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Research Article

The Intracellular Scots Pine Shoot Symbiont Methylobacterium extorquens DSM13060 Aggregates around the Host Nucleus and Encodes Eukaryote-Like Proteins

Janne J. Koskimäki, Anna Maria Pirttilä, Emmi-Leena Ihantola, Outi Halonen, A. Carolin Frank
Angela Sessitsch, Invited Editor, Nicole Dubilier, Editor
Janne J. Koskimäki
Department of Biology, University of Oulu, Oulu, Finland
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Anna Maria Pirttilä
Department of Biology, University of Oulu, Oulu, Finland
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Emmi-Leena Ihantola
Department of Biology, University of Oulu, Oulu, FinlandInstitute of Clinical Microbiology, University of Eastern Finland, Kuopio, Finland
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Outi Halonen
Department of Biology, University of Oulu, Oulu, Finland
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A. Carolin Frank
Life and Environmental Sciences and Sierra Nevada Research Institute, University of California, Merced, California, USA
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Angela Sessitsch
AIT Austrian Institute of Technology GmbH
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Nicole Dubilier
Max Planck Institute for Marine Microbiology
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DOI: 10.1128/mBio.00039-15
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  • FIG 1 
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    FIG 1 

    Transmission electron micrographs of bud cells of Scots pine displaying intracellular bacteria (b) and yeasts (y) residing near the nucleus (n). The cells are rich with dark chromoplasts (c). Scale bar, 2 µm.

  • FIG 2 
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    FIG 2 

    Laser scanning confocal microscopy of Scots pine seedlings colonized by M. extorquens 13061. Bacterial cells carrying a fluorescent GFP reporter under the control of a constitutive promoter are visualized in green. Cryosections of the pine seedlings colonized by M. extorquens 13061 were analyzed 15 to 90 days after inoculation. (A) Lateral section of a pine root. Arrow indicates a microcolony of 13061 actively penetrating through the root epidermis. Individual bacterial cells are visible in the root cortex. (B) Cross section of a pine stem where a 13061 colony is penetrating an epidermal cell. (C) Cross section of a pine shoot with an intracellular 13061 microcolony inside an epidermal cell. (D) Lateral section of a pine root where epidermal cells are colonized by 13061, with bacteria aggregated around the nucleus. (E) Lateral section of a root, where the arrow indicates 13061 cells congregated around the nucleus of an epidermal cell. (F) Lateral section of a pine root where epidermal cells are completely colonized by 13061 and bacteria surround the nucleus. (G) Lateral section of a pine stem where individual 13061 cells reside close to the nucleus of a cortical cell. (H) Lateral section of a lower-stem (transition zone between root and stem) cortex, where the arrow indicates 13061 cells within a parenchymatic cell. (I) Cross section of a pine shoot where intracellular 13061 cells are visible inside parenchymatic cells. (J) Magnification of the boxed area from panel I, displaying the intracellular 13061 cells near the nucleus. Co, cortex; E, epiderm; n, nucleus; S, cylindrical sheath; Xy, xylem; scale bars, 10 µm.

  • FIG 3 
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    FIG 3 

    Morphological identification of cell viability in M. extorquens 13061- and 13062-colonized tissues of Scots pine seedlings. (A) Confocal micrographs representing the spectrum of nuclei observed in the acridine orange (AO)-ethidium bromide (EB)-stained tissue. Morphology of nuclei and increased incorporation of EB (red) over AO (green) correspond with advancing programmed cell death (PCD). (B to G) Representative merged confocal micrographs of Scots pine seedling tissues. In addition to AOEB stains, bacterial cells carrying a fluorescent GFP reporter are visualized in bright green and endogenous autofluorescence of plant cells is shown in purple. (B) Cross section of an uninoculated pine root incubated in 20 mM H2O2 for 2 h at room temperature. (C) Lateral section of an uninoculated root incubated in PBS for 2 h at room temperature. (D) Lateral section of a root where an individual bacterial cell resides close to the healthy nucleus of a cortical cell. (E) Cross section of a root where intracellular 13061 cells are visible inside a parenchymatic cell. (F) Lateral section of a root where a single bacterial cell is localized in the cytoplasm of a viable cortical cell. (G) Lateral section of a root where 13062 cells are congregated around the nucleus of a cortical cell. Bacterial cells carrying fluorescent GFP and mCherry reporters under the control of constitutive promoters are visualized in white-red to distinguish them from the green nucleus. Bacteria are indicated with white arrows, and arrowheads highlight cells with dead nuclei. Co, cortex; E, epiderm; n, nucleus; Xy, xylem; scale bars, 10 µm.

