Single Molecule Imaging of T-DNA Intermediates Following Agrobacterium tumefaciens Infection in Nicotiana benthamiana

Plant transformation mediated by Agrobacterium tumefaciens is a well-studied phenomenon in which a bacterial DNA fragment (T-DNA), is transferred to the host plant cell, as a single strand, via type IV secretion system and has the potential to reach the nucleus and to be integrated into its genome. While Agrobacterium-mediated transformation has been widely used for laboratory-research and in breeding, the time-course of its journey from the bacterium to the nucleus, the conversion from single- to double-strand intermediates and several aspects of the integration in the genome remain obscure. In this study, we sought to follow T-DNA infection directly using single-molecule live imaging. To this end, we applied the LacO-LacI imaging system in Nicotiana benthamiana, which enabled us to identify double-stranded T-DNA (dsT-DNA) molecules as fluorescent foci. Using confocal microscopy, we detected progressive accumulation of dsT-DNA foci in the nucleus, starting 23 h after transfection and reaching an average of 5.4 and 8 foci per nucleus at 48 and 72 h post-infection, respectively. A time-course diffusion analysis of the T-DNA foci has demonstrated their spatial confinement.

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Faculty Opinions recommendation of Single Molecule Imaging of T-DNA Intermediates Following Agrobacterium tumefaciens Infection in Nicotiana benthamiana.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.737059789.793568445 ◽

2019 ◽

Author(s):

Vitaly Citovsky ◽

Benoît Lacroix

Keyword(s):

Agrobacterium Tumefaciens ◽

Single Molecule ◽

Nicotiana Benthamiana ◽

Single Molecule Imaging

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Transcriptomic Analysis of Arabidopsis Seedlings in Response to an Agrobacterium-Mediated Transformation Process

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-10-17-0249-r ◽

2018 ◽

Vol 31 (4) ◽

pp. 445-459 ◽

Cited By ~ 5

Author(s):

Kaixuan Duan ◽

Christopher J. Willig ◽

Joann R. De Tar ◽

William G. Spollen ◽

Zhanyuan J. Zhang

Keyword(s):

Agrobacterium Tumefaciens ◽

Defense Response ◽

Time Course ◽

Basic Research ◽

Defense Responses ◽

Transformation Process ◽

Host Responses ◽

Nucleic Acid Metabolism ◽

Agrobacterium Strain ◽

Agrobacterium Mediated Transformation

Agrobacterium tumefaciens is a plant pathogen that causes crown gall disease. This pathogen is capable of transferring the T-DNA from its Ti plasmid to the host cell and, then, integrating it into the host genome. To date, this genetic transformation ability has been harnessed as the dominant technology to produce genetically modified plants for both basic research and crop biotechnological applications. However, little is known about the interaction between Agrobacterium tumefaciens and host plants, especially the host responses to Agrobacterium infection and its associated factors. We employed RNA-seq to follow the time course of gene expression in Arabidopsis seedlings infected with either an avirulent or a virulent Agrobacterium strain. Gene Ontology analysis indicated many biological processes were involved in the Agrobacterium-mediated transformation process, including hormone signaling, defense response, cellular biosynthesis, and nucleic acid metabolism. RNAseq and quantitative reverse transcription-polymerase chain reaction results indicated that expression of genes involved in host plant growth and development were repressed but those involved in defense response were induced by Agrobacterium tumefaciens. Further analysis of the responses of transgenic Arabidopsis lines constitutively expressing either the VirE2 or VirE3 protein suggested Vir proteins act to enhance plant defense responses in addition to their known roles facilitating T-DNA transformation.

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The Agrobacterium tumefaciens Ti Plasmid Virulence Gene virE2 Reduces Sri Lankan Cassava Mosaic Virus Infection in Transgenic Nicotiana benthamiana Plants

Viruses ◽

10.3390/v7052641 ◽

2015 ◽

Vol 7 (5) ◽

pp. 2641-2653 ◽

Cited By ~ 5

Author(s):

Thulasi Resmi ◽

Thomas Hohn ◽

Barbara Hohn ◽

Karuppannan Veluthambi

Keyword(s):

Agrobacterium Tumefaciens ◽

Virus Infection ◽

Mosaic Virus ◽

Nicotiana Benthamiana ◽

Virulence Gene ◽

Ti Plasmid ◽

Sri Lankan ◽

Cassava Mosaic Virus ◽

Mosaic Virus Infection

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Faculty Opinions recommendation of Detergent extraction identifies different VirB protein subassemblies of the type IV secretion machinery in the membranes of Agrobacterium tumefaciens.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1009044.117507 ◽

