Plant genome complexity may be a factor limiting in situ the transfer of transgenic plant genes to the phytopathogen Ralstonia solanacearum

Abstract

The development of natural competence by bacteria in situ is considered one of the main factors limiting transformation-mediated gene exchanges in the environment. Ralstonia solanacearum is a plant pathogen that is also a naturally transformable bacterium that can develop the competence state during infection of its host. We have attempted to determine whether this bacterium could become the recipient of plant genes. We initially demonstrated that plant DNA was released close to the infecting bacteria. We constructed and tested various combinations of transgenic plants and recipient bacteria to show that the effectiveness of such transfers was directly related to the ratio of the complexity of the plant genome to the number of copies of the transgene.

FIG. 1

(A) Light microscope observations of a longitudinal section of tomato xylem tissues infected by R. solanacearum. Bacteria were mainly inside the tracheids and associated parenchyma cells, some of which still contained a cytoplasm and a nucleus. (B, C, D and E) Transmission electron microscope views of tomato parenchyma cells infected by R. solanacearum. (B) R. solanacearum bacteria in a parenchyma cell, the contents of which were necrotic but which still had a recognizable nucleus, are shown. (C) R. solanacearum bacteria are shown in differentiating vascular tissues; an organelle could be identified as a degraded nucleus. (D) Heavy infection of a parenchyma cell, which still contained an organelle that could be a nucleus, is shown. (E) R. solanacearum bacteria are shown in the extraplasmatic space of a plasmolysed parenchyma cell containing living organelles, such as a chloroplast, mitochondria, and a nucleus. Abbreviations: b, bacteria; c, chloroplast; ep, extraplasmatic space; m, mitochondria; n, nucleus; pm, plasma membrane; pn, putative nucleus. Bars indicate lengths of 10 μm (A), 5 μm (B, C, and D), and 1 μm (E).

FIG. 2

Physical map of the T-DNA of plasmid pZpop1 used to transform plants. The gentamycin resistance gene aacC3-IV that was extracted from pUC1813AM/Gm (9) after a SalI digest was cloned into the XhoI site of the pFB2 plasmid to give pFB21. A SacII cassette containing the antibiotic resistance genes flanked by 719 and 907 bp of the popA gene from the R. solanacearum GMI1000 strain was cloned into the SmaI site of the binary vector pPZP212 (17). LB and RB are the left and right borders of the T-DNA.

FIG. 3

Construction of the pFB3 plasmid containing a defective T-DNA. The BglII-BglII T-DNA fragment from pBin19 (6) was ligated into the BamHI linearized pBluescript vector to create plasmid pT-DNA. This plasmid was digested with NcoI and BalI to generate a deletion of 354 bp within the nptII gene before being ligated to the gentamycin resistance cassette resulting from the digestion of pMGm with NcoI-SmaI to give plasmid pΔT-DNA. The SacII-SacII popA gene that was recovered from pKSpopA (1) was ligated into a SacII linearized pBluescript whose BamHI restriction site was deleted previously to give plasmid pFB1. pFB1 was modified by the ligation of the BamHI-BglII aad gene (40) into the unique BamHI site of the popA gene, creating pFB2 in order to provide suitable conditions for the integration of this defective T-DNA. Plasmid pFB3 was constructed by cloning the 5.5-kb SpeI-XhoI fragment of defective T-DNA from pΔT-DNA into the SpeI-XhoI sites of the vector pFB2.

FIG. 4

Influence of the concentration of pZpop1 on the transformation frequencies of R. solanacearum GMI1000. R. solanacearum GMI1000 cells were transformed with 100, 10, 1, 0.1, and 0.01 ng of a pure pZpop1 solution (open squares). Transformations were also conducted with the same amounts of plasmid diluted in 5 μg of wild-type tomato DNA (solid circles). The open circles indicate transformation frequencies below the detection limit (dotted line). Error bars show standard deviations of triplicate experiments. The mean value symbols from three replicate experiments occasionally obscure the smaller standard error bars.