Strain-dependent temperature-sensitive phase in crown gall tumorigenesis

The effect of high temperature treatments on the early stages of crown gall tumorigenesis in sunflowers was investigated. Treatments of 32 C initiated at various times during the first ten days after infection had a similar effect on tumors induced by Agrobacterium tumefaciens strains B₆ and C58. Tumor growth was sensitive to 32 C until 60 to 72 hours after infection and was stimulated by 32 C after that time. Therefore, the “inception phase” for both C58 and B₆-induced tumors ends between 60 to 72 hours after infection. In contrast, B₆ and C58 tumors varied in their response to 37 C treatments during the first 168 hours after infection. Both C58 and B₆ tumors were sensitive to 37 C during the first 72 hours; however, B₆ tumors became resistant to 37 C after 96 hours, whereas C58-induced tumors remained sensitive until 144 to 168 hours after infection.     

Agrobacterium tumefaciens mutants affected in crown gall tumorigenesis and octopine catabolism.

Mutants of Agrobacterium tumefaciens which affect virulence or the ability to catabolize octopine were isolated after Tn5-induced mutagenesis. Of 8,900 colonies tested, 7 mutants with Tn5 insertions in a specific region of other Ti plasmid unable to catabolize octopine were isolated. Thirty-seven mutants affected in tumorigenesis resulted from insertions in the Ti plasmid and the Agrobacterium chromosome. Of these mutations, 12 were chromosomal and 25 mapped on the plasmid. Twenty-three mapped within a 20-megadalton region, which is distinct from the Ti plasmid sequences found stably integrated into the plant cell genome T-deoxyribonucleic acid). Included in these were mutants that were either a virulent or produced tumors with unusual morphologies. Three mutants contained insertions in the T-deoxyribonucleic acid. These three mutants incited tumors which synthesized octopine but had an altered morphology due to either extensive proliferation of shoots or roots from the tumor callus. Three additional mutants not caused by Tn5 contained mutations in the Ti plasmid.     

The host range of crown gall

Crown gall is a plant tumor disease caused by the specific action of the bacteriumAgrobacterium tumefaciens. In the current literature its host range is not clearly defined or is thought to be restricted to the dicotyledonous class of the angiosperms. We reviewed the susceptibility of 1193 species belonging to 588 genera and 138 families; 643 are host plants belonging to 331 genera and 93 families. Our list seems to be so far the most extensive source of information on crown gall susceptibility of plants. We attempted to correlate the susceptibility of plants to crown gall with known and/or presumed taxonomic relationships (according to the taxonomic systems of Engler and Takhtajan). No lower plant is known to be a host for crown gall. About 60% of the gymnosperms and the dicotyledonous angiosperms examined were sensitive for crown gall. In the latter class, there is no significant relationship between the taxonomic position of a plant family and its susceptibility. According to the literature, the susceptible monocots are limited to theLiliales andArales. The common opinion that the host range of crown gall is restricted to the dicotyledonous plants, is thus incorrect.     

Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis

Evidence is presented that crown gall tumors are caused by the incorporation of part of a virulence plasmid carried by the inciting bacterium, Agrobacterium tumefaciens. The rate of reassociation of labeled plasmid DNA was slightly accelerated in the presence of tobacco crown gall tumor DNA, but not normal tobacco DNA. Treatment of tumor DNA with DNAase abolished the acceleration. To determine whether all plasmid sequences are represented in tumor DNA, the labeled plasmid DNA was separated into specific fragments after digestion with restriction endonuclease Sma I. Renaturation rates for DNA from bands 1, 2, 7, 8, 9, 12 and 14 were not affected by tumor DNA. DNA from band 3 showed a slight rate increase in the presence of tumor DNA, indicating 21–27 copies of 14–18% of the DNA sequences in this (doublet) band. The band 3 doublet was separated by electrophoresis into bands 3a and 3b. Tumor DNA had little effect on the rate of reassociation of labeled band 3a DNA. Band 3b DNA renatured rapidly in the presence of tumor DNA, and its rate increase indicated that approximately 18 copies of 40% of band 3b DNA sequences are present per diploid tumor cell. This amounts to 3.7 × 10⁶ daltons of foreign genetic information and represents a contribution of 0.0011% to the DNA content of the tumor cell. The relationship between this plant tumor and virally induced animal tumor systems is discussed.     

