Transposable Element Regulation in Rice and Arabidopsis: Diverse Patterns of Active Expression and siRNA-mediated Silencing

Transposable elements (TEs) are DNA segments that can mediate or cause movement within genomes. We performed a comprehensive, whole-genome analysis of annotated TEs in rice (Oryza sativa L.) and Arabidopsis thaliana, focusing on their expression (mRNA data) and silencing (small RNA data), and we compared these data with annotated genes that are not annotated as transposons. TEs demonstrated higher levels of antisense mRNA expression in comparison to non-TE genes. The majority of the TEs were silenced, as demonstrated by higher levels of small RNAs and a lack of mRNA MPSS data. When TEs were expressed, their activity was usually limited to just one or a few of the mRNA libraries. When we examined TE expression at the whole-genome level and across the complete mRNA dataset, we observed that most activity was contributed by a few highly expressed transposable elements. These TEs were characterized by their low copy number and few matching small RNAs. Our results help define the relationship between gene expression and gene silencing for TEs, and indicate that TE silencing can impact neighboring genes, perhaps via a mechanism of heterochromatin formation and spreading. These data may be used to define active TEs and families of transposable elements that continue to shape plant genomes.     

Structure and Evolution of the Actin Gene Family in Arabidopsis Thaliana

Higher plants contain families of actin-encoding genes that are divergent and differentially expressed. Progress in understanding the functions and evolution of plant actins has been hindered by the large size of the actin gene families. In this study, we characterized the structure and evolution of the actin gene family in Arabidopsis thaliana. DNA blot analyses with gene-specific probes suggested that all 10 of the Arabidopsis actin gene family members have been isolated and established that Arabidopsis has a much simpler actin gene family than other plants that have been examined. Phylogenetic analyses suggested that the Arabidopsis gene family contains at least two ancient classes of genes that diverged early in land plant evolution and may have separated vegetative from reproductive actins. Subsequent divergence produced a total of six distinct subclasses of actin, and five showed a distinct pattern of tissue specific expression. The concordance of expression patterns with the phylogenetic structure is discussed. These subclasses appear to be evolving independently, as no evidence of gene conversion was found. The Arabidopsis actin proteins have an unusually large number of nonconservative amino acid substitutions, which mapped to the surface of the actin molecule, and should effect protein-protein interactions.     

Spider Glue Proteins Have Distinct Architectures Compared with Traditional Spidroin Family Members

Adhesive spider glues are required to perform a variety of tasks, including web construction, prey capture, and locomotion. To date, little is known regarding the molecular and structural features of spider glue proteins, in particular bioadhesives that interconnect dragline or scaffolding silks during three-dimensional web construction. Here we use biochemical and structural approaches to identify and characterize two aggregate gland specific gene products, AgSF1 and AgSF2, and demonstrate that these proteins co-localize to the connection joints of both webs and wrapping silks spun from the black widow spider, Latrodectus hesperus. Protein architectures are markedly divergent between AgSF1 and AgSF2, as well as traditional spider silk fibroin family members, suggesting connection joints consist of a complex proteinaceous network. AgSF2 represents a nonglycosylated 40-kDa protein that has novel internal amino acid block repeats with the consensus sequence NVNVN embedded in a glycine-rich matrix. Analysis of the amino acid sequence of AgSF1 reveals pentameric QPGSG iterations that are similar to conserved modular elements within mammalian elastin, a rubber-like elastomeric protein that interfaces with collagen. Wet-spinning methodology using purified recombinant proteins show AgSF1 has the potential to self-assemble into fibers. X-ray fiber diffraction studies performed on these synthetic fibers reveal the presence of noncrystalline domains that resemble classical rubber networks. Collectively, these data support that the aggregate gland serves to extrude a protein mixture that contains substances that allow for the self-assembly of fiber-like structures that interface with dragline silks to mediate prey capture.     

