The visible touch: in planta visualization of protein-protein interactions by fluorophore-based methods

Non-invasive fluorophore-based protein interaction assays like fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC, also referred to as "split YFP") have been proven invaluable tools to study protein-protein interactions in living cells. Both methods are now frequently used in the plant sciences and are likely to develop into standard techniques for the identification, verification and in-depth analysis of polypeptide interactions. In this review, we address the individual strengths and weaknesses of both approaches and provide an outlook about new directions and possible future developments for both techniques

A Modular Plasmid Assembly Kit for Multigene Expression, Gene Silencing and Silencing Rescue in Plants

The Golden Gate (GG) modular assembly approach offers a standardized, inexpensive and reliable way to ligate multiple DNA fragments in a pre-defined order in a single-tube reaction. We developed a GG based toolkit for the flexible construction of binary plasmids for transgene expression in plants. Starting from a common set of modules, such as promoters, protein tags and transcribed regions of interest, synthetic genes are assembled, which can be further combined to multigene constructs. As an example, we created T-DNA constructs encoding multiple fluorescent proteins targeted to distinct cellular compartments (nucleus, cytosol, plastids) and demonstrated simultaneous expression of all genes in Nicotiana benthamiana, Lotus japonicus and Arabidopsis thaliana. We assembled an RNA interference (RNAi) module for the construction of intron-spliced hairpin RNA constructs and demonstrated silencing of GFP in N. benthamiana. By combination of the silencing construct together with a codon adapted rescue construct into one vector, our system facilitates genetic complementation and thus confirmation of the causative gene responsible for a given RNAi phenotype. As proof of principle, we silenced a destabilized GFP gene (dGFP) and restored GFP fluorescence by expression of a recoded version of dGFP, which was not targeted by the silencing construct

The provenance of late Cenozoic East Asian Red Clay : Tectonic-metamorphic history of potential source regions and a novel combined zircon-rutile approach

Constraining the provenance of aeolian mineral dust is critical in understanding past climate changes, atmospheric dust activity, circulation, and sediment generation. On the Chinese Loess Plateau (CLP), use of detrital zircon U-Pb age data as source tracers for the dust has seen a huge growth and lead to breakthroughs in understanding dust provenance. However, significant ambiguities remain especially regarding the provenance of the aeolian Neogene Red Clay (RC). To address this, here we review the state of the art of understanding of Neogene RC provenance, with a focus on single-grain analyses, and introduce detrital rutile geochemistry as a tool to complement zircon U-Pb dating. Furthermore, to better utilise the link between the detrital minerals and their primary origin, we compile primary source region geologic background and single-grain data relevant for use of geochronological and metamorphic provenance proxy minerals. We discuss four major tectonic divisions in northern China and southern Mongolia: North China Craton (NCC), Tarim Craton (TC), Central China Orogen (CCO), parts of the Central Asian Orogenic Belt (CAOB), and briefly summarize the Tibetan-Himalayan orogen. Many of these regions have been tectonically active during the same time periods in the Earth's history, and our analysis demonstrates how use of zircon age data alone has limitations in differentiating between a number of key potential dust sources to the CLP. Addition of a metamorphic source tracer such as rutile allows some of these possible source areas to be distinguished. For example, the proximal northern NCC regions that show high-/ ultrahigh-temperature metamorphic conditions can potentially be diagnostic of a northerly source component to CLP dust. Our combined zircon-rutile data analysis of ca. 4 Ma Nihewan RC in northern CLP verifies the utility of the novel rutile provenance proxy in sourcing CLP sediments. The zircon and rutile data suggest similar dust provenance: the dominant sources are proximal areas on the NCC, while contributions from the dry areas in parts of the CAOB, central deserts, and the Yellow River are also likely. Our results also hint at a minor source component deriving from distal western source regions in the TC, and/or in the central parts of the CCO, but rutile data from potential secondary source areas are needed to verify this possibility. We also conclude that multi-proxy single-grain provenance analyses are needed for more reliable provenance analyses.Peer reviewe

The role of the Kupferschiefer in the formation of hydrothermal base metal mineralization in the Spessart ore district, Germany: insight from detailed sulfur isotope studies

