Comparison of octopine, histopine, lysopine, and octopinic acid synthesizing activities in sunflower crown gall tissues.

Extracts of sunflower crown gall tissues induced by $\text{Agrobacterium tumefaciens}$ strain B₆ catalyze the synthesis of octopine, histopine, lysopine and octopinic acid. These compounds are not synthesized either in extracts of crown gall tissues induced by strains AT1 and C58 or in extracts of habituated sunflower callus. All four synthetic activities require NADPH or NADH, pyruvate, and the appropriate basic amino acid. Incorporation of radioactivity from any one of the four labeled, basic amino acids into its product is inhibited by the other three basic amino acids. All the reactions are inhibited by ε-aminocaproic acid but none are inhibited by the neutral amino acids alanine and phenylalanine.     

Intracellular accumulation of mannopine, an opine produced by crown gall tumors, transiently inhibits growth of Agrobacterium tumefaciens

pYDH208, a cosmid clone from the octopine-mannityl opine-type tumor-inducing (Ti) plasmid pTi15955 confers utilization of mannopine (MOP) and agropine (AGR) on Agrobacterium tumefaciens strain NT1. NT1 harboring pYDH208 with an insertion mutation in mocC, which codes for MOP oxidoreductase, not only fails to utilize MOP as a sole carbon source, but also was inhibited in its growth by MOP and AGR. In contrast, the growth of mutants with insertions in other tested moc genes was not inhibited by either opine. Growth of strains NT1 or UIA5, a derivative of C58 that lacks pAtC58, was not inhibited by MOP, but growth of NT1 or UIA5 harboring pRE10, which codes for the MOP transport system, was inhibited by the opine. When a clone expressing mocC was introduced, the growth of strain NT1(pRE10) was not inhibited by MOP, although UIA5(pRE10) was still weakly inhibited. In strain NT1(pRE10, mocC), santhopine (SOP), produced by the oxidation of MOP by MocC, was further degraded by functions encoded by pAtC58. These results suggest that MOP and, to a lesser extent, SOP are inhibitory when accumulated intracellularly. The growth of NT1(pRE10), as measured by turbidity and viable cell counts, ceased upon the addition of MOP but restarted in a few hours. Regrowth was partly the result of the outgrowth of spontaneous MOP-resistant mutants and partly the adaptation of cells to MOP in the medium. Chrysopine, isochrysopine, and analogs of MOP in which the glutamine residue is substituted with other amino acids were barely taken up by NT1(pRE10) and were not inhibitory to growth of the strain. Sugar analogs of MOP were inhibitory, and those containing sugars in the D form were more inhibitory than those containing sugars in the L form. MOP analogs containing hexose sugars were more inhibitory than those containing sugars with three, four, or five carbon atoms. Mutants of NT1(pRE10) that are resistant to MOP arose in the zone of growth inhibition. Genetic and physiological analyses indicate that the mutations are located on pRE10 and abolish uptake of the opine.     

A useful synthesis of nopaline,a crown gall tumor metabolite

Nopaline, a reductive conjugate of arginine and α-ketoglutaric acid found in plant tumors incited by $\text{Argobacterium tumefaciens}$, has been synthesized. Three routes were studied. Reduction of L-arginine and α-ketoglutaric acid with sodium cyanoborohydride gave an 80% yield of the diastereomers nopaline and isonopaline. This was by far the best method. The synthetic mixture of diastereomers is capable of replacing purified natural nopaline in biological experiments.     

Phytohormones in the formation of crown gall tumors

Crown gall tumors were initiated in a variety of plant species by infection with Agrobacterium tumefaciens strain B6 and the concomitant changes in the tissue levels of phytohormones, mainly indole-3-acetic acid (IAA) and cytokinins, were analyzed. A comparison was made of these hormones with those produced by virulent and avirulent strains of the bacterium in liquid culture and with those of bacteria-free crown gall callus cultures. Specific radioimmunoassays were employed for hormone determinations. An assay for the quantitation of femto-mol amounts of isopentenyladenosine and related cytokinins was newly developed and is described in detail. The results can be summarized as follows: Virulence in strain B 6 is associated with the ability to release trans-zeatin and increased amounts of IAA into the surrounding environment. In many, but not all plants analyzed, the development of crown gall tumors is also associated with a sharp rise in the levels of trans-zeatin-type zytokinins and IAA (e.g., Euphorbia lathyris, Catharanthus roseus). Crown gall calli growing on hormone-free media varied greatly in their cytokinin levels. In a culture of Nicotiana tabacum, both trans-zeatin and isopentenyladenine or related cytokinins were not detected. Thus, tumor growth cannot be explained on the basis of elevated levels of IAA and/or cytokinins alone.Abbreviations ABA abscisic acid - GC-MS gas chromatography-mass spectroscopy - HPLC high pressure liquid chromatography - IAA indole-3-acetic acid - RIA radioimmunoassay - TLC thin layer chromatography Part 19 in the series Use of immunoassay in plant science     

