Genome structure of Ri plasmid (3). Sequencing analysis of the vir region of pRi1724 in Japanese Agrobacterium rhizogenes.

The entire genome of the pRi1724 (217.6-kb) in the mikimopine type Agrobacterium rhizogenes strain MAFF03-01724 has been completely sequenced. The vir region covering 30.2-kb has found to be composed of 21 genes resembling virH1, virA, virB1-11, virG, virC1-2, and virD1-5. The structural organization of the pRi1724 vir operons in this study is exactly the same as that of the previously reported vir operons of other Ri or Ti plasmids, although the size of some ORFs showed little variations among the plasmids. We also found virE3 gene in the pRi1724 (1), but different from Ti plasmids, virE1 and virE2 that are also important for the virulence do not exist in the vir region of pRi1724.     

Genome structure of Ri plasmid (2). Sequencing analysis of T-DNA and its flanking regions of pRi1724 in Japanese Agrobacterium rhizogenes.

We sequenced 42.6 kb including T-DNA and its flanking regions which corresponds to about 1/5 of entire length of a mikimopine-type Ri plasmid, pRi1724 in A. rhizogenes. We identified 37 ORFs (Open Reading Frames) including genes in total. Among them, 20 ORFs are probably new genes. Those ORFs have similarity with those in Agrobacterium and 9 ORFs of them was newly found on Ri plasmids.     

Expression analysis of the NgORF13 promoter during the development of tobacco genetic tumors

We investigated the expression pattern of the promoter of Nicotiana glauca (Ng) ORF13 in the hybrids between N. glauca and N. langsdorffii harboring the NgORF13-beta-glucuronidase (GUS) chimeric gene. The promoter of NgORF13 of N. glauca had lower activities than the promoter of RiORF13 of Agrobacterium rhizogenes agropine-type root-inducing (Ri) plasmid. However, the localization of GUS activity in the NgORF13 transgenic plants was similar to that in the RiORF13 transgenic plants. The GUS activity of NgORF13-GUS was high in genetic tumors cultured in vitro or developed spontaneously on F1 plants with aging or by wounding. The GUS activity in tumors was observed in bud primordia, vascular bundles and leaves in the buds. While the activity was lower than in tumors, NgORF13-GUS was also expressed in vascular bundles and the parenchymatous tissues in plants regenerated from tumors. Furthermore, the promoter activity of NgORF13 was induced by wounding and activated by exogenous application of methyl jasmonate. During tumorization, NgORF13 was induced at an early stage and showed expression patterns similar to both NgrolB and NgrolC whose expression were investigated by Nagata et al. (1996) Plant Cell Physiol. 37: 489-498. It is thought that Ngrol genes might be involved in the formation of genetic tumors, and, moreover, NgORF13 might work in cooperation with NgrolB and NgrolC.     

Horticultural characterization of<Emphasis Type="Italic"> Angelonia salicariifolia</Emphasis> plants transformed with wild-type strains of<Emphasis Type="Italic"> Agrobacterium rhizogenes</Emphasis>

Genetic transformation was carried out with wild-type strains of Agrobacterium rhizogenes for introducing a dwarf trait into the Scrophulariaceous ornamental plant, angelonia (Angelonia salicariifolia). Leaf segments of two angelonia genotypes (Ang.1 and Ang.2) were co-cultivated with mikimopine-type strains of A. rhizogenes. Adventitious roots that showed vigorous growth and increased lateral branching when cultured on half-strength Murashige and Skoog's (MS) basal salts medium lacking plant growth regulators (PGRs) after co-cultivation were selected as putatively transformed lines. All of these selected lines produced mikimopine. Adventitious shoots were efficiently induced from putatively transformed root segments on half-strength MS basal salts medium containing 1 mg l(-1) benzyladenine (BA) under continuous illumination (24-h photoperiod), and the shoots easily rooted following their transfer to half-strength MS basal salts medium lacking PGRs. The transgenic nature of regenerated plants was confirmed by Southern hybridization. Transformed plants frequently died during their acclimatization, and acclimatized plants of eight transformed lines grew very slowly for 1-5 months after transplantation to the greenhouse. Plants of two transformed lines of Ang.2 flowered 4-6 months after transplantation. These transformed plants exhibited phenotypic alterations such as dwarfness and smaller leaves. There were no apparent alterations observed in the number, shape, and size of the flowers. Pollen fertility of the transformed plants was 60-80% based on aceto-carmine staining. These results indicate the possibility of applying A. rhizogenes-mediated transformation for introducing a dwarf trait into angelonia.     