The Spessart district (SW Germany), located at the southwestern margin of the Permian Kupferschiefer basin in Central Europe, hosts abundant stratabound and structurally controlled base metal mineralization. The mineralization styles identified are (1) stratabound Cu-Pb-Zn-(Ag) ores in Zechstein sedimentary rocks, (2) structurally controlled Cu-As-(Ag) ores in Zechstein sedimentary rocks, (3) crosscutting Co-Ni-(Bi)-As and Cu-Fe-As veins, (4) stratabound metasomatic Fe-Mn carbonate ores in Zechstein dolomite, (5) barren barite veins, and (6) Fe-Mn-As veins in Permian rhyolites. Building on previous work that involved mineralogical, textural, and chemical characterization of the major mineralization types, we have performed a comprehensive sulfur isotope study that applied both conventional and novel laser-ablation multi-collector inductively coupled plasma mass spectrometry techniques. The δ34S values of sulfide minerals from the different ore types are consistently negative and highly variable, in the range between −44.5‰ and −3.9‰, whereas the δ34S values of barite are all positive in the range between 4.7‰ and 18.9‰. Remarkably, stratabound and structurally controlled mineralization in Zechstein sedimentary rocks has the least negative δ34S values, whereas vein-type deposits have consistently more negative δ34S values. The observed pattern of sulfide δ34S values can be best interpreted in terms of fluid mixing at the basement-cover interface. Hydrothermal fluids originating from the crystalline basement migrated upward along subvertical fault zones and were periodically injected into groundwaters that were flowing in the post-Variscan sedimentary cover. These groundwaters had interacted with the Zechstein sedimentary rocks, resulting in fluids characterized by elevated concentrations of reduced sulfur (with negative δ34S values) and alkaline pH. Repeated mixing between both chemically contrasting fluids caused rapid and efficient precipitation of sulfide ore minerals in hydrothermal veins with highly variable but distinctly negative δ34S value

TALENs facilitate targeted genome editing in human cells with high specificity and low cytotoxicity

Designer nucleases have been successfully employed to modify the genomes of various model organisms and human cell types. While the specificity of zinc-finger nucleases (ZFNs) and RNA-guided endonucleases has been assessed to some extent, little data are available for transcription activator-like effector-based nucleases (TALENs). Here, we have engineered TALEN pairs targeting three human loci (CCR5, AAVS1 and IL2RG) and performed a detailed analysis of their activity, toxicity and specificity. The TALENs showed comparable activity to benchmark ZFNs, with allelic gene disruption frequencies of 15-30% in human cells. Notably, TALEN expression was overall marked by a low cytotoxicity and the absence of cell cycle aberrations. Bioinformatics-based analysis of designer nuclease specificity confirmed partly substantial off-target activity of ZFNs targeting CCR5 and AAVS1 at six known and five novel sites, respectively. In contrast, only marginal off-target cleavage activity was detected at four out of 49 predicted off-target sites for CCR5- and AAVS1-specific TALENs. The rational design of a CCR5-specific TALEN pair decreased off-target activity at the closely related CCR2 locus considerably, consistent with fewer genomic rearrangements between the two loci. In conclusion, our results link nuclease-associated toxicity to off-target cleavage activity and corroborate TALENs as a highly specific platform for future clinical translation

Targeting and tracing of specific DNA sequences with dTALEs in living cells

Epigenetic regulation of gene expression involves, besides DNA and histone modifications, the relative positioning of DNA sequences within the nucleus. To trace specific DNA sequences in living cells, we used programmable sequence-specific DNA binding of designer transcription activator-like effectors (dTALEs). We designed a recombinant dTALE (msTALE) with variable repeat domains to specifically bind a 19-bp target sequence of major satellite DNA. The msTALE was fused with green fluorescent protein (GFP) and stably expressed in mouse embryonic stem cells. Hybridization with a major satellite probe (3D-fluorescent in situ hybridization) and co-staining for known cellular structures confirmed in vivo binding of the GFP-msTALE to major satellite DNA present at nuclear chromocenters. Dual tracing of major satellite DNA and the replication machinery throughout S-phase showed co-localization during mid to late S-phase, directly demonstrating the late replication timing of major satellite DNA. Fluorescence bleaching experiments indicated a relatively stable but still dynamic binding, with mean residence times in the range of minutes. Fluorescently labeled dTALEs open new perspectives to target and trace DNA sequences and to monitor dynamic changes in subnuclear positioning as well as interactions with functional nuclear structures during cell cycle progression and cellular differentiation

Promoter elements of rice susceptibility genes are bound and activated by specific TAL effectors from the bacterial blight pathogen, Xanthomonas oryzae pv. oryzae.

