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

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.

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.

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.