of these motifs for ZP domain-mediated protein assembly. Proteolytic cleavage separating the external motif from the mature monomer is necessary to release the inhibitory function and allow protein polymerization. Moreover, we report absent or abnormal assembly into filaments of GPI-anchored uromodulin mutated in either the internal or the external motif. This effect is due to altered processing on the plasma membrane, demonstrating that the presence of the two modules has not only an inhibitory function but also can positively regulate protein polymerization. Our data expand previous knowledge on the control of ZP domain function and suggest a common mechanism regulating polymerization of ZP domain proteins.     

Pathway Analysis of GWAS Provides New Insights into Genetic Susceptibility to 3 Inflammatory Diseases

Although the introduction of genome-wide association studies (GWAS) have greatly increased the number of genes associated with common diseases, only a small proportion of the predicted genetic contribution has so far been elucidated. Studying the cumulative variation of polymorphisms in multiple genes acting in functional pathways may provide a complementary approach to the more common single SNP association approach in understanding genetic determinants of common disease. We developed a novel pathway-based method to assess the combined contribution of multiple genetic variants acting within canonical biological pathways and applied it to data from 14,000 UK individuals with 7 common diseases. We tested inflammatory pathways for association with Crohn''s disease (CD), rheumatoid arthritis (RA) and type 1 diabetes (T1D) with 4 non-inflammatory diseases as controls. Using a variable selection algorithm, we identified variants responsible for the pathway association and evaluated their use for disease prediction using a 10 fold cross-validation framework in order to calculate out-of-sample area under the Receiver Operating Curve (AUC). The generalisability of these predictive models was tested on an independent birth cohort from Northern Finland. Multiple canonical inflammatory pathways showed highly significant associations (p 10⁻³–10⁻²⁰) with CD, T1D and RA. Variable selection identified on average a set of 205 SNPs (149 genes) for T1D, 350 SNPs (189 genes) for RA and 493 SNPs (277 genes) for CD. The pattern of polymorphisms at these SNPS were found to be highly predictive of T1D (91% AUC) and RA (85% AUC), and weakly predictive of CD (60% AUC). The predictive ability of the T1D model (without any parameter refitting) had good predictive ability (79% AUC) in the Finnish cohort. Our analysis suggests that genetic contribution to common inflammatory diseases operates through multiple genes interacting in functional pathways.     

Comparative Genome Analysis Provides Insights into the Pathogenicity of Flavobacterium psychrophilum

Flavobacterium psychrophilum is a fish pathogen in salmonid aquaculture worldwide that causes cold water disease (CWD) and rainbow trout fry syndrome (RTFS). Comparative genome analyses of 11 F. psychrophilum isolates representing temporally and geographically distant populations were used to describe the F. psychrophilum pan-genome and to examine virulence factors, prophages, CRISPR arrays, and genomic islands present in the genomes. Analysis of the genomic DNA sequences were complemented with selected phenotypic characteristics of the strains. The pan genome analysis showed that F. psychrophilum could hold at least 3373 genes, while the core genome contained 1743 genes. On average, 67 new genes were detected for every new genome added to the analysis, indicating that F. psychrophilum possesses an open pan genome. The putative virulence factors were equally distributed among isolates, independent of geographic location, year of isolation and source of isolates. Only one prophage-related sequence was found which corresponded to the previously described prophage 6H, and appeared in 5 out of 11 isolates. CRISPR array analysis revealed two different loci with dissimilar spacer content, which only matched one sequence in the database, the temperate bacteriophage 6H. Genomic Islands (GIs) were identified in F. psychrophilum isolates 950106-1/1 and CSF 259–93, associated with toxins and antibiotic resistance. Finally, phenotypic characterization revealed a high degree of similarity among the strains with respect to biofilm formation and secretion of extracellular enzymes. Global scale dispersion of virulence factors in the genomes and the abilities for biofilm formation, hemolytic activity and secretion of extracellular enzymes among the strains suggested that F. psychrophilum isolates have a similar mode of action on adhesion, colonization and destruction of fish tissues across large spatial and temporal scales of occurrence. Overall, the genomic characterization and phenotypic properties may provide new insights to the mechanisms of pathogenicity in F. psychrophilum.     

