Horizontal transfer of chloroplast genomes between plant species

Abstract

The genomes of DNA-containing cell organelles (mitochondria, chloroplasts) can be laterally transmitted between organisms, a process known as organelle capture. Organelle capture often occurs in the absence of detectable nuclear introgression, and the capture mechanism is unknown. Here, we have considered horizontal genome transfer across natural grafts as a mechanism underlying chloroplast capture in plants. By grafting sexually incompatible species, we show that complete chloroplast genomes can travel across the graft junction from one species into another. We demonstrate that, consistent with reported phylogenetic evidence, replacement of the resident plastid genome by the alien genome occurs in the absence of intergenomic recombination. Our results provide a plausible mechanism for organelle capture in plants and suggest natural grafting as a path for horizontal gene and genome transfer between sexually incompatible species.

Fig. 1.

Grafting and experimental selection for horizontal transfer of chloroplast genomes between different species. (A) Natural graft between an oak (Left) and a birch (Right) in a forest between Potsdam-Golm and Wildpark-West, Germany. The two trees are fused at two different sites. (B and C) Species used for experimental reconstruction of chloroplast capture by grafting. (B) A young tree of N. glauca, the tree tobacco, growing in San Francisco, a village in the northern Argentinian Andes. (C) Nine-week-old plants of N. tabacum (Left), N. glauca (two plants in the middle), and N. benthamiana (Right). Note that the herbaceous species N. tabacum and N. benthamiana flower after 2 mo, whereas the tree tobacco N. glauca is still in its early vegetative growth. (D) Graft of N. tabacum (scion) onto N. glauca (stock) growing under aseptic conditions. The silicon sleeve holds scion and stock together before tissue fusion. (E and F) Selection of putative chloroplast capture lines by exposing stem sections from the graft site to double selection for spectinomycin resistance and kanamycin resistance. Whereas cell division in leaf explants and stem sections from the two grafting partners is fully suppressed (left part of the Petri dish), explants from graft sites frequently give rise to growing calli that are resistant to both antibiotics (right part of the Petri dish; arrows). (E) Selection from a N. tabacum (N.t.)/N. glauca (N.g.) graft. (F) Selection from a N. tabacum (N.t.)/N. benthamiana (N.b.) graft. (G) Plant regeneration from a putative chloroplast capture line selected from a N. tabacum/N. glauca graft.

Fig. 2.

Expression of both fluorescent reporter proteins in the same cell after horizontal chloroplast DNA transfer from N. tabacum. GFP fluorescence, YFP fluorescence, and chlorophyll (Chl) fluorescence and the three pairwise overlays are shown for both wild types, the two grafting partners, and putative chloroplast capture lines. (A) Analysis of two independent lines obtained from N. tabacum (N.t.)/N. glauca (N.g.) grafts. Line KY/SG-08 was selected from a graft with N. glauca as scion and N. tabacum as stock, line SG/KY-24 was selected from a reciprocal graft. In both selected lines, GFP-expressing chloroplasts reside in N. glauca cells that accumulate YFP in the cytosol and the nucleus. (B) Analysis of a putative chloroplast capture line obtained from an N. tabacum/N. benthamiana (N.b.) graft.

Fig. 3.

Detection of the presence of all four transgenes after grafting-mediated chloroplast capture by PCR. (A) Analysis of three independent chloroplast capture lines obtained from N. tabacum (N.t.)/N. glauca (N.g.) grafts. Presence of the aadA, gfp, nptII, and yfp transgenes was tested by PCR assays with gene-specific primer pairs. The two wild types (Wt) and the two grafting partners were included as negative and positive controls, respectively. M, DNA size marker (sizes given in kilobases). (B) Analysis of three independent chloroplast capture lines obtained from N. tabacum (N.t.)/N. benthamiana (N.b.) grafts. Expected sizes of PCR products are indicated at the right of each gel in base pairs. M, DNA size marker (fragment sizes given at the left of the gel in kilobases).

Fig. 4.

Morphology of chloroplast capture plants. (A and B) The phenotype of a chloroplast capture plant selected from a graft between N. tabacum and N. glauca (KY/SG-06) is compared with a N. tabacum plant and a N. glauca plant of similar age. The glauca phenotype of the chloroplast capture plant is already visible early during growth under aseptic conditions on synthetic medium (A) and becomes even more apparent after transfer to soil and growth under greenhouse conditions (B). (C and D) Phenotypic comparison of a chloroplast capture plant selected from a graft between N. tabacum and N. benthamiana (KY/SG-60), a N. tabacum plant and a N. benthamiana plant. The benthamiana phenotype of the chloroplast capture plant is clearly visible both in in vitro culture (C) and upon growth in soil under greenhouse conditions (D).