  • FIG 4 
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    FIG 4 

    Structure of the M. extorquens DSM13060 genome and origins of genes. Large circle, DSM13060 chromosome; small circle, DSM13060 megaplasmid (not to scale). Successive circles from inside to outside are as follows for both chromosome and megaplasmid. First circle, AM1 BLAST hits (E < 1e−30); colors denote percentages of identity between orthologous proteins, with red denoting 100%, orange <100% and >85%, and yellow <85%. Second to 6th circles, OrthoMCL orthologs in M. extorquens strains AM1, PA1, CM4, DM4, and BJ001. Seventh to 10th circles, inferred gene origins (see Table S1 in the supplemental material). Eleventh circle, HGT to Methylobacterium spp., M. extorquens spp., or DSM13060. The outermost circle on the chromosome shows the arrangement of the 5 largest contigs in the DSM13060 scaffold, assuming colinearity with strain AM1. The outermost circles on the megaplasmid show regions that differ in their degrees of similarity to the AM1 megaplasmid (see “Genome sequence and structure,” in the text). The color coding keyed to the phylogenic tree shows when genes in different categories may have been imported, as follows: red denotes genes of the DSM13060 lineage, purple genes of the DSM13060/AM1 lineages, and green genes present before the diversification of the genus. TFSS, type IV secretion system.

  • FIG 5 
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    FIG 5 

    Phylogeny of a 73-bp conserved region of an alignment of the M. extorquens DSM13060 PLA2 protein, orthologs in the Methylobacterium spp. under study (Fig. S1), and all 60 similar sequences in the BLAST nr database (E < 0.001), excluding other Methylobacterium hits. Only bootstrap values above 50 are shown in the tree.

Tables

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  • TABLE 1 

    Proteins with eukaryote-like domains

    Eukaryote-like protein #Pfam identifierPfam symbolPercentage of sequences belonging to this Pfam family that are bacterialProposed function in host cell
    1pfam00777Glyco_transf_291.6Sialyltransferase; host immune evasion
    2, 3, 4pfam00068Phospholip_A2_12.1Host growth promotion
    5pfam06839zf-GRF2.8DNA binding; host transcription
    6pfam05686Glyco_transf_904.4None/various
    7pfam02229PC44.6DNA binding; host transcription
    8, 9pfam00400WD404.8Various (possibly host growth and development)
    10pfam13964Kelch_64.8None/various
    11pfam13202EF_hand_35.4Signaling; cell division, cell elongation, cell differentiation, and plant defense and stress responses
    12pfam13202EF_hand_35.4Signaling; cell division, cell elongation, cell differentiation, and plant defense and stress responses
    13pfam03098An_peroxidase5.8Systemic resistance
    14, 15pfam00069Pkinase6.5Protein kinase; host signal transduction
    16pfam12796Ank_27.4Protein or DNA binding; possibly host transcription
    17, 18pfam02201SWIB8.8Host chromatin remodeling
    19pfam00782DSPc9.3Tyrosine phosphatase; host signal transduction
    20pfam00450Peptidase_S109.5Defense
    21, 22pfam14226DIOX_N10.4Gibberellin biosynthesis
    23pfam11721Malectin10.6None/various
    24pfam11523DUF322310.9None/various
    25pfam07250Glyoxal_oxid_N12.3Lignin degradation
    26, 27, 28pfam04577DUF56313.1None/various
    29–34pfam00067p45013.7Gibberellin biosynthesis
    35pfam12799LRR_417.3Host defense suppression

Supplemental Material

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  • Movie S1 

    Merged confocal z-stack images showing intracellular M. extorquens 13061 cells in the parenchymal cells of a shoot of Scots pine. The red dashed line highlights the GFP-tagged bacterial cells (bright green), and the plant tissue morphology visible in the green and red channels is due to autofluorescence. Images were collected at intervals of 1 µm (z axis), depicted by the numbering in the top left corner. Co, cortex; n, nucleus. Images were processed and prepared with Zen lite 2012 software (Carl Zeiss). Download Movie S1, AVI file, 4.4 MB.

    Copyright © 2015 Koskimäki et al.

    This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

  • Movie S2 

    Merged confocal z-stack images of AOEB-stained Scots pine roots showing M. extorquens 13061 inside healthy parenchymal cells. Nuclear morphology of the plant cells with uniform incorporation of AO (green) over EB (red) and without condensation of chromatin corresponds with cellular viability. Bacterial cells carrying a fluorescent GFP reporter are visualized in bright green, and endogenous autofluorescence of plant cells is shown in gray/purple. The white arrows indicate intracellular bacteria in living cortical cells of the pine root. Images were collected at intervals of 1 µm (z axis), depicted by the numbering in the top left corner. Co, cortex; E, epiderm; n, nucleus; Xy, xylem. Images were processed and prepared with Zen lite 2012 software (Carl Zeiss). Download Movie S2, AVI file, 2.9 MB.

    Copyright © 2015 Koskimäki et al.

    This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

  • Movie S3 

    Merged confocal z-stack images of AOEB-stained pine roots showing M. extorquens 13062 residing close to the nucleus of a cortical cell. Nuclear morphology of the plant cells with uniform incorporation of AO (green) over EB (red) and without condensation of chromatin corresponds with cellular viability. Bacterial cells carrying fluorescent GFP and mCherry reporters under the control of constitutive promoters are visualized in white-red to differentiate them from the green nucleus. The white arrow indicates intracellular bacteria congregated around the nucleus of a living cortical cell. Images were collected at intervals of 1 µm (z axis), depicted by the numbering in the top left corner. Co, cortex; E, epiderm; n, nucleus. Images were processed and prepared with Zen lite 2012 software (Carl Zeiss). Download Movie S3, AVI file, 5.2 MB.