dGTP Starvation in Escherichia coli Provides New Insights into the Thymineless-Death Phenomenon

Starvation of cells for the DNA building block dTTP is strikingly lethal (thymineless death, TLD), and this effect is observed in all organisms. The phenomenon, discovered some 60 years ago, is widely used to kill cells in anticancer therapies, but many questions regarding the precise underlying mechanisms have remained. Here, we show for the first time that starvation for the DNA precursor dGTP can kill E. coli cells in a manner sharing many features with TLD. dGTP starvation is accomplished by combining up-regulation of a cellular dGTPase with a deficiency of the guanine salvage enzyme guanine-(hypoxanthine)-phosphoribosyltransferase. These cells, when grown in medium without an exogenous purine source like hypoxanthine or adenine, display a specific collapse of the dGTP pool, slow-down of chromosomal replication, the generation of multi-branched nucleoids, induction of the SOS system, and cell death. We conclude that starvation for a single DNA building block is sufficient to bring about cell death.     

Comprehensive Analysis of Pan-African Mitochondrial DNA Variation Provides New Insights into Continental Variation and Demography

Africa is the cradle of all human beings, and although it has been the focus of a number of genetic studies, there are many questions that remain unresolved. We have performed one of the largest and most comprehensive meta-analyses of mitochondrial DNA(mt DNA)lineages carried out in the African continent to date. We generated high-throughput mtDNA single nucleotide polymorphism(SNP) data(230 SNPs) from 2024 Africans, where more than 500 of them were additionally genotyped for the control region. These data were analyzed together with over 12,700 control region profiles collected from the literature, representing more than 300 population samples from Africa. Insights into the African homeland of humans are discussed. Phylogeographic patterns for the African continent are shown at a high phylogeographic resolution as well as at the population and regional levels. The deepest branch of the mtDNA tree, haplogroup L0,shows the highest sub-haplogroup diversity in Southeast and East Africa, suggesting this region as the homeland for modern humans.Several demographic estimates point to the coast as a facilitator of human migration in Africa, but the data indicate complex patterns,perhaps mirroring the effect of recent continental-scaled demographic events in re-shaping African mtDNA variability.     

Molecular Taxonomy Provides New Insights into Anopheles Species of the Neotropical Arribalzagia Series

Phylogenetic analysis of partial mitochondrial cytochrome oxidase c subunit I (COI) and nuclear internal transcribed spacer 2 (ITS2) sequences were used to evaluate initial identification and to investigate phylogenetic relationships of seven Anopheles morphospecies of the Arribalzagia Series from Colombia. Phylogenetic trees recovered highly supported clades for An. punctimaculas.s., An. calderoni, An. malefactor s.l., An. neomaculipalpus, An. apicimacula s.l., An. mattogrossensis and An. peryassui. This study provides the first molecular confirmation of An. malefactorfrom Colombia and discovered conflicting patterns of divergence for the molecular markers among specimens from northeast and northern Colombia suggesting the presence of two previously unrecognized Molecular Operational Taxonomic Units (MOTUs). Furthermore, two highly differentiated An. apicimacula MOTUs previously found in Panama were detected. Overall, the combined molecular dataset facilitated the detection of known and new Colombian evolutionary lineages, and constitutes the baseline for future research on their bionomics, ecology and potential role as malaria vectors.     

Ancient DNA Provides New Insights into the Evolutionary History of New Zealand's Extinct Giant Eagle

Prior to human settlement 700 years ago New Zealand had no terrestrial mammals—apart from three species of bats—instead, approximately 250 avian species dominated the ecosystem. At the top of the food chain was the extinct Haast's eagle, Harpagornis moorei. H. moorei (10–15 kg; 2–3 m wingspan) was 30%–40% heavier than the largest extant eagle (the harpy eagle, Harpia harpyja), and hunted moa up to 15 times its weight. In a dramatic example of morphological plasticity and rapid size increase, we show that the H. moorei was very closely related to one of the world's smallest extant eagles, which is one-tenth its mass. This spectacular evolutionary change illustrates the potential speed of size alteration within lineages of vertebrates, especially in island ecosystems.     