Expression of Distinct Self-Incompatibility Specificities in Arabidopsis thaliana

The interplay of balancing selection within a species and rapid gene evolution between species can confound our ability to determine the functional equivalence of interspecific and intergeneric pairs of alleles underlying reproduction. In crucifer plants, mating specificity in the barrier to self-fertilization called self-incompatibility (SI) is controlled by allele-specific interactions between two highly polymorphic and co-evolving proteins, the S-locus receptor kinase (SRK) and its S-locus cysteine rich (SCR) ligand. These proteins have diversified both within and between species such that it is often difficult to determine from sequence information alone if they encode the same or different SI specificity. The self-fertile Arabidopsis thaliana was derived from an obligate outbreeding ancestor by loss of self-incompatibility, often in conjunction with inactivation of SRK or SCR. Nevertheless, some accessions of A. thaliana can express self-incompatibility upon transformation with an SRK–SCR gene pair isolated from its self-incompatible close relative A. lyrata. Here we show that several additional and highly diverged SRK/SCR genes from A. lyrata and another crucifer plant, Capsella grandiflora, confer self-incompatibility in A. thaliana, either as intact genes isolated from genomic libraries or after manipulation to generate chimeric fusions. We describe how the use of this newly developed chimeric protein strategy has allowed us to test the functional equivalence of SRK/SCR gene pairs from different taxa and to assay the functionality of endogenous A. thaliana SRK and SCR sequences.MATING reactions in plants, fungi, and animals are strongly influenced by molecular recognition machineries that act as gauges of genetic relatedness (Brown and Casselton 2001; Nasrallah 2005; Yamazaki and Beauchamp 2007). Many plants with hermaphroditic flowers have evolved inbreeding avoidance mechanisms, known as self-incompatibility (SI) systems. These systems are based on the ability of the female reproductive apparatus (the pistil) to discriminate among genetically distinct pollen grains, resulting in the failure of self-pollination despite functional female and male reproductive structures. In the Brassicaceae (crucifers), specific recognition of pollen by the epidermal cells of the stigma (a structure located at the tip of the pistil) is controlled by haplotypes of the S locus, and activation of the SI response leading to inhibition of pollen tube growth occurs if pollen and stigma are derived from plants that express the same S-locus haplotype (S haplotype). Within self-incompatible crucifer species, the number of S haplotypes and corresponding SI specificities is usually high, with >50 reported in some species (Watanabe et al. 2000), and SI dictates that self-incompatible plants are typically heterozygous and carry two S haplotypes. Each S haplotype is composed of two highly polymorphic genes that are the determinants of SI specificity in stigma and pollen (Stein et al. 1991; Schopfer et al. 1999). The S-locus receptor kinase (SRK) gene encodes a single-pass transmembrane serine/threonine kinase localized on the surface of stigma epidermal cells, and the S-locus cysteine-rich protein (SCR) gene encodes a small peptide localized in the pollen coat. SCR is the ligand for SRK and will bind to the extracellular domain of SRK (hereafter eSRK) only if both proteins are encoded by the same S-locus haplotype (Kachroo et al. 2001; Takayama et al. 2001; Chookajorn et al. 2004). The binding of SCR to its cognate eSRK triggers an intracellular phosphorylation cascade that results in pollen rejection by a poorly understood mechanism.A mechanistic understanding of the recognition phase of SI requires detailed structure–function analyses of SRK and SCR aimed at identifying the amino acid residues that determine their allele-specific interaction and explaining the puzzling dominance/recessive interactions exhibited by different SRK alleles in the heterozygous stigmas of self-incompatible plants (Hatakeyama et al. 2001; Mable et al. 2003; Prigoda et al. 2005). Such structure–function studies require an experimental system that allows efficient in vivo functional analysis of large numbers of SRK and SCR sequence variants generated in vitro by site-directed mutagenesis or domain swapping between proteins that determine different SI specificities. The recent transfer of the SI trait into Arabidopsis thaliana has established this species as a model organism for mechanistic and evolutionary studies of mating systems in crucifers (Nasrallah et al. 2002, 2004). However, to date, only one SI specificity, that which is determined by the Sb haplotype of A. lyrata, has been successfully introduced into A. thaliana and shown to alter the plant''s mating reaction from strict autogamy to full SI. To exploit fully the A. thaliana transgenic SI model, additional S haplotypes must be introduced into this species. In addition to facilitating mechanistic studies of the SRK–SCR interaction and dominance relationships, the expression of multiple SI specificities in A. thaliana promises to shed light on processes underlying the diversification of SRK and SCR genes. For example, expression in A. thaliana of SI specificities derived from different crucifer species will allow direct assays of the functional equivalence or nonequivalence of the corresponding S haplotypes, an issue that is difficult to resolve on the basis of sequence information alone.Although conceptually simple, expressing different SI specificities by transformation with different SRK/SCR gene pairs is not a straightforward proposition. Difficulties stem largely from the availability of appropriate cloned SRK/SCR variants for use in transformation experiments. A large number of SRK/SCR gene pairs are available from Brassica species as a result of extensive and long-standing studies of SI. However, attempts to restore SI in transgenic A. thaliana using Brassica S-locus genes had met with failure (Bi et al. 2000; J. B. Nasrallah, unpublished data), possibly because of the inability of Brassica SRKs to interact productively with A. thaliana components of the SI signal transduction pathway. In the past few years, studies of SI were initiated in self-incompatible species more closely related to A. thaliana, such as A. lyrata, A. halleri, and Capsella grandiflora. However, with a few exceptions, these studies produced only partial SRK and SCR sequences amplified from genomic DNA (Schierup et al. 2001; Prigoda et al. 2005; Bechsgaard et al. 2006; Paetsch et al. 2006). The challenging task of cloning the very highly polymorphic SCR sequences and complete SRK and SCR genes, which requires genomic library construction and in many cases chromosome walking, has only been accomplished for two S haplotypes of A. lyrata, Sb (hereafter AlSb, which was used in previous transformation studies (Nasrallah et al. 2002, 2004), and Sa (AlSa; Kusaba et al. 2001), and for the S7 haplotype of C. grandiflora (CgS7; Nasrallah et al. 2007).In this article, we report the isolation of two new SRK/SCR gene pairs from genomic libraries of A. lyrata and expression of the corresponding SI specificities in A. thaliana. We also describe a novel strategy for rapid and efficient transfer of several distinct SI specificities into A. thaliana, which only requires knowledge of the eSRK sequence and SCR second-exon sequences that encode the mature SCR protein.     