Cytokinins in tobacco crown gall tumors

Bacteria-free tobacco ($\text{Nicotiana tabacum}$ cv. Wisconsin #38) crown gall strains incited by $\text{Agrobacterium tumefaciens}$ C58, 27, B6, CGIC, and AT4 have been analyzed for cytokinin content with the tobacco callus bioassay. All tumor strains contained high total levels of cytokinins ranging from 4–810 kinetin equivalents per kg fresh weight compared to 0.5 kinetin equivalents per kg for normal callus growing on medium with 0.1 μM N6-benzyladenine. Fractionation on a column of Sephadex LH-20 separated cytokinin activity from B6 tumors into a number of components among which ribosyl-$\text{trans}$-zeatin has been purified and characterized based on uv spectrum, biological activity and mass spectrum.     

Polyamines and crown gall tumor growth

The effect of the polyamine spermidine on the growth of crown gall tumors was determined using the potato disc bioassay. Addition of lmM spermidine resulted in a 30–50% increase in tumor growth. The spermidine effect was found to be biphasic, with lmM being optimal. Closely related polyamines including spermine, as well as other nitrogen containing compounds such as arginine and alanine, failed to promote tumor growth or inhibited the growth of these tumors. Endogenous levels of spermidine in crown gall tumor tissue were consistently greater than those of corresponding normal potato tissue. Rapidly dividing normal potato tissue derived from buds also contained elevated spermidine levels.     

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.     

The specificity of carrier-mediated auxin transport by suspension-cultured crown gall cells

1. The specificity of the auxin transport system of suspension-cultured crown gall cells from Parthenocissus tricuspidata Planch- is examined with regard to 2,4-Dichlorophenoxyacetic acid (2,4 D), l-Naphthylacetic acid (NAA) and Benzoic acid (BA) as well as for indole-3-acetic acid (IAA). — 2. All four weak acids can be accumulated by the cells from a medium more acidic than the cytoplasm. This is by virtue of non-specific passive diffusion of their lipid-soluble protonated forms down a concentration gradient. The corresponding anionic species are much less permeant. The extent of the accumulation is dependent on the pH difference that is maintained by the cells between their cytoplasm and the incubation medium. Studies of the concentration dependence of BA and NAA net uptake at a series of external pHs suggest that an acidification of the cytoplasm can be eventually brought about by the entry of weak acid into the cells. — 3. The uptake of 2,4 D, as well as that of IAA, has a saturable carrier-mediated component in addition to the passive diffusion of the undissociated acid. These saturable components probably represent anion uptake and appear to be mediated by a common carrier. The kinetic studies provided no evidence for the participation of carriers in the transport of BA or NAA. — 4. It was shown that the efflux of 2,4 D also has a carrier-mediated component and it is suggested that both the influx and efflux of IAA and 2,4 D occur on a common carrier. — 5. The inhibitor of polar auxin transport, 2,3,5-triiodobenzoic acid (TIBA), stimulates the net uptake of IAA by inhibiting carrier-mediated efflux of IAA from the cells. However, TIBA could not be demonstrated to have a significant effect on 2,4 D transport and any perturbation that occurs is very small in comparison with its effect on IAA movement. To account for this, the proposed common carrier could exhibit some difference in its internal binding characteristics betweend 2,4 D and IAA. An alternative explanation is that a second carrier is present, which mediates IAA efflux only, and which is inhibited by TIBA. — 6. TIBA has no significant effect on the transport of either BA or NAA, except to bring about an inhibition of net uptake, and a corresponding stimulation of efflux, when it is present at concentrations sufficient to acidify the cytoplasm. —7. The crown gall cells are compared to intact plant tissues capable of polar auxin transport with regard to the specificities exhibited for the transport of the auxins IAA, 2,4 D and NAA and the non-auxin BA.Abbreviations IAA indol-3-yl acetic acid - 2,4 D 2,4-Dichlorophenoxyacetic acid - NAA 1-Naphthylacetic acid - BA Benzoic acid - TIBA 2,3,5-triiodobenzoic acid     