Peroxisome Size Provides Insights into the Function of Autophagy-related Proteins

Autophagy is a major pathway of intracellular degradation mediated by formation of autophagosomes. Recently, autophagy was implicated in the degradation of intracellular bacteria, whose size often exceeds the capacity of normal autophagosomes. However, the adaptations of the autophagic machinery for sequestration of large cargos were unknown. Here we developed a yeast model system to study the effect of cargo size on the requirement of autophagy-related (Atg) proteins. We controlled the size of peroxisomes before their turnover by pexophagy, the selective autophagy of peroxisomes, and found that peroxisome size determines the requirement of Atg11 and Atg26. Small peroxisomes can be degraded without these proteins. However, Atg26 becomes essential for degradation of medium peroxisomes. Additionally, the pexophagy-specific phagophore assembly site, organized by the dual interaction of Atg30 with functionally active Atg11 and Atg17, becomes essential for degradation of large peroxisomes. In contrast, Atg28 is partially required for all autophagy-related pathways independent of cargo size, suggesting it is a component of the core autophagic machinery. As a rule, the larger the cargo, the more cargo-specific Atg proteins become essential for its sequestration.     

基因共表达网络分析揭示马铃薯薯块发育特征及原始抗性

转录组测序(RNA-seq)技术提供的全基因组数据信息已广泛应用于研究多个样本之间的基因表达模式和调控机制.通过构建种间或种内基因共表达网络(GCNs)挖掘的表达相关基因在功能上通常是相似的.对于马铃薯(Solanum tuberosum)而言,目前有大量的公共转录组测序数据,但是缺乏针对这些高通量数据构建的GCN网络,因此也无法探索在不同基因型、不同组织以及不同环境条件下基因的表达模式及规律.本研究选取16个公共转录组测序数据库构建了 GCN网络,这些数据库涵盖了来自全球各地的11个马铃薯栽培种.基于两两间基因表达相关性,我们在GCN网络中发现了一些具有特定生物学意义的基因模块.该网络共由14个基因模块组成并富集到植物光合形态建成、薯块休眠解除等多个生理过程,其中一个模块的134个基因在原始栽培种(ssp.Andigena)中特异性高表达,且通过功能富集发现这些基因与马铃薯病害和逆境的抗性相关.该结果揭示了在马铃薯人工驯化期间基因进化压力出现遗传漂移.本研究中基于GCN网络分析揭示了马铃薯种间和种内基因共表达模块的聚类以及不同模块基因间在进化上的分化,为马铃薯基因功能研究提供了新的视角.     

Pan-Genomic Analysis Provides Insights into the Genomic Variation and Evolution of Salmonella Paratyphi A

Salmonella Paratyphi A (S. Paratyphi A) is a highly adapted, human-specific pathogen that causes paratyphoid fever. Cases of paratyphoid fever have recently been increasing, and the disease is becoming a major public health concern, especially in Eastern and Southern Asia. To investigate the genomic variation and evolution of S. Paratyphi A, a pan-genomic analysis was performed on five newly sequenced S. Paratyphi A strains and two other reference strains. A whole genome comparison revealed that the seven genomes are collinear and that their organization is highly conserved. The high rate of substitutions in part of the core genome indicates that there are frequent homologous recombination events. Based on the changes in the pan-genome size and cluster number (both in the core functional genes and core pseudogenes), it can be inferred that the sharply increasing number of pseudogene clusters may have strong correlation with the inactivation of functional genes, and indicates that the S. Paratyphi A genome is being degraded.     