Analysis of unique variable region of a plant root inducing plasmid, pRi1724, by the construction of its physical map and library.

Ri plasmids in Agrobacterium rhizogenes specifically induce the hairy root syndrome on various dicotyledonous plants. Its T-DNA transfer system as well as those of Ti plasmids have successfully provided the fundamental technique to introduce exogenous genes into plants. To study the Ri genome structure, we constructed a complete BamHI physical map and a lambda library of pRi1724 of A. rhizogenes strain 1724. By using these, we carried out the complete sequence of the 74-kb region between the right border of T-DNA and tra operon, which is the highly variable region (VAR) among Ri and Ti plasmids. As a result, we found three kinds of putative ABC-type transport operons, histidine utilization operon, glycerol utilization operon and two chemoreceptor genes. In addition, a virulence-related gene, tzs was located independently of the vir region.     

Nuclear DNA codes for Nicotiana ferredoxin.

Ferredoxin was purified from 10 species of Nicotiana and spinach leaves. Fingerprints showed all to contain five major tryptic peptides. Some of the spinach peptides were different in RF and mobility from the Nicotiana peptides, but none of the Nicotiana ferredoxins had peptides which could distinguish one species of ferredoxin from another. Electrofocusing S-carbaminomethylcysteinyl ferredoxins showed spinach ferredoxin to have a more acidic and N. glutinosa ferredoxin a slightly more acidic isoelectric point than the other 9 Nicotiana species which were alike. Electro-focusing ferredoxin from the hybrid N. glutinosa female times N. glauca male resolved two bands or isozymes of ferredoxin, one corresponding to N. glutinosa, the other to N. glauca, the code for the latter having come from the DNA in the N. glauca pollen used to form the hybrid plant. N. glutinosa ferredoxin does not contain methionine and is different from N. tabacum and N. glauca ferredoxins which contain methionine. The N. glutinosa female times N. glauca male ferredoxin contained one-half the methionine found in N. glauca ferredoxin, thus confirming that some of the genetic information for ferredoxin in the hybrid was originally contained in the nuclear DNA of N. glauca.     

Homology studies demonstrate colinear organization of the transferred regions of plasmids pRi 1855 and pRi 8196 from Agrobacterium rhizogenes

Agrobacterium rhizogenes, a pathogenic bacterium determining for hairy-root tumors in plants, acts by insertion of a fragment (T-DNA) of its Ri plasmid into the plant nuclear DNA. Two A. rhizogenes strains, pRi 1855 and pRi 8196, responsible for similar disease symptoms, differ when compared at the structural level. However, some morphogenetic loci previously identified by insertion mutagenesis in either one of the two T-DNAs seem physiologically equivalent. The possibility that these morphogenetic loci are structurally similar was tested by cross-hybridization studies. Our data allow establishment of an unequivocal correspondence between the two T-DNA maps where apparently equivalent morphogenic loci occupy similar positions which suggests that the observed structural homologies also reflect physiological similarities between both T-DNAs.     