Summary • Plant pathogenic bacteria of the genus Xanthomonas inject transcription activator-like effector (TALe) proteins that bind to and activate host promoters, thereby promoting disease or inducing plant defense. TALes bind to corresponding UPT (up-regulated by TALe) promoter boxes via tandemly arranged 34 ⁄ 35-amino acid repeats. Recent studies uncovered the TALe code in which two amino acid residues of each repeat define specific pairing to UPT boxes. • Here we employed the TALe code to predict potential UPT boxes in TALeinduced host promoters and analyzed these via b-glucuronidase (GUS) reporter and electrophoretic mobility shift assays (EMSA). • We demonstrate that the Xa13, OsTFX1 and Os11N3 promoters from rice are induced directly by the Xanthomonas oryzae pv. oryzae TALes PthXo1, PthXo6 and AvrXa7, respectively. We identified and functionally validated a UPT box in the corresponding rice target promoter for each TALe and show that box mutations suppress TALe-mediated promoter activation. Finally, EMSA demonstrate that code-predicted UPT boxes interact specifically with corresponding TALes. • Our findings show that variations in the UPT boxes of different rice accessions correlate with susceptibility or resistance of these accessions to the bacterial blight pathogen

The eINTACT system dissects bacterial exploitation of plant osmosignalling to enhance virulence

Bacteria inject effector proteins into host cells to manipulate cellular processes that promote disease. Since bacteria deliver minuscule amounts of effectors only into targeted host cells, it is technically challenging to capture effector-dependent cellular changes from bulk-infected host tissues. Here, we report a new technique called effector-inducible isolation of nuclei tagged in specific cell types (eINTACT), which facilitates affinity-based purification of nuclei from Arabidopsis plant cells that have received Xanthomonas bacterial effectors. Analysis of purified nuclei reveals that the Xanthomonas effector XopD manipulates the expression of Arabidopsis abscisic acid signalling-related genes and activates OSCA1.1, a gene encoding a calcium-permeable channel required for stomatal closure in response to osmotic stress. The loss of OSCA1.1 causes leaf wilting and reduced bacterial growth in infected leaves, suggesting that OSCA1.1 promotes host susceptibility. eINTACT allows us to uncover that XopD exploits host OSCA1.1/abscisic acid osmosignalling-mediated stomatal closure to create a humid habitat that favours bacterial growth and opens up a new avenue for accurately elucidating functions of effectors from numerous gram-negative plant bacteria in native infection contexts.Fil: You, Yuan. Eberhard Karls Universität Tübingen; AlemaniaFil: Koczyk, Grzegorz. Polish Academy of Sciences; ArgentinaFil: Nuc, Maria. Polish Academy of Sciences; ArgentinaFil: Morbitzer, Robert. Eberhard Karls Universität Tübingen; AlemaniaFil: Holmes, Danalyn R.. Eberhard Karls Universität Tübingen; AlemaniaFil: von Roepenack Lahaye, Edda. Eberhard Karls Universität Tübingen; AlemaniaFil: Hou, Shiji. Huazhong Agricultural University; ChinaFil: Giudicatti, Axel Joel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Gris, Carine. Université de Toulouse; FranciaFil: Manavella, Pablo Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Noël, Laurent D.. Université de Toulouse; FranciaFil: Krajewski, Paweł. Polish Academy of Sciences; ArgentinaFil: Lahaye, Thomas. Eberhard Karls Universität Tübingen; Alemani

Neogene global climate change and East Asian dust sources: Combined rutile geochemistry and zircon U-Pb analysis from the northern Chinese Loess Plateau

Peer reviewe

VII Jornadas de Expania

Sección: Noticias. Noticias externasLos días 27 y 28 de mayo se celebraron en Santiago de Compostela las VII Jornadas de Expania, la Asociación de Usuarios de Ex Libris en España.N