J. Schell

Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity.

A Ti plasmid mutant was constructed in which all the on‐cogenic functions of the T‐DNA have been deleted and replaced by pBR322. This Ti plasmid, pGV3850, still mediates efficient transfer and stabilization of its truncated T‐DNA into infected plant cells. Moreover, integration and expression of this minimal T‐DNA in plant cells does not interfere with normal plant cell differentiation. A DNA fragment cloned in a pBR vector can be inserted in the pGV3850 T‐region upon a single recombination event through the pBR322 region of pGV3850 producing a co‐integrate useful for the transformation of plant cells. Based upon these properties, pGV3850 is proposed as an extremely versatile vector for the introduction of any DNA of interest into plant cells.

Single genes from Agrobacterium rhizogenes influence plant development

The combined expression of the rol A, B and C loci of Agrobacterium rhizogens Ri‐plasmids establishes, in transgenic tobacco plants, a pathological state called hairy‐root syndrome. However, when expressed separately they provoke distinct developmental abnormalities characteristic for each rol gene. Moreover, changes in their mode of expression obtained by replacing the promoters of the rol B and C genes with the cauliflower mosaic virus 35S promoter elicit new and distinct developmental patterns. These results indicate that the different rol gene products have either different targets, or have a qualitatively different effect on the same target. The target(s) must be involved in the control of plant development. Although each of the three rol genes are independently able to promote root formation in tobacco, efficient root initiation and growth is best achieved through the combined activities of more than a single rol gene. Models explaining the biological effects of A. rhizogenes‐derived TL‐DNA genes are discussed.

Independent and synergistic activity of rol A, B and C loci in stimulating abnormal growth in plants

The Ri plasmid A4 of Agrobacterium rhizogenes contains within its T‐DNA genetic information able to trigger root formation in infected plants. Tobacco plants regenerated from transformed roots display the hairy root (hr) syndrome. We show that DNA fragments containing the rol B locus alone are able to induce root formation both in tobacco and kalanchoe tissues. The rol A and the rol C loci by themselves are also able to induce root formation in tobacco but not in kalanchoe. This capacity to induce root formation in either host is greatly increased when the rol A and/or C loci are combined with the rol B locus. Root induction is shown to be correlated with the expression of the rol loci. Transgenic plants exhibit all the characteristics of the hairy root syndrome only when all three loci are present and expressed. Although the activity of the rol encoded functions is synergistic, each of them appears to independently influence host functions involved in the determination of root differentiation.

Characterization of different plaque-forming and defective temperate phages in Agrobacterium strains

Four Agrobacterium tumefaciens temperate phages (PB2A, PB6(omega), PV-1(LV-1) and PS8), were shown to have the same genome size. Moreover hybridization experiments by the heteroduplex method and electron microscopy showed a 100% homology between these four phage genomes. Indications for lysogeny were found by direct means for the Agrobacterum timefaciens strain 396, Agrobacterium radiobacter strain 8149 and Agrobacterium species 0362 and by the electron microscope negative staining technique for the A.tumefaciens strains b6-806,b6-6,b6s3,b2as,cv-1,4452,11156,11158,396, and 925; for A.radiobacter strains tr-1 and 8149, the latter being bi-lysogenic, and for the A. species 0362. These isolated phage particles, most of which appear to be defective, could be grouped into different classes. No particles could be detected in the lysates of A. tumefaciens RV3, A. radiobacter strains 4718 and S1005, and A. species 0363. Further characterization by genome size was carried out for the defective temperate phages PB6-806, P4452,P8149 and P0362. No evidence for homology between PB6-806 and PB6 omega could be found. The defective phages PB6-806 and P4452 showed the same morphology but a different genome size, whereas the two phages P0362 and P8149 had a very different morphology and genome size.

T-DNA integration: a mode of illegitimate recombination in plants.

Transferred DNA (T‐DNA) insertions of Agrobacterium gene fusion vectors and corresponding insertional target sites were isolated from transgenic and wild type Arabidopsis thaliana plants. Nucleotide sequence comparison of wild type and T‐DNA‐tagged genomic loci showed that T‐DNA integration resulted in target site deletions of 29–73 bp. In those cases where integrated T‐DNA segments turned out to be smaller than canonical ones, the break‐points of target deletions and T‐DNA insertions overlapped and consisted of 5–7 identical nucleotides. Formation of precise junctions at the right T‐DNA border, and DNA sequence homology between the left termini of T‐DNA segments and break‐points of target deletions were observed in those cases where full‐length canonical T‐DNA inserts were very precisely replacing plant target DNA sequences. Aberrant junctions were observed in those transformants where termini of T‐DNA segments showed no homology to break‐points of target sequence deletions. Homology between short segments within target sites and T‐DNA, as well as conversion and duplication of DNA sequences at junctions, suggests that T‐DNA integration results from illegitimate recombination. The data suggest that while the left T‐DNA terminus and both target termini participate in partial pairing and DNA repair, the right T‐DNA terminus plays an essential role in the recognition of the target and in the formation of a primary synapsis during integration.

