The exchange of small RNAs (sRNAs) between hosts and pathogens can lead to gene silencing in the recipient organism, a mechanism termed cross-kingdom RNAi (ck-RNAi). While fungal sRNAs promoting ...virulence are established, the significance of ck-RNAi in distinct plant pathogens is not clear. Here, we describe that sRNAs of the pathogen
, which represents the kingdom of oomycetes and is phylogenetically distant from fungi, employ the host plant's Argonaute (AGO)/RNA-induced silencing complex for virulence. To demonstrate
sRNA (
sRNA) functionality in ck-RNAi, we designed a novel CRISPR endoribonuclease Csy4/GUS reporter that enabled in situ visualization of
sRNA-induced target suppression in Arabidopsis. The significant role of
sRNAs together with
AGO1 in virulence was revealed in plant
mutants and by transgenic Arabidopsis expressing a short-tandem-target-mimic to block
sRNAs, that both exhibited enhanced resistance.
sRNA-targeted plant genes contributed to host immunity, as Arabidopsis gene knockout mutants displayed quantitatively enhanced susceptibility.
Ralstonia solanacearum is a devastating bacterial phytopathogen with a broad host range. Ralstonia solanacearum injected effector proteins (Rips) are key to the successful invasion of host plants. We ...have characterized Brg11(hrpB‐regulated 11), the first identified member of a class of Rips with high sequence similarity to the transcription activator‐like (TAL) effectors of Xanthomonas spp., collectively termed RipTALs. Fluorescence microscopy of in planta expressed RipTALs showed nuclear localization. Domain swaps between Brg11 and Xanthomonas TAL effector (TALE) AvrBs3 (avirulence protein triggering Bs3 resistance) showed the functional interchangeability of DNA‐binding and transcriptional activation domains. PCR was used to determine the sequence of brg11 homologs from strains infecting phylogenetically diverse host plants. Brg11 localizes to the nucleus and activates promoters containing a matching effector‐binding element (EBE). Brg11 and homologs preferentially activate promoters containing EBEs with a 5′ terminal guanine, contrasting with the TALE preference for a 5′ thymine. Brg11 and other RipTALs probably promote disease through the transcriptional activation of host genes. Brg11 and the majority of homologs identified in this study were shown to activate similar or identical target sequences, in contrast to TALEs, which generally show highly diverse target preferences. This information provides new options for the engineering of plants resistant to R. solanacearum.
This paper reports on the deposition and characterization of piezoelectric AlXSc1-XN (further: AlScN) films on Si substrates using AlSc alloy targets with 30 at.% Sc. Films were deposited on a Ø200 ...mm area with deposition rates of 200 nm/min using a reactive magnetron sputtering process with a unipolar–bipolar hybrid pulse mode of FEP. The homogeneity of film composition, structural properties and piezoelectric properties were investigated depending on process parameters, especially the pulse mode of powering in unipolar–bipolar hybrid pulse mode operation. Characterization methods include energy-dispersive spectrometry of X-ray (EDS), X-ray diffraction (XRD), piezoresponse force microscopy (PFM) and double-beam laser interferometry (DBLI). The film composition was Al0.695Sc0.295N. The films showed good homogeneity of film structure with full width at half maximum (FWHM) of AlScN(002) rocking curves at 2.2 ± 0.1° over the whole coating area when deposited with higher share of unipolar pulse mode during film growth. For a higher share of bipolar pulse mode, the films showed a much larger c-lattice parameter in the center of the coating area, indicating high in-plane compressive stress in the films. Rocking curve FWHM also showed similar values of 1.5° at the center to 3° at outer edge. The piezoelectric characterization method revealed homogenous d33,f of 11–12 pm/V for films deposited at a high share of unipolar pulse mode and distribution of 7–10 pm/V for a lower share of unipolar pulse mode. The films exhibited ferroelectric switching behavior with coercive fields of around 3–3.5 MV/cm and polarization of 80–120 µC/cm².
AvrBs3, the archetype of the family of transcription activator-like (TAL) effectors from phytopathogenic Xanthomonas bacteria, is translocated by the type III secretion system into the plant cell. ...AvrBs3 localizes to the plant cell nucleus and activates the transcription of target genes. Crucial for this is the central AvrBs3 region of 17.5 34-amino acid repeats that functions as a DNA-binding domain mediating recognition in a "one-repeat-to-one base pair" manner. Although AvrBs3 forms homodimers in the plant cell cytosol prior to nuclear import, it binds DNA as a monomer. Here, we show that complex formation of AvrBs3 proteins negatively affects their DNA-binding affinity in vitro. The conserved cysteine residues at position 30 of each repeat facilitate AvrBs3 complexes via disulfide bonds in vitro but are also required for the gene-inducing activity of the AvrBs3 monomer, i.e., activation of plant gene promoters. Our data suggest that the latter is due to a contribution to protein plasticity and that cysteine substitutions to alanine or serine result in a different DNA-binding mode. In addition, our studies revealed that extended parts of both the N-terminal and C-terminal regions of AvrBs3 contribute to DNA binding and, hence, gene-inducing activity in planta.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Avalanche-safe LINQ compilation Grust, Torsten; Rittinger, Jan; Schreiber, Tom
Proceedings of the VLDB Endowment,
09/2010, Letnik:
3, Številka:
1-2
Journal Article
Recenzirano
Odprti dostop
We report on a query compilation technique that enables the construction of alternative efficient query providers for Microsoft's Language Integrated Query (LINQ) framework. LINQ programs are mapped ...into an intermediate algebraic form, suitable for execution on any SQL:1999-capable relational database system.