拟南芥MATE家族成员DTX18 表达调控

拟南芥多药物和有毒化合物排出家族(MATE)属次级转运蛋白家族, 此类转运蛋白与解毒内源的次生代谢物和外源的有毒化合物有关.通过 PCR 的方法从拟南芥基因组中扩增到该家族成员DTX18的启动子序列,构建重组质粒后,通过农杆菌介导的方法获得转基因植物.GUS 组织化学染色发现此基因的表达受到伤害和茉莉酸甲酯(MJ)诱导.同时结合半定量 PCR 的方法检测该基因在伤害及 MJ 处理下转录本丰度的变化,进一步证实了此结果.另外,此基因在突变体coi1,ein2中的表达量明显降低,这一点揭示了此基因表达的调控机制,即与植物激素JA/ET的信号传导密切相关.     

Interactions with Actin Monomers,Actin Filaments,and Arp2/3 Complex Define the Roles of WASP Family Proteins and Cortactin in Coordinately Regulating Branched Actin Networks

Arp2/3 complex is an important actin filament nucleator that creates branched actin filament networks required for formation of lamellipodia and endocytic actin structures. Cellular assembly of branched actin networks frequently requires multiple Arp2/3 complex activators, called nucleation promoting factors (NPFs). We recently presented a mechanism by which cortactin, a weak NPF, can displace a more potent NPF, N-WASP, from nascent branch junctions to synergistically accelerate nucleation. The distinct roles of these NPFs in branching nucleation are surprising given their similarities. We biochemically dissected these two classes of NPFs to determine how their Arp2/3 complex and actin interacting segments modulate their influences on branched actin networks. We find that the Arp2/3 complex-interacting N-terminal acidic sequence (NtA) of cortactin has structural features distinct from WASP acidic regions (A) that are required for synergy between the two NPFs. Our mutational analysis shows that differences between NtA and A do not explain the weak intrinsic NPF activity of cortactin, but instead that cortactin is a weak NPF because it cannot recruit actin monomers to Arp2/3 complex. We use TIRF microscopy to show that cortactin bundles branched actin filaments using actin filament binding repeats within a single cortactin molecule, but that N-WASP antagonizes cortactin-mediated bundling. Finally, we demonstrate that multiple WASP family proteins synergistically activate Arp2/3 complex and determine the biochemical requirements in WASP proteins for synergy. Our data indicate that synergy between WASP proteins and cortactin may play a general role in assembling diverse actin-based structures, including lamellipodia, podosomes, and endocytic actin networks.     