Regeneration of tobacco plants from crown gall tumors

Summary Tissue culture methods have been developed for regeneration of normal appearing tobacco plants from bacteria-free crown gall strains incited byAgrobacterium tumefaciens C58, IIBV7, B6, CGIC, A6NC, 27, and AT4. Regenerants fall into two categories depending on the properties of tissues from these plants. The first type of regenerant was obtained from tumors incited byA. tumefaciens C58 and it retained the potential for expression of tumor characteristics such as a nonrequirement for phytohormones (auxin and cytokinin) by explants in vitro and the presence of detectable concentrations of nopaline. Normal appearing plants obtained from C58 tumors had much lower concentrations of nopaline than the corresponding tumor tissue (130 versus 1700 μg per g dry wt) indicating a parallel repression of abnormal growth and nopaline concentrations in regenerants. The second type of regenerant was obtained from tumors incited by the otherA. tumefaciens strains and was characterized by requirements for phytohormones by explants in vitro and the apparent lack of octopine or nopaline in regenerant tissues.     

Bioassay and attributes of a growth factor associated with crown gall tumors

An improved bioassay is described for a factor that promotes tumor growth which was first obtained from extracts of pinto bean leaves with crown gall tumors. Sixteen primary pinto bean leaves per sample are inoculated with sufficient Agrobacterium tumefaciens to initiate about 5 to 10 tumors per leaf and treated with tumor growth factor at day 3 after inoculation. The diameters of 30 to 48 round tumors (no more than 3 randomly selected per leaf) are measured per test sample at day 6. Mean tumor diameter increased linearly with the logarithm of the concentration of tumor growth factor applied. The tumor growth factor was separated by column chromatography from an ultraviolet light-absorbing compound previously reported to be associated with fractions having maximal tumor growth factor activity. Partly purified tumor growth factor showed no activity in a cytokinin bioassay or an auxin bioassay, and negligible activity in gibberellin bioassays. Representatives of these three classes of growth factors did not promote tumor growth. Extracts from crown gall tumors on primary pinto bean leaves, primary castor bean leaves, Bryophyllum leaves, carrot root slices, and tobacco stems showed tumor growth factor activity, whereas extracts from healthy control tissues did not. Extracts from actively growing parts of healthy pinto beans, Bryophyllum, and tobacco, however, showed tumor growth factor activity. Tumor growth factor is proposed to be a normal plant growth factor associated with rapidly growing tissues. Its synthesis may be activated in nongrowing tissues by infection with Agrobacterium sp.     

Comparison of hairy root and crown gall tumors ofArabidopsis thaliana

The phenotype appearance ofArabidopsis thaliana hairy roots and crown galls their teratomas and regenerated plants were compared. Several differences were found, which correlate with T-DNA differences between Ti and Ri plasmids. Presented at the International Symposium “Plant Growth Regulators” held on June 18–22, 1984 at Liblice, Czechoslovakia.     

Induction and in vitro culture of soybean crown gall tumors

Induction of crown galls on 4–6 week old soybean (Glycine max (L.) Merr.) plants cultured in growth chambers was obtained with Agrobacterium tumefaciens strains C58, T37 and ACH5. The crown galls were isolated and cultured in vitro as sterile callus and liquid suspension cultures. Transformation was tested by opine tests and by molecular hybridization of restricted cell DNA with T-DNA fragments. Protoplasts were isolated from suspension cultures. Transformed protoplasts regenerate cell walls, divide and form calli without an exogenous supply of hormones.     

Aeromonas, a possible etiological agent in the formation of secondary tumors of crown gall

Abstract From a secondary tumor in a bean stem we have isolated a Gram-negative bacteria, named by us T.2. These bean stems had crown gall tumors induced by the ATV strain of Agrobacterium tumefaciens . This bacterium was classified as belonging to the genus Aeromonas and possesses the capacity of inducing overgrowths in plants, synthesizing indole acetic acid (IAA). The codified phenotypic characteristics of bacterium T.2. via the Ti-plasmid of A. tumefaciens , such as opine utilization and sensitivity to agrocin 84, have been studied. Neither octopine nor nopaline is utilized by T.2. and it is resistant to agrocin 84, whereas the strain ATV of A. tumefaciens utilizes nopaline, and is sensitive to agrocin 84.     

A new amino acid derivative present in crown gall tumor tissue

A new amino acid derivative has been found in primary and secondary sunflower crown gall tissue cultures and in fresh crown gall tumors from sunflower plants wound-inoculated with Agrobacterium tumefaciens B₆. Normal plant tissue does not contain detectable levels of the compound. Radioactive labeling and cochromatography experiments strongly suggest that the natural derivative is identical to synthetic N²-(1-carboxyethyl)-L-histidine (histopine). Crown gall tissue cultures contain 1 μmole of histopine/20 g fresh weight. A. tumefaciens strain B₆, but not strain C₅₈, can utilize natural histopine and incorporate the products into macromolecules.     

