Horizontal gene transfer and mutation: ngrol genes in the genome of Nicotiana glauca

Abstract

Ngrol genes (NgrolB, NgrolC, NgORF13, and NgORF14) that are similar in sequence to genes in the left transferred DNA (TL-DNA) of Agrobacterium rhizogenes have been found in the genome of untransformed plants of Nicotiana glauca. It has been suggested that a bacterial infection resulted in transformation of Ngrol genes early in the evolution of the genus Nicotiana. Although the corresponding four rol genes in TL-DNA provoked hairy-root syndrome in plants, present-day N. glauca and plants transformed with Ngrol genes did not exhibit this phenotype. Sequenced complementation analysis revealed that the NgrolB gene did not induce adventitious roots because it contained two point mutations. Single-base site-directed mutagenesis at these two positions restored the capacity for root induction to the NgrolB gene. When the NgrolB, with these two base substitutions, was positioned under the control of the cauliflower mosaic virus 35S promoter (P35S), transgenic tobacco plants exhibited morphological abnormalities that were not observed in P35S-RirolB plants. In contrast, the activity of the NgrolC gene may have been conserved after an ancient infection by bacteria. Discussed is the effect of the horizontal gene transfer of the Ngrol genes and mutations in the NgrolB gene on the phenotype of ancient plants during the evolution of N. glauca.

Figure 1

(A) Schematic representation of constructs. A restriction map of the NgrolB-NgORF14 region (Top, solid line) is compared with the RirolB-RiORF14 region (Bottom, open box). The various fragments and chimeric constructs are indicated by solid bars (Ngrol genes) and open boxes (Rirol genes). P, PvuI; Sa, SalI; Sm, SmaI; Sp, SpeI; X, XhoI. (B) Promoter and ORF cassettes. Pnb, NgrolB promoter cassette; Prb, RirolB promoter cassette; NB, cassette harboring the coding and 3′ untranslated region of NgrolB; NB212Q242E, NB cassette under the modifications of site-directed mutagenesis; RB, cassette harboring the coding and untranslated region of RirolB; Xb (PCR), new restriction sites for XbaI that were created by PCR. (C) Comparison of the amino acid sequences deduced from the RirolB and NgrolB genes (4, 11). Amino acids encoded by NgrolB that are the same as those encoded by RirolB are not shown. Asterisks indicate termination codons. Arrows indicate the termination codons that were changed by site-directed mutagenesis. The restored region in the amino acid sequence encoded by NgrolB212Q242E is indicated by a box.

Figure 2

Root Induction by Ngrol or Rirol genes. The frequency of root production was determined by calculating the number of segments that formed roots as a percentage of the total number of segments in each flask. Binary vector pMM454-Km was used as the control. (A) Leaf segments were infected with A. tumefaciens harboring constructs of Ngrol and Rirol genes. Sixty leaf segments in ten flasks were used for each construct. Bars indicate SE (n = 10). (B) Leaf segments were inoculated with the constructs of rolB homologues. Fifty-four leaf segments in nine flasks were examined for each construct. Bars indicate SE (n = 9).

Figure 3

RNA gel blot analysis of NgrolB (lanes 1–4) and NgrolC (lanes 5–8) in plant tissues. Lanes 1 and 5, genetic tumor of the hybrids of N. glauca and N. langsdorffii. Lanes 2 and 6, leaf tissue of N. glauca. Lanes 3 and 7, stem tissue of N. glauca. Lanes 4 and 8, callus tissue of N. glauca cultured in MS medium supplemented with 1-mg liter^-1 kinetin. Poly(A)⁺RNA (3 μg for lanes 1–3 and 5–7, and 10 μg for lanes 4 and 8) was subjected to electrophoresis. The probes for analysis of expression of the NgrolB and NgrolC were a 1.1-kbp PstI-HindIII fragment and a 0.8-kbp HincII-EcoRI fragment of pLJ1, respectively (13).

Figure 4

Phenotypes of transgenic plants harboring rolB homologues. (A) A transgenic plant carrying the P35S-NgrolB212Q242E chimeric gene (Left) and a control plant (Right). Bar = 10 cm. (B) Leaves of a NgrolB212Q242E-overexpressing plant. (E) Leaves of a NgrolB-overexpressing plant. (F) Leaves of a RirolB-overexpressing plant. (C) Comparison of floral leaves from transgenic plants. (B, C, E, and F) Bars = 1 cm. (D) Transcripts of the NgrolB212Q242E (lanes 1–6) and NgrolB (lanes 7–9) genes in transgenic plants. Lanes 1–3, T0 transgenic plants; lanes 4–9, T1 plants; lane 10, a control plant. The probe was a 0.8-kbp XbaI-SacI fragment of λNg31. Fifteen micrograms of total RNA per lane was resolved on a formamide gel and blotted onto a nylon membrane.

Figure 5

Comparison of plants transformed with rolC homologues. (A) Whole plants harboring P35S-RirolC chimeric gene (Left) or P35S-NgrolC gene (Center) and a control plant (Right). Bar = 10 cm. (B) Leaves from transgenic plants harboring P35S-RirolC (Left) or P35S-NgrolC (Center) and from a control plant (Right). (C) Abnormal chimeric morphology of leaves from transgenic plants harboring P35S-RirolC gene (Left) or the P35S-NgrolC gene (Center). A normal leaf of wild-type tobacco is shown as a control (Right). (B and C) Bars = 1 cm. (D) Transcripts of the NgrolC in leaf tissues of transgenic plants. Lanes 1–5, T0 plants infected with A. tumefaciens harboring the P35S-NgrolC gene; lane 6, a T1 plant; lanes 7–9, wild-type and control tobacco plants. The probe was a 1.1-kbp EcoRV-EcoRI fragment of λNg31.