LIM蛋白KyoT与PcG蛋白的相互作用

KyoT为LIM结构域蛋白 ,可与转录因子RBP J相互作用并抑制RBP J介导的转录激活。为了研究其功能 ,用酵母双杂交法筛选了与KyoT相互作用的蛋白质 ,其中包括同属于PcG (polycombgroup)家族的蛋白质Ring1和hPc2 (humanpolycomb2 )。为进一步验证KyoT2与Ring1和hPc2的相互作用 ,首先在酵母中证实了KyoT2与它们的相互作用并得到阳性结果。然后做GST pulldown实验在体外验证了KyoT2与Ring1和hPc2在体外的相互作用。进而又构建了KyoT2与Ring1和hPc2不同的截短体 ,利用酵母双杂交进一步验证了它们相互作用的区段 ,发现KyoT的LIM结构域与Ring1的全长相互作用 ,与hPc2的C末端和全长相互作用。本研究验证了KyoT2与Ring1和hPc2的相互作用 ,对探讨KyoT的功能以及分析Notch信号途径与PcG家族的关系提供一定的依据。     

pAUL: A Gateway-Based Vector System for Adaptive Expression and Flexible Tagging of Proteins in Arabidopsis

Determination of protein function requires tools that allow its detection and/or purification. As generation of specific antibodies often is laborious and insufficient, protein tagging using epitopes that are recognized by commercially available antibodies and matrices appears more promising. Also, proper spatial and temporal expression of tagged proteins is required to prevent falsification of results. We developed a new series of binary Gateway cloning vectors named pAUL1-20 for C- and N-terminal in-frame fusion of proteins to four different tags: a single (i) HA epitope and (ii) Strep-tagIII, (iii) both epitopes combined to a double tag, and (iv) a triple tag consisting of the double tag extended by a Protein A tag possessing a 3C protease cleavage site. Expression can be driven by either the 35 S CaMV promoter or, for C-terminal fusions, promoters from genes encoding the chloroplast biogenesis factors HCF107, HCF136, or HCF173. Fusions of the four promoters to the GUS gene showed that endogenous promoter sequences are functional and drive expression more moderately and consistently throughout different transgenic lines when compared to the 35 S CaMV promoter. By testing complementation of mutations affected in chloroplast biogenesis factors HCF107 and HCF208, we found that the effect of different promoters and tags on protein function strongly depends on the protein itself. Single-step and tandem affinity purification of HCF208 via different tags confirmed the integrity of the cloned tags.     

Ancient Out-of-Africa Mitochondrial DNA Variants Associate with Distinct Mitochondrial Gene Expression Patterns

Mitochondrial DNA (mtDNA) variants have been traditionally used as markers to trace ancient population migrations. Although experiments relying on model organisms and cytoplasmic hybrids, as well as disease association studies, have served to underline the functionality of certain mtDNA SNPs, only little is known of the regulatory impact of ancient mtDNA variants, especially in terms of gene expression. By analyzing RNA-seq data of 454 lymphoblast cell lines from the 1000 Genomes Project, we found that mtDNA variants defining the most common African genetic background, the L haplogroup, exhibit a distinct overall mtDNA gene expression pattern, which was independent of mtDNA copy numbers. Secondly, intra-population analysis revealed subtle, yet significant, expression differences in four tRNA genes. Strikingly, the more prominent African mtDNA gene expression pattern best correlated with the expression of nuclear DNA-encoded RNA-binding proteins, and with SNPs within the mitochondrial RNA-binding proteins PTCD1 and MRPS7. Our results thus support the concept of an ancient regulatory transition of mtDNA-encoded genes as humans left Africa to populate the rest of the world.     