    Copyright © 2015 Koskimäki et al.

    This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

  • Table S1 

    BLAST-based assignments of gene origins, showing the number of hits in each of the following taxonomic categories: non-extorquens Methylobacterium (m), non-Methylobacterium Rhizobiales (r), non-Rhizobiales Alphaproteobacteria, and non-Alphaproteobacteria (n), excluding hits classified as “unclassified sequences” and “other sequences.” Table S1, PDF file, 0.1 MB.

    Copyright © 2015 Koskimäki et al.

    This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

  • Figure S1 

    Maximum-likelihood genome phylogeny of DSM13060 and related species. Download Figure S1, PDF file, 0.2 MB.

    Copyright © 2015 Koskimäki et al.

    This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

  • Figure S2 

    (A) Phylogeny of the RepA protein carried on the strain DSM13060 megaplasmid and homologs in members of the alphaproteobacteria. Red font shows Methylobacterium spp. (B) Synteny between the DSM13060 and AM1 megaplasmids. Colors denote the percentage of identity between orthologous proteins as follows: red, 100%; orange, <100% and >85%; yellow, <85%. Download Figure S2, PDF file, 0.9 MB.

    Copyright © 2015 Koskimäki et al.

    This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

  • Text S1

    Supplemental methods. Download Text S1, PDF file, 0.2 MB.

    Copyright © 2015 Koskimäki et al.

    This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

  • Figure S3 

    Reporter gene analysis of bacteriophytochrome (bphP), ACC deaminase (acdS), and cobalamin synthase (cobS) promoter activity in M. extorquens 13061 during colonization of Scots pine seedlings. M. extorquens strain 13061 with an mCherry reporter controlled by the promoter of the bphP, acdS, or cobS gene was used for laser scanning confocal microscopic analysis. The left image in each panel is a merged image of a pine tissue section infected by bacteria with both tags, and the middle and right images show bacteria carrying the mCherry (white) or GFP tag (green), respectively. In panels A and B, the middle and right images are magnifications of the boxed area in the left image. Cryosections of pine seedlings colonized by M. extorquens 13061 were analyzed 20 to 60 days after inoculation. (A) Cross section of a root displaying activated bphP in M. extorquens 13061 cells during active colonization of the outer cortex. (B) Lateral section of the lower part of a Scots pine shoot with a microcolony of M. extorquens 13061 on the epidermis without clear bphP activation. (C) Cross section of a Scots pine root. Arrows indicate M. extorquens 13061 cells without acdS promoter activity in the cortex and on the epidermis. (D) Cross section of a pine root where arrow shows an M. extorquens 13061 colony penetrating through the root epidermis without visible activity of the cobS promoter. (E) Cross section of a pine stem where arrows depict advancing 13061 colonies in the cortex without cobS promoter activity. Co, cortex; E, epiderm; En, endoderm; n, nucleus; Xy, xylem; scale bars, 10 µm. Download Figure S3, PDF file, 1.9 MB.

    Copyright © 2015 Koskimäki et al.

    This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

  • Figure S4 

    Maximum-likelihood phylogeny of the DotC protein in DSM13060 and homologs from various genera, showing relationship to the Legionella DotC protein from the dot/icm Legionella TFSS. Download Figure S4, PDF file, 0.1 MB.

    Copyright © 2015 Koskimäki et al.

    This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

  • Table S2 

    Strains, plasmids, and primers used in the study. Table S2, PDF file, 0.02 MB.

    Copyright © 2015 Koskimäki et al.

    This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

Additional Files

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    Supplementary Data

    Files in this Data Supplement:

    • Figure sf1, PDF - Figure sf1, PDF
    • Figure sf2, PDF - Figure sf2, PDF
    • Figure sf3, PDF - Figure sf3, PDF
    • Figure sf4, PDF - Figure sf4, PDF
    • Table st1, PDF - Table st1, PDF
    • Table st2, PDF - Table st2, PDF
    • Text s1, PDF - Text s1, PDF
    • Text sm1, AVI - Text sm1, AVI
    • Text sm2, AVI - Text sm2, AVI
    • Text sm3, AVI - Text sm3, AVI
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The Intracellular Scots Pine Shoot Symbiont Methylobacterium extorquens DSM13060 Aggregates around the Host Nucleus and Encodes Eukaryote-Like Proteins
Janne J. Koskimäki, Anna Maria Pirttilä, Emmi-Leena Ihantola, Outi Halonen, A. Carolin Frank
mBio Mar 2015, 6 (2) e00039-15; DOI: 10.1128/mBio.00039-15

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The Intracellular Scots Pine Shoot Symbiont Methylobacterium extorquens DSM13060 Aggregates around the Host Nucleus and Encodes Eukaryote-Like Proteins
Janne J. Koskimäki, Anna Maria Pirttilä, Emmi-Leena Ihantola, Outi Halonen, A. Carolin Frank
mBio Mar 2015, 6 (2) e00039-15; DOI: 10.1128/mBio.00039-15
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