Mobility and Translocation of Heavy Metals from Mine Tailings in Three Plant Species after Amendment with Compost and Biosurfactant

An experiment was performed to determine the effects of mine tailings alone mixed with compost or with compost plus crude biosurfactant on the accumulation of heavy metals (Pb, Zn, Cu, Cr, Cd, and Ni) in Acacia retinodes, Nicotiana glauca, and Echinochloa polystachya. The plants were grown in soil, mine tailings, and mine tailings containing compost over a period of seven and five months for shrubs or grass, respectively. The plants Acacia retinodes and Nicotiana glauca grown in mine tailings containing compost showed increases in dry biomass (from 62 to 79%) compared with plants in only tailings. Heavy metals accumulated in the roots and leaves showed high translocation rates of Cr in N. glauca, Cd in A. retinodes, and Ni in E. polystachya. Concentrations of heavy metals in the three plants irrigated with crude biosurfactant were not significantly different from those irrigated with water. Zn and Cd fractions within mine tailings containing compost were bound to carbonate, Pb was bound to residues, and Cu was bound to Fe-oxides. Cd had the highest mobility factor followed in order by Zn, Pb, and Cu. The elevated concentrations of Pb in roots and the low translocation rate for N. glauca and A. retinodes indicate that they are suitable for phytostabilizing Pb and Zn.     

A century of tobamovirus evolution in an Australian population of Nicotiana glauca.

The evolution over the past century of two tobamoviruses infecting populations of the immigrant plant Nicotiana glauca in New South Wales (NSW), Australia, has been studied. This plant species probably entered Australia in the 1870s. Isolates of the viruses were obtained from N. glauca specimens deposited in the NSW Herbarium between 1899 and 1972, and others were obtained from living plants in 1985 and 1993. It was found that the NSW N. glauca population was infected with tobacco mosaic tobamovirus (TMV) and tobacco mild green mosaic tobamovirus (TMGMV) before 1950 but only with TMGMV after that date. Half the pre-1950 infections were mixtures of the two viruses, and one was a recombinant. Remarkably, sequence analyses showed no increase in the genetic diversity among the TMGMV isolates over the period. However, for TMV, the genetic diversity of synonymous (but not of nonsynonymous) differences between isolates varied and was correlated with their time of isolation. TMV accumulated to smaller concentrations than TMGMV in N. glauca plants, and in mixed experimental infections, the accumulation of TMV, but not of TMGMV, was around 1/10 that in single infections. However, no evidence was found of isolate-specific interaction between the viruses. We conclude that although TMV may have colonized N. glauca in NSW earlier or faster than TMGMV, the latter virus caused a decrease of the TMV population below a threshold at which deleterious mutations were eliminated. This phenomenon, called Muller's ratchet, or a "mutational meltdown," probably caused the disappearance of TMV from the niche.     

Shoot regeneration from GUS-transformed tomato (Lycopersicon esculentum) hairy root

To study the influence of genetic background on the transformation and regeneration of cultivated tomato plants, hairy root lines of tomato (Lycopersicon esculentum) were obtained by inoculating the hypocotyl explants of three tomato cultivars with the Agrobacterium rhizogenes strain DCAR-2, which harbors the pBI-121 binary vector. The Ri-T-DNA transformation into the plant DNA was confirmed by both of mikimopine and GUS assay analyses. The regeneration efficiency from hairy root explants was assessed. The data indicated that white embryonic calli were formed within two weeks in the presence of 2 mgl(-1) 2, 4-D plus 0.25 mgl(-1) kinetin. Adventitious shoots emerged from the embryonic callus in the presence of 1 mgl(-1) GA3 along with 0.5 mgl(-1) NAA. The regeneration frequency was higher in the cultivar UC-97, followed by Momotaro and then Edkawi. Molecular confirmation of the integration of the GUS gene into the hairy root-derived plants genomes was done via PCR using GUS-specific primers and also using Southern blotting analysis. Our data shows that regeneration is possible from hairy roots of the cultivated tomato and this system could be used to produce transgenic tomato plants expressing the genes present in Agrobacterium rhizogenes binary vectors.     