2002 ◽

Author(s):

Patricia Zambryski

Keyword(s):

Agrobacterium Tumefaciens ◽

Type Iv Secretion ◽

Type Iv ◽

Detergent Extraction

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Quantifying accuracy and heterogeneity in single-molecule super-resolution microscopy

10.1101/721837 ◽

2019 ◽

Author(s):

Hesam Mazidi ◽

Tianben Ding ◽

Arye Nehorai ◽

Matthew D. Lew

Keyword(s):

Single Molecule ◽

Computational Models ◽

Amyloid Fibrils ◽

Imaging System ◽

Simulated Data ◽

Super Resolution ◽

Ground Truth ◽

Localization Algorithm ◽

Reconstruction Algorithms ◽

Imaging Data

The resolution and accuracy of single-molecule localization micro-scopes (SMLMs) are routinely benchmarked using simulated data, calibration “rulers,” or comparisons to secondary imaging modalities. However, these methods cannot quantify the nanoscale accuracy of an arbitrary SMLM dataset. Here, we show that by computing localization stability under a well-chosen perturbation with accurate knowledge of the imaging system, we can robustly measure the confidence of individual localizations without ground-truth knowledge of the sample. We demonstrate that our method, termed Wasserstein-induced flux (WIF), measures the accuracy of various reconstruction algorithms directly on experimental 2D and 3D data of microtubules and amyloid fibrils. We further show that WIF confidences can be used to evaluate the mismatch between computational models and imaging data, enhance the accuracy and resolution of recon-structed structures, and discover hidden molecular heterogeneities. As a computational methodology, WIF is broadly applicable to any SMLM dataset, imaging system, and localization algorithm.

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Membrane and Core Periplasmic Agrobacterium tumefaciens Virulence Type IV Secretion System Components Localize to Multiple Sites around the Bacterial Perimeter during Lateral Attachment to Plant Cells

mBio ◽

10.1128/mbio.00218-11 ◽

2011 ◽

Vol 2 (6) ◽

Cited By ~ 33

Author(s):

Julieta Aguilar ◽

Todd A. Cameron ◽

John Zupan ◽

Patricia Zambryski

Keyword(s):

Agrobacterium Tumefaciens ◽

Plant Cells ◽

Secretion System ◽

Type Iv Secretion ◽

Host Cells ◽

Type Iv Secretion System ◽

Secretion Systems ◽

Content Type ◽

Protein Substrates ◽

Type Iv

ABSTRACTType IV secretion systems (T4SS) transfer DNA and/or proteins into recipient cells. Here we performed immunofluorescence deconvolution microscopy to localize the assembled T4SS by detection of its native components VirB1, VirB2, VirB4, VirB5, VirB7, VirB8, VirB9, VirB10, and VirB11 in the C58 nopaline strain ofAgrobacterium tumefaciens, following induction of virulence (vir) gene expression. These different proteins represent T4SS components spanning the inner membrane, periplasm, or outer membrane. Native VirB2, VirB5, VirB7, and VirB8 were also localized in theA. tumefaciensoctopine strain A348. Quantitative analyses of the localization of all the above Vir proteins in nopaline and octopine strains revealed multiple foci in single optical sections in over 80% and 70% of the bacterial cells, respectively. Green fluorescent protein (GFP)-VirB8 expression followingvirinduction was used to monitor bacterial binding to live host plant cells; bacteria bind predominantly along their lengths, with few bacteria binding via their poles or subpoles.vir-induced attachment-defective bacteria or bacteria without the Ti plasmid do not bind to plant cells. These data support a model where multiplevir-T4SS around the perimeter of the bacterium maximize effective contact with the host to facilitate efficient transfer of DNA and protein substrates.IMPORTANCETransfer of DNA and/or proteins to host cells through multiprotein type IV secretion system (T4SS) complexes that span the bacterial cell envelope is critical to bacterial pathogenesis. Early reports suggested that T4SS components localized at the cell poles. Now, higher-resolution deconvolution fluorescence microscopy reveals that all structural components of theAgrobacterium tumefaciens vir-T4SS, as well as its transported protein substrates, localize to multiple foci around the cell perimeter. These results lead to a new model ofA. tumefaciensattachment to a plant cell, whereA. tumefacienstakes advantage of the multiplevir-T4SS along its length to make intimate lateral contact with plant cells and thereby effectively transfer DNA and/or proteins through thevir-T4SS. The T4SS ofA. tumefaciensis among the best-studied T4SS, and the majority of its components are highly conserved in different pathogenic bacterial species. Thus, the results presented can be applied to a broad range of pathogens that utilize T4SS.

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