Deciphering the Physalis floridana Double-Layered-Lantern1 Mutant Provides Insights into Functional Divergence of the GLOBOSA Duplicates within the Solanaceae

Glycosylphosphatidylinositol-anchor biosynthesis and glycosylphosphatidylinositol modification of proteins are central to coordinated plant development.Since their discovery (Low and Saltiel, 1988), glycosylphosphatidylinositol-anchored proteins (GPI-APs) have provoked intense interest as crucial regulators for growth, morphogenesis, reproduction, and disease pathogenesis in organisms ranging from yeast and trypanosomes to animals and plants. The lipid moiety, the glycosylphosphatidylinositol (GPI) anchor, is synthesized in the endoplasmic reticulum (ER); the protein component is cotranslationally inserted into the ER and posttranslationally modified by the addition of a GPI anchor (Kinoshita et al., 2013; Fig. 1). GPI-APs are then transported via the Golgi to the outer surface of the plasma membrane. The lipid anchor mediates stable attachment of these proteins to the cell surface, where some play important roles as signaling regulators from sphingolipid- and sterol-enriched membrane microdomains (Simons and Gerl, 2010). Some GPI-APs are released from the cell membrane by phosphatidylinositol-specific phospholipases to the extracellular matrix, where they might engage in processes such as cell adhesion and cell-cell communication. In Arabidopsis (Arabidopsis thaliana), there are about 250 predicted GPI-APs (Borner et al., 2003), a relatively large number compared with about 150 in mammals and 50 in the budding yeast (Saccharomyces cerevisiae). Important functions for plant GPI-APs have been elucidated through the study of individual proteins, such as the COBRA family in cell expansion and cell wall biosynthesis (Brady et al., 2007), ARABINOGALACTAN PROTEIN18 in megagametogenesis (Demesa-Arévalo and Vielle-Calzada, 2013), and LORELEI in the pollen tube-female gametophyte interaction (Capron et al., 2008; Tsukamoto et al., 2010; Duan et al., 2014). However, it is the studies of mutants defective in GPI biosynthesis that underscore the general importance of GPI-APs as a class: lacking the capacity to assemble the anchor is lethal.Open in a separate windowFigure 1.GPI anchor biosynthesis pathway. Ten or 11 stepwise modifications of phosphoinositide occur, starting from the synthesis of N-glucosamine-phosphoinositide on the cytoplasmic surface of the ER, followed by its flipping to the ER lumenal side for additional modifications, ending with the addition of the terminal ethanolamine phosphate. Proteins destined for GPI modification are synthesized with a C-terminal signature sequence recognized by the GPI transamidase (a five-protein-enzyme complex) that concomitantly cleaves the peptide at what is designated as the ω and ω+1 amino acids and attaches the GPI anchor in a transamination reaction (red arrows). The GPI-modified proteins are then sorted and transported via the Golgi apparatus to the cell membrane. The established biosynthetic proteins from Arabidopsis and their mammalian homologs are indicated; the galactosylation step appears to be plant specific. The diagram is modeled after figure 3 in Ellis et al. (2010), which also provided a complete list of potential plant orthologs of the human and yeast proteins in the pathway.The GPI anchor is synthesized by an elaborate biosynthetic pathway, starting on the cytoplasmic side of the ER and ending with a completely assembled core anchor on the lumenal surface of the ER (Fig. 1). Prior to their transport out of the ER, proteins destined for GPI modification are cleaved at a C-terminal signature sequence by a GPI transamidase complex that in two enzymatic steps concomitantly attaches a GPI anchor to the C terminus of processed proteins (Kinoshita, 2014). Most of the knowledge on GPI biosynthesis and GPI-AP modification is derived from studies in mammals and yeast, but the pathway is likely to be conserved in plants (Ellis et al., 2010). In a recent article in Plant Physiology, Dai et al. (2014) reported that a GPI anchor biosynthesis mutant, abnormal pollen tube guidance1 (atpg1), displays both embryo lethality and severely depressed male fertility. They determined that APTG1 is homologous to the yeast GPI10 and human PIG-B (for phosphatidylinositol glycan anchor biosynthesis) proteins, the last of three distinct mannosyltransferases that modify the