Sequence of the cellular T-DNA in the untransformed genome of Nicotiana glauca that is homologous to ORFs 13 and 14 of the Ri plasmid and analysis of its expression in genetic tumors of N. glauca x N. langsdorffii

A region homologous to the TL-DNA of Agrobacterium rhizogenes was previously detected in the genome of untransformed Nicotiana glauca and designated cellular T-DNA (cT-DNA). Subsequently, part of this region was sequenced and two genes, which corresponded to rolB and rolC and were named NgrolB and NgrolC, were found. We have now sequenced a region of the cT-DNA other than the region that includes NgrolB and C and we have found two other open reading frames (ORFs), NgORF13 and NgORF14. These ORFs correspond to ORFs 13 and 14 of the TL-DNA of A. rhizogenes and exhibit a high degree of homology to these ORFs, without having a nonsense codon. We have not found any sequence homologous to rolD (ORF15). The two genes, NgORF13 and 14, as well as the NgrolB and C genes, are expressed in genetic tumors of hybrids between N. glauca and N. langsdorffii but not in leaf tissues of the hybrid.     

Molecular evolutionary analysis of the psbP gene family of the photosystem II oxygen-evolving complex in Nicotiana.

Photosystem II psbP protein of the oxygen-evolving complex is involved in the photosynthetic oxygen evolution in plants. Four psbP polypeptides were detected in Nicotiana tabacum on a two-dimensional gel by immunostaining the proteins with antiserum against the pea psbP Comparison of the protein patterns of psbP from N. tabacum and its ancestral parents, N. sylvestris and N. tomentosiformis, indicated that each of the ancestral parents has contributed a pair of psbP proteins. This was supported by Southern hybridization results, which suggested that psbP in Nicotiana is encoded by a gene family consisting of four members in N. tabacum and two members each in N. glauca, N. langsdorffii, N. sylvestris, and N. tomentosiformis. A scheme of molecular evolution of the psbP genes in Nicotiana is also proposed.     

Cloning and morphological properties of Nicgl;CYCD3;1 gene in genetic tumors from interspecific hybrid of N. langsdorffii and N. glauca

Plant genetic tumors represent neoplastic growths, which arise spontaneously in hybrid plants without apparent external induction. To understand the molecular nature of unregulated cell proliferation, a cyclin D cDNA clone encoding a cyclin D of 1104bp was isolated from a genetic tumor and designated Nicgl;CYCD3;1 gene. DNA gel blot analysis suggested that there are two copies of Nicgl;CYCD3;1 in the genetic tumors. Northern analysis showed that this gene had the highest expression level in genetic tumor compared to Nicotiana glauca, N. langsdorffii and hybrid plants. Plant morphology of hybrid plant was an intermediate between N. glauca and N. langsdorffii and was altered in the genetic tumors. The cell cycle distribution in N. glauca was G0/G1, 90.59; S, 0.60; G2/M, 8.81; in N. langsdorffii it was G 0/G1, 86.22; S, 6.90; G2/M, 6.88; in hybrid plants it was G 0/G1, 96.40; S, 1.79; G2/M, 1.81; and in genetic tumors G 0/G1, 74.70; S, 2.35; G2/M, 22.94. These data provide new insights into the molecular mechanisms underlying genetic tumor formation from interspecific hybrid between N. langsdorffii and N. glauca.     

Sequence of the cellular T-DNA in the untransformed genome of Nicotiana glauca that is homologous to ORFs 13 and 14 of the Ri plasmid and analysis of its expression in genetic tumors of N. glauca x N. langsdorffii

A region homologous to the TL-DNA of Agrobacterium rhizogenes was previously detected in the genome of untransformed Nicotiana glauca and designated cellular T-DNA (cT-DNA). Subsequently, part of this region was sequenced and two genes, which corresponded to rolB and rolC and were named NgrolB and NgrolC, were found. We have now sequenced a region of the cT-DNA other than the region that includes NgrolB and C and we have found two other open reading frames (ORFs), NgORF13 and NgORF14. These ORFs correspond to ORFs 13 and 14 of the TL-DNA of A. rhizogenes and exhibit a high degree of homology to these ORFs, without having a nonsense codon. We have not found any sequence homologous to rolD (ORF15). The two genes, NgORF13 and 14, as well as the NgrolB and C genes, are expressed in genetic tumors of hybrids between N. glauca and N. langsdorffii but not in leaf tissues of the hybrid.     