Arabidopsis VILLIN1 and VILLIN3 Have Overlapping and Distinct Activities in Actin Bundle Formation and Turnover

Actin filament bundles are higher-order cytoskeletal structures that are crucial for the maintenance of cellular architecture and cell expansion. They are generated from individual actin filaments by the actions of bundling proteins like fimbrins, LIMs, and villins. However, the molecular mechanisms of dynamic bundle formation and turnover are largely unknown. Villins belong to the villin/gelsolin/fragmin superfamily and comprise at least five isovariants in Arabidopsis thaliana. Different combinations of villin isovariants are coexpressed in various tissues and cells. It is not clear whether these isovariants function together and act redundantly or whether they have unique activities. VILLIN1 (VLN1) is a simple filament-bundling protein and is Ca²⁺ insensitive. Based on phylogenetic analyses and conservation of Ca²⁺ binding sites, we predict that VLN3 is a Ca²⁺-regulated villin capable of severing actin filaments and contributing to bundle turnover. The bundling activity of both isovariants was observed directly with time-lapse imaging and total internal reflection fluorescence (TIRF) microscopy in vitro, and the mechanism mimics the “catch and zipper” action observed in vivo. Using time-lapse TIRF microscopy, we observed and quantified the severing of individual actin filaments by VLN3 at physiological calcium concentrations. Moreover, VLN3 can sever actin filament bundles in the presence of VLN1 when calcium is elevated to micromolar levels. Collectively, these results demonstrate that two villin isovariants have overlapping and distinct activities.     

Genome-Wide Identification and Expression of Xenopus F-Box Family of Proteins

Protein degradation via the multistep ubiquitin/26S proteasome pathway is a rapid way to alter the protein profile and drive cell processes and developmental changes. Many key regulators of embryonic development are targeted for degradation by E3 ubiquitin ligases. The most studied family of E3 ubiquitin ligases is the SCF ubiquitin ligases, which use F-box adaptor proteins to recognize and recruit target proteins. Here, we used a bioinformatics screen and phylogenetic analysis to identify and annotate the family of F-box proteins in the Xenopus tropicalis genome. To shed light on the function of the F-box proteins, we analyzed expression of F-box genes during early stages of Xenopus development. Many F-box genes are broadly expressed with expression domains localized to diverse tissues including brain, spinal cord, eye, neural crest derivatives, somites, kidneys, and heart. All together, our genome-wide identification and expression profiling of the Xenopus F-box family of proteins provide a foundation for future research aimed to identify the precise role of F-box dependent E3 ubiquitin ligases and their targets in the regulatory circuits of development.     

Expression of SFRP Family Proteins in Human Keratoconus Corneas

We investigated the expression of the secreted frizzled-related proteins (SFRPs) in keratoconus (KC) and control corneas. KC buttons (∼8 mm diameter) (n = 15) and whole control corneas (n = 7) were fixed in 10% formalin or 2% paraformaldehyde and subsequently paraffin embedded and sectioned. Sections for histopathology were stained with hematoxylin and eosin, or Periodic Acid Schiff’s reagent. A series of sections was also immunolabelled with SFRP 1 to 5 antibodies, visualised using immunofluorescence, and examined with a Zeiss LSM700 scanning laser confocal microscope. Semi-quantitative grading was used to compare SFRP immunostaining in KC and control corneas. Overall, KC corneas showed increased immunostaining for SFRP1 to 5, compared to controls. Corneal epithelium in all KC corneas displayed heterogeneous moderate to strong immunoreactivity for SFRP1 to 4, particularly in the basal epithelium adjacent to cone area. SFRP3 and 5 were localised to epithelial cell membranes in KC and control corneas, with increased SFRP3 cytoplasmic expression observed in KC. Strong stromal expression of SFRP5, including extracellular matrix, was seen in both KC and control corneas. In control corneas we observed differential expression of SFRP family proteins in the limbus compared to more central cornea. Taken together, our results support a role for SFRPs in maintaining a healthy cornea and in the pathogenesis of epithelial and anterior stromal disruption observed in KC.