Absolute stereochemistry of leucinopine,a crown gall opine

Crown gall tumors incited by Agrobacterium tumefaciens strain Bo542 have been reported to synthesize a tumor-specific substance identified as N-(1,3-dicarboxypropyl)-leucine (leucinopine), a compound with two centers of asymmetry. We report here evidence that leucinopine is indeed a crown gall opine, in that it is specifically catabolized by A. tumefaciens strains carrying the tumor-inducing plasmid pTi Bo542, as well as strains carrying closely related plasmids pTi AT1 and pTi AT4. We further report catabolism of leucinopine by the succinamopine-type strains A518, A519 and A532, carrying pTi EU6, pTi AT181 and pTi T10/73, respectively. Strains lacking any virulence plasmid, as well as those carrying octopine or nopaline type Ti plasmids or mannopine type Ri plasmids, did not catabolize leucinopine. On the basis of specificity of catabolism by bacteria carrying pTi Bo542, we conclude that the stereochemistry of natural leucinopine is l-threo, i.e. l^glu,l^leu. Such stereochemistry is novel in the opines known thus far: octopine, nopaline and succinamopine have d,l-stereochemistry: d^ala,l^arg (octopine), d^glu,l^arg (nopaline) and d^glu,l^asn (succinamopine).     

Highly tumorigenic Agrobacterium tumefaciens strain from crown gall tumors of chrysanthemum

A wild-type Agrobacterium tumefaciens strain CNI5 isolated from crown gall of chrysanthemum (Dendranthema grandiflora Tzvelev) was characterized. Strain CNI5 was classified into biovar 1, based on physiological and biochemical characteristics, and was resistant to ampicillin. Strain CNI5 induced tumors at a higher frequency and on a larger area of explants in most tested plant species, especially in chrysanthemum cultivars, than the octopine-type strain C58C1cmr (pTiB6S3). Agropine and mannopine were detected in tumors induced by strain CNI5 and were specifically catabolized by this strain. Strain CNI5 harbored five plasmids including one plasmid that shared sequence similarity to TL-DNA of the octopine-type Ti plasmid and four cryptic plasmids.     

Succinamopine: a new crown gall opine

Agrobacterium tumefaciens strains can incite plant tumors consisting of transformed cells that synthesize novel metabolites called opines. The pattern of opine synthesis is dictated by plasmid-borne genes in the pathogen; additional plasmid genes confer on the pathogen the ability to catabolize the same pattern of opines synthesized. One group of A. tumefaciens strains, AT181, EU6, and T10/73, contains closely related tumor-inducing (Ti) plasmids that encode the ability to degrade the opine nopaline; but tumors incited by these strains do not synthesize nopaline. We demonstrated by Southern blot hybridization that AT181(pTi) has no DNA homologous to the nopaline synthase gene of pTi T37, a nopaline Ti plasmid that appears to be most closely related to this group based on fingerprint analysis. Tumors incited by these seemingly anomalous strains contain a new opine that we designate succinamopine. Its structure is analogous to that of nopaline, with asparagine replacing arginine. Evidence for the structure of succinamopine, as well as those of two related metabolites, succinamopine lactam and succinopine lactam, will be published elsewhere. Ability to catabolize succinamopine, succinamopine lactam, and succinopine lactam is encoded by pTi AT181, pTi EU6, and pTi T10/73, but not by any of 15 other Ti and root-inducing plasmids tested. Three avirulent strains tested did not catabolize succinamopine, succinamopine lactam, or succinopine lactam. We propose that pTi AT181, pTi EU6, and pTi T10/73 be designated the succinamopine Ti plasmids.     

Biological control of crown gall, Agrobacterium tumefaciens

Biological control of crown gall caused by Agrobacteriurn turnefaciens (Smith & Townsend) Conn, pioneered by Dr A. Kerr in South Australia, is effected through the establishment of a high population of the related non-pathogen A. radiobacter (Beijerinck & van Delden) Conn, strain 84 in the rhizosphere of susceptible plants. Strain 84 produces a bacteriocin to which many strains of the pathogen in Australasia, North America and Britain are sensitive. The disease is present in Britain on a variety of hosts including cherry. At East Malling cherry leaf scars, invaluable as an avenue of infection for bacterial canker infectivity titrations, have been used successfully in crown gall studies. Live cells of strain 84, but neither an avirulent strain of the pathogen nor a soil bacterium highly antagonistic to A. tumefaciens in vitro, inhibited gall formation in cherry leaf scars. Heat-killed cells had no effect. In a field experiment at East Malling hardwood cuttings of the new rootstock Colt have been dipped in strain 84 and total inhibition of crown gall is expected to ensue. The results of other experiments where the disease is already established on cherry-rootstock layer-beds and in blackberry plantations are less predictable. In time we hope to solve this problem. Only time will show whether this method of biological control is long lasting or will eventually break down.     

Enterobacter cloacae, not Aeromonas species, a possible etiological agent in the formation of secondary tumors of crown gall

Abstract A novel aminotransferase catalysing the first step of lysine catabolism, the oxidative transamination of the ϵ-group of L -lysine, was found and characterised in the yeast Pichia guilliermondii . The enzyme, L -lysine : pyruvate aminotransferase (Lys-AT), is strongly derepressed in cells grown on L -lysine as sole nitrogen source and its activity is highly specific for both L -lysine and pyruvate. We could successfully isolate a regulatory mutant which is unable to use lysine as sole nitrogen source based on its inability to derepress the Lys-AT.