Genetic identification of functions of TL-DNA transcripts in octopine crown galls

The TL‐DNA in octopine crown galls encodes seven transcripts most, if not all, expressed from individual promoters. Site‐specific deletions and substitutions in the T‐region of the octopine plasmid pTiB6S3 indicate some of the functions of the TL‐DNA transcripts. Two of the seven genes are sufficient to allow tumorous growth. T‐DNA transfer and oncogenicity are controlled by different and independently acting functions. None of the transcripts of TL‐DNA appear to be essential for T‐DNA transfer. Four, possibly five, of the TL‐DNA transcripts act by suppressing organ development. Shoot and root formation are suppressed by the action of different transcripts.

The use of nuclear-encoded sequences to direct the light-regulated synthesis and transport of a foreign protein into plant chloroplasts

The light‐inducible nuclear gene coding for the small subunit of ribulose‐1,5‐bisphosphate carboxylase (Rubisco), produces a precursor protein with an amino‐terminal transit peptide which is transported into the plastids and cleaved by a specific proteinase. To test whether the promoter and transit peptide‐coding sequences of the small subunit gene can be used to direct the light‐inducible synthesis and transport of a foreign protein into chloroplasts, a chimaeric gene was constructed consisting of the promoter, first exon and intron as well as part of the second exon of the small subunit Rubisco gene fused to the amino‐terminal end of the neomycin phosphotransferase II gene, (nptII) of Tn5. Tobacco tissue, as well as whole plants, into which this chimaeric gene was introduced, were resistant to kanamycin. The transcription of the chimaeric gene as well as the NPTII activity of the resulting fusion protein were shown to be light inducible. The fusion protein is processed and located within the chloroplasts of the transformed plants.

Genetic analysis of the individual T-DNA genes of Agrobacterium tumefaciens: further evidence that two genes are involved in indole-3-acetic acid synthesis

SummaryThe T-DNA genes of Ti plasmids of Agrobacterium tumefaciens can induce tumorous growth on a wide range of dicotyledonous plants. We subcloned the individual onc genes of the pTiC58 T-DNA and reintroduced them in the T-region of the Ti plasmid gene vector pGV3850 (from which the onc genes had been removed (Zambryski et al. 1983)). These experiments were designed to analyze the contribution of each onc gene to the development of a tumor and have fulfilled two purposes. First, it was found that only the strains carrying gene 4 produced tumors without the aid of other T-DNA genes; in cell culture these tumors sprout shoots. Second, the shoot-forming phenotype of tumors induced by agrobacteria carrying Ti plasmids defective in either gene 1 or gene 2 can be restored to wildtype phenotype by simple coinfection with Agrobacterium strains whose Ti plasmids contain respectively only gene 2, or only gene 1 in their T-region. A parallel experiment demonstrated that the combined action of genes 1 and 2 is sufficient to induce tumor formation on tobacco plantlets.The external addition of α-naphthalene acetic acid (NAA) restores to wild-type the phenotype of tumors induced by mutants in gene 1 or in gene 2. However, α-naphthalene acetamide can only restore to wild-type the phenotype of mutants in gene 1. These data indicate that the product of the T-DNA gene 2 participates in the conversion of α-naphthalene acetamide to a biologically active auxin, presumably NAA, and suggest that gene 1 codes for an enzyme involved in the synthesis of an indole-3-acetyl derivative.

Transgenic Plants as Tools to Study the Molecular Organization of Plant Genes

Transgenic plants are generated in nature by Agrobacterium tumefaciens, a pathogen that produces disease through the transfer of some of its own DNA into susceptible plants. The genes are carried on a plasmid. Much has been learned about how the plasmid is transferred, how the plasmid-borne genes are organized, regulated, and expressed, and how the bacterias pathogenic effects are produced. The A. tumefaciens plasmid has been manipulated for use as a general vector for the transfer of specific segments of foreign DNA of interest (from plants and other sources) into plants; the activities of various genes and their regulation by enhancer and silencer sequences have been assessed. Future uses of the vector (or others like it that have different host ranges) by the agriculture industry are expected to aid in moving into vulnerable plants specific genes that will protect them from such killers as nonselective herbicides, insects, and viruses.

Physical Identification of Bacteriocinogenic, Nodulation and Other Plasmids in Strains of Rhizobium leguminosarm

Summary: Plasmids obtained from four field isolates of Rhizobium leguminosarum were visualized following electrophoresis on agarose gels. The relative mobilities of the bands observed corresponded to plasmids with molecular weights of about 100 × 106 and greater. Each field isolate examined had a different pattern of plasmids. Lysates from R. leguminosarum strain 300 normally produced three plasmid bands, although in some preparations two much larger plasmids were also visible. The smallest plasmid band seen in strain 300 probably contains two co-migrating plasmids, because in one derivative of strain 300 it was replaced by a doublet, presumably as a result of the presence of a small deletion in one of the co-migrating plasmids. No apparent symbiotic defects were associated with the presence of this deletion. However, a non-nodulating derivative of strain 300 (strain 6015) was found to have suffered a deletion in the third-smallest plasmid of this strain. Transfer of the determinant of medium-sized bacteriocin production pRL1JI (from isolate 248) was correlated with the appearance of an extra plasmid with a molecular weight of about 130 × 106. Another determinant of medium bacteriocinogenicity, pRL4JI (from isolate 309), was correlated with the presence of an extra plasmid of the same size (about 160 × 106) as one seen in the donor strain 309. The third determinant, pRL3JI (from isolate 306), could not be correlated with the presence of an extra plasmid of the same size as any in strain 306, and it appears that recombination occurred between pRL3JI and plasmids resident in strain 300.