Methylation of chloroplast DNA does not affect viability and maternal inheritance in tobacco and may provide a strategy towards transgene containment

Abstract

We report the integration of a type II restriction-methylase, mFokI, into the tobacco chloroplast genome and we demonstrate that the introduced enzyme effectively directs the methylation of its target sequence in vivo and does not affect maternal inheritance. We further report the transformation of tobacco with an E. coli dcm methylase targeted to plastids and we demonstrate efficient cytosine methylation of the plastid genome. Both adenosine methylation of FokI sites and cytosine methylation of dcm sites appeared phenotypically neutral. The ability to tolerate such plastid genome methylation is a pre-requisite for a proposed plant transgene containment system. In such a system, a chloroplast located, maternally inherited restriction methylase would provide protection from a nuclear-encoded, plastid targeted restriction endonuclease. As plastids are not paternally inherited in most crop species, pollen from such plants would carry the endonuclease transgene but not the corresponding methylase; the consequence of this should be containment of all nuclear transgenes, as pollination will only be viable in crosses to the appropriate transplastomic maternal background.

Fig. 1

Chloroplast transformation with the FokI methylase. The transformation vector pFaadMet2 targets transgene insertion to the small single copy region at a site proximal to the ndhF gene (a). Insertion at this site results in an increase in size of 3,278 bp for the BglII fragment containing the ndhF probe sequence (b). Following Southern analysis, the absence of the smaller band derived from the wild type (WT) plastome indicates that all six transplastomic plants are homoplastomic (lanes 1–6)

Fig. 2

Southern analysis indicates that FokI sites are protected from digestion to varying degrees in transplastomic plants. a Digestion with BglII gives ndhF hybridizing fragments of 4,746 bp and 8,024 bp for the wild type (WT) and transplastomic (T) plants respectively. Digestion with both BglII and FokI gives bands of 2,531 and 1,255 bp for wild type but protection of all six FokI sites in the transplastomic plant means that the 8,024 bp fragment is not digested further. b FokI sites within the LSC were assayed using the accD probe indicated. In both wild type and transplastomic plants, digestion with BglII gives a fragment of 2,666 bp. Digestion with both BglII and FokI, gives the two predicted bands of 1,287 and 578 bp for wild type, but protection of the two FokI sites leaves the 2,666 bp fragment unaltered in the transplastomic plant. c FokI sites within the IR were tested using the chIRE probe indicated. Digestion of both wild type and transplastomic cpDNA with BglII gives a single band of 1,685 bp. Digestion of wild type with both BglII and FokI gives a band of 673 (the smaller 414 bp and 223 bp fragments have been run out of the gel). Digestion of transplastomic DNA with both enzymes gives bands of 1,685 (no FokI digestion) and 673 bp (full digestion). A number of additional partial digestion products are evident and their derivation can be explained as follows; 1,310 bp (A + B + C), 1,271 bp (B + C + D), 1,048 bp (C + D), 896 bp (B + C) B BglII, F FokI, WT wild type, T transplastomic

Fig. 3

The FaadMet2 transgene is maternally inherited. Reciprocal crosses were performed between transplastomic FaadMet2 and wild type plants and seedlings were screened for spectinomycin resistance. 100% of germinating seeds were spectinomycin resistant when FaadMet2 was the maternal parent (a) and no spectinomycin resistant progeny were seen when it was the pollen donor (b)

Fig. 4

Southern analysis shows cpDNA from the transgenic dcm methylase plant is resistant to ScrFI cleavage at CCWGG sites. a In the absence of methylation, the rbcL probe (LSC) detects a fragment of 797 bp following digestion with ScrFI (WT lane). Protection of CCWGG sites gives a fragment of 1,810 bp (DCM). b Probe chlA was used to assay eight more CCWGG sites within the LSC. In the wild type, ScrFI digestion gives fragments of 505, 461, 360 and 27 bp (not visible). In the Dcm line, two fragments of 874 and 2,738 bp are generated as a result of protection of all eight CCWGG sites. The presence of additional intermediate bands may indicate partial methylation. c Probe chlB (SSC) hybridizes to a single 1,082 bp ScrFI fragment when the two internal CCWGG sites are methylated in the Dcm plant. In the wild type plants, the absence of CCWGG methylation results in fragments of 731, 290 and 61 bp. d Probe chlRD (IR) detects ScrFI fragments of 424, 364, 317, 311 and 161 bp in wild type cpDNA. In the Dcm plant, methylation of all six CCWGG sites results in two hybridizing bands of 583and 2,151 bp. The presence of additional intermediate bands may indicate partial methylation in some DNA molecules