precursor anchor (Fig. 1), and showed that APTG1 can functionally substitute for GPI10 in a conditionally lethal gpi10 yeast mutant. Previous studies have demonstrated that Arabidopsis SETH1 (a male fertility god in Egyptian mythology), SETH2, and PEANUT1 (PNT1), encoding homologs of mammalian PIG-C, PIG-A, and PIG-M (Fig. 1) and their corresponding yeast counterparts, respectively, are important for male fertility (Lalanne et al., 2004; Gillmor et al., 2005). In addition, loss of the first mannosyltransferase in the pathway in pnt1 results in early seedling lethality. pnt1 embryos are severely defective, displaying various cell division anomalies and exhibiting altered levels and ectopic deposition of cell wall polymers. The results reported by Dai et al. (2014), therefore, further demonstrate the conservation of the GPI biosynthesis pathway and the importance of GPI anchoring in plant development and reproduction.The aptg1 mutant was isolated in a search for mutants defective in pollen tube targeting of ovules (Fig. 2), an intriguing and crucial step in plant reproduction. A pollen tube is guided to an ovule by attractants, and upon reaching the target, the female gametophyte, the pollen tube ruptures, ejecting its cytoplasm and releasing sperm for fertilization (Dresselhaus and Franklin-Tong, 2013). aptg1 pollen tubes either fail to target ovules or undertake a more twisted pathway before entering an ovule. In an earlier study, Li et al. (2013) showed that a GPI-AP, COBRA-LIKE10 (COBL10), is required to maintain normal pollen tube growth rates and ovule targeting efficiency. In aptg1 pollen tubes, citrine fluorescent protein-COBL10 was absent from its normal apical membrane location while the citrine fluorescent signal in the cytoplasm was more intense, implying that the diminished presence of COBL10 on the apical membrane could be an underlying cause for the ovule-targeting phenotype. This observation also demonstrates that GPI anchoring is important for the subsequent sorting and transport of these proteins to their destined locations (Kinoshita et al., 2013) and consistent with a wholesale failure of GPI-APs to reach their functional locations as underlying lethality in GPI biosynthesis mutants.Open in a separate windowFigure 2.Pollen tube targeting of ovules in an Arabidopsis pistil. GUS-expressing pollen grains pollinated the pistil. Each blue dot represents discharged cytoplasm from a pollen tube that, in response to attractants, has successfully targeted the ovule and penetrated the female gametophyte and was induced to burst. The cytoplasmic discharge releases sperm for fertilization.While it is clear that major biological roles are played by GPI-APs, many questions remain. Most constituents of the plant GPI anchor biosynthetic pathway remain to be functionally established (Fig. 1). Much has been said about the membrane environments where GPI-APs are localized, but we are far from understanding the precise roles they play in assembling these domains and regulating their functional dynamics. Advances in high-resolution imaging at the cell surface and biochemical approaches to determine the constituents in these membrane microdomains (Simons and Gerl, 2010) should accelerate our understanding of the importance of GPI anchoring as a conserved strategy among eukaryotes to control a wide range of processes.     

A Proteomic Approach Provides New Insights into the Control of Soil-Borne Plant Pathogens by Bacillus Species

Beneficial microorganisms (also known as biopesticides) are considered to be one of the most promising methods for more rational and safe crop management practices. We used Bacillus strains EU07, QST713 and FZB24, and investigated their inhibitory effect on Fusarium. Bacterial cell cultures, cell-free supernatants and volatiles displayed varying degrees of suppressive effect. Proteomic analysis of secreted proteins from EU07 and FZB24 revealed the presence of lytic enzymes, cellulases, proteases, 1,4-β-glucanase and hydrolases, all of which contribute to degradation of the pathogen cell wall. Further proteomic investigations showed that proteins involved in metabolism, protein folding, protein degradation, translation, recognition and signal transduction cascade play an important role in the control of Fusarium oxysporum. Our findings provide new knowledge on the mechanism of action of Bacillus species and insight into biocontrol mechanisms.