The potential for biological control of crown gall disease on grapevines

Crown gall is caused by the bacterium Agrobacterium tumefaciens. Non-pathogenic isolates of this species which produce pathogen-specific toxins, called agrocins, have been used successfully as biological control agents on a variety of plants. To date, however, no biocontrol agent has been isolated which can protect grapevines from this economically important disease. A better understanding of the genetic bases of agrocin production and pathogen—host interaction, aided by the application of genetic engineering techniques, may lead to the development of effective control measures.     

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.     

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.     

Hormonal control of tobacco crown gall tumor morphology

The endogenous levels of auxin and cytokinin in teratoma and unorganized tobacco (Nicotiana tabacum L. var Wisconsin #38) crown gall tumor tissues were determined. Teratoma tissues contain levels of auxin and cytokinin favorable for shoot formation, whereas unorganized tumors contain levels of auxin that suppress shoot formation. This conclusion is based upon the observation that when levels of auxin and cytokinin similar to those found in a teratoma were added to the growth medium of nontumorous tobacco tissue, shoot formation resulted; when levels similar to those found in unorganized tumors were added, the normal tissue grew as unorganized callus.     

Ribosylzeatin and zeatin in tobacco crown gall tissue

Cytokinins were extracted from two cultures of tobacco crown gall tumor tissue: an unorganized tissue and a teratoma which produced leafy shoots. On Sephadex LH-20 column chromatography, extracts of both types of tissue yielded two peaks of cytokinin activity with elution volumes similar to ribosylzeatin and zeatin. Ribosylzeatin and zeatin were detected and quantified by coupled gas chromatography — mass spectrometry selected ion monitoring (GC/MS SIM), comparable quantities being found in the two extracts. Full mass spectral evidence for the presence of ribosylzeatin in both tissues was obtained. No evidence was found for the presence of N⁶-(²-isopentenyl)adenosine (i⁶Ade) or N⁶-(²-isopentenyl)adenine (i⁶Ade) although these compounds have been reported to occur in cytokinin-habituated tobacco callus tissues.Abbreviations BAP 6-benzylaminopurine - GC gas chromatography - GC/MS coupled gas chromatography-mass spectrometry - i⁶ Ade N⁶-(²-isopentenyl)adenine - i⁶ Ado N⁶-(²-isopentenyl)adenosine - RFE rotary film evaporation - SIM selected ion monitoring - TLC thin-layer chromatography - TMS trimethylsilyl     

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).     

Membrane phospholipids of normal and crown gall callus cultures

The relative proportions of different phospholipid species were determined in membrane-enriched fractions from normal and crown-gall callus cultures. In general the pattern of phospholipids was similar, but the tumour cultures contained relatively more phosphatidyl choline and less phosphatidic acid and phosphatidyl inositol than the corresponding normal cultures. These differences are probably not caused by phospholipase-D activity during homogenization.     

An electron microscope study of sunflower crown gall tumor

Summary The inception and developmental phases of sunflower (Helianthus annuus) crown gall tumor growth induced byAgrobacterium tumefaciens were studied submicroscopically. Bacteria were found only in the inception phase. Chloroplasts from tumor cells of both phases showed several kinds of structural variation. Other cytoplasmic organelles remained more or less normal.This investigation was supported in part by an American Cancer Society Institutional Grant.