Polypeptide composition of fraction I protein from Nicotiana glauca and from cultivars of Nicotiana tabacum,including a male sterile line

The polypeptide compositions of fraction I protein isolated from six collections of Nicotiana glauca and from ten cultivars of N. tabacum, as well as a polyploid series and a male sterile line, have been analyzed by isoelectric focusing in polyacrylamide gel slabs containing 8 M urea. Apart from the male sterile line, none of the plants showed any variation from the species pattern of polypeptides. Fraction I protein from the male sterile Burley 21 cultivar of N. tabacum contained the two small subunit polypeptides of N. tabacum and the three large subunit polypeptides of an Australian species, probably N. megalosiphon. This indicates a changed chloroplast genome in the male sterile line in comparison to the normal fertile N. tabacum.     

A new vector derived from Agrobacterium rhizogenes plasmids: a micro-Ri plasmid and its use to construct a mini-Ri plasmid

A new binary vector system has been constructed, based on agropine-type root-inducing plasmid (pRi) left transferred-region border sequences cloned in a plasmid containing the replication origin of another A. rhizogenes plasmid (pArA4a). This micro-pRi has been used to introduce a chimeric kanamycin resistance gene into tobacco plants, vir functions being provided by either octopine or nopaline tumor-inducing plasmids deleted of their own transferred regions. In addition, we show that cloning of pRi EcoRI fragment 15, which contains three open reading frames (which may correspond to loci rolA, B and C), in the micro-Ri vector generates a mini-pRi capable of inducing the proliferation of transformed roots.     

Transgenic plants regenerated from hairy roots of Nicotiana benthamiana: a promising host for transient expression of foreign proteins

Hairy root cultures of Nicotiana benthamiana have been obtained by co-cultivation of leaf explants with Agrobacterium rhizogenes strain A4 harboring a binary vector plasmid, and transgenic nature of the obtained cultures was confirmed by PCR analysis. Transgenic plants were regenerated from hairy roots. The biomass yield of transgenic plants grown in vitro was almost two-fold higher than those of wild-type N. benthamiana plants. They differed from untransformed plants by short internodes, reinforced stem, thick and wrinkled leaves and more developed root system. The level of Agrobacterium-mediated transient expression of green fluorescent protein (GFP) in the regenerated plants was similar to that of untransformed plants.     

Expression of Kanamycin resistance introduced byAgrobacteriutn binary vector intoNicotiana tabacum andAtropa belladonna

Kanamycin resistance gene was introduced into tobacco and Atropa belladonna cells by binary vectors, based on Agrobacterium, by means of inoculation of seedlings. The plasmid pGA472, which carries chimaeric kanamycin resistance gene expressed in plants was introduced by transformation into A. tumefaciens Bo542, harbouring pTiBo542 plasmid and A. rhizogenes 8196, carrying pRi8196 plasmids and the resulting two strains were used as binary vectors. Tobacco tumors induced by A. tumefaciens Bo542(pGA472) grew as undifferentiated, kanamycin resistant tissues. Those induced by A. rhizogenes 8196(pGA472) differentiated into transformed plants. When cultivated in vitro on 200 μg ml^-1 kanamycin medium, they showed yellow green sectoring, which was not selected out during vegetative propagation. Atropa belladonna tissues transformed by both A. tumefaciens Bo542(pGA472) and A. rhizogenes 8196(pGA472) differentiated plants which grew well on 200 μg ml^-1 kanamycin as green, non-sectoring plants; sensitive cells obviously did not divide at all. Selection of Atropa belladonna transformed tissues on kanamycin medium is much more efficient than selection of transformed tobacco tissues with introduced kanamycin resistance gene.