This compilation technique leads to query providers that (1) faithfully preserve list order and nesting, both being core features of the LINQ data model, (2) support the complete family of LINQ's Standard Query Operators, (3) bring database support to LINQ to XML where the original provider performs in-memory query evaluation, and, most importantly, (4) emit SQL statement sequences whose size is only determined by the input query's result
type
(and thus independent of the database size).
A sample query scenario uses this LINQ provider to marry database-resident TPC-H and XMark data---resulting in a unique query experience that exhibits quite promising performance characteristics, especially for large data instances.
SUMMARY RNA‐guided endonucleases originating from the bacterial CRISPR/Cas system are a versatile tool for targeted gene editing. To determine the functional relevance of a gene of interest, deletion ...of the entire open reading frame (ORF) by two independent double‐strand breaks (DSBs) is particularly attractive. This strategy greatly benefits from high editing efficiency, which is strongly influenced by the Cas endonuclease version used. We developed two reporter switch‐on assays, for quantitative comparison and optimization of Cas constructs. The assays are based on four components: (i) A reporter gene, the mRNA of which carries a hairpin (HP) loop targeted by (ii) the endoribonuclease Csy4. Cleavage of the mRNA at the HP loop by Csy4 abolishes the translation of the reporter. Csy4 was used as the target for full deletion. (iii) A Cas system targeting sites flanking the Csy4 ORF with a 20‐bp spacer either side to preferentially detect full‐deletion events. Loss of functional Csy4 would lead to reporter gene expression, allowing indirect quantification of Cas‐mediated deletion events. (iv) A reference gene for normalization. We tested these assays on Nicotiana benthamiana leaves and Lotus japonicus calli induced on hypocotyl sections, using Firefly luciferase and mCitrine as reporter genes and Renilla luciferase and hygromycin phosphotransferase II as reference genes, respectively. We observed a >90% correlation between reporter expression and full Csy4 deletion events, demonstrating the validity of these assays. The principle of using the Csy4 –HP module as Cas target should be applicable to other editing goals including single DSBs in all organisms.
Significance Statement Cas endonuclease systems have revolutionized genome editing but rapid quantitative assays for the optimization of this process that circumvent the need for DNA extraction from individual plants are rare or lacking. Here, we describe two quantitative reporter switch‐on assays for the direct side‐by‐side comparison of the editing efficiency of different Cas systems, like Cas9 and Cas12a, the principles of which can be applied for optimizing gene editing in any plant species.
Transcription factors with programmable DNA-binding specificity constitute valuable tools for the design of orthogonal gene regulatory networks for synthetic biology. Transcription activator-like ...effectors (TALEs), as natural transcription regulators, were used to design, build, and test libraries of synthetic TALE-activated promoters (STAPs) that show a broad range of expression levels in plants. In this chapter, we present protocols for the construction of artificial TALEs and corresponding STAPs.
Transcription activator-like effectors (TALEs) are bacterial Type-III effector proteins from phytopathogenic
species that act as transcription factors in plants. The modular DNA-binding domain of ...TALEs can be reprogrammed to target nearly any DNA sequence. Here, we designed and optimized a two-component AND-gate system for synthetic circuits in plants based on TALEs. In this system, named split-TALE (sTALE), the TALE DNA binding domain and the transcription activation domain are separated and each fused to protein interacting domains. Physical interaction of interacting domains leads to TALE-reconstitution and can be monitored by reporter gene induction. This setup was used for optimization of the sTALE scaffolds, which result in an AND-gate system with an improved signal-to-noise ratio. We also provide a toolkit of ready-to-use vectors and single modules compatible with Golden Gate cloning and MoClo syntax. In addition to its implementation in synthetic regulatory circuits, the sTALE system allows the analysis of protein-protein interactions in planta.
AvrBs3, the founding member of the Xanthomonas transcription-activator-like effectors (TALEs), is translocated into the plant cell where it localizes to the nucleus and acts as transcription factor. ...The DNA-binding domain of AvrBs3 consists of 17.5 nearly-identical 34 amino acid-repeats. Each repeat specifies binding to one base in the target DNA via amino acid residues 12 and 13 termed repeat variable diresidue (RVD). Natural target sequences of TALEs are generally preceded by a thymine (T0), which is coordinated by a tryptophan residue (W232) in a degenerated repeat upstream of the canonical repeats. To investigate the necessity of T0 and the conserved tryptophan for AvrBs3-mediated gene activation we tested TALE mutant derivatives on target sequences preceded by all possible four bases. In addition, we performed domain swaps with TalC from a rice pathogenic Xanthomonas because TalC lacks the tryptophan residue, and the TalC target sequence is preceded by cytosine. We show that T0 works best and that T0 specificity depends on the repeat number and overall RVD-composition. T0 and W232 appear to be particularly important if the RVD of the first repeat is HD ('rep1 effect'). Our findings provide novel insights into the mechanism of T0 recognition by TALE proteins and are important for TALE-